WO2014131934A1 - Combined genetic polymorphisms of ldlr and il28b for predicting response to treatment with pegylated interferon plus ribavirin in patients infected with the hepatitis c virus - Google Patents

Combined genetic polymorphisms of ldlr and il28b for predicting response to treatment with pegylated interferon plus ribavirin in patients infected with the hepatitis c virus Download PDF

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WO2014131934A1
WO2014131934A1 PCT/ES2014/070158 ES2014070158W WO2014131934A1 WO 2014131934 A1 WO2014131934 A1 WO 2014131934A1 ES 2014070158 W ES2014070158 W ES 2014070158W WO 2014131934 A1 WO2014131934 A1 WO 2014131934A1
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hcv
treatment
genotype
ldlr
response
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PCT/ES2014/070158
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Spanish (es)
French (fr)
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Karin Isolde NEUKAM
Antonio José Caruz Arcos
Luis Miguel REAL NAVARRETE
Juan Antonio PINEDA VERGARA
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Servicio Andaluz De Salud
Universidad De Jaen
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Priority claimed from ES201330294A external-priority patent/ES2490492B1/en
Priority claimed from ES201330292A external-priority patent/ES2502017B1/en
Application filed by Servicio Andaluz De Salud, Universidad De Jaen filed Critical Servicio Andaluz De Salud
Publication of WO2014131934A1 publication Critical patent/WO2014131934A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention is within medicine and molecular biology, and refers to a method of obtaining useful data to predict the response to treatment with pegylated interferon plus ribavirin, of hepatitis virus infection. C (HCV), and specifically, the use of the low density lipoprotein receptor (LDLR) gene.
  • C hepatitis virus infection
  • LDLR low density lipoprotein receptor
  • the viral replication cycle is affected by cholesterol and fatty acid biosynthesis.
  • Infection with HCV increases lipid biosynthesis and its storage in hepatocytes, contributing to HCV-associated steatosis, hypobetalipoproteinemia and insulin resistance.
  • Inhibition of cholesterol production by blocking HMG-CoA reductase or 3 beta-hydroxysteroid-delta 7-reductase induces a marked reduction in HCV RNA levels in vitro.
  • the HCV virus assembly is closely linked to the synthesis of very low density lipoproteins (VLDL) and its secretory pathway.
  • VLDL very low density lipoproteins
  • the HCV particles in serum are complexed with VLDL; Although the exact molecular structure is not known, indirect data suggests that HCV can hide in the lipid nucleus of VLDL, making HCV undetectable to antibodies and serum complement. This association with VLDL allows the virus to bind to target cells through lipoprotein receptors, after partial processing by hepatic lipoprotein lipase, which hydrolyzes triglycerides in VLDL resulting in IDL and LDL.
  • LDLR LDL receptor
  • HCV RNA in primary hepatocytes is related to the expression of LDLR mRNA and the efficiency of LDL uptake.
  • Soluble LDLR can inhibit HCV infectivity.
  • Higher plasma LDL levels are associated with sustained virologic response (VS) after treatment with pegylated interferon (Peg-IFN) plus ribavirin (RBV), probably due to competitive blockage of HCV entry.
  • Peg-IFN pegylated interferon
  • RBV ribavirin
  • LDLR polymorphisms which are associated with plasma LDL levels, have a synergistic impact on the likelihood of achieving SVR with Peg-IFN and RBV in patients infected with viral genotypes 1-4, as well as in kinetics viral after treatment.
  • LDLR has a greater role in the post-entry phases of the viral life cycle, probably increasing uptake, an essential factor for HCV replication. Additionally, changes in the intracellular distribution of cholesterol from the plasma membrane to the membranous network can disrupt the rafts of cholesterol-rich lipid surface, leading to repression of the JAK-STAT signaling pathway regulated by interferon. In the case of West Nile virus, the exogenous addition of cholesterol can rescue interferon signaling and restrict viral replication.
  • the authors of the present invention have evaluated the association of polymorphisms in cholesterol synthesis genes, as well as their transport pathways, in the sustained post-treatment viral response and HCV viral load.
  • the present invention provides a method of obtaining useful data for predicting or predicting the sustained viral response (VS) after treatment with pegylated interferon plus ribavirin for HCV.
  • the authors of the present invention have found a biomarker useful for predicting the response to hepatitis C treatment.
  • a first aspect of the invention relates to the use of LDLR gene polymorphisms to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV.
  • Another aspect of the invention relates to the use of any of the polymorphisms (SN Ps) that are selected from: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099, rs6413504, or any combination thereof, to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV.
  • SN Ps polymorphisms
  • a preferred embodiment relates to the simultaneous use of polymorphisms (SNPs) rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504 to predict or predict the response to treatment with pegylated interferon plus ribavirin infected patients.
  • SNPs polymorphisms
  • the response to treatment is the sustained virological response (SVR).
  • SVR sustained virological response
  • the HCV genotype is selected from: genotype 1, genotype 2, genotype 3, genotype 4, or any combination thereof.
  • the LDLR gene is located on the small arm of chromosome 19, and contains 18 exons encoding the six functional domains of the mature protein: signal peptide, ligand binding domain, epidermal growth precursor type (EGF), transmembrane and cytoplasmic domains .
  • Antiviral therapy is basically aimed at achieving virological, biochemical and histological objectives.
  • the evaluation of the response to the treatment is based on the effect of the same on the virological state, not only at the end of the treatment but, more importantly, in its follow-up after the end of the treatment.
  • it is called "sustained virological response” or “sustained viral response” (SVR) viremia negativization (viral RNA negative by polymerase chain reaction techniques -PC -) that is maintained at least 24 weeks after the end of treatment; In other words, the absence of HCV-RNA in the serum 6 months after the end of treatment.
  • the absence of viremia negativization at the end of treatment defines the "virological non-response" to the treatment.
  • Interferon alfa is an immunomodulatory cytokine that has antiviral activity, antiangiogenic and antiproliferative properties.
  • IFN-Peg is obtained by the physicochemical binding of polyethylene glycol (PEG) to recombinant interferon alfa (peginterferon a-2a and a-2b), which increases its permanence in the blood requiring a lower frequency of injections.
  • PEG polyethylene glycol
  • peginterferon alfa-2a Pulsys ® , Roche
  • peginterferon alfa-2b Pegintron ® , Schering-Plow
  • Ribavirin ⁇ - ⁇ -D-ribofuranosyl-lH-l, 2,4-triazol-3-carboxamide
  • Ribavirin is a synthetic nucleotide analogous to guanidine, with antiviral properties and immunoregulatory activity. It was one of the first drugs that demonstrated its effectiveness in reducing the ability to reproduce viruses.
  • Viramidine is a prodrug of RBV, in the liver it is converted to RBV by the action of hepatic adenosinedeaminase.
  • RBV and viramidine are rapidly eliminated, their molecules and metabolites are excreted by the kidney and have a Tmax of 1.5-3 h.
  • hepatic retention of RBV derived from an oral dose of viramidine is 3 times higher than oral RBV.
  • the concentration of RBV phosphates in red blood cells is much lower, hence it can be maintain a more stable concentration of hemoglobin. It is a safe drug and is well tolerated.
  • the adverse effects with doses of 200, 600 and 1,200 mg were: 0, 26 and 50%, respectively; mostly mild and disappeared without sequelae.
  • Levovirin is the L-enantiomer of BV and does not cause hemolytic anemia. It has an immunomodulatory activity similar to RBV, but it has no direct antiviral activity.
  • Ribavirin alone is not effective against HCV, but when combined with interferon, viral eradication rates are much better compared to those achieved only with interferon.
  • the viral load is the amount of HCV RNA in a milliliter of blood.
  • HCV RNA genetic code
  • Quantitative viral load tests measure the amount of virus in a milliliter of blood. They are generally used to assess whether interferon or interferon plus ribavirin treatment will have a chance of succeeding and then, if the treatment is working.
  • PCR Polymerase chain reaction
  • bDNA Branched chain of DNA
  • TMA Transcription mediated amplification
  • the "HCV genotype”, or "viral strain” is determined by a blood test.
  • the hepatitis C virus has been classified into six different genotypes based on differences in their genomes, these genotypes in turn are subdivided. The preponderance and distribution of these genotypes varies globally. For example, in North America, genotype 1 predominates, followed by 2 and 3. In Europe, genotype 1 predominates, followed by 3, 4 and 2. Genotype 4 predominates in Egypt, 5 occurs almost exclusively in Africa, and genotype 6 in the Southeast Asian.
  • sustained virological response or “sustained viral response” (VS) the viremia negativization (negative viral RNA by polymerase chain reaction techniques -PCR-) that is maintained at least 24 weeks after the end of treatment; In other words, the absence of HCV-RNA in the serum 6 months after the end of treatment.
  • nucleotide sequences of the polymorphisms are as follows: rs2738456
  • Another aspect of the invention relates to the simultaneous use of the LDLR and IL28B genes to predict or predict the viral load of the individual. METHOD OF OBTAINING USEFUL DATA, METHOD OF FORECAST AND PREDICTION OF THE SUSTAINED VIRAL RESPONSE.
  • Another aspect of the invention relates to a method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV, hereinafter the first method of the invention, comprising: a) obtaining an isolated biological sample from an individual, and b) detect the genetic polymorphisms: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504 of the LDLR gene, in the isolated biological sample of (a).
  • the response to treatment is the sustained virological response of HCV.
  • the first method of the invention further comprises measuring the viral load of HCV in an individual.
  • it further comprises determining the polymorphism of the IL28B gene.
  • the polymorphisms of the IL28B gene are rsl2979860 and / or rs8099917.
  • nucleotide sequences of the polymorphisms are: rsl2979860 CTGAACCAGGGAGCTCCCCGAAGGCG [C / T] GAACCAGGGTTGAATTGCACTCCGC rs8099917
  • the LDL receptor (LDLR) can act as a co-receptor for HCV.
  • the authors of the present invention have determined that different genotypes of the LDLR gene can influence the viral load of HCV and SVR, such as for example the C allele of SN P rs2738459 of LDLR is a risk factor and is related to a greater VL in HCV genotypes 2-3. Certain genotypes of this gene in Combined genotypes of the IL28B gene, they also have an influence on the sustained viral response.
  • LDLR and IL28B genotypes showed a synergistic effect on SVR in patients infected with HCV genotypes 1-4, when homozygous individuals carrying protective IL28B genotypes were classified according to LDLR rs2738457 genotypes, the frequency of SVR it was 0.73 for AA versus 0.26 in AG + GG.
  • Another aspect of the invention relates to a method for predicting or predicting the sustained virological response of HCV, hereafter referred to as the second method of the invention, comprising steps (a) and (b) of the first method of the invention, and It also includes: c) classifying individuals according to the LDLR genotype they present and their sustained virological response of HCV to treatment with pegylated interferon plus ribavirin.
  • this method further comprises: d) classifying individuals according to the LDLR genotype and their HCV viral load.
  • step (b) of any of the methods of the invention further comprises determining the genotype of the IL28B gene.
  • genotyping is determined by PCR.
  • haplotype analysis is used.
  • steps (b) and / or (c) and / or (d) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the amount in step (b ) or the computerized comparison in steps (c) and (d).
  • isolated biological sample includes, but is not limited to, cells, tissues and / or biological fluids of an organism, such as blood, serum, urine, saliva, cerebrospinal fluid, or a solid or semi-solid sample, such as tissues, feces, and similar, or alternatively, a solid tissue such as those commonly used in histological diagnosis, obtained by any method known to a person skilled in the art.
  • genotyping SNPs there are more than 100 different methods of genotyping SNPs, whichever is applicable in the present invention. For example, without limiting our, it can be detected using restriction enzymes or by simple sequencing by PCR, ASO probes and hybridization in DNA microarrays or spheres. So, for example, as amplification methods of SN Ps are PC, Invader method, amplification by rolling circle DNA, etc. As allelic discrimination methods, allele-specific hybridization, allele-specific oligonucleotide ligation, probe mini-sequencing or extension (SBE, Single Base Extension), allele-specific invasive breakage (Invader), etc. can be used, but not limited to.
  • a haplotype is a set of single nucleotide polymorphisms (SN Ps) on a particular chromosome that are physically associated inheriting as a block with very little or no genetic recombination between them. The genetic analysis association of haplotypes happens to be a powerful strategy towards the discovery of genes that predispose to complex human diseases.
  • the term “individual” is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
  • kit or device of the invention comprising the elements necessary to analyze the genotype of the LDLR gene.
  • kits may contain all those reagents necessary to analyze the genotype of the LDLR gene by means of any of the methods described hereinbefore.
  • the kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit further comprises instructions for carrying out any of the methods of the invention.
  • the kit or device comprising one or more nucleotide sequences necessary to detect the polymorphisms of the LDLR gene. More preferably the polymorphisms of the LDLR gene are selected from: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099, rs6413504, or any combination thereof. It is also possible that the oligonucleotide (s) or probes are immobilized in spots on a (preferably solid) surface. In one of its embodiments, the kit comprises a microarray, or microarray of the invention.
  • An AN microarray is a matrix on a solid substrate (usually a glass holder or a cell of a thin silicon film) that evaluates large amounts of different RNAs that are detectable by specific probes immobilized on spots on a solid substrate.
  • Each stain contains a specific nucleic acid sequence, which normally corresponds to a specific DNA sequence.
  • the microarray is customized for the methods of the invention. Therefore, another aspect of the invention relates to a microarray, hereafter referred to as a microarray of the invention, comprising at least one probe for detecting the genotype of the LDLR gene.
  • oligonucleotide sequences are constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography.
  • the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask.
  • the photomask can be physical or virtual.
  • oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides.
  • oligonucleotides per gene are preferably used.
  • Synthesis in situ on a solid support could be done using ink-jet technology, which requires longer probes.
  • the supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy.
  • the probe is each of the chip samples.
  • the target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
  • the kit or device, and / or the microarray further comprises one or more probes necessary to detect the haplotype of the IL28B gene. It preferably comprises the probes necessary to detect the rsl2979860 and rs8099917 polymorphisms.
  • kits or devices, the microarray, or the microarray of the invention to predict or predict the response, in patients infected with HCV, to treatment with Pegylated interferon plus ribavirin.
  • the kit or device, the microarray or the microarray further comprises the ability to predict or predict the viral load of HCV.
  • the individual is infected by HCV genotypes 1, 2, 3 or 4, 15 or any combination thereof.
  • Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).
  • Another aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
  • amino acid sequence referring to polymeric forms of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified
  • Fig. 1 Gene structure of the LDLR gene and pattern of linkage imbalance of the analyzed population.
  • Fig. 1 Gene structure of the LDLR gene and pattern of linkage imbalance of the analyzed population.
  • FIG. 2 Comparison of VL between patients with LDL polymorphisms rsl433099 (left side) and rs2569549 (right side).
  • Fig. 5 Association between HCV VL and the presence of 1 or 2 copies of the AGGCGGAG extended haplotype of SNPs rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rs6413504, rsl4158 in patients infected with genotypes.
  • Fig. 7 Association between the compact LDLR haplotype that includes SNPs rs2738456, rs2738457, rs2569540, rs2738459 and the IL28B genotype with SVR.
  • a) All IL28b genotypes (p 0.016).
  • c) risk genotypes of IL28B (p 0.055). Fisher exact test.
  • Study population A population of 442 patients infected with HCV were subsequently recruited into infectious disease units of 5 hospitals in Spain (Seville, Cordoba, Madrid, Malaga and Huelva) from May 2000 to December 2010. All of them were without treatment. HCV Clinical and laboratory checks at the initial visit and blood samples and / or plasma were collected and stored at -70 Q C, for subsequent genetic, biochemical and virological determinations they were developed. 239 patients were infected with HCV genotype 1, 62 with genotype 4, 141 with genotype 3, and one individual with genotype 2. During treatment, patients were evaluated every 4 weeks during the first 24 weeks of treatment, every 8 12 weeks later, including a visit 24 weeks after completing therapy to evaluate the sustained virological response (VS). Clinical and laboratory checks each visit were performed, and samples of blood and / or plasma were collected and stored at -70 Q C for further genetic and virological determinations.
  • Serum HCV antibodies were determined by an enzyme immunoassay (EIA) (ADVIA Centaur XP, Siemens Healthcare Diagnostics S. L. Tarrytown, NY, USA).
  • EIA enzyme immunoassay
  • the HCV RNA plasma load was measured by quantitative real-time PCR assays, according to the technique available at the time each patient was treated (Cobas Amplicor HCV monitor; Roche Diagnostic Systems Inc., Branchburg, NJ, USA: detection limit 600UI / ml; Cobas AmpliPrep-Cobas TaqMan; Roche Diagnostics Systems Inc., Meylan, France: detection limit 50 Ul / ml; Cobas TaqMan; Roche Diagnostics Systems Inc., Pleasanton, CA, USA: detection limit 10 Ul / ml).
  • the HCV genotype was determined with the Versant HVC genotype 2.0 LIPA; Siemens (Tarrytown, NY, USA). LDL, HDL, total cholesterol (tCol) and triglycerides were measured by routine standard procedures in the clinical biochemistry departments of the participating hospitals.
  • SNPs of cholesterol metabolism and transport (table 1), previously associated with SVR or liver fibrosis, were selected according to 2 criteria, first, the genotype data of the Hapmap-CEU population were directly downloaded from the database Hapmap (www.hapmap.org) and the SNP2 tag was identified using the Haploview's V4.1 software tagging option (www.broad.harvard.edu/haploview/haploview); second, the lists of SNPs were completed with potentially functional polymorphisms, previously associated with either spontaneous HCV or liver fibrosis.
  • the DNA was extracted from fresh mononuclear cells from peripheral blood or from frozen whole blood, using the Quick Puré Blood DNA extraction kit (Macherey-Nagel, Düren, Germany) or the Magna Puré system (Roche, Basel, Switzerland). SNPs were genotyped using a Golden Gate Veracode genotyping assay (lllumina, San Diego, California USA) in the structural genomics department of Neocodex Inc. (Seville, Spain) according to the manufacturer's instructions.
  • Biostatistics and bioinformatics Linkage disequilibrium (LD) values between the genetic markers studied (measured in the Lewontin standardized imbalance coefficient), haplotype frequencies, and haplotype-based association analyzes were calculated using the 10 Haploview v4 software .1, Plink VI.07 and SPSS vl4. Haplotype effects are expressed as odds ratios (for binary analysis of cases and controls) or as mean difference (for numerical values of viral load). Haplotype association tests were adjusted using a Bonferroni correction for multiple tests by division of the level of significance (.05) by the number of main haplotypes. (> 5%).
  • a value of p ⁇ 0.0025 was considered statistically significant (corrected for 20 tests) for haplotype analysis assuming a risk of underestimation given the inherent lack of independence of haplotype analysis.
  • the Hardy-Weinberg equilibrium was calculated using Haploview V4.1 software and the adapted Sasieni test (1997) from the online resource of the Institute for Human Genomics, Kunststoff, Germany (www.ihg.gsf.de).
  • Unique markers, haplotype frequencies and chromosome phases were estimated and compared using PLINK software, supplemented with Java-based gPLINK implementation software (http://pngu.mgh.harvard.edu/ ⁇ purcell/plink/gplink. shtml).
  • RESULTS Genotyping statistics Five SNPs did not pass the genotyping quality controls of the Veracode Golden Gate lllumine protocol: rsl800796 (IL6), rs5742911 (LDLR), rs673 (TN FA), rs7412 (APOE) and rs7491 (LDLRAP1).
  • 8 SN Ps were mono morphic in our population with only one allele detected rsl2713911, rsl2720791, rsl2720794, rsl801703, rs7589300 (APOB), rs2069830 (IL6), rs6880589 (HAVCRl) and rs71638390 (LDLRAP1).
  • SN Ps rs4418583 (LDLRAP1) and rsll31454 (OAS1) were discarded due to the significant deviation from Hardy-Weinberg equilibrium.
  • the average genotyping rates of the rest of the SNPs were 99.2% (SD2.09).
  • a haplotype-based association test was developed by a multi-marker regression analysis using Plink software. Haplotype frequencies were estimated and compared independently for patients infected with genotypes 1-4 or 2-3 (Table 2).
  • the LDLR SNPs included in this study cover 92.6 Kb (from rsl529729 to 25 rs4804146).
  • the linkage disequilibrium of the LDLR gene was calculated in patients infected with viral genotypes 1-4 and 2-3 and showed that in both populations the SNPs with significant univariate association with VL were independently informative and contained within 3 haplotypic blocks, which were similar to the block structure in the HapMap Phase II database (Figure Ib).
  • Table 2 shows the frequencies of important haplotypes in our cohort for the 3 identified haplotypes.
  • LDLR haplotypes modify the effect of IL28R in SVR
  • IL28B genotype a notable impact on LDLR haplotypes is observed.
  • Table 1 SNPs included in this study.
  • the present invention is within the field of biomedicine and biotechnology, and refers to a method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with hepatitis C virus ( HCV) genotype 3, using as a predictor a polymorphism in the proprotein convertase subtilisin / kexin type 9 gene (PCSK9).
  • HCV hepatitis C virus
  • the duration of dual therapy can be shortened in carriers of genotype 3 that achieve rapid virological response.
  • a therapy of 24 weeks before a 48-week one is recommended in HCV-3 carriers, which show an undetectable burden of A N-HCV after 4 weeks of double therapy .
  • the duration of commonly used therapy guidelines still vary from 12 to 48 weeks, shorter regimens that include direct antivirals will be available in the near future.
  • SN Ps single nucleotide polymorphism
  • LDLR low density lipoprotein receptor
  • Proprotein convertase subtilisin / kexin type 9 is a proprotein convertase that belongs to the sub-family of proteinase K, which impacts plasma lipoprotein levels by intra and extra-hepatic modulation of LDLR and CD81 levels. . Variations in this gene interfere with the LDL / LDLR system and could thus affect the life cycle of HCV and, consequently, on SVR. However, there is no data on the impact of variations on the PCSK9 gene.
  • the present invention describes the influence of SN Ps of the PCSK9 gene on the viral response of treatment against HCV infection, which includes Peg-IFN plus RBV in a cohort of individuals infected with HCV genotype 3.
  • the present invention provides a method of obtaining useful data to predict or predict the response to treatment of HCV genotype 3, and specifically, to treatment with Peg-I FN / RBV.
  • the authors of the present invention have found a useful biomarker to predict the response to the treatment of hepatitis C genotype 3, which could lead doctors to detect those patients with a higher probability of responding to Peg-I FN / RBV therapy.
  • a first aspect of the invention relates to the use of the PCSK9 gene to predict an individual's response to HCV treatment with IFN-Peg plus a guanosine analog.
  • the guanosine analog is ribavirin (RBV).
  • the individual is also infected with HIV.
  • HCV is genotype 3.
  • Antiviral therapy is basically aimed at achieving virological, biochemical and histological objectives.
  • the evaluation of the response to the treatment is based on the effect of the same on the virological state, not only at the end of the treatment but, more importantly, in its follow-up after the end of the treatment.
  • sustained virological response or “sustained viral response” (SVR) the viremia negativization (negative viral RNA by polymerase chain reaction techniques -PCR-) that is maintained at least 24 weeks after the end of treatment; In other words, the absence of HCV-RNA in the serum 6 months after the end of treatment.
  • the absence of viremia negativization at the end of treatment defines the "virological non-response" to the treatment.
  • Interferon alfa is an immunomodulatory cytokine that has antiviral activity, antiangiogenic and antiproliferative properties.
  • I FN-Peg is obtained by the physicochemical binding of polyethylene glycol (PEG) to recombinant interferon alfa (peginterferon a-2a and a-2b), which increases its permanence in the blood requiring less frequency of injections.
  • PEG polyethylene glycol
  • peginterferon alfa-2a (Pegasys ® , Roche) and peginterferon alfa-2b (Pegintron ® , Schering-Plow). These Two products are similar with respect to their efficacy and safety but their dosage regimens differ slightly.
  • Ribavirin ( ⁇ - ⁇ -D-ribofuranosyl-lH-l, 2,4-triazol-3-carboxamide) is a synthetic nucleotide analogous to guanidine, with antiviral properties and immunoregulatory activity. It was one of the first drugs that demonstrated its effectiveness in reducing the ability to reproduce viruses. It is described in the Merck index, compound N 9 8199, eleventh edition. Its manufacture and formulation are described in US Pat. n. No. 4,211,771.
  • ribavirin The rapid erythrocyte uptake of ribavirin and its phosphorylation are responsible for significant dose-dependent hemolytic anemia.
  • the design of the new drugs contemplates that they are not captured or phosphorylated by erythrocytes or that they are rapidly captured and phosphorylated in the liver. Both approaches have the potential disadvantage that they can override fundamental steps that contribute to the therapeutic efficacy of ribavirin.
  • ribavirin derivatives, prodrugs or analogs of ribavirin are viramidine and levovirin.
  • Viramidine is a prodrug of BV, in the liver it is converted to RBV by hepatic adenosinedeaminase. RBV and viramidine are rapidly eliminated, their molecules and metabolites are excreted by the kidney and have a Tmax of 1.5-3 h.
  • hepatic retention of RBV derived from an oral dose of viramidine is 3 times higher than oral RBV.
  • concentration of RBV phosphates in red blood cells is much lower, hence a more stable hemoglobin concentration can be maintained. It is a safe drug and is well tolerated.
  • the adverse effects with doses of 200, 600 and 1,200 mg were: 0, 26 and 50%, respectively; mostly mild and disappeared without sequelae.
  • Levovirin is the L-enantiomer of RBV and does not cause hemolytic anemia. It has an immunomodulatory activity similar to RBV, but it has no direct antiviral activity.
  • Ribavirin alone is not effective against HCV, but when combined with interferon, viral eradication rates are much better compared to those achieved only with interferon.
  • the "HCV genotype”, or "viral strain” is determined by a blood test.
  • the hepatitis C virus has been classified into six different genotypes based on differences in their genomes, these genotypes in turn are subdivided. The preponderance and distribution of these genotypes varies globally. For example, in North America, genotype 1 predominates, followed by 2 and 3. In Europe, genotype 1 predominates followed by 3, 4 and 2. Genotype 4 predominates in Egypt, 5 occurs almost exclusively in Africa and genotype 6 in Southeast Asia.
  • Genotypes 1 and 4 respond less to this treatment than the others (genotypes 2, 3, 5 and 6).
  • the duration of standard interferon-based therapy for genotypes 1 and 4 is 48-72 weeks, while for genotypes 2 and 3 it is only 12-24 weeks.
  • some HCV genotypes, such as types 2 and 3, are easier to eradicate than type 1.
  • the amount of HCV RNA in the blood (serum) is called "basal plasma viral load of HCV A N". Its presence is indicative of an active infection with ongoing viral reproduction.
  • the viral load is usually expressed in logarithmic units [log IU / ml].
  • the response to antiviral treatment is evaluated with a baseline viral load (pre-treatment) and at least one viral load at the end of it and at 24 weeks post treatment.
  • a response to antiviral treatment is considered if an undetectable viral load is obtained at the end of the treatment and an undetectable viral load and / or qualitative negative RNA at 24 weeks (sustained viral response).
  • a fall in the early viral load (early viral response), with a decrease of 2 loglO Ul / ml or more at 12 weeks intra-treatment, allows an early evaluation of the probability of obtaining a sustained virological response at the end of the therapy.
  • the genotyping of the "PCKS9" gene can be obtained, preferably, but not limited to, by sequencing the entire gene but even more preferably, it is obtained by analyzing the single nucleotide polymorphism (single nucleotide polymorphism, SN P) rs2479409, located within the region 5 'up.
  • nucleotide sequence of SN P rs2479409 is: TAGAATTCTGAATGTACCTATATGAC [A / G] TCTTTGCAAACTTAAAACCTGAATC
  • PCSK9 gene is the proprotein convertase subtilisin / kexin type 9 gene, found on chromosome 1 (lp32.3).
  • Another aspect of the invention relates to a method of obtaining useful data for predicting or predicting SVR to the treatment of HCV with I FN-Peg plus a guanosine analog, hereafter referred to as the first method of the invention, comprising: a ) obtain an isolated biological sample from an individual, and b) determine the genotype of the PCSK9 gene in the sample isolated from (a).
  • the genotyping of the PSCK9 gene yields an SPN rs2479409 that can predict or predict the response to the treatment of HCV genotype 3, with I FN-Peg and Ribavirin (or analogues).
  • the authors of the present invention have determined that:
  • Another aspect of the invention relates to a method for predicting or predicting SVR to the treatment of HCV with I FN-Peg plus a guanosine analogue, hereafter referred to as the second method of the invention, comprising steps (a) and (b) of the first method of the invention, and further comprises: c) classifying individuals according to the genotype of PCSK9 they present and their response to treatment with IFN-peg plus a guanosine analog.
  • the guanosine analog is ribavirin (RBV).
  • the individual is coinfected by the VI H and the HCV.
  • HCV is genotype 3.
  • Steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the amount in step (b) or the computerized comparison in step (c).
  • isolated biological sample includes, but is not limited to, cells, tissues and / or biological fluids of an organism, such as blood, serum, urine, saliva, cerebrospinal fluid, or a solid or semi-solid sample, such as tissues, feces, and similar, or alternatively, a solid tissue such as those commonly used in histological diagnosis, obtained by any method known to a person skilled in the art.
  • a solid or semi-solid sample such as tissues, feces, and similar, or alternatively, a solid tissue such as those commonly used in histological diagnosis, obtained by any method known to a person skilled in the art.
  • genotyping SNPs There are more than 100 different methods of genotyping SNPs, whichever is applicable in the present invention. For example, without limiting our, it can be detected using restriction enzymes or by simple sequencing by PC, ASO probes and hybridization in DNA microarrays or spheres.
  • PCR methods of amplification of SN Ps
  • Invader methods of amplification of SN Ps
  • allelic discrimination methods allele-specific hybridization, allele-specific oligonucleotide ligation, probe mini-sequencing or extension (SBE, Single Base Extension), allele-specific invasive breakage (Invader), etc. can be used, but not limited to.
  • SBE Single Base Extension
  • Invader allele-specific invasive breakage
  • PCSK9 genotyping is performed by PCR, and more preferably, the determination is rs2479409. In an even more preferred embodiment, the determination is made of genotype AA of SN P rs2479409. In another preferred embodiment, the AG genotype of SN P rs2479409 is determined. In another preferred embodiment, the determination is of the GG genotype of SN P rs2479409.
  • prognosis is understood as the expected evolution of a disease and refers to the assessment of the probability according to which a subject suffers from a disease as well as the assessment of its onset, developmental status, evolution, or its regression, and / or the prognosis of the course of the disease in the future. As those skilled in the art will understand, such assessment, although preferred, may not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant part of the subjects can be identified as having the disease or having a predisposition to it.
  • a part is statistically significant, it can be determined by the person skilled in the art using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test , etc.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%.
  • P values are preferably 0.2, 0.1, 0.05.
  • "Prediction of the response” means, in the context of the present invention, the determination of the probability that the patient responds favorably or unfavorably to a given therapy or treatment.
  • the term "prediction" refers to a Individual evaluation of any parameter that may be useful in determining the evolution of a patient.
  • the prediction of the clinical response to treatment does not need to be correct for 100% of the subjects to be diagnosed or evaluated.
  • the term requires that a statistically significant part of the subjects can be identified as having an increased probability of having a positive response.
  • the person skilled in the art can easily determine if a subject is statistically significant using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann Whitney test, etc. .
  • Preferred confidence intervals are at least 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%), at least 95%>.
  • P values are preferably 0.2, 0.1 or 0.05.
  • the prediction of the clinical response can be made using any assessment criteria used in oncology and known to the person skilled in the art.
  • the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.
  • the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001.
  • the present invention makes it possible to correctly detect the disease differentially by at least 60%, more preferably at least 70%, much more preferably at least 80%, or even much more preferably at least 90%. of the subjects of a certain group or population analyzed.
  • the term “individual” is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
  • kit or device of the invention comprising the elements necessary to analyze the genotype of the PCSK9 gene.
  • the kit may contain one or more nucleotide sequences to analyze the genotype of SNP rs2479409.
  • the kit may contain one or more nucleotide sequences to analyze the AA genotype of SNP rs2479409. Another preferred embodiment relates to the determination of genotype AG of SNP rs2479409. In another preferred embodiment, the determination is of the GG genotype of SNP rs2479409. Said kit may contain all those reagents necessary to analyze the genotype of the PCSK9 gene by means of any of the methods described hereinabove.
  • the kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc.
  • the kit can include all the supports and containers necessary for commissioning and optimization.
  • the kit further comprises instructions for carrying out any of the methods of the invention.
  • the kit comprises a microarray, or microarray of the invention.
  • An A N microarray is a matrix on a solid substrate (usually a glass holder or a cell of a thin silicon film) that evaluates large amounts of different RNAs that are detectable by specific probes immobilized on spots on a solid substrate.
  • Each stain contains a specific nucleic acid sequence, which normally corresponds to a specific DNA sequence.
  • the microarray is customized for the methods of the invention. Therefore, another aspect of the invention relates to a microarray, hereafter referred to as a microarray of the invention, comprising at least one probe for detecting the genotype of the PCSK9 gene.
  • oligonucleotide sequences are constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography.
  • the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask.
  • the photomask can be physical or virtual.
  • oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides.
  • oligonucleotides per gene are preferably used.
  • Synthesis in situ on a solid support could be done using ink-jet technology, which requires longer probes.
  • the supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy.
  • the probe is each of the chip samples.
  • the target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
  • kits or devices to predict or predict an individual's response to HCV treatment with I FN-Peg plus a guanosine analog.
  • the guanosine analog is ribavirin (RBV).
  • RBV ribavirin
  • HCV is genotype 3.
  • Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
  • amino acid sequence referring to polymeric forms of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
  • Fig. 8 Graphic representation of the PSCK9 gene with the positions of genotyped polymorphisms. Untranslated regions of exons are represented in gray. * Coding SN Ps. Fig. 9. Genomic position of the SNPs and the linkage imbalance blocks analyzed.
  • Fig. 10 Results of treatment according to rs2479409 genotypes.
  • the patients were selected from a cohort of patients infected with HCV who were subsequently followed by four units of infectious diseases in Spain, between June 2000 and December 2010. The inclusion criteria for this cohort were: i) Over 18 years of age. age. ii) Prior gentle treatment. iii) The start of dual therapy with Peg-IFN plus RBV.
  • All patients included in this cohort provided whole blood or peripheral blood samples for genetic determination.
  • the samples were cryopreserved at -80 degrees Celsius. Those patients infected with HCV genotype 3 were selected from this study.
  • the responses were determined in treatment, that is, those who discontinued treatment due to adverse effects, or who voluntarily dropped out, were excluded.
  • Peg-IFN alpha 2a or alpha 2b was administered at doses of 180 ⁇ g or 1.5 ⁇ g / kg once a week, respectively, in combination with daily doses of 800-1200 mg ribavirin (RBV).
  • the duration of treatment in monoinfected HCV patients was 12 weeks in those who presented a baseline HCV RNA load ⁇ 600,000 lU / mL, absence of cirrhosis and rapid virological response (RVR), and 24 weeks the rest. Patients co-infected with HCV and HIV were treated for 24 to 48 weeks, depending on whether they achieved RVR or not.
  • the suspension rules were applied following the international guidelines in force at the time of treatment. Answer definition
  • SVR was defined as undetectable HCV RNA in plasma, 24 weeks after treatment termination. RVR was considered as the undetectability of HCV RNA at the fourth week of treatment.
  • Early virological response (RVT) was defined as either undetectability or a decrease> 2 log of HCV RNA at week 12.
  • HCV RNA detectable in plasma during treatment after reaching undetectability was defined as virological progress.
  • HCV RNA detectable after presenting a decrease> 2 logs at 12 weeks, or the lack of RVT was defined as non-response.
  • the end-of-treatment response (RFT) was considered when HCV RNA may not be detected at the end of scheduled therapy. Relapse was defined as RFT without achieving SVR.
  • Genomic DNA was isolated from the general blood sample or from peripheral blood isolated from mononuclear cells using the MagNa Puré DNA automated extraction method (Roche Diagnostic Corporation, Indianapolis, IN, USA) or the Quick Puré Blood DNA extraction kit (Macherey- Nagel, Düren, Germany).
  • PCSK9 SNPs were identified as SNPs tags, using Haploview's V4.1 software, labeling option (https://www.broad.harvard.edu/haploview/haploview) and genotype data of the Hapmap-CEU population (Table 1 ).
  • Table 1 Single nucleotide polymorphism analyzed in PCSK9 genes.
  • ISNP single nucleotide polymorphism
  • 2BBP even base position
  • the PCSK9 gene is shown in Figure 1. All SNPs were genotyped using a Golden Gate Veracode genotyping assay (lllumina, San Diego, CA, USA), according to the manufacturer's instructions. Those SNPs that showed the following exclusion criteria were discarded: i) Less allele frequency of ⁇ 0.25. ii) Genotyping rate ⁇ 80% iii) P ⁇ 0.05 for Hardy-Weinberg equilibrium. Finally, 10 SNPs of the 19 previously selected were analyzed, IL28B genotyping by PC and plasma HCV-RNA determination were carried out as described elsewhere.
  • the Hardy-Weinberg equilibrium was calculated for SNPs using the online resource of the Institute for Human Genetics of the Technical University of Kunststoff, Germany (http://ihg.gsf.de).
  • Those SNPs that showed an association with SVR in this study were selected to enter deeper analyzes.
  • Continuous variables are presented as median (interquartile range, IQR) and compared by means of the student's t test or the Mann-Whitney U test, when applicable.
  • the ⁇ 2 test (chi-square) and the exact Fischer test were used to analyze the categorical variables in 2x2 tables.
  • the ⁇ 2 test for RxC tables was applied for variables classified in more than 2 categories, using standardized residual (ASR) for the interpretation of significance.
  • Categorical variables are expressed as numbers (percentages).
  • the primary variable outcome was RVS.
  • Characteristics of the study population 132 individuals were selected for the study. The mean age (interquartile range) was 41.5 (37.6-45.1) years and 99 (75%) were men. Advanced fibrosis was diagnosed in 44 (41.1%) of the 107 patients in whom the determination of the stages of fibrosis by means of a liver biopsy (F> 3 according to the Scheuer index) or transient elastometry (liver stiffness value > 11 kPa) was possible. The mean baseline viral load was 5.94 (5.42-6.49) log IU / mL. 99 (75%) individuals were coinfected with VI H, presenting an average CD4 cell count of 470 (350-680) cells / ⁇ .
  • Table 2 Baseline characteristics of the study population according to rs2479409.
  • Triglycerides (mg / dL) * 113 (66-185) 97 (77-125) 0.643
  • CD4 cell count (cells / ⁇ .) * 463 (351-664) 564 (272-746) 0.634
  • the sustained virological response (SVR) was achieved in 108 patients (81.8%).
  • the rapid viral response (RVR) could be evaluated in 78 (67.8%) of the 115 patients (87.1%) Four individuals (3%) did not respond to therapy and 6 (4.5%) experienced virological progress. (10.6%) of the patients relapsed.
  • the details of the SVR and RVR rates according to the characteristics of the patients are shown in Table 3. r-.
  • a method for predicting or predicting an individual's response to HCV treatment with I FN-peg plus a guanosine analogue comprising steps a) and b) of the preceding claim, and further comprising: c) classifying a the individuals according to the genotype of PCSK9 that they present and their response to IFN-peg treatment plus a guanosine analog.
  • kits or devices comprising one or more nucleotide sequences necessary to determine the genotype of the PCKS9 gene.
  • a kit or device according to the preceding claim comprising one or more nucleotide sequences necessary to determine the SN P rs2479409 of the PCKS9 gene.
  • kits or devices according to claims 18-22 to predict or predict the response of an individual with HCV to treatment with IFN-Peg plus a guanosine analog.
  • guanosine analog is ribavirin (RBV).

Abstract

The invention relates to the use of the genetic polymorphisms rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, and rs6413504 of the gene LDLR, for predicting or forecasting response to treatment with pegylated interferon plus ribavirin in patients infected with HCV.

Description

Polimorfismos genéticos combinados de LDLR e IL28B para la predicción de la respuesta al tratamiento con interferon pegilado más ribavirina en pacientes infectados con el virus de la hepatitis C  Combined genetic polymorphisms of LDLR and IL28B for predicting the response to treatment with pegylated interferon plus ribavirin in patients infected with hepatitis C virus
CAMPO DE LA INVENCIÓN La presente invención se encuentra dentro de la medicina y la biología molecular, y se refiere a un método de obtención de datos útiles para predecir la respuesta al tratamiento con interferon pegilado más ribavirina, de la infección por el virus de la hepatitis C (VHC), y en concreto, al empleo del gen del receptor de las lipoproteínas de baja densidad (LDLR). La predicción a priorí, con datos genéticos, de los pacientes con mayor probabilidad de curación permitiría una mejor selección de los mismos, con el consecuente ahorro económico y mejora de la calidad de vida de dichos pacientes. FIELD OF THE INVENTION The present invention is within medicine and molecular biology, and refers to a method of obtaining useful data to predict the response to treatment with pegylated interferon plus ribavirin, of hepatitis virus infection. C (HCV), and specifically, the use of the low density lipoprotein receptor (LDLR) gene. The a priori prediction, with genetic data, of the patients with greater probability of cure would allow a better selection of them, with the consequent economic saving and improvement of the quality of life of said patients.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El ciclo de replicación viral está afectado por el colesterol y la biosíntesis de ácidos grasos. La infección con VHC incrementa la biosíntesis lipídica y su almacenamiento en los hepatocitos, contribuyendo a la esteatosis asociada a VHC, hipobetalipoproteinemia y resistencia a la insulina. La inhibición de la producción de colesterol por medio del bloqueo de la HMG-CoA reductasa o de la 3 beta- hidroxiesteroide-delta 7-reductasa induce una marcada reducción en los niveles de ARN de VHC in vitro. The viral replication cycle is affected by cholesterol and fatty acid biosynthesis. Infection with HCV increases lipid biosynthesis and its storage in hepatocytes, contributing to HCV-associated steatosis, hypobetalipoproteinemia and insulin resistance. Inhibition of cholesterol production by blocking HMG-CoA reductase or 3 beta-hydroxysteroid-delta 7-reductase induces a marked reduction in HCV RNA levels in vitro.
El ensamblado del virus VHC está estrechamente enlazado con la síntesis de lipoproteínas de muy baja densidad (VLDL) y su vía secretora. Las partículas de VHC en suero se complejan con VLDL; aunque la estructura molecular exacta no es conocida, datos indirectos sugieren que VHC puede esconderse en el núcleo lipídico de VLDL, haciendo al VHC indetectable a anticuerpos y complemento del suero. Esta asociación con VLDL permite al virus unirse a células diana a través de los receptores de lipoproteínas, después del procesamiento parcial por la lipoproteín-lipasa hepática, la cual hidroliza los triglicéridos en la VLDL dando lugar a IDL y LDL. Datos clínicos y experimentales sugieren que el receptor LDL (LDLR) es una molécula de acoplamiento o un co-receptor para VHC: The HCV virus assembly is closely linked to the synthesis of very low density lipoproteins (VLDL) and its secretory pathway. The HCV particles in serum are complexed with VLDL; Although the exact molecular structure is not known, indirect data suggests that HCV can hide in the lipid nucleus of VLDL, making HCV undetectable to antibodies and serum complement. This association with VLDL allows the virus to bind to target cells through lipoprotein receptors, after partial processing by hepatic lipoprotein lipase, which hydrolyzes triglycerides in VLDL resulting in IDL and LDL. Clinical and experimental data suggest that the LDL receptor (LDLR) is a coupling molecule or a co-receptor for HCV:
1) El nivel de ARN de VHC en hepatocitos primarios se relaciona con la expresión de ARNm de LDLR y la eficiencia en la captación de LDL. 1) The level of HCV RNA in primary hepatocytes is related to the expression of LDLR mRNA and the efficiency of LDL uptake.
2) LDLR soluble puede inhibir la infectividad de VHC. 3) Los niveles plasmáticos más elevados de LDL están asociados con la respuesta virológica sostenida ( VS) después del tratamiento con interferon pegilado (Peg-IFN) más ribavirina (RBV), probablemente debido al bloqueo competitivo de la entrada de VHC. 2) Soluble LDLR can inhibit HCV infectivity. 3) Higher plasma LDL levels are associated with sustained virologic response (VS) after treatment with pegylated interferon (Peg-IFN) plus ribavirin (RBV), probably due to competitive blockage of HCV entry.
4) Los polimorfismos en LDLR, los cuales están asociados con los niveles plasmáticos de LDL, tienen un impacto sinérgico en la probabilidad de lograr RVS con Peg-IFN y RBV en pacientes infectados con los genotipos virales 1-4, así como en la cinética viral después del tratamiento. 4) LDLR polymorphisms, which are associated with plasma LDL levels, have a synergistic impact on the likelihood of achieving SVR with Peg-IFN and RBV in patients infected with viral genotypes 1-4, as well as in kinetics viral after treatment.
Sin embargo se mantienen algunas discrepancias. De esta manera, los experimentos tiempo de adición' con un anticuerpo monoclonal contra LDLR revela que LDLR tiene un mayor papel en las fases de postentrada del ciclo de vida viral, probablemente incrementando la captación, un factor esencial para la replicación del VHC. Adicionalmente, cambios en la distribución intracelular del colesterol de la membrana plasmática a la red membranosa puede interrumpir las balsas de superficie lipídica rica en colesterol, llevando a la represión de la vía de señalización JAK-STAT regulada por interferon. En el caso del virus West Nile, la adición exógena de colesterol puede rescatar la señalización por interferon y restringir la replicación viral. Cuatro estudios anteriores revelaron datos con respecto a la evolución clínica del VHC y de los polimorfismos genéticos de LDLR, no obstante, el papel de los polimorfismos genéticos en el metabolismo del colesterol, en la replicación del VHC, no está claro. Las principales ventajas de la presente invención están en la profundidad de los análisis genéticos de los genes más esenciales de las vías de transporte del colesterol, un denso reportaje de los polimorfismos de un solo nucleótido (SNPs) en los genes LDLR, y el hecho de que no existen estudios previos que hayan estudiado una asociación de LDLR con el pre-tratamiento VL (carga viral). La identificación de los factores críticos del hospedador que modulan la replicación y la propagación del VHC, tiene el potencial de llegar a ser una nueva diana para medicamentos antivirales, centrada en la inhibición de la maquinaria celular destruido por la replicación viral. El tratamiento de interferon pegilado más ribavirina presenta fuertes efectos secundario, y además tiene un coste medio de unos 8-10.000 euros por paciente. Sin embargo, la tasa media de curación alcanza sólo el 50-60% de los pacientes. Por ello, la predicción a príori, con datos genéticos, de los pacientes con mayor probabilidad de curación permitiría una mejor selección de los mismos; con el consecuente ahorro económico y mejora de la calidad de vida de dichos pacientes. Es necesario, por tanto, encontrar un método y un kit de predicción de respuesta al tratamiento Peg-IFN y RBV. DESCRIPCIÓN DE LA INVENCIÓN However, some discrepancies remain. In this way, the addition time experiments with a monoclonal antibody against LDLR reveals that LDLR has a greater role in the post-entry phases of the viral life cycle, probably increasing uptake, an essential factor for HCV replication. Additionally, changes in the intracellular distribution of cholesterol from the plasma membrane to the membranous network can disrupt the rafts of cholesterol-rich lipid surface, leading to repression of the JAK-STAT signaling pathway regulated by interferon. In the case of West Nile virus, the exogenous addition of cholesterol can rescue interferon signaling and restrict viral replication. Four previous studies revealed data regarding the clinical evolution of HCV and genetic LDLR polymorphisms, however, the role of genetic polymorphisms in cholesterol metabolism, in HCV replication, is unclear. The main advantages of the present invention are in the depth of the genetic analyzes of the most essential genes of cholesterol transport pathways, a dense report of single nucleotide polymorphisms (SNPs) in LDLR genes, and the fact of that there are no previous studies that have studied an association of LDLR with the pre-treatment VL (viral load). The identification of the critical host factors that modulate the replication and spread of HCV has the potential to become a new target for antiviral drugs, focused on the inhibition of cellular machinery destroyed by viral replication. The treatment of pegylated interferon plus ribavirin has strong side effects, and also has an average cost of about 8-10,000 euros per patient. However, the average cure rate reaches only 50-60% of patients. Therefore, the prediction of prori, with genetic data, of patients with a greater probability of cure would allow a better selection of them; with the consequent economic saving and improvement of the quality of life of these patients. It is necessary, therefore, to find a method and a kit for predicting the response to the Peg-IFN and RBV treatment. DESCRIPTION OF THE INVENTION
Los autores de la presente invención han evaluado la asociación de polimorfismos en genes de la síntesis del colesterol, así como de sus vías de transporte, en la respuesta viral sostenida pos-tratamiento y la carga viral del VHC. La presente invención proporciona un método de obtención de datos útiles para predecir o pronosticar la respuesta viral sostenida ( VS) después del tratamiento con interferon pegilado más ribavirina del VHC. Los autores de la presente invención han encontrado un biomarcador útil para predecir la respuesta al tratamiento de la hepatitis C. The authors of the present invention have evaluated the association of polymorphisms in cholesterol synthesis genes, as well as their transport pathways, in the sustained post-treatment viral response and HCV viral load. The present invention provides a method of obtaining useful data for predicting or predicting the sustained viral response (VS) after treatment with pegylated interferon plus ribavirin for HCV. The authors of the present invention have found a biomarker useful for predicting the response to hepatitis C treatment.
Por tanto, un primer aspecto de la invención se refiere al uso de los polimorfismos del gen LDLR para predecir o pronosticar la respuesta al tratamiento con interferon pegilado más ribavirina en pacientes infectados con VHC. Therefore, a first aspect of the invention relates to the use of LDLR gene polymorphisms to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV.
Otro aspecto de la invención se refiere al uso de cualquiera de los polimorfismos (SN Ps) que se seleccionan de entre: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099, rs6413504, o cualquiera de sus combinaciones, para predecir o pronosticar la respuesta al tratamiento con interferon pegilado más ribavirina en pacientes infectados con VHC. Una realización preferida se refiere al uso simultáneo de los polimorfismos (SNPs) rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 y rs6413504 para predecir o pronosticar la respuesta al tratamiento con interferon pegilado más ribavirina en pacientes infectados con VHC. Another aspect of the invention relates to the use of any of the polymorphisms (SN Ps) that are selected from: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099, rs6413504, or any combination thereof, to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV. A preferred embodiment relates to the simultaneous use of polymorphisms (SNPs) rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504 to predict or predict the response to treatment with pegylated interferon plus ribavirin infected patients.
En otra realización preferida de este aspecto de la invención, la respuesta al tratamiento es la respuesta virológica sostenida (RVS). In another preferred embodiment of this aspect of the invention, the response to treatment is the sustained virological response (SVR).
En otra realización preferida de este aspecto de la invención, el genotipo del VHC se selecciona de entre: genotipo 1, genotipo 2, genotipo 3, genotipo 4, o cualquiera de sus combinaciones. In another preferred embodiment of this aspect of the invention, the HCV genotype is selected from: genotype 1, genotype 2, genotype 3, genotype 4, or any combination thereof.
El gen LDLR se localiza en el brazo pequeño del cromosoma 19, y contiene 18 exones codificando los seis dominios funcionales de la proteína madura: péptido señal, dominio de unión del ligando, factor tipo precursor de crecimiento epidermal (EGF), dominios transmembrana y citoplasmático. The LDLR gene is located on the small arm of chromosome 19, and contains 18 exons encoding the six functional domains of the mature protein: signal peptide, ligand binding domain, epidermal growth precursor type (EGF), transmembrane and cytoplasmic domains .
La terapia antiviral va dirigida básicamente a lograr unos objetivos virológicos, bioquímicos e histológicos. La evaluación de la respuesta al tratamiento, se basa en el efecto del mismo sobre el estado virológico, no sólo al terminar el tratamiento sino, lo que es más importante, en su seguimiento después de finalizado el mismo. Tal y como se entiende en esta memoria, se denomina "respuesta virológica sostenida" o "respuesta viral sostenida" (RVS) la negativización de la viremia (ARN viral negativo mediante técnicas de reacción en cadena de la polimerasa -PC -) que se mantiene al menos 24 semanas después de finalizado el tratamiento; o dicho de otra manera, la ausencia en el suero del ARN-VHC a los 6 meses de finalizado el tratamiento. Antiviral therapy is basically aimed at achieving virological, biochemical and histological objectives. The evaluation of the response to the treatment, is based on the effect of the same on the virological state, not only at the end of the treatment but, more importantly, in its follow-up after the end of the treatment. As understood herein, it is called "sustained virological response" or "sustained viral response" (SVR) viremia negativization (viral RNA negative by polymerase chain reaction techniques -PC -) that is maintained at least 24 weeks after the end of treatment; In other words, the absence of HCV-RNA in the serum 6 months after the end of treatment.
La negativización de la viremia al final del tratamiento, con reaparición del ARN una vez terminado el mismo, define la "respuesta virológica con recaída". La ausencia de negativización de la viremia al final del tratamiento define la "no respuesta virológica" al tratamiento. The negativization of the viremia at the end of the treatment, with the reappearance of the RNA once it is finished, defines the "virological response with relapse". The absence of viremia negativization at the end of treatment defines the "virological non-response" to the treatment.
El "interferón alfa" (IFN-α) es una citocina inmunomoduladora que posee actividad antiviral, propiedades antiangiogénicas y antiproliferativas. El IFN-Peg se obtiene mediante la unión fisicoquímica de polietilenglicol (PEG) al interferón alfa (peginterferón a-2a y a-2b) recombinante, lo que aumentan su permanencia en la sangre requiriendo menor frecuencia de inyecciones. "Interferon alfa" (IFN-α) is an immunomodulatory cytokine that has antiviral activity, antiangiogenic and antiproliferative properties. IFN-Peg is obtained by the physicochemical binding of polyethylene glycol (PEG) to recombinant interferon alfa (peginterferon a-2a and a-2b), which increases its permanence in the blood requiring a lower frequency of injections.
Actualmente existen dos tipos de interferones pegilados comerciales cuya aplicación se ha extendido: peginterferón alfa-2a (Pegasys®, Roche) y peginterferón alfa-2b (Pegintron®, Schering-Plough). Estos dos productos son semejantes con respecto a su eficacia y seguridad pero sus regímenes de dosificación difieren levemente. La ribavirina (Ι-β-D-ribofuranosil-lH-l, 2,4-triazol-3-carboxamida) es un nucleótido sintético análogo de la guanidina, con propiedades antivirales y actividad inmunorreguladora. Fue una de las primeras drogas que demostró su eficacia para reducir la capacidad de reproducción de virus. Está descrita en el índice Merck, compuesto N 9 8199, undécima edición. Su fabricación y formulación se describen en la patente de EE.UU. n. No. 4.211.771. La rápida captación eritrocitaria de la ribavirina y su fosforilación son responsables de la importante anemia hemolítica dependiente de la dosis. Con el fin de evitar este efecto indeseado, el diseño de los nuevos fármacos contempla que no sean captados o fosforilados por los eritrocitos o que sean captados rápidamente y fosforilados en el hígado. Ambos enfoques tienen la desventaja potencial de que pueden anular pasos fundamentales que contribuyen con la eficacia terapéutica de la ribavirina. Otros derivados, profármacos o análogos de la ribavirina son la viramidina y la levovirina. There are currently two types of commercial pegylated interferons whose application has been extended: peginterferon alfa-2a (Pegasys ® , Roche) and peginterferon alfa-2b (Pegintron ® , Schering-Plow). These two products are similar with respect to their efficacy and safety but their dosage regimens differ slightly. Ribavirin (Ι-β-D-ribofuranosyl-lH-l, 2,4-triazol-3-carboxamide) is a synthetic nucleotide analogous to guanidine, with antiviral properties and immunoregulatory activity. It was one of the first drugs that demonstrated its effectiveness in reducing the ability to reproduce viruses. It is described in the Merck index, compound N 9 8199, eleventh edition. Its manufacture and formulation are described in US Pat. n. No. 4,211,771. The rapid erythrocyte uptake of ribavirin and its phosphorylation are responsible for significant dose-dependent hemolytic anemia. In order to avoid this unwanted effect, the design of the new drugs contemplates that they are not captured or phosphorylated by erythrocytes or that they are rapidly captured and phosphorylated in the liver. Both approaches have the potential disadvantage that they can override fundamental steps that contribute to the therapeutic efficacy of ribavirin. Other derivatives, prodrugs or analogs of ribavirin are viramidine and levovirin.
La viramidina es un profármaco de la RBV, en el hígado se convierte en RBV por acción de la adenosindeaminasa hepática. La RBV y viramidina son rápidamente eliminadas, sus moléculas y los metabolitos son excretados por el riñon y tienen una Tmáx de 1,5-3 h. Experimentalmente, la retención hepática de la RBV derivada de una dosis oral de viramidina es 3 veces mayor que la RBV oral. En cambio, la concentración de fosfatos de RBV en los hematíes es mucho menor, de ahí que se pueda mantener una concentración más estable de hemoglobina. Es un fármaco seguro y se tolera bien. Los efectos adversos con dosis de 200, 600 y 1.200 mg fueron: 0, 26 y 50%, respectivamente; la mayoría leves y desaparecieron sin secuelas. Viramidine is a prodrug of RBV, in the liver it is converted to RBV by the action of hepatic adenosinedeaminase. RBV and viramidine are rapidly eliminated, their molecules and metabolites are excreted by the kidney and have a Tmax of 1.5-3 h. Experimentally, hepatic retention of RBV derived from an oral dose of viramidine is 3 times higher than oral RBV. In contrast, the concentration of RBV phosphates in red blood cells is much lower, hence it can be maintain a more stable concentration of hemoglobin. It is a safe drug and is well tolerated. The adverse effects with doses of 200, 600 and 1,200 mg were: 0, 26 and 50%, respectively; mostly mild and disappeared without sequelae.
La levovirina es la L-enantiomero de la BV y no origina anemia hemolítica. Tiene una actividad inmunomoduladora similar a la RBV, pero no tiene actividad antiviral directa. Levovirin is the L-enantiomer of BV and does not cause hemolytic anemia. It has an immunomodulatory activity similar to RBV, but it has no direct antiviral activity.
Otros posibles análogos se describen, por ejemplo, pero sin limitarnos, en la patente WO 2010/135520. Other possible analogs are described, for example, but not limited to, in WO 2010/135520.
La ribavirina por sí sola no es eficaz contra el VHC, pero cuando se la combina con interferón, los índices de erradicación viral son mucho mejores comparados con aquellos logrados solamente con interferón. Ribavirin alone is not effective against HCV, but when combined with interferon, viral eradication rates are much better compared to those achieved only with interferon.
La carga viral es la cantidad de ARN del VHC en un mililitro de sangre. Hay dos tipos principales de pruebas de carga viral para el VHC: The viral load is the amount of HCV RNA in a milliliter of blood. There are two main types of viral load tests for HCV:
- Las pruebas cualitativas de carga viral determinan la presencia de ARN del VHC (código genético) en la sangre. Este tipo de prueba se usa generalmente para confirmar una infección crónica con el VHC. Si se detecta ARN viral, se reporta un resultado positivo. Si no se detecta ARN viral, el resultado es negativo. Hasta un 15% de las personas infectadas con el VHC eliminan la infección sin tratamiento. Estas personas tienen una prueba de anticuerpos positiva y una prueba de carga viral negativa. - Qualitative viral load tests determine the presence of HCV RNA (genetic code) in the blood. This type of test is usually used to confirm a chronic infection with HCV. If viral RNA is detected, a positive result is reported. If viral RNA is not detected, the result is negative. Up to 15% of people infected with HCV eliminate the infection without treatment. These people have a positive antibody test and a negative viral load test.
- Las pruebas de carga viral cuantitativas miden la cantidad de virus en un mililitro de sangre. Generalmente se usan para evaluar si el tratamiento con interferón o con interferón más ribavirina tendrá posibilidades de tener éxito y luego, si el tratamiento está funcionando. - Quantitative viral load tests measure the amount of virus in a milliliter of blood. They are generally used to assess whether interferon or interferon plus ribavirin treatment will have a chance of succeeding and then, if the treatment is working.
Existen técnicas diferentes, sin limitarnos, para la cuantificación del virus VHC: - Reacción en cadena de polimerasa (PCR). Esta prueba mide copias del código genético del VHC. La prueba PCR es muy sensible. There are different techniques, without limiting ourselves, for the quantification of the HCV virus: - Polymerase chain reaction (PCR). This test measures copies of the HCV genetic code. The PCR test is very sensitive.
- Cadena ramificada de ADN (bDNA, siglas en inglés). Esta prueba es menos sensible que la PCR. Sin embargo, es más barata. - Branched chain of DNA (bDNA). This test is less sensitive than PCR. However, it is cheaper.
- Amplificación mediada por la transcripción (TMA, siglas en inglés). Esta tecnología es extremadamente sensible. Se está volviendo más rápida y menos cara. - Transcription mediated amplification (TMA). This technology is extremely sensitive. It is becoming faster and less expensive.
Las diferentes pruebas pueden dar resultados diferentes en una misma muestra. Debido a que las pruebas son diferentes, debería usar el mismo tipo de pruebas para medir su carga viral a través del tiempo. El "genotipo del VHC", o "cepa viral" se determina por una prueba de sangre. El virus de la hepatitis C se ha clasificado en seis genotipos distintos en base a diferencias en sus genomas, estos genotipos a su vez se subdividen. La preponderancia y distribución de dichos genotipos varía de modo global. Por ejemplo, en Norte América predomina el genotipo 1, seguido de 2 y 3. En Europa predomina el genotipo 1 seguido de 3, 4 y 2. Los genotipos 4 predomina en Egipto, 5 se da casi exclusivamente en África y genotipo 6 en el sudeste asiático. Different tests may give different results in the same sample. Because the tests are different, you should use the same type of tests to measure your viral load over time. The "HCV genotype", or "viral strain" is determined by a blood test. The hepatitis C virus has been classified into six different genotypes based on differences in their genomes, these genotypes in turn are subdivided. The preponderance and distribution of these genotypes varies globally. For example, in North America, genotype 1 predominates, followed by 2 and 3. In Europe, genotype 1 predominates, followed by 3, 4 and 2. Genotype 4 predominates in Egypt, 5 occurs almost exclusively in Africa, and genotype 6 in the Southeast Asian.
Tal y como se entiende en esta memoria, se denomina "respuesta virológica sostenida" o "respuesta viral sostenida" ( VS) la negativización de la viremia (ARN viral negativo mediante técnicas de reacción en cadena de la polimerasa -PCR-) que se mantiene al menos 24 semanas después de finalizado el tratamiento; o dicho de otra manera, la ausencia en el suero del ARN-VHC a los 6 meses de finalizado el tratamiento. As is understood herein, it is called "sustained virological response" or "sustained viral response" (VS) the viremia negativization (negative viral RNA by polymerase chain reaction techniques -PCR-) that is maintained at least 24 weeks after the end of treatment; In other words, the absence of HCV-RNA in the serum 6 months after the end of treatment.
Las secuencias nucleotídicas de los polimorfismos son las siguientes: rs2738456 The nucleotide sequences of the polymorphisms are as follows: rs2738456
GACAAGAGTGAAACTCCATTCCCCTC [C/T] GCAAAGAAAAGGAATATTATCAGAT rs2738457 GACAAGAGTGAAACTCCATTCCCCTC [C / T] GCAAAGAAAAGGAATATTATCAGAT rs2738457
ATTCATTCATTCATTTATGTAGAGAC [A/G] AGGGGGATCTGGCTATATTGCCTAG rs2569540 ATTCATTCATTCATTTATGTAGAGAC [A / G] AGGGGGATCTGGCTATATTGCCTAG rs2569540
CAGGTGAAATTCTTGTCAACCTACTT [C/G] TG C ATTTT ATTA AA ATG AG C ATATT rs2738459 TAGCCAGACCTCTGTTTCTATCCCCC [A/C] CAAAAAGAACCTTCTTAAACCGGAA 15 rs2738460 CAGGTGAAATTCTTGTCAACCTACTT [C / G] TG C ATTTT ATTA AA ATG AG C ATATT rs2738459 TAGCCAGACCTCTGTTTCTATCCCCC [A / C] CAAAAAGAACCTTCTTAAACCGGAA 15 rs2738460
CCAGCGTCCCCCAGGTCACAGCCTCC [C/T] GCTATGTGACCTCGTGCCTGGCTGG rs2116898 CCAGCGTCCCCCAGGTCACAGCCTCC [C / T] GCTATGTGACCTCGTGCCTGGCTGG rs2116898
TTCCCCCAGGAGCCGCCAGAGGGAGA [C/T] GGTGAGTACCGGATTCAGTCACTTC rsl433099 TTCCCCCAGGAGCCGCCAGAGGGAGA [C / T] GGTGAGTACCGGATTCAGTCACTTC rsl433099
GCACTTAATAAATATTAAGGGTGACC[A/G]GTGACTCAGGCTCTGCCTCTGGGAA rs6413504 GCACTTAATAAATATTAAGGGTGACC [A / G] GTGACTCAGGCTCTGCCTCTGGGAA rs6413504
AGGTGGCCTCCAGCCGTGTTTCCTGA [A/G] TGCTGGACTGATAGTTTCCGCTGTT AGGTGGCCTCCAGCCGTGTTTCCTGA [A / G] TGCTGGACTGATAGTTTCCGCTGTT
Otro aspecto de la invención se refiere al uso simultáneo de los genes LDLR e IL28B para predecir o pronosticar la carga viral del individuo. MÉTODO DE OBTENCIÓN DE DATOS ÚTILES, MÉTODO DE PRONÓSTICO Y PREDICCIÓN DE LA RESPUESTA VIRAL SOSTENIDA. Another aspect of the invention relates to the simultaneous use of the LDLR and IL28B genes to predict or predict the viral load of the individual. METHOD OF OBTAINING USEFUL DATA, METHOD OF FORECAST AND PREDICTION OF THE SUSTAINED VIRAL RESPONSE.
Otro aspecto de la invención se refiere a un método de obtención de datos útiles para predecir o pronosticar la respuesta al tratamiento con interferón pegilado más ribavirina en pacientes infectados con VHC, de ahora en adelante primer método de la invención, que comprende: a) obtener una muestra biológica aislada de un individuo, y b) detectar los polimorfismos genéticos: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 y rs6413504 del gen LDLR, en la muestra biológica aislada de (a). Another aspect of the invention relates to a method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV, hereinafter the first method of the invention, comprising: a) obtaining an isolated biological sample from an individual, and b) detect the genetic polymorphisms: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504 of the LDLR gene, in the isolated biological sample of (a).
En una realización preferida, la respuesta al tratamiento es la respuesta virológica sostenida del VHC. In a preferred embodiment, the response to treatment is the sustained virological response of HCV.
En otra realización preferida, el primer método de la invención además comprende medir la carga viral del VHC en un individuo. In another preferred embodiment, the first method of the invention further comprises measuring the viral load of HCV in an individual.
Más preferiblemente, además comprende determinar los polimorfismo del gen IL28B. aún más preferiblemente los polimorfismos del gen IL28B son el rsl2979860 y/o rs8099917. More preferably, it further comprises determining the polymorphism of the IL28B gene. even more preferably the polymorphisms of the IL28B gene are rsl2979860 and / or rs8099917.
Las secuencias nucleotídicas de los polimorfismos son: rsl2979860 CTGAACCAGGGAGCTCCCCGAAGGCG [C/T] GAACCAGGGTTGAATTGCACTCCGC rs8099917 The nucleotide sequences of the polymorphisms are: rsl2979860 CTGAACCAGGGAGCTCCCCGAAGGCG [C / T] GAACCAGGGTTGAATTGCACTCCGC rs8099917
CTTTTGTTTTCCTTTCTGTGAGCAAT [G/T] TCACCCAAATTGGAACCATGCTGTA CTTTTGTTTTCCTTTCTGTGAGCAAT [G / T] TCACCCAAATTGGAACCATGCTGTA
El receptor de LDL (LDLR) puede actuar como co-receptor para el VHC. Los autores de la presente invención han determinado que distintos genotipos del gen LDLR, pueden influir en la carga viral del VHC y la RVS, como por ejemplo el alelo C del SN P rs2738459 de LDLR es un factor de riesgo y está relacionado con una mayor VL en los genotipos 2-3 de VHC. Ciertos genotipos de este gen en combinación a genotipos del gen IL28B, también tienen influencia en la respuesta viral sostenida. La combinación entre los genotipos de LDLR e IL28B mostraron un efecto sinérgico sobre la RVS en pacientes infectados con los genotipos 1-4 de VHC, cuando individuos homocigoticos portadores de genotipos IL28B protectores fueron clasificados de acuerdo a los genotipos LDLR rs2738457, la frecuencia de RVS fue 0.73 para AA versus 0.26 en AG+GG. The LDL receptor (LDLR) can act as a co-receptor for HCV. The authors of the present invention have determined that different genotypes of the LDLR gene can influence the viral load of HCV and SVR, such as for example the C allele of SN P rs2738459 of LDLR is a risk factor and is related to a greater VL in HCV genotypes 2-3. Certain genotypes of this gene in Combined genotypes of the IL28B gene, they also have an influence on the sustained viral response. The combination between LDLR and IL28B genotypes showed a synergistic effect on SVR in patients infected with HCV genotypes 1-4, when homozygous individuals carrying protective IL28B genotypes were classified according to LDLR rs2738457 genotypes, the frequency of SVR it was 0.73 for AA versus 0.26 in AG + GG.
Otro aspecto de la invención se refiere a un método para predecir o pronosticar la respuesta virológica sostenida del VHC, de ahora en adelante segundo método de la invención, que comprende los pasos (a) y (b) del primer método de la invención, y además comprende: c) clasificar a los individuos según el genotipo LDLR que presentan y su respuesta virológica sostenida del VHC al tratamiento con interferón pegilado más ribavirina. Another aspect of the invention relates to a method for predicting or predicting the sustained virological response of HCV, hereafter referred to as the second method of the invention, comprising steps (a) and (b) of the first method of the invention, and It also includes: c) classifying individuals according to the LDLR genotype they present and their sustained virological response of HCV to treatment with pegylated interferon plus ribavirin.
En una realización preferida, este método, además comprende: d) clasificar a los individuos según el genotipo LDLR y su carga viral del VHC. In a preferred embodiment, this method further comprises: d) classifying individuals according to the LDLR genotype and their HCV viral load.
En otra realización preferida el individuo está infectado por los genotipos 1, 2, 3 y 4 del VHC, o cualquiera de sus combinaciones. En otra realización preferida, el paso (b) de cualquiera de los métodos de la invención, además comprende determinar el genotipo del gen IL28B. In another preferred embodiment the individual is infected by HCV genotypes 1, 2, 3 and 4, or any combination thereof. In another preferred embodiment, step (b) of any of the methods of the invention further comprises determining the genotype of the IL28B gene.
En otra realización preferida la determinación del genotipado se realiza mediante PCR. En una realización aún más preferida, se usa el análisis de haplotipos. In another preferred embodiment, genotyping is determined by PCR. In an even more preferred embodiment, haplotype analysis is used.
Los pasos (b) y/o (c) y/o (d) de los métodos descritos anteriormente pueden ser total o parcialmente automatizados, por ejemplo, por medio de un equipo robótico sensor para la detección de la cantidad en el paso (b) o la comparación computarizada en los pasos (c) y (d). The steps (b) and / or (c) and / or (d) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the amount in step (b ) or the computerized comparison in steps (c) and (d).
El término "muestra biológica aislada" incluye, pero sin limitarnos a, células, tejidos y/o fluidos biológicos de un organismo, como sangre, suero, orina, saliva, fluido cerebroespinal, o a una muestra sólida o semisólida, como tejidos, heces, y similar, o alternativamente, un tejido sólido tales como aquellos usados habitualmente en diagnóstico histológico, obtenidos mediante cualquier método conocido por un experto en la materia. The term "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, such as blood, serum, urine, saliva, cerebrospinal fluid, or a solid or semi-solid sample, such as tissues, feces, and similar, or alternatively, a solid tissue such as those commonly used in histological diagnosis, obtained by any method known to a person skilled in the art.
Existen más de 100 métodos distintos de genotipado de SNPs, siendo cualquiera aplicable en la presente invención. Por ejemplo, sin limitarnos, puede detectarse empleando desde enzimas de restricción o por secuenciación simple mediante PCR, sondas ASO e hibridación en Micromatrices de ADN o esferas. Así, por ejemplo, como métodos de amplificación de SN Ps se encuentran la PC , el método Invader, la amplificación por ADN de círculo rodante, etc. Como métodos de discriminación alélica, pueden emplearse, pero sin limitarnos, hibridación específica de alelo, ligamiento de oligonucleótidos específicos de alelo, minisecuenciación o extensión de sondas (SBE, Single Base Extensión), rotura invasiva específica de alelo (Invader), etc. Según el formato de detección, podemos citar, también sin limitarnos, el uso de sondas Taqman, de molecular beacons, scorpion, etc. Respecto a reacciones en un soporte sólido, tenemos, por ejemplo, biochips y microarrays de ADN, u otros métodos que emplean partículas, como por ejemplo, Luminex 100, BeadArray, etc. También existen métodos de detección masiva, como la pirosecuenciación, o la miniaturización. Un haplotipo es un conjunto de polimorfismos de un solo nucleótido (SN Ps) en un cromosoma particular que están físicamente asociados heredándose como un bloque con muy poca o nula recombinación genética entre ellos. Los análisis genéticos asociación de haplotipos pasan por ser una poderosa estrategia hacia el descubrimiento de genes que predisponen a las enfermedades humanas complejas. There are more than 100 different methods of genotyping SNPs, whichever is applicable in the present invention. For example, without limiting ourselves, it can be detected using restriction enzymes or by simple sequencing by PCR, ASO probes and hybridization in DNA microarrays or spheres. So, for example, as amplification methods of SN Ps are PC, Invader method, amplification by rolling circle DNA, etc. As allelic discrimination methods, allele-specific hybridization, allele-specific oligonucleotide ligation, probe mini-sequencing or extension (SBE, Single Base Extension), allele-specific invasive breakage (Invader), etc. can be used, but not limited to. Depending on the detection format, we can mention, also without limiting ourselves, the use of Taqman probes, molecular beacons, scorpion, etc. Regarding reactions on a solid support, we have, for example, biochips and microarrays of DNA, or other methods that employ particles, such as, for example, Luminex 100, BeadArray, etc. There are also methods of mass detection, such as pyrosequencing, or miniaturization. A haplotype is a set of single nucleotide polymorphisms (SN Ps) on a particular chromosome that are physically associated inheriting as a block with very little or no genetic recombination between them. The genetic analysis association of haplotypes happens to be a powerful strategy towards the discovery of genes that predispose to complex human diseases.
El término "individuo", tal y como se utiliza en la descripción, se refiere a animales, preferiblemente mamíferos, y más preferiblemente, humanos. El término "individuo" no pretende ser limitativo en ningún aspecto, pudiendo ser éste de cualquier edad, sexo y condición física. The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
KIT O DISPOSITIVO DE DIAGNÓSTICO, MICRO ARRAY Y USOS KIT OR DEVICE FOR DIAGNOSIS, MICRO ARRAY AND USES
Otro aspecto de la presente invención se refiere a un kit o dispositivo, de aquí en adelante kit o dispositivo de la invención, que comprende los elementos necesarios para analizar el genotipo del gen LDLR. Another aspect of the present invention relates to a kit or device, hereinafter kit or device of the invention, comprising the elements necessary to analyze the genotype of the LDLR gene.
Dicho kit puede contener todos aquellos reactivos necesarios para analizar el genotipo del gen LDLR por medio de cualquiera de los métodos descritos anteriormente en este documento. El kit además puede incluir, sin ningún tipo de limitación, tampones, agentes para prevenir la contaminación, inhibidores de la degradación de las proteínas, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo cualquiera de los métodos de la invención. Said kit may contain all those reagents necessary to analyze the genotype of the LDLR gene by means of any of the methods described hereinbefore. The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out any of the methods of the invention.
Preferiblemente, el kit o dispositivo que comprende una o varias secuencias nucleotídicas necesarias para detectar los polimorfismos del gen LDLR. Más preferiblemente los polimorfismos del gen LDLR se seleccionan de entre: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099, rs6413504, o cualquiera de sus combinaciones. Es también posible que el (los) oligonucleótido(s) o sondas estén inmovilizados en manchas sobre una superficie (preferiblemente sólida). En una de sus realizaciones, el kit comprende una micromatriz, o micromatriz de la invención. Una micromatriz de A N es una matriz sobre un sustrato sólido (normalmente un porta de vidrio o una celda de una película fina de silicio) que evalúa grandes cantidades de diferentes ARN que son detectables mediante sondas específicas inmovilizadas sobre manchas sobre un sustrato sólido. Cada mancha contiene una secuencia específica de ácido nucleico, que normalmente corresponde a una secuencia concreta de ADN. Aunque el número de manchas no está limitado de manera alguna, existe una realización preferida en la que la micromatriz se personaliza para los procedimientos de la invención. Por tanto, otro aspecto de la invención se refiere a un microarray, de ahora en adelante microarray de la invención, que comprende al menos una sonda para detectar el genotipo del gen LDLR. Preferably, the kit or device comprising one or more nucleotide sequences necessary to detect the polymorphisms of the LDLR gene. More preferably the polymorphisms of the LDLR gene are selected from: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099, rs6413504, or any combination thereof. It is also possible that the oligonucleotide (s) or probes are immobilized in spots on a (preferably solid) surface. In one of its embodiments, the kit comprises a microarray, or microarray of the invention. An AN microarray is a matrix on a solid substrate (usually a glass holder or a cell of a thin silicon film) that evaluates large amounts of different RNAs that are detectable by specific probes immobilized on spots on a solid substrate. Each stain contains a specific nucleic acid sequence, which normally corresponds to a specific DNA sequence. Although the number of spots is not limited in any way, there is a preferred embodiment in which the microarray is customized for the methods of the invention. Therefore, another aspect of the invention relates to a microarray, hereafter referred to as a microarray of the invention, comprising at least one probe for detecting the genotype of the LDLR gene.
Así, por ejemplo, las secuencias de oligonucleotidos son construidas en la superficie de un chip mediante el elongamiento secuencial de una cadena en crecimiento con un sólo nucleótido utilizando fotolitografía. Así, los oligonucleotidos son anclados por el extremo 3' mediante un método de activación selectiva de nucleotidos, protegidos por un reactivo fotolábil, mediante la incidencia selectiva de luz a través de una fotomáscara. La fotomáscara puede ser física o virtual. Thus, for example, oligonucleotide sequences are constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography. Thus, the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask. The photomask can be physical or virtual.
Así, las sondas oligonucleotidos pueden ser de entre 10 y 100 nucleotidos, más preferiblemente, de entre 20 y 70 nucleotidos, y aún más preferiblemente, de entre 24 y 30 nucleotidos. Para la cuantificación de la expresión génica, preferiblemente se emplean aproximadamente unos 40 oligonucleotidos por gen. Thus, oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides. For the quantification of gene expression, preferably about 40 oligonucleotides per gene are preferably used.
La síntesis in situ sobre un soporte sólido (por ejemplo, vidrio), podría hacerse mediante tecnología chorro de tinta (ink-jet), lo que requiere sondas más largas. Los soportes podrían ser, pero sin limitarse, filtros o membranas de NC o nylon (cargadas), silicio, o Portas de vidrio para microscopios cubiertos con aminosilanos, polilisina, aldehidos o epoxy. La sonda es cada una de las muestras del chip. El target es la muestra a analizar: RNA mensajero, RNA total, un fragmento de PCR, etc. Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy. The probe is each of the chip samples. The target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
Más preferiblemente, el kit o dispositivo, y/o el microarray además comprende una o varias sondas necesarias para detectar el haplotipo del gen IL28B. Preferiblemente comprende las sondas necesarias para detectar los polimorfismos rsl2979860 y el rs8099917. More preferably, the kit or device, and / or the microarray further comprises one or more probes necessary to detect the haplotype of the IL28B gene. It preferably comprises the probes necessary to detect the rsl2979860 and rs8099917 polymorphisms.
Otro aspecto de la invención se refiere al uso del kit o dispositivo, la micromatriz, o el microarray de la invención, para predecir o pronosticar la respuesta, en pacientes infectados con VHC, al tratamiento con interferón pegilado más ribavirina. En una realización preferida, el kit o dispositivo, la micromatriz o el microarray, comprende además la capacidad de predecir o pronosticar la carga viral del VHC. Another aspect of the invention relates to the use of the kit or device, the microarray, or the microarray of the invention, to predict or predict the response, in patients infected with HCV, to treatment with Pegylated interferon plus ribavirin. In a preferred embodiment, the kit or device, the microarray or the microarray, further comprises the ability to predict or predict the viral load of HCV.
En otra realización preferida el individuo está infectado por los genotipos 1, 2, 3 o 4 del VHC, 15 o cualquiera de sus combinaciones. Otro aspecto de la invención se refiere a un medio de almacenamiento legible por un ordenador que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos de cualquiera de los métodos de la invención (del primer o del segundo método de la invención). In another preferred embodiment the individual is infected by HCV genotypes 1, 2, 3 or 4, 15 or any combination thereof. Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).
Otro aspecto de la invención se refiere a una señal transmisible que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos de cualquiera de los métodos de la invención. Another aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer perform the steps of any of the methods of the invention.
Los términos "polinucleótido" y "ácido nucleico" se usan aquí de manera intercambiable, refiriéndose a formas poliméricas de nucleótidos de cualquier longitud, tanto ribonucleótidos 25 (ARN ó RNA) como desoxirribonucleótidos (ADN ó DNA). Los términos "secuencia aminoacídica", "péptido", "oligopéptido", "polipéptido" y "proteína" se usan aquí de manera intercambiable, y se refieren a una forma polimérica de aminoácidos de cualquier longitud, que pueden ser codificantes o no codificantes, química o bioquímicamente modificados The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA). The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no 30 pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
BREVE DESCRIPCIÓN DE LAS FIGURAS BRIEF DESCRIPTION OF THE FIGURES
Fig. 1: Estructura génica del gen LDLR y patrón del desequilibrio de ligamiento de la población analizada. Una representación gráfica del gen LDLR con las posiciones de los polimorfismos genotipados. La codificación de los SNPs está subrayada, los SNPs asociados con la carga viral están en cajas. '3 UTR en gris, y el resto de los exones en negro. B, patrones de desequilibrio de seguimiento en la población de estudio. Los valores D' fueron calculados y trazados con Haploview. Los números dentro de las cajas, corresponden a D' x 100; D'= no están representados. Una estructura de bloque fue considerada para pares de marcadores, mostrando D' > 0.8, seguido de la definición de bloque de columna sólida en Haploview. Fig. 2: Comparación de VL entre pacientes portadores de polimorfismos LDL rsl433099 (lado izquierdo) y rs2569549 (lado derecho). A, pacientes infectados con los genotipos 1-4 de VHC (p=0.0001 rsl433099; p=0.007 rs2569540 prueba de Mann Whitney). B, pacientes infectados con los genotipos 2-3 de VHC (p=0.18 rsl433099; p=0.82 rs2569540, prueba de Mann Whitney). Fig. 3: Relación entre el polimorfismo rs28416813 de IL28B y respuesta viral sostenida (p=0.000003, Fisher exact test). Fig. 1: Gene structure of the LDLR gene and pattern of linkage imbalance of the analyzed population. A graphic representation of the LDLR gene with the positions of genotyped polymorphisms. The coding of the SNPs is underlined, the SNPs associated with the viral load are in boxes. '3 UTR in gray, and the rest of the exons in black. B, follow-up imbalance patterns in the study population. D 'values were calculated and plotted with Haploview. The numbers inside the boxes correspond to D 'x 100; D '= are not represented. A block structure was considered for pairs of markers, showing D '> 0.8, followed by the definition of solid column block in Haploview. Fig. 2: Comparison of VL between patients with LDL polymorphisms rsl433099 (left side) and rs2569549 (right side). A, patients infected with HCV genotypes 1-4 (p = 0.0001 rsl433099; p = 0.007 rs2569540 Mann Whitney test). B, patients infected with HCV 2-3 genotypes (p = 0.18 rsl433099; p = 0.82 rs2569540, Mann Whitney test). Fig. 3: Relationship between rs28416813 polymorphism of IL28B and sustained viral response (p = 0.000003, Fisher exact test).
Fig. 4: Interacción entre rs2738457 de LDLR y rs28416813 de IL28B en relación con la respuesta viral sostenida, a) IL28B GG fondo genético protector p=0.0006, Fisher exact test. B) IL28B CC+CG fondo genético de riesgo, P=0.72, Fisher exact test. Fig. 5: Asociación entre VHC VL y la presencia de 1 o 2 copias del haplotipo extendido AGGCGGAG de los SNPs rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rs6413504, rsl4158 en pacientes infectados con los genotipos 1-4 de VHC. a), asociación con el nivel umbral de VL de 60000 Ul/ml (ρ=9·10~6, prueba exacta de Fischer). b), asociación del haplotipo AGGCGGAG con el loglO de carga viral del VHC (ρ=8·10~5, prueba de Mann Whitney). Fig. 6: Asociación entre VHC VL y la presencia de 1 o 2 copias del haplotipo compacto AGGC/GGCC de los SNPs rs2738456, rs2738457, rs2569540, rs2738459 en pacientes infectados con los genotipos 1-4 del VHC. a), asociación con el nivel umbral de VL de 60000 Ul/ml (ρ=7· 10"4, prueba exacta de Fischer). b), asociación con el loglO de carga viral del VHC (ρ=8· 10"4, prueba de Mann Whitney). Fig. 4: Interaction between rs2738457 of LDLR and rs28416813 of IL28B in relation to the sustained viral response, a) IL28B GG protective genetic background p = 0.0006, Fisher exact test. B) IL28B CC + CG genetic risk fund, P = 0.72, Fisher exact test. Fig. 5: Association between HCV VL and the presence of 1 or 2 copies of the AGGCGGAG extended haplotype of SNPs rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rs6413504, rsl4158 in patients infected with genotypes. a), association with the VL threshold level of 60000 Ul / ml (ρ = 9 · 10 ~ 6 , Fischer's exact test). b), association of the AGGCGGAG haplotype with the HCV viral load log (ρ = 8 · 10 ~ 5 , Mann Whitney test). Fig. 6: Association between HCV VL and the presence of 1 or 2 copies of the AGGC / GGCC compact haplotype of SNPs rs2738456, rs2738457, rs2569540, rs2738459 in patients infected with HCV genotypes 1-4. a), association with the VL threshold level of 60000 Ul / ml (ρ = 7 · 10 "4 , Fischer's exact test). b), association with the HCV viral load log (ρ = 8 · 10 " 4 , test by Mann Whitney).
Fig. 7: Asociación entre el haplotipo compacto de LDLR que incluye los SNPs rs2738456, rs2738457, rs2569540, rs2738459 y el genotipo de la IL28B con RVS. a) Todos los genotipos de IL28b (p=0,016). b) genotipos protectores de IL28B (0,005) c) genotipos de riesgo de IL28B (p=0,055). Fisher exact test. Fig. 7: Association between the compact LDLR haplotype that includes SNPs rs2738456, rs2738457, rs2569540, rs2738459 and the IL28B genotype with SVR. a) All IL28b genotypes (p = 0.016). b) protective genotypes of IL28B (0.005) c) risk genotypes of IL28B (p = 0.055). Fisher exact test.
EJEMPLOS DE LA INVENCIÓN EXAMPLES OF THE INVENTION
Métodos Methods
Población de estudio Una población de 442 pacientes infectados con VHC fue reclutada posteriormente en unidades de enfermedades infecciosas de 5 hospitales en España (Sevilla, Córdoba, Madrid, Málaga y Huelva) desde Mayo de 2000 a Diciembre de 2010. Todos ellos estaban sin tratamiento de VHC. Se desarrollaron controles clínicos y de laboratorio en la visita inicial y las muestras de sangre y/o plasma fueron recogidas y almacenadas a -70QC, para posteriores determinaciones genéticas, bioquímicas y virológicas. 239 pacientes estaban infectados con VHC genotipo 1, 62 con el genotipo 4, 141 con el genotipo 3, y un individuo con el genotipo 2. Durante el tratamiento, los pacientes fueron evaluados cada 4 semanas durante las primeras 24 semanas de tratamiento, cada 8 a 12 semanas más tarde, incluyendo una visita 24 semanas después de completar la terapia para evaluar la respuesta virológica sostenida ( VS). Se realizaron controles clínica y de laboratorio en cada visita, y las muestras de sangre y/o plasma fueron recogidas y almacenadas a -70QC para posteriores determinaciones genéticas y virológicas. Study population A population of 442 patients infected with HCV were subsequently recruited into infectious disease units of 5 hospitals in Spain (Seville, Cordoba, Madrid, Malaga and Huelva) from May 2000 to December 2010. All of them were without treatment. HCV Clinical and laboratory checks at the initial visit and blood samples and / or plasma were collected and stored at -70 Q C, for subsequent genetic, biochemical and virological determinations they were developed. 239 patients were infected with HCV genotype 1, 62 with genotype 4, 141 with genotype 3, and one individual with genotype 2. During treatment, patients were evaluated every 4 weeks during the first 24 weeks of treatment, every 8 12 weeks later, including a visit 24 weeks after completing therapy to evaluate the sustained virological response (VS). Clinical and laboratory checks each visit were performed, and samples of blood and / or plasma were collected and stored at -70 Q C for further genetic and virological determinations.
Aspectos éticos Ethical aspects
El estudio fue diseñado y desarrollado de acuerdo con la declaración de Helsinki, y fue aprobado por el comité ético del Hospital Universitario de Valme. Todos los pacientes firmaron un informe de consentimiento para participar en el estudio. The study was designed and developed in accordance with the Helsinki declaration, and was approved by the ethical committee of the Valme University Hospital. All patients signed a consent report to participate in the study.
Determinaciones clínicas en laboratorio Clinical laboratory determinations
Los anticuerpos séricos de VHC fueron determinados por un inmunoensayo enzimático (EIA) (ADVIA Centaur XP, Siemens Healthcare Diagnostics S. L. Tarrytown, NY, USA). La carga plasmática de ARN de VHC fue medida por ensayos de PCR cuantitativa a tiempo real, de acuerdo a la técnica disponible en el momento que cada paciente fue tratado (Cobas Amplicor VHC monitor; Roche Diagnostic Systems Inc., Branchburg, NJ, USA: límite de detección 600UI/ml; Cobas AmpliPrep-Cobas TaqMan; Roche Diagnostics Systems Inc., Meylan, Francia: límite de detección 50 Ul/ml; Cobas TaqMan; Roche Diagnostics Systems Inc., Pleasanton, CA, USA: límite de detección 10 Ul/ml). El genotipo del VHC se determinó con el Versant HVC genotype 2.0 LIPA; Siemens (Tarrytown, NY, USA). LDL, HDL, colesterol total (tCol) y triglicéridos fueron medidos por procedimientos estándar rutinarios en los departamentos de bioquímica clínica de los hospitales participantes. Serum HCV antibodies were determined by an enzyme immunoassay (EIA) (ADVIA Centaur XP, Siemens Healthcare Diagnostics S. L. Tarrytown, NY, USA). The HCV RNA plasma load was measured by quantitative real-time PCR assays, according to the technique available at the time each patient was treated (Cobas Amplicor HCV monitor; Roche Diagnostic Systems Inc., Branchburg, NJ, USA: detection limit 600UI / ml; Cobas AmpliPrep-Cobas TaqMan; Roche Diagnostics Systems Inc., Meylan, France: detection limit 50 Ul / ml; Cobas TaqMan; Roche Diagnostics Systems Inc., Pleasanton, CA, USA: detection limit 10 Ul / ml). The HCV genotype was determined with the Versant HVC genotype 2.0 LIPA; Siemens (Tarrytown, NY, USA). LDL, HDL, total cholesterol (tCol) and triglycerides were measured by routine standard procedures in the clinical biochemistry departments of the participating hospitals.
Selección del polimorfismo y genotipado Polymorphism and genotyping selection
Los SNPs del metabolismo y transporte del colesterol (tabla 1), previamente asociados con RVS o fibrosis hepática, fueron seleccionados de acuerdo a 2 criterios, primero, los datos del genotipo de la población de Hapmap-CEU fueron directamente descargados de la base de datos Hapmap (www.hapmap.org) y la etiqueta SNP2 fue identificada usando la opción etiquetadora del software Haploview's V4.1 (www.broad.harvard.edu/haploview/haploview); segundo, la listas de SNPs fueron completadas con polimorfismos potencialmente funcionales, previamente asociados tanto con VHC espontáneo o con fibrosis hepática. El ADN fue extraído de células frescas mononucleares de sangre periférica o de sangre total congelada, usando el kit de extracción Quick Puré Blood DNA extraction kit (Macherey-Nagel, Düren, Germany) o el sistema Magna Puré (Roche, Basel, Switzerland). Los SNPs fueron genotipados usando un Golden Gate Veracode genotyping assay (lllumina, San Diego, California USA) en el departamento de genómica estructural de Neocodex Inc. (Sevilla, España) de acuerdo a las indicaciones del fabricante. SNPs of cholesterol metabolism and transport (table 1), previously associated with SVR or liver fibrosis, were selected according to 2 criteria, first, the genotype data of the Hapmap-CEU population were directly downloaded from the database Hapmap (www.hapmap.org) and the SNP2 tag was identified using the Haploview's V4.1 software tagging option (www.broad.harvard.edu/haploview/haploview); second, the lists of SNPs were completed with potentially functional polymorphisms, previously associated with either spontaneous HCV or liver fibrosis. The DNA was extracted from fresh mononuclear cells from peripheral blood or from frozen whole blood, using the Quick Puré Blood DNA extraction kit (Macherey-Nagel, Düren, Germany) or the Magna Puré system (Roche, Basel, Switzerland). SNPs were genotyped using a Golden Gate Veracode genotyping assay (lllumina, San Diego, California USA) in the structural genomics department of Neocodex Inc. (Seville, Spain) according to the manufacturer's instructions.
Bioesta dística y bioinformática Los valores de desequilibrio de ligamiento (LD) entre los marcadores genéticos estudiados (medidos en el coeficiente de desequilibrio estandarizado de Lewontin), las frecuencias de haplotipo, y análisis de asociación basados en haplotipo fueron calculados usando los 10 software Haploview v4.1, Plink VI.07 y SPSS vl4. Los efectos de haplotipo son expresados como odds ratio (para análisis binario de casos y controles) o como diferencia media (para valores numéricos de carga viral). Los test de asociación de haplotipos fueron ajustados usando una corrección de Bonferroni para test múltiples por división del nivel de significación (.05) por el número de haplotipos principales. (>5%). Un valor de p<0.0025 fue considerado significativo estadísticamente (corregido para 20 tests) para el análisis de haplotipos asumiendo un riesgo de subestimación dado la falta inherente de independencia de análisis de haplotipos. El equilibrio de Hardy-Weinberg fue calculado usando el software Haploview V4.1 y el test adaptado de Sasieni (1997) del recurso on-line del instituto para la genómica humana, Munich, Alemania (www.ihg.gsf.de). Marcadores únicos, frecuencias de haplotipo y las fases del cromosoma fueron estimados y comparados usando el software PLINK, complementado con el software de implementación gPLINK basado en Java (http://pngu.mgh.harvard.edu/~purcell/plink/gplink.shtml). La asociación alélica estándar caso/control (1 grado de libertad) así como diferentes modelos genéticos: dominante, recesivo y general (2 grados de libertad), fue hecho usando el test exacto de Fischer, adicionalmente fue usado el test de tendencias Cochran-Armitage para probar la asociación genotípica. Los modelos genéticos aditivos fueron probados usando el test de tendencias Armitage. Log 10 VHC VLs fueron probados por asociación, usando el test ratio de probabilidad asintótica y el test Wald (software Plink) así como la prueba de Mann-Whitney. Se desarrollaron análisis complementarios usando el software SPSS (versión 14.0). Los valores p de asociación de marcador único, fueron corregidos por múltiples pruebas (múltiple testing) seguidas de un enfoque de descomposición espectral de SNP, en todo el experimento el umbral de significación estadística requerido para mantener la tasa de error al 5% fue 0.0005. Biostatistics and bioinformatics Linkage disequilibrium (LD) values between the genetic markers studied (measured in the Lewontin standardized imbalance coefficient), haplotype frequencies, and haplotype-based association analyzes were calculated using the 10 Haploview v4 software .1, Plink VI.07 and SPSS vl4. Haplotype effects are expressed as odds ratios (for binary analysis of cases and controls) or as mean difference (for numerical values of viral load). Haplotype association tests were adjusted using a Bonferroni correction for multiple tests by division of the level of significance (.05) by the number of main haplotypes. (> 5%). A value of p <0.0025 was considered statistically significant (corrected for 20 tests) for haplotype analysis assuming a risk of underestimation given the inherent lack of independence of haplotype analysis. The Hardy-Weinberg equilibrium was calculated using Haploview V4.1 software and the adapted Sasieni test (1997) from the online resource of the Institute for Human Genomics, Munich, Germany (www.ihg.gsf.de). Unique markers, haplotype frequencies and chromosome phases were estimated and compared using PLINK software, supplemented with Java-based gPLINK implementation software (http://pngu.mgh.harvard.edu/~purcell/plink/gplink. shtml). The standard allele association case / control (1 degree of freedom) as well as different genetic models: dominant, recessive and general (2 degrees of freedom), was made using the exact Fischer test, additionally the Cochran-Armitage trend test was used to test genotypic association. Additive genetic models were tested using the Armitage trend test. Log 10 HCV VLs were tested by association, using the asymptotic probability ratio test and the Wald test (Plink software) as well as the Mann-Whitney test. Complementary analyzes were developed using SPSS software (version 14.0). The p values of single marker association were corrected by multiple tests (multiple testing) followed by a SNP spectral decomposition approach, throughout the experiment the statistical significance threshold required to maintain the 5% error rate was 0.0005.
RESULTADOS Estadísticas de genotipado. Cinco SNPs no pasaron los controles de calidad del genotipado del protocolo de lllumina Veracode Golden Gate: rsl800796 (IL6), rs5742911 (LDLR), rs673 (TN FA), rs7412 (APOE) and rs7491 (LDLRAP1). Además, 8 SN Ps fueron mono mórficos en nuestra población con solo un alelo detectado rsl2713911, rsl2720791, rsl2720794, rsl801703, rs7589300 (APOB), rs2069830 (IL6), rs6880589 (HAVCRl) y rs71638390 (LDLRAP1 ). Los SN Ps rs4418583 (LDLRAP1) y rsll31454 (OAS1 ) fueron descartados debido a la desviación significativa del equilibrio de Hardy-Weinberg. Las tasas medias de genotipado del resto de los SNPs fueron 99.2% (SD2.09). RESULTS Genotyping statistics. Five SNPs did not pass the genotyping quality controls of the Veracode Golden Gate lllumine protocol: rsl800796 (IL6), rs5742911 (LDLR), rs673 (TN FA), rs7412 (APOE) and rs7491 (LDLRAP1). In addition, 8 SN Ps were mono morphic in our population with only one allele detected rsl2713911, rsl2720791, rsl2720794, rsl801703, rs7589300 (APOB), rs2069830 (IL6), rs6880589 (HAVCRl) and rs71638390 (LDLRAP1). SN Ps rs4418583 (LDLRAP1) and rsll31454 (OAS1) were discarded due to the significant deviation from Hardy-Weinberg equilibrium. The average genotyping rates of the rest of the SNPs were 99.2% (SD2.09).
Análisis de asociaciones genéticas univariante de VL Analysis of univariate genetic associations of VL
Los valores plasmáticos de VHC VL fueron caracterizados de acuerdo a nivel umbral de corte de 600000 Ul/ml, en paralelo se realizaron también análisis de asociación cuantitativa. Cuando se analizaron las variables continuas, los valores VL fueron expresados como LoglO. Solo el marcador rs2569540 de LDLR mostró una asociación límite bajo el modelo dominante en el uso del nivel de corte de 600000 U l/ml (odds ratio=1.74; p=0.009). Cuando los pacientes clasificados de acuerdo al genotipo VHC (tabla 2), encontramos asociación entre pacientes infectados con los genotipo 1-4 de VHC bajo el modelo dominante con los genotipos AA+AG de rsll672123 localizados en la región distal promotora de LDLR (p=0.001; OR: 2.3; 95% Cl [1.359-3.917]), los genotipos AA+AG de rsl433099 posicionados en el extremo 3' a la región no traducida de LDLR (p=0.0004, OR=0.41, 95% Cl [0.22-0.66]) y rs2569540 GG+GC (p=0.0023; OR=0.45, 95% Cl [0.277-0.761]) localizado en el intrón cerca del extremo 3' terminal del gen (Figura 1§). El alelo C de rs2738459 de LDLR es un factor de riesgo y está relacionado con una mayor VL en los genotipos 2-3 (OR: 4.29; p=0.0002, 95% Cl [1.930-9.558]. Asociaciones de rsl433099 y rs2738459 con VL alcanzaron significación estadística después de la corrección por múltiple testing. Las localizaciones de los SNPs en el gen de LDLR están representadas en la Figura 1. The plasma HCV VL values were characterized according to the cut-off threshold level of 600000 Ul / ml, in parallel, quantitative association analysis was also performed. When the continuous variables were analyzed, the VL values were expressed as LoglO. Only the rs2569540 marker of LDLR showed a boundary association under the dominant model in the use of the cut-off level of 600,000 U l / ml (odds ratio = 1.74; p = 0.009). When patients classified according to the HCV genotype (table 2), we found an association between patients infected with HCV genotype 1-4 under the dominant model with the AA + AG genotypes of rsll672123 located in the LDLR distal promoter region (p = 0.001; OR: 2.3; 95% Cl [1,359-3,917]), the AA + AG genotypes of rsl433099 positioned at the 3 'end to the untranslated region of LDLR (p = 0.0004, OR = 0.41, 95% Cl [0.22 -0.66]) and rs2569540 GG + GC (p = 0.0023; OR = 0.45, 95% Cl [0.277-0.761]) located in the intron near the 3 'end of the gene (Figure 1 § ). The C allele of rs2738459 of LDLR is a risk factor and is related to a higher VL in genotypes 2-3 (OR: 4.29; p = 0.0002, 95% Cl [1.930-9.558]. Associations of rsl433099 and rs2738459 with VL reached statistical significance after correction by multiple testing The locations of SNPs in the LDLR gene are represented in Figure 1.
El análisis de asociación cuantitativa del loglO de VL, demostró asociación solo con el rs2569540 de LDLR (mediaiSD; CC 6.22±0.9 vs. GG+CG 5.8±0.9, p= 0.0007, Prueba de Mann-Whitney) y rsl433099 (mediaiSD; GG 6.2±0.9 vs. AA+AG 5.8±0.9, p=0.0001, Prueba de Mann-Whitney), pero solo para los genotipos virales 1-4 (Figura 2). La asociación de rs2738459 con la VL estratificada de acuerdo al nivel de corte de 600000 U l/ml para el genotipo 3, no fue confirmada en el análisis cuantitativo. The quantitative association analysis of the VL log showed association only with the LDLR rs2569540 (mediaiSD; CC 6.22 ± 0.9 vs. GG + CG 5.8 ± 0.9, p = 0.0007, Mann-Whitney Test) and rsl433099 (mediaiSD; GG 6.2 ± 0.9 vs. AA + AG 5.8 ± 0.9, p = 0.0001, Mann-Whitney Test), but only for viral genotypes 1-4 (Figure 2). The association of rs2738459 with the stratified VL according to the cut-off level of 600000 U l / ml for genotype 3, was not confirmed in the quantitative analysis.
Ninguno de los demás genes estudiados (tabla 1) parece estar relacionados con el fenotipo VHC VL. La relación entre la respuesta virológica sostenida fue analizada también, pero ninguno de los SN Ps mostró nivel significativo de asociación bajo condiciones de múltiple testing, los mejores SNPs fueron rs2738457 cercano a rs2569540 (alelo A, 0.275 no-RVS, 0.199 RVS; p=0.037). Por el contrario, la combinación genotípica CC + CG del SNP de I L28B rs28416813 tiene una frecuencia del 0.30 en RVS versus 0.69 en no respondedores (figura 3, p=2xl0-6). La combinación entre los genotipos de LDLR e IL28B mostraron un efecto remarcable sobre la RVS en los genotipos 1-4, cuando individuos homocigóticos portadores de genotipos IL28B protectores fueron clasificados de acuerdo a los genotipos 15 LDLR rs2738457, la frecuencia de RVS fue 0.73 para AA versus 0.26 en AG+GG (p=6xl0-4) (figura 4). Esta interacción de IL28B y LDLR no fue observada en pacientes con genotipos de riesgo IL28B que mostraron una frecuencia de RVS de 0.29 en AA versus 0.29 en AG+GG p=0.72 (figura 4). None of the other genes studied (table 1) seems to be related to the HCV VL phenotype. The relationship between the sustained virological response was also analyzed, but none of the SN Ps showed a significant level of association under multiple testing conditions, the best SNPs were rs2738457 close to rs2569540 (allele A, 0.275 non-RVS, 0.199 RVS; p = 0.037). In contrast, the genotypic combination CC + CG of the SNP of I L28B rs28416813 has a frequency of 0.30 in SVR versus 0.69 in non responders (figure 3, p = 2xl0-6). The combination between LDLR and IL28B genotypes showed a remarkable effect on SVR in genotypes 1-4, when homozygous individuals carrying protective IL28B genotypes were classified according to genotypes 15 LDLR rs2738457, the frequency of SVR was 0.73 for AA versus 0.26 in AG + GG (p = 6xl0-4) (figure 4). This interaction of IL28B and LDLR was not observed in patients with IL28B risk genotypes that showed an SVR frequency of 0.29 in AA versus 0.29 in AG + GG p = 0.72 (Figure 4).
Análisis haplotípicos Haplotype analysis
Una prueba de asociación basada en haplotipo fue desarrollada por un análisis de regresión multi- marcador usando el software Plink. Las frecuencias de haplotipo fueron estimadas y comparadas independientemente para pacientes infectados con genotipos 1-4 o 2-3 (tabla 2). Los SNPs de LDLR incluidos en este estudio abarcan 92.6 Kb (de rsl529729 a 25 rs4804146). El desequilibrio de ligamiento del gen LDLR fue calculado en pacientes infectados con genotipos virales 1-4 y 2-3 y mostró que en ambas poblaciones los SNPs con asociación significativa univariante con VL eran independientemente informativos y estaban contenidos en el interior de 3 bloques haplotípicos, los cuales eran similares a la estructura del bloque en el basa de datos HapMap Phase II (Figura Ib). La tabla 2 muestra las frecuencias de haplotipos importantes en nuestra cohorte para los 3 haplotipos identificados. La presencia (1 o 2 copias) de un haplotipo extendido que abarca las últimos 3 exones de LDLR, incluyendo la combinación AGGCGGAG de los SNPs rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rs6413504, rsl4158 mostraron una fuerte asociación con el nivel de carga viral con umbral de corte de 600000 Ul/ml para los genotipos 1-4 (OR=0.27; p=3xl0"6, Figura 3a), así como una cuantitativa VL (mediaiSD; 6.19±0.9 vs. CC+CG 5.58±l.l,p=0.0001), figura 5). Sin embargo, este haplotipo no tiene efectos significativos en múltiple testing para los genotipos virales 2-3. A haplotype-based association test was developed by a multi-marker regression analysis using Plink software. Haplotype frequencies were estimated and compared independently for patients infected with genotypes 1-4 or 2-3 (Table 2). The LDLR SNPs included in this study cover 92.6 Kb (from rsl529729 to 25 rs4804146). The linkage disequilibrium of the LDLR gene was calculated in patients infected with viral genotypes 1-4 and 2-3 and showed that in both populations the SNPs with significant univariate association with VL were independently informative and contained within 3 haplotypic blocks, which were similar to the block structure in the HapMap Phase II database (Figure Ib). Table 2 shows the frequencies of important haplotypes in our cohort for the 3 identified haplotypes. The presence (1 or 2 copies) of an extended haplotype covering the last 3 exons of LDLR, including the AGGCGGAG combination of SNPs rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rs6413504, rsl4158 showed a strong association viral load with cut-off threshold of 600000 Ul / ml for genotypes 1-4 (OR = 0.27; p = 3xl0 "6 , Figure 3a), as well as a quantitative VL (mediaiSD; 6.19 ± 0.9 vs. CC + CG 5.58 ± ll, p = 0.0001), figure 5) However, this haplotype has no significant effects on multiple testing for 2-3 viral genotypes.
Un haplotipo compacto, incluyendo los primeros 4 SNPs rs2738456, rs2738457, rs2569540, rs2738459 del largo haplotipo, permite capturar la mayor parte de la información de la región; los haplotipos AGGC y GGCC de estos 4 marcadores están asociados con cargas virales más bajas según el nivel umbral de 600.000 copias (OR=0.32; P=2xl0"5) así como con la carga viral cuantitativa (p=8xl0~4) en pacientes infectados con los genotipos 1-4. Los genotipos IL28B no modifican el efecto de haplotipos LDLR en carga viral (p=0.33 para haplotipos AGGC+GGCC y p=0.055 para haplotipos AGCA+GACC) (figura 6). A compact haplotype, including the first 4 SNPs rs2738456, rs2738457, rs2569540, rs2738459 of the long haplotype, allows to capture most of the information in the region; the AGGC and GGCC haplotypes of these 4 markers are associated with lower viral loads according to the threshold level of 600,000 copies (OR = 0.32; P = 2xl0 "5 ) as well as with the quantitative viral load (p = 8xl0 ~ 4 ) in patients Infected with genotypes 1-4 IL28B genotypes do not modify the effect of LDLR haplotypes on viral load (p = 0.33 for AGGC + GGCC haplotypes and p = 0.055 for AGCA + GACC haplotypes) (Figure 6).
Los haplotipos de LDLR modifican el efecto de IL28R en RVS, los pacientes portadores de los haplotipos AGGC/GGC desarrollaron tasas más altas de RVS comparadas con los pacientes AGCA/GACC (0.52 versus 0.36 p=0.016), sin embargo, si los pacientes están clasificados de acuerdo al genotipo IL28B, se observa un notable impacto en los haplotipos de LDLR. Si los portadores del genotipo protector IL28B están clasificados de acuerdo a los haplotipos LDLR, el grupo AGGC/GGCC alcanzaron 78.9% de RVS comparados al 48.6% del grupo AGCA/GACC (p=0.005). Esta tendencia no fue observada en pacientes portadores del genotipo de riesgo de IL28B, donde el genotipo de LDLR no tienen influencia en la probabilidad de RVS (p=0.55) (figura 7). Tabla 1: SNPs incluidos en este estudio. LDLR haplotypes modify the effect of IL28R in SVR, patients with AGGC / GGC haplotypes developed higher rates of SVR compared to AGCA / GACC patients (0.52 versus 0.36 p = 0.016), however, if patients are classified according to the IL28B genotype, a notable impact on LDLR haplotypes is observed. If the carriers of the IL28B protective genotype are classified according to LDLR haplotypes, the AGGC / GGCC group reached 78.9% of SVR compared to 48.6% of the AGCA / GACC group (p = 0.005). This trend was not observed in patients with the IL28B risk genotype, where the LDLR genotype has no influence on the probability of SVR (p = 0.55) (Figure 7). Table 1: SNPs included in this study.
Figure imgf000018_0001
rs 11676704 2 21244358 APOB INTRON
Figure imgf000018_0001
rs 11676704 2 21244358 APOB INTRON
rs 12720791 2 21244617 APOB INTRON rs 12720791 2 21244617 APOB INTRON
rs7589300 2 21244760 APOB INTRON rs7589300 2 21244760 APOB INTRON
rs 12720804 2 21254997 APOB INTRON rs 12720804 2 21254997 APOB INTRON
rs 12720832 2 21256713 APOB INTRON rs 12720832 2 21256713 APOB INTRON
rs 12720842 2 21257927 APOB INTRON rs 12720842 2 21257927 APOB INTRON
rs579826 2 21261507 APOB INTRON rs579826 2 21261507 APOB INTRON
rs 12720796 2 21261998 APOB INTRON rs 12720796 2 21261998 APOB INTRON
rs 10205849 2 21262190 APOB INTRON rs 10205849 2 21262190 APOB INTRON
rs 12720794 2 21262375 APOB INTRON rs 12720794 2 21262375 APOB INTRON
rs531819 2 21263639 APOB INTRON rs531819 2 21263639 APOB INTRON
rs512535 2 21267782 APOB INTERGENIC rs512535 2 21267782 APOB INTERGENIC
rs7575840 2 21273490 APOB INTERGENIC rs7575840 2 21273490 APOB INTERGENIC
rs 1267059 2 162018643 TANK INTRON rs 1267059 2 162018643 TANK INTRON
rs5742909 2 204732347 CTLA4 INTERGENIC rs5742909 2 204732347 CTLA4 INTERGENIC
rs231775 2 204732714 CTLA4 CODING SYNON rs919178 2 231044464 SP110 INTRON rs231775 2 204732714 CTLA4 CODING SYNON rs919178 2 231044464 SP110 INTRON
rs4571051 2 234091158 INPP5D INTRON rs4571051 2 234091158 INPP5D INTRON
rs 17740066 3 121 100283 STXBP5L CODING SYNON rs 12654264 5 74648603 HMGCR INTRON rs 17740066 3 121 100283 STXBP5L CODING SYNON rs 12654264 5 74648603 HMGCR INTRON
rs2070721 5 131825842 IRF1 INTRON rs2070721 5 131825842 IRF1 INTRON
rs6880589 5 156460440 HAVCR1 INTRON rs6880589 5 156460440 HAVCR1 INTRON
rs953569 5 156477400 HAVCR1 INTRON rs953569 5 156477400 HAVCR1 INTRON
rs 1800629 6 31543031 TNF INTERGENIC rs 1800629 6 31543031 TNF INTERGENIC
rs673 6 31543095 TNF INTERGENIC rs673 6 31543095 TNF INTERGENIC
rs3093665 6 31545391 TNF UTR rs3093665 6 31545391 TNF UTR
rs 1800796 7 22766246 IL6 INTERGENIC rs 1800796 7 22766246 IL6 INTERGENIC
rs 1800795 7 22766645 IL6 INTERGENIC rs 1800795 7 22766645 IL6 INTERGENIC
rs2069830 7 22767137 IL6 CODING SYNON rs 13242262 7 128591364 IRF5 INTERGENIC rs2069830 7 22767137 IL6 CODING SYNON rs 13242262 7 128591364 IRF5 INTERGENIC
rs 10488631 7 128594183 IRF5 INTERGENIC rs 10488631 7 128594183 IRF5 INTERGENIC
rs2280714 7 128594725 IRF5 INTERGENIC rs2280714 7 128594725 IRF5 INTERGENIC
rs328 8 19819724 LPL CODING NONSYN rs 1454626 9 2621030 VLDLR INTERGENIC rs328 8 19819724 LPL CODING NONSYN rs 1454626 9 2621030 VLDLR INTERGENIC
rs7043199 9 2621145 VLDLR INTERGENIC rs7043199 9 2621145 VLDLR INTERGENIC
Rs7852409 9 2621482 VLDLR INTERGENIC  Rs7852409 9 2621482 VLDLR INTERGENIC
rs7032549 9 2623615 VLDLR INTERGENIC rs7032549 9 2623615 VLDLR INTERGENIC
rs7022122 9 2624186 VLDLR INTERGENIC rs7022122 9 2624186 VLDLR INTERGENIC
rs 10967213 9 2625172 VLDLR INTERGENIC rs 10967213 9 2625172 VLDLR INTERGENIC
rs 1545566 9 2626031 VLDLR INTERGENIC rs 1545566 9 2626031 VLDLR INTERGENIC
rs4741747 9 2626654 VLDLR INTERGENIC rs4741747 9 2626654 VLDLR INTERGENIC
rs12551418 9 2627365 VLDLR INTERGENIC rs12551418 9 2627365 VLDLR INTERGENIC
rs 1869592 9 2632250 VLDLR INTERGENIC rs 1869592 9 2632250 VLDLR INTERGENIC
rs 10812379 9 2633548 VLDLR INTERGENIC rs 10812379 9 2633548 VLDLR INTERGENIC
rs4740698 9 2637551 VLDLR INTRON rs4740698 9 2637551 VLDLR INTRON
rs3890182 9 107647655 ABCA 1 INTRON rs3890182 9 107647655 ABCA 1 INTRON
rs4986791 9 120475602 TLR4 UTR rs4986791 9 120475602 TLR4 UTR
rs886277 11 2439767 TRPM5 CODING NONSYN rs2298455 1 1 71710478 IL18BP INTRON rs886277 11 2439767 TRPM5 CODING NONSYN rs2298455 1 1 71710478 IL18BP INTRON
rs 187238 1 1 1 12034988 IL18 INTERGENIC rs 187238 1 1 1 12034988 IL18 INTERGENIC
rs 1946518 1 1 1 12035458 IL18 INTERGENIC rs2878771 12 50352393 AQP2 UTR rs 1946518 1 1 1 12035458 IL18 INTERGENIC rs2878771 12 50352393 AQP2 UTR
rs7303577 12 64874102 TBK1 INTRON rs7303577 12 64874102 TBK1 INTRON
rs1131454 12 1 13348870 0AS1 COMPLEX rs1131454 12 1 13348870 0AS1 COMPLEX
rs2295977 14 24657226 IP04 INTRON rs2295977 14 24657226 IP04 INTRON
rs 1800588 15 58723675 LIPC INTERGENIC rs 1800588 15 58723675 LIPC INTERGENIC
rs2290351 15 90374781 AP3S2 UTR rs2290351 15 90374781 AP3S2 UTR
rs 1800775 16 56995236 CETP INTERGENIC rs 1800775 16 56995236 CETP INTERGENIC
rs 1529729 19 1 1163562 LDLR INTRON rs 1529729 19 1 1163562 LDLR INTRON
rs1041581 1 19 1 1177408 LDLR INTERGENIC rs1041581 1 19 1 1177408 LDLR INTERGENIC
rs8102273 19 1 1180047 LDLR INTERGENIC rs8102273 19 1 1180047 LDLR INTERGENIC
rs9305020 19 1 1186711 LDLR INTERGENIC rs9305020 19 1 1186711 LDLR INTERGENIC
rs11672123 19 1 1194823 LDLR INTERGENIC rs11672123 19 1 1194823 LDLR INTERGENIC
rs651 1720 19 1 1202306 LDLR INTRON rs651 1720 19 1 1202306 LDLR INTRON
rs2228671 19 1 1210912 LDLR CODING NONSYN rs 12983082 19 1 1216561 LDLR INTRON rs2228671 19 1 1210912 LDLR CODING NONSYN rs 12983082 19 1 1216561 LDLR INTRON
rs 11669576 19 1 1222300 LDLR CODING SYNON rs 1003723 19 1 1224181 LDLR INTRON rs 11669576 19 1 1222300 LDLR CODING SYNON rs 1003723 19 1 1224181 LDLR INTRON
rs5930 19 1 1224265 LDLR CODING NONSYN rs4508523 19 1 1226944 LDLR INTRON rs5930 19 1 1224265 LDLR CODING NONSYN rs4508523 19 1 1226944 LDLR INTRON
rs2738446 19 1 1227326 LDLR INTRON rs2738446 19 1 1227326 LDLR INTRON
rs688 19 1 1227602 LDLR CODING NONSYN rs2738452 19 1 1229218 LDLR INTRON rs688 19 1 1227602 LDLR CODING NONSYN rs2738452 19 1 1229218 LDLR INTRON
rs5925 19 1 1230881 LDLR CODING NONSYN rs2738456 19 1 1236804 LDLR INTRON rs5925 19 1 1230881 LDLR CODING NONSYN rs2738456 19 1 1236804 LDLR INTRON
rs2738457 19 1 1237269 LDLR INTRON rs2738457 19 1 1237269 LDLR INTRON
rs2569540 19 1 1238239 LDLR INTRON rs2569540 19 1 1238239 LDLR INTRON
rs2738459 19 1 1238473 LDLR INTRON rs2738459 19 1 1238473 LDLR INTRON
rs2738460 19 1 1238807 LDLR INTRON rs2738460 19 1 1238807 LDLR INTRON
rs21 16898 19 1 1241810 LDLR INTRON rs21 16898 19 1 1241810 LDLR INTRON
rs6413504 19 1 1241915 LDLR INTRON rs6413504 19 1 1241915 LDLR INTRON
rs14158 19 1 1242044 LDLR UTR rs14158 19 1 1242044 LDLR UTR
rs3826810 19 1 1242133 LDLR UTR rs3826810 19 1 1242133 LDLR UTR
rs2738464 19 1 1242307 LDLR UTR rs2738464 19 1 1242307 LDLR UTR
rs2738465 19 1 1242496 LDLR UTR rs2738465 19 1 1242496 LDLR UTR
rs 1433099 19 1 1242658 LDLR UTR rs 1433099 19 1 1242658 LDLR UTR
rs2738466 19 1 1242765 LDLR UTR rs2738466 19 1 1242765 LDLR UTR
rs5742911 19 1 1243445 LDLR UTR rs5742911 19 1 1243445 LDLR UTR
rs7258950 19 1 1250139 LDLR INTERGENIC rs7258950 19 1 1250139 LDLR INTERGENIC
rs4804570 19 1 1256059 LDLR INTERGENIC rs4804570 19 1 1256059 LDLR INTERGENIC
rs4804146 19 1 1256176 LDLR INTERGENIC rs4804146 19 1 1256176 LDLR INTERGENIC
rs28416813 19 39735644 IL28B INTERGENIC rs28416813 19 39735644 IL28B INTERGENIC
rs8099917 19 39743165 IL28B INTERGENIC rs8099917 19 39743165 IL28B INTERGENIC
rs1800471 19 41858876 TGFB1 CODING NONSYN rs 1800469 19 41860296 TGFB1 INTERGENIC rs1800471 19 41858876 TGFB1 CODING NONSYN rs 1800469 19 41860296 TGFB1 INTERGENIC
rs405509 19 45408836 APOE INTERGENIC rs405509 19 45408836 APOE INTERGENIC
rs429358 19 45411941 APOE CODING NONSYN rs7412 19 45412079 APOE CODING NONSYN rs439401 19 45414451 AP0C1 INTERGENIC rs429358 19 45411941 APOE CODING NONSYN rs7412 19 45412079 APOE CODING NONSYN rs439401 19 45414451 AP0C1 INTERGENIC
rs445925 19 45415640 AP0C1 INTERGENIC rs445925 19 45415640 AP0C1 INTERGENIC
rs4420638 19 45422946 AP0C1 INTERGENIC rs4420638 19 45422946 AP0C1 INTERGENIC
rs12104272 19 50148052 SCAF1/IRF3 INTERGENIC rs12104272 19 50148052 SCAF1 / IRF3 INTERGENIC
rs2229207 21 34614250 IFNAR2 CODING NONSYN rs2243592 21 34707808 IFNAR1 I NTRON rs2229207 21 34614250 IFNAR2 CODING NONSYN rs2243592 21 34707808 IFNAR1 I NTRON
Tabla 2. Frecuencia de los haplotipos mayores (>5%) y carga viral de VHC en pacientes infectados con virus de genotipos 1 y 4 Table 2. Frequency of major haplotypes (> 5%) and HCV viral load in patients infected with genotype viruses 1 and 4
Figure imgf000021_0001
Figure imgf000021_0001
INVENCIÓN 2 INVENTION 2
Método de obtención de datos útiles para predecir o pronosticar la respuesta al tratamiento con interferón pegilado más ribavirina en pacientes infectados por el virus de la hepatitis C genotipo 3. Method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with the hepatitis C virus genotype 3.
La presente invención se encuentra dentro del campo de la biomedicina y la biotecnología, y se refiere a un método de obtención de datos útiles para predecir o pronosticar la respuesta al tratamiento con interferón pegilado más ribavirina en pacientes infectados por el virus de la hepatitis C (VHC) genotipo 3, usando como predictor un polimorfismo en el gen de la proproteína convertasa subtilisina/kexina de tipo 9 (PCSK9). The present invention is within the field of biomedicine and biotechnology, and refers to a method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with hepatitis C virus ( HCV) genotype 3, using as a predictor a polymorphism in the proprotein convertase subtilisin / kexin type 9 gene (PCSK9).
ESTADO DE LA TÉCNICA ANTERIOR Las tasas de respuesta virológica sostenida ( VS) a interferón pegilado (Peg-IFN) más ribavirina (RBV) dependen en gran medida del virus y de factores relacionados con el hospedador, así como en la cinética viral durante la terapia. Este último es comúnmente usado para decidir la duración del tratamiento. Así, el tratamiento en pacientes monoinfectados con VHC-1 con baja carga viral de base y respuesta virológica rápida puede ser acortado a 24 semanas. STATE OF THE PRIOR ART The rates of sustained virological response (VS) to pegylated interferon (Peg-IFN) plus ribavirin (RBV) depend largely on the virus and host-related factors, as well as on viral kinetics during therapy . The latter is commonly used to decide the duration of the treatment. Thus, treatment in monoinfected patients with HCV-1 with low base viral load and rapid virological response can be shortened to 24 weeks.
Del mismo modo, la duración de la terapia dual puede ser acortada en portadores del genotipo 3 que alcancen respuesta virológica rápida. En el marco de la co-infección con VI H, una terapia de 24 semanas antes que una de 48 semanas es recomendada en portadores de VHC-3, los cuales muestran una indetectable carga de A N-VHC después de 4 semanas de terapia doble. Mientras que la duración de las pautas de terapia usadas comúnmente todavía varían de 12 a 48 semanas, regímenes más cortos que incluyen antivirales directos, estarán disponibles en un futuro cercano. En pacientes con los genotipos 1 o 4 de VHC, polimorfismo de un solo nucleótido (SN Ps) en los genes de la interleuquina 28B y del receptor de la lipoproteína de baja densidad (LDLR), han sido identificados para predecir RVS y la cinética viral. Sin embargo, el impacto de estas variaciones genéticas en RVS es mucho menos relevante en los pacientes infectados con el genotipo 3, y las herramientas predictivas para la respuesta al tratamiento específico para VHC genotipo 3, son escasas. Una asociación entre los niveles plasmáticos de LDL y RVS ha sido descrita por pacientes con o sin coinfección con VI H. Aunque menos significativo que pacientes infectados con los genotipos 1 y 4 de VHC, este efecto es observado también en la sub-población de pacientes portadores de los genotipos 2 y 3 de VHC. Sin embargo, el mecanismo subyacente para este descubrimiento, no está dilucidado del todo. Hay una evidencia de que LDLR es usado por VHC para entrar en el hepatocito después de la formación de partículas lipovirales principalmente con LDL. Este paso es seguido por un complejo progreso que implica al co- receptor de superficie celular de tetraspanina CD81. La proproteína convertasa subtilisina/kexina de tipo 9 (PKCS9) es una proproteína convertasa que pertenece a la sub-familia de la proteinasa K, que impacta en los niveles plasmáticos de lipoproteínas por modulación intra y extra- hepática de los niveles de LDLR y CD81. Las variaciones en este gen interfieren con el sistema LDL/LDLR y podrían así afectar al ciclo vital de VHC y, en consecuencia, sobre la RVS. Sin embargo, no hay datos sobre el impacto de las variaciones en el gen PCSK9. Similarly, the duration of dual therapy can be shortened in carriers of genotype 3 that achieve rapid virological response. Within the framework of co-infection with VI H, a therapy of 24 weeks before a 48-week one is recommended in HCV-3 carriers, which show an undetectable burden of A N-HCV after 4 weeks of double therapy . While the duration of commonly used therapy guidelines still vary from 12 to 48 weeks, shorter regimens that include direct antivirals will be available in the near future. In patients with HCV genotypes 1 or 4, single nucleotide polymorphism (SN Ps) in the interleukin 28B and low density lipoprotein receptor (LDLR) genes have been identified to predict SVR and viral kinetics . However, the impact of these genetic variations on SVR is much less relevant in patients infected with genotype 3, and the predictive tools for the response to the specific treatment for HCV genotype 3 are scarce. An association between plasma levels of LDL and SVR has been described by patients with or without coinfection with HV. Although less significant than patients infected with HCV genotypes 1 and 4, this effect is also observed in the sub-population of patients. carriers of HCV genotypes 2 and 3. However, the underlying mechanism for this discovery is not fully elucidated. There is evidence that LDLR is used by HCV to enter the hepatocyte after the formation of lipoviral particles mainly with LDL. This step is followed by a complex progress involving the CD81 tetraspanin cell surface receptor. Proprotein convertase subtilisin / kexin type 9 (PKCS9) is a proprotein convertase that belongs to the sub-family of proteinase K, which impacts plasma lipoprotein levels by intra and extra-hepatic modulation of LDLR and CD81 levels. . Variations in this gene interfere with the LDL / LDLR system and could thus affect the life cycle of HCV and, consequently, on SVR. However, there is no data on the impact of variations on the PCSK9 gene.
Puesto que las herramientas predictivas para la respuesta al tratamiento específico para VHC genotipo 3, son escasas, es necesario encontrar marcadores adecuados. Since the predictive tools for the response to the specific treatment for HCV genotype 3 are scarce, it is necessary to find suitable markers.
DESCRIPCIÓN DE LA INVENCIÓN En la presente invención se describe la influencia de los SN Ps del gen PCSK9 en la respuesta viral del tratamiento contra la infección por VCH, que incluye Peg-IFN más RBV en una cohorte de individuos infectados por VHC genotipo 3. DESCRIPTION OF THE INVENTION The present invention describes the influence of SN Ps of the PCSK9 gene on the viral response of treatment against HCV infection, which includes Peg-IFN plus RBV in a cohort of individuals infected with HCV genotype 3.
La presente invención proporciona un método de obtención de datos útiles para predecir o pronosticar la respuesta al tratamiento del VHC genotipo 3, y específicamente, al tratamiento con Peg-I FN/RBV. Los autores de la presente invención han encontrado un biomarcador útil para predecir la respuesta al tratamiento de la hepatitis C genotipo 3, que podría conducir a los médicos a detectar aquellos pacientes con una probabilidad mayor a responder a la terapia Peg-I FN/RBV. The present invention provides a method of obtaining useful data to predict or predict the response to treatment of HCV genotype 3, and specifically, to treatment with Peg-I FN / RBV. The authors of the present invention have found a useful biomarker to predict the response to the treatment of hepatitis C genotype 3, which could lead doctors to detect those patients with a higher probability of responding to Peg-I FN / RBV therapy.
Por tanto, un primer aspecto de la invención se refiere al uso del gen PCSK9 para predecir la respuesta de un individuo al tratamiento del VHC con IFN-Peg más un análogo de guanosina. Therefore, a first aspect of the invention relates to the use of the PCSK9 gene to predict an individual's response to HCV treatment with IFN-Peg plus a guanosine analog.
En una realización preferida, el análogo de la guanosina es la ribavirina (RBV). In a preferred embodiment, the guanosine analog is ribavirin (RBV).
En otra realización preferida de este aspecto de la invención, el individuo está infectado también por el VIH. En otra realización preferida, el VHC es del genotipo 3. In another preferred embodiment of this aspect of the invention, the individual is also infected with HIV. In another preferred embodiment, HCV is genotype 3.
La terapia antiviral va dirigida básicamente a lograr unos objetivos virológicos, bioquímicos e histológicos. La evaluación de la respuesta al tratamiento, se basa en el efecto del mismo sobre el estado virológico, no sólo al terminar el tratamiento sino, lo que es más importante, en su seguimiento después de finalizado el mismo. Tal y como se entiende en esta memoria, se denomina "respuesta virológica sostenida" o "respuesta viral sostenida" (RVS) la negativización de la viremia (ARN viral negativo mediante técnicas de reacción en cadena de la polimerasa -PCR-) que se mantiene al menos 24 semanas después de finalizado el tratamiento; o dicho de otra manera, la ausencia en el suero del ARN-VHC a los 6 meses de finalizado el tratamiento. Antiviral therapy is basically aimed at achieving virological, biochemical and histological objectives. The evaluation of the response to the treatment, is based on the effect of the same on the virological state, not only at the end of the treatment but, more importantly, in its follow-up after the end of the treatment. As understood herein, it is called "sustained virological response" or "sustained viral response" (SVR) the viremia negativization (negative viral RNA by polymerase chain reaction techniques -PCR-) that is maintained at least 24 weeks after the end of treatment; In other words, the absence of HCV-RNA in the serum 6 months after the end of treatment.
La negativización de la viremia al final del tratamiento, con reaparición del ARN una vez terminado el mismo, define la "respuesta virológica con recaída". La ausencia de negativización de la viremia al final del tratamiento define la "no respuesta virológica" al tratamiento. The negativization of the viremia at the end of the treatment, with the reappearance of the RNA once it is finished, defines the "virological response with relapse". The absence of viremia negativization at the end of treatment defines the "virological non-response" to the treatment.
El "interferón alfa" (IFN-α) es una citocina inmunomoduladora que posee actividad antiviral, propiedades antiangiogénicas y antiproliferativas. El I FN-Peg se obtiene mediante la unión fisicoquímica de polietilenglicol (PEG) al interferón alfa (peginterferón a-2a y a-2b) recombinante, lo que aumentan su permanencia en la sangre requiriendo menor frecuencia de inyecciones. "Interferon alfa" (IFN-α) is an immunomodulatory cytokine that has antiviral activity, antiangiogenic and antiproliferative properties. I FN-Peg is obtained by the physicochemical binding of polyethylene glycol (PEG) to recombinant interferon alfa (peginterferon a-2a and a-2b), which increases its permanence in the blood requiring less frequency of injections.
Actualmente existen dos tipos de interferones pegilados comerciales cuya aplicación se ha extendido: peginterferón alfa-2a (Pegasys®, Roche) y peginterferón alfa-2b (Pegintron®, Schering-Plough). Estos dos productos son semejantes con respecto a su eficacia y seguridad pero sus regímenes de dosificación difieren levemente. There are currently two types of commercial pegylated interferons whose application has been extended: peginterferon alfa-2a (Pegasys ® , Roche) and peginterferon alfa-2b (Pegintron ® , Schering-Plow). These Two products are similar with respect to their efficacy and safety but their dosage regimens differ slightly.
La ribavirina (Ι-β-D-ribofuranosil-lH-l, 2,4-triazol-3-carboxamida) es un nucleótido sintético análogo de la guanidina, con propiedades antivirales y actividad inmunorreguladora. Fue una de las primeras drogas que demostró su eficacia para reducir la capacidad de reproducción de virus. Está descrita en el índice Merck, compuesto N 9 8199, undécima edición. Su fabricación y formulación se describen en la patente de EE.UU. n. No. 4.211.771. Ribavirin (Ι-β-D-ribofuranosyl-lH-l, 2,4-triazol-3-carboxamide) is a synthetic nucleotide analogous to guanidine, with antiviral properties and immunoregulatory activity. It was one of the first drugs that demonstrated its effectiveness in reducing the ability to reproduce viruses. It is described in the Merck index, compound N 9 8199, eleventh edition. Its manufacture and formulation are described in US Pat. n. No. 4,211,771.
La rápida captación eritrocitaria de la ribavirina y su fosforilación son responsables de la importante anemia hemolítica dependiente de la dosis. Con el fin de evitar este efecto indeseado, el diseño de los nuevos fármacos contempla que no sean captados o fosforilados por los eritrocitos o que sean captados rápidamente y fosforilados en el hígado. Ambos enfoques tienen la desventaja potencial de que pueden anular pasos fundamentales que contribuyen con la eficacia terapéutica de la ribavirina. The rapid erythrocyte uptake of ribavirin and its phosphorylation are responsible for significant dose-dependent hemolytic anemia. In order to avoid this unwanted effect, the design of the new drugs contemplates that they are not captured or phosphorylated by erythrocytes or that they are rapidly captured and phosphorylated in the liver. Both approaches have the potential disadvantage that they can override fundamental steps that contribute to the therapeutic efficacy of ribavirin.
Otros derivados, profármacos o análogos de la ribavirina son la viramidina y la levovirina. Other derivatives, prodrugs or analogs of ribavirin are viramidine and levovirin.
La viramidina es un profármaco de la BV, en el hígado se convierte en RBV por acción de la adenosindeaminasa hepática. La RBV y viramidina son rápidamente eliminadas, sus moléculas y los metabolitos son excretados por el riñon y tienen una Tmáx de 1,5-3 h. Experimentalmente, la retención hepática de la RBV derivada de una dosis oral de viramidina es 3 veces mayor que la RBV oral. En cambio, la concentración de fosfatos de RBV en los hematíes es mucho menor, de ahí que se pueda mantener una concentración más estable de hemoglobina. Es un fármaco seguro y se tolera bien. Los efectos adversos con dosis de 200, 600 y 1.200 mg fueron: 0, 26 y 50%, respectivamente; la mayoría leves y desaparecieron sin secuelas. Viramidine is a prodrug of BV, in the liver it is converted to RBV by hepatic adenosinedeaminase. RBV and viramidine are rapidly eliminated, their molecules and metabolites are excreted by the kidney and have a Tmax of 1.5-3 h. Experimentally, hepatic retention of RBV derived from an oral dose of viramidine is 3 times higher than oral RBV. In contrast, the concentration of RBV phosphates in red blood cells is much lower, hence a more stable hemoglobin concentration can be maintained. It is a safe drug and is well tolerated. The adverse effects with doses of 200, 600 and 1,200 mg were: 0, 26 and 50%, respectively; mostly mild and disappeared without sequelae.
La levovirina es la L-enantiomero de la RBV y no origina anemia hemolítica. Tiene una actividad inmunomoduladora similar a la RBV, pero no tiene actividad antiviral directa. Levovirin is the L-enantiomer of RBV and does not cause hemolytic anemia. It has an immunomodulatory activity similar to RBV, but it has no direct antiviral activity.
Otros posibles análogos se describen, por ejemplo, pero sin limitarnos, en la patente WO 2010/135520. La ribavirina por sí sola no es eficaz contra el VHC, pero cuando se la combina con interferón, los índices de erradicación viral son mucho mejores comparados con aquellos logrados solamente con interferón. Other possible analogs are described, for example, but not limited to, in WO 2010/135520. Ribavirin alone is not effective against HCV, but when combined with interferon, viral eradication rates are much better compared to those achieved only with interferon.
El "genotipo del VHC", o "cepa viral" se determina por una prueba de sangre. El virus de la hepatitis C se ha clasificado en seis genotipos distintos en base a diferencias en sus genomas, estos genotipos a su vez se subdividen. La preponderancia y distribución de dichos genotipos varía de modo global. Por ejemplo, en Norte América predomina el genotipo 1, seguido de 2 y 3. En Europa predomina el genotipo 1 seguido de 3, 4 y 2. Los genotipos 4 predomina en Egipto, 5 se da casi exclusivamente en África y genotipo 6 en el sudeste asiático. The "HCV genotype", or "viral strain" is determined by a blood test. The hepatitis C virus has been classified into six different genotypes based on differences in their genomes, these genotypes in turn are subdivided. The preponderance and distribution of these genotypes varies globally. For example, in North America, genotype 1 predominates, followed by 2 and 3. In Europe, genotype 1 predominates followed by 3, 4 and 2. Genotype 4 predominates in Egypt, 5 occurs almost exclusively in Africa and genotype 6 in Southeast Asia.
El genotipo tiene importancia clínica ya que determina la respuesta potencial de la terapia actual y la duración que va a requerir la misma. Los genotipos 1 y 4 responden menos a dicho tratamiento que los otros (genotipos 2, 3, 5 y 6). La duración de la terapia estándar basada en el interferón para los genotipos 1 y 4 es de 48-72 semanas, mientras que para los genotipos 2 y 3 es solo de 12-24 semanas. En resumen, algunos genotipos del VHC, tales como los tipos 2 y 3, son más fáciles de erradicar que el tipo 1. The genotype is of clinical importance since it determines the potential response of the current therapy and the duration that it will require. Genotypes 1 and 4 respond less to this treatment than the others (genotypes 2, 3, 5 and 6). The duration of standard interferon-based therapy for genotypes 1 and 4 is 48-72 weeks, while for genotypes 2 and 3 it is only 12-24 weeks. In summary, some HCV genotypes, such as types 2 and 3, are easier to eradicate than type 1.
Se denomina "carga viral plasmática basal de A N del VHC", a la cantidad de ARN del VHC en la sangre (suero). Su presencia es indicativa de una infección activa con reproducción viral en curso. La carga viral suele expresarse en unidades logarítmicas [loglO IU/ml] . The amount of HCV RNA in the blood (serum) is called "basal plasma viral load of HCV A N". Its presence is indicative of an active infection with ongoing viral reproduction. The viral load is usually expressed in logarithmic units [log IU / ml].
Para el pronóstico se considera que a mayor carga viral basal, menor probabilidad de una respuesta sostenida. La respuesta al tratamiento antiviral se evalúa con una carga viral basal (pre-tratamiento) y al menos una carga viral al término de éste y a las 24 semanas post tratamiento. Se considera una respuesta al tratamiento antiviral si se obtiene una carga viral indetectable al término del tratamiento y una carga viral indetectable y/o ARN cualitativo negativo a las 24 semanas (respuesta viral sostenida). Igualmente se ha determinado que una caída de la carga viral temprana (respuesta viral precoz), con descenso de 2 loglO Ul/ml o más a las 12 semanas intra-tratamiento, permite evaluar anticipadamente la probabilidad de obtener una respuesta virológica sostenida al término de la terapia. La genotipación del gen "PCKS9" se puede obtener, preferiblemente, pero sin limitarnos, secuenciando el gen completo pero aún más preferiblemente, se obtiene analizando el polimorfismo de un solo nucleótido (single nucleotide polymorphism, SN P) rs2479409, localizado dentro de la región 5' hacia arriba. For the prognosis, the higher the baseline viral load, the lower the likelihood of a sustained response. The response to antiviral treatment is evaluated with a baseline viral load (pre-treatment) and at least one viral load at the end of it and at 24 weeks post treatment. A response to antiviral treatment is considered if an undetectable viral load is obtained at the end of the treatment and an undetectable viral load and / or qualitative negative RNA at 24 weeks (sustained viral response). Likewise, it has been determined that a fall in the early viral load (early viral response), with a decrease of 2 loglO Ul / ml or more at 12 weeks intra-treatment, allows an early evaluation of the probability of obtaining a sustained virological response at the end of the therapy. The genotyping of the "PCKS9" gene can be obtained, preferably, but not limited to, by sequencing the entire gene but even more preferably, it is obtained by analyzing the single nucleotide polymorphism (single nucleotide polymorphism, SN P) rs2479409, located within the region 5 'up.
La secuencia nucleotídica del SN P rs2479409 es: TAGAATTCTGAATGTACCTATATGAC [A/G] TCTTTGCAAACTTAAAACCTGAATC The nucleotide sequence of SN P rs2479409 is: TAGAATTCTGAATGTACCTATATGAC [A / G] TCTTTGCAAACTTAAAACCTGAATC
En esta memoria el "gen PCSK9" es el gen de la proprotein convertase subtilisin/kexin type 9, se encuentra en el cromosoma 1 (lp32.3). In this report, the "PCSK9 gene" is the proprotein convertase subtilisin / kexin type 9 gene, found on chromosome 1 (lp32.3).
MÉTODO DE OBTENCIÓN DE DATOS ÚTILES, MÉTODO DE PRONÓSTICO Y PREDICCIÓN DE LA RESPUESTA VIRAL SOSTENIDA AL TRATAMIENTO DEL VHC CON QUIMIOTERAPIA. Otro aspecto de la invención se refiere a un método de obtención de datos útiles para predecir o pronosticar la RVS al tratamiento del VHC con I FN-Peg más un análogo de guanosina, de ahora en adelante primer método de la invención, que comprende: a) obtener una muestra biológica aislada de un individuo, y b) determinar el genotipo del gen PCSK9 en la muestra aislada de (a). METHOD OF OBTAINING USEFUL DATA, METHOD OF FORECAST AND PREDICTION OF THE VIRAL RESPONSE SUSTAINED TO THE TREATMENT OF HCV WITH CHEMOTHERAPY. Another aspect of the invention relates to a method of obtaining useful data for predicting or predicting SVR to the treatment of HCV with I FN-Peg plus a guanosine analog, hereafter referred to as the first method of the invention, comprising: a ) obtain an isolated biological sample from an individual, and b) determine the genotype of the PCSK9 gene in the sample isolated from (a).
El genotipado del gen PSCK9, arroja un SPN rs2479409 que puede pronosticar o predecir la respuesta al tratamiento de VHC genotipo 3, con I FN-Peg y Ribavirina (o análogos). Los autores de la presente invención han determinado que: The genotyping of the PSCK9 gene yields an SPN rs2479409 that can predict or predict the response to the treatment of HCV genotype 3, with I FN-Peg and Ribavirin (or analogues). The authors of the present invention have determined that:
1) Los genotipos AA y AG del SNP rs2479409 del gen PCSK9, muestran unos buenos resultados de RVS al tratamiento. 1) The AA and AG genotypes of the SNP rs2479409 of the PCSK9 gene show good results of SVR at treatment.
2) El genotipo GG del SNP rs2479409 del gen PCSK9, responde peor al tratamiento que los dos anteriores. 2) The GG genotype of SNP rs2479409 of the PCSK9 gene responds worse to treatment than the previous two.
Otro aspecto de la invención se refiere a un método para predecir o pronosticar la RVS al tratamiento del VHC con I FN-Peg más un análogo de guanosina, de ahora en adelante 25 segundo método de la invención, que comprende los pasos (a) y (b) del primer método de la invención, y además comprende: c) clasificar a los individuos según el genotipo de PCSK9 que presentan y su respuesta al tratamiento con IFN-peg más un análogo de guanosina. Another aspect of the invention relates to a method for predicting or predicting SVR to the treatment of HCV with I FN-Peg plus a guanosine analogue, hereafter referred to as the second method of the invention, comprising steps (a) and (b) of the first method of the invention, and further comprises: c) classifying individuals according to the genotype of PCSK9 they present and their response to treatment with IFN-peg plus a guanosine analog.
En una realización preferida, el análogo de guanosina es la ribavirina (RBV). In a preferred embodiment, the guanosine analog is ribavirin (RBV).
En otra realización preferida de este aspecto de la invención, el individuo está coinfectado por el VI H y el VHC. En otra realización preferida, el VHC es del genotipo 3. In another preferred embodiment of this aspect of the invention, the individual is coinfected by the VI H and the HCV. In another preferred embodiment, HCV is genotype 3.
Los pasos (b) y/o (c) de los métodos descritos anteriormente pueden ser total o parcialmente automatizados, por ejemplo, por medio de un equipo robótico sensor para la detección de la cantidad en el paso (b) o la comparación computarizada en el paso (c). Steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the amount in step (b) or the computerized comparison in step (c).
El término "muestra biológica aislada" incluye, pero sin limitarnos a, células, tejidos y/o fluidos biológicos de un organismo, como sangre, suero, orina, saliva, fluido cerebroespinal, o a una muestra sólida o semisolida, como tejidos, heces, y similar, o alternativamente, un tejido sólido tales como aquellos usados habitualmente en diagnóstico histológico, obtenidos mediante cualquier método conocido por un experto en la materia. Existen más de 100 métodos distintos de genotipado de SNPs, siendo cualquiera aplicable en la presente invención. Por ejemplo, sin limitarnos, puede detectarse empleando desde enzimas de restricción o por secuenciación simple mediante PC , sondas ASO e hibridación en Micromatrices de ADN o esferas. Así, por ejemplo, como métodos de amplificación de SN Ps se encuentran la PCR, el método Invader, la amplificación por ADN de círculo rodante, etc. Como métodos de discriminación alélica, pueden emplearse, pero sin limitarnos, hibridación específica de alelo, ligamiento de oligonucleótidos específicos de alelo, minisecuenciación o extensión de sondas (SBE, Single Base Extensión), rotura invasiva específica de alelo (Invader), etc. Según el formato de detección, podemos citar, también sin limitarnos, el uso de sondas Taqman, de molecular beacons, scorpion, etc. Respecto a reacciones en un soporte sólido, tenemos, por ejemplo, biochips y microarrays de ADN, u otros métodos que emplean partículas, como por ejemplo, Luminex 100, BeadArray, etc. También existen métodos de detección masiva, como la pirosecuenciación, o la miniaturización. The term "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, such as blood, serum, urine, saliva, cerebrospinal fluid, or a solid or semi-solid sample, such as tissues, feces, and similar, or alternatively, a solid tissue such as those commonly used in histological diagnosis, obtained by any method known to a person skilled in the art. There are more than 100 different methods of genotyping SNPs, whichever is applicable in the present invention. For example, without limiting ourselves, it can be detected using restriction enzymes or by simple sequencing by PC, ASO probes and hybridization in DNA microarrays or spheres. Thus, for example, as methods of amplification of SN Ps are PCR, the Invader method, amplification by rolling circle DNA, etc. As allelic discrimination methods, allele-specific hybridization, allele-specific oligonucleotide ligation, probe mini-sequencing or extension (SBE, Single Base Extension), allele-specific invasive breakage (Invader), etc. can be used, but not limited to. Depending on the detection format, we can mention, also without limiting ourselves, the use of Taqman probes, molecular beacons, scorpion, etc. Regarding reactions on a solid support, we have, for example, biochips and microarrays of DNA, or other methods that employ particles, such as, for example, Luminex 100, BeadArray, etc. There are also methods of mass detection, such as pyrosequencing, or miniaturization.
En otra realización preferida, el genotipado de PCSK9 se realiza mediante PCR, y más preferiblemente, la determinación es del rs2479409. En una realización aún mas preferida, la determinación se realiza del genotipo AA del SN P rs2479409. En otra realización preferida, se determina el genotipo AG del SN P rs2479409. En otra realización preferida, la determinación es del genotipo GG del SN P rs2479409. In another preferred embodiment, PCSK9 genotyping is performed by PCR, and more preferably, the determination is rs2479409. In an even more preferred embodiment, the determination is made of genotype AA of SN P rs2479409. In another preferred embodiment, the AG genotype of SN P rs2479409 is determined. In another preferred embodiment, the determination is of the GG genotype of SN P rs2479409.
En la presente invención "pronóstico" se entiende como la evolución esperada de una enfermedad y se refiere a la valoración de la probabilidad según la cual un sujeto padece una enfermedad así como a la valoración de su inicio, estado de desarrollo, evolución, o de su regresión, y/o el pronóstico del curso de la enfermedad en el futuro. Como entenderán los expertos en la materia, tal valoración, aunque se prefiere que sea, normalmente puede no ser correcta para el 100% de los sujetos que se va a diagnosticar. El término, sin embargo, requiere que una parte estadísticamente significativa de los sujetos se pueda identificar como que padecen la enfermedad o que tienen predisposición a la misma. Si una parte es estadísticamente significativa se puede determinar sin más por el experto en la materia usando varias herramientas de evaluación estadística bien conocidas, por ejemplo, determinación de intervalos de confianza, determinación de valores p, prueba t de Student, prueba de Mann-Whitney, etc. Los intervalos de confianza preferidos son al menos el 50%, al menos el 60%, al menos el 70%, al menos el 80%, al menos el 90%, al menos el 95%. Los valores de p son, preferiblemente, 0,2, 0,1, 0,05. Por "predicción de la respuesta" se entiende, en el contexto de la presente invención, la determinación de la probabilidad de que el paciente responda de forma favorable o desfavorable a una terapia o a un tratamiento determinado. Especialmente, el término "predicción", como se usa aquí, se refiere a una evaluación individual de cualquier parámetro que pueda ser útil en determinar la evolución de un paciente. Como entenderán los expertos en la materia, la predicción de la respuesta clínica al tratamiento, aunque se prefiere que sea, no necesita ser correcta para el 100% de los sujetos a ser diagnosticados o evaluados. El término, sin embargo, requiere que se pueda identificar una parte estadísticamente significativa de los sujetos como que tienen una probabilidad aumentada de tener una respuesta positiva. El experto en la materia puede determinar fácilmente si un sujeto es estadísticamente significativo usando varias herramientas de evaluación estadística bien conocidas, por ejemplo, determinación de intervalos de confianza, determinación de los valores de p, prueba t de Student, prueba de Mann Whitney, etc. Los intervalos de confianza preferidos son al menos del 50%>, al menos del 60%>, al menos del 70%>, al menos del 80%>, al menos del 90%), al menos del 95%>. Los valores de p son, preferiblemente, 0,2, 0,1 ó 0,05. La predicción de la respuesta clínica se puede hacer utilizando cualquier criterio de valoración usado en oncología y conocido por el experto en la materia. In the present invention "prognosis" is understood as the expected evolution of a disease and refers to the assessment of the probability according to which a subject suffers from a disease as well as the assessment of its onset, developmental status, evolution, or its regression, and / or the prognosis of the course of the disease in the future. As those skilled in the art will understand, such assessment, although preferred, may not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant part of the subjects can be identified as having the disease or having a predisposition to it. If a part is statistically significant, it can be determined by the person skilled in the art using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann-Whitney test , etc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. P values are preferably 0.2, 0.1, 0.05. "Prediction of the response" means, in the context of the present invention, the determination of the probability that the patient responds favorably or unfavorably to a given therapy or treatment. Especially, the term "prediction", as used here, refers to a Individual evaluation of any parameter that may be useful in determining the evolution of a patient. As those skilled in the art will understand, the prediction of the clinical response to treatment, although it is preferred, does not need to be correct for 100% of the subjects to be diagnosed or evaluated. The term, however, requires that a statistically significant part of the subjects can be identified as having an increased probability of having a positive response. The person skilled in the art can easily determine if a subject is statistically significant using several well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t-test, Mann Whitney test, etc. . Preferred confidence intervals are at least 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%), at least 95%>. P values are preferably 0.2, 0.1 or 0.05. The prediction of the clinical response can be made using any assessment criteria used in oncology and known to the person skilled in the art.
A su vez, atendiendo al método de la presente invención, se podrían establecer otras subclasificaciones dentro de esta principal, facilitando, por tanto, la elección y el establecimiento de regímenes terapéuticos o tratamiento adecuados. Esta discriminación tal y como es entendida por un experto en la materia no pretende ser correcta en un 100% de las muestras analizadas. Sin embargo, requiere que una cantidad estadísticamente significativa de las muestras analizadas sean clasificadas correctamente. La cantidad que es estadísticamente significativa puede ser establecida por un experto en la materia mediante el uso de diferentes herramientas estadísticas, por ejemplo, pero sin limitarse, mediante la determinación de intervalos de confianza, determinación del valor significación P, test de Student o funciones discriminantes de Fisher, medidas no paramétricas de Mann Whitney, correlación de Spearman, regresión logística, regresión lineal, área bajo la curva de OC (AUC). Preferiblemente, los intervalos de confianza son al menos del 90%, al menos del 95%, al menos del 97%, al menos del 98% o al menos del 99%. Preferiblemente, el valor de p es menor de 0,1, de 0,05, de 0,01, de 0,005 o de 0,0001. Preferiblemente, la presente invención permite detectar correctamente la enfermedad de forma diferencial en al menos el 60%, más preferiblemente en al menos el 70%, mucho más preferiblemente en al menos el 80%, o aún mucho más preferiblemente en al menos el 90% de los sujetos de un determinado grupo o población analizada. In turn, according to the method of the present invention, other subclassifications could be established within this principal, thus facilitating the choice and establishment of appropriate therapeutic regimens or treatment. This discrimination, as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. The amount that is statistically significant can be established by a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the significance value P, Student test or discriminant functions. Fisher, non-parametric measurements of Mann Whitney, Spearman correlation, logistic regression, linear regression, area under the OC curve (AUC). Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention makes it possible to correctly detect the disease differentially by at least 60%, more preferably at least 70%, much more preferably at least 80%, or even much more preferably at least 90%. of the subjects of a certain group or population analyzed.
El término "individuo", tal y como se utiliza en la descripción, se refiere a animales, preferiblemente mamíferos, y más preferiblemente, humanos. El término "individuo" no pretende ser limitativo en ningún aspecto, pudiendo ser éste de cualquier edad, sexo y condición física. The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
KIT O DISPOSITIVO DE DIAGNÓSTICO, MICROARRAY Y USOS Otro aspecto de la presente invención se refiere a un kit o dispositivo, de aquí en adelante kit o dispositivo de la invención, que comprende los elementos necesarios para analizar el genotipo del gen PCSK9. KIT OR DEVICE FOR DIAGNOSIS, MICROARRAY AND USES Another aspect of the present invention relates to a kit or device, hereinafter kit or device of the invention, comprising the elements necessary to analyze the genotype of the PCSK9 gene.
En otra realización preferida el kit puede contener una o varias secuencias nucleotídicas para analizar el genotipo del SNP rs2479409. In another preferred embodiment, the kit may contain one or more nucleotide sequences to analyze the genotype of SNP rs2479409.
En otra realización aún mas preferida, el kit puede contener una o varias secuencias nucleotídicas para analizar el genotipo AA del SNP rs2479409. Otra realización preferida se refiere a la determinación del genotipo AG del SNP rs2479409. En otra realización preferida, la determinación es del genotipo GG del SNP rs2479409. Dicho kit puede contener todos aquellos reactivos necesarios para analizar el genotipo del gen PCSK9 por medio de cualquiera de los métodos descritos anteriormente en este documento. El kit además puede incluir, sin ningún tipo de limitación, tampones, agentes para prevenir la contaminación, inhibidores de la degradación de las proteínas, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimización. Preferiblemente, el kit comprende además las instrucciones para llevar a cabo cualquiera de los métodos de la invención. In another even more preferred embodiment, the kit may contain one or more nucleotide sequences to analyze the AA genotype of SNP rs2479409. Another preferred embodiment relates to the determination of genotype AG of SNP rs2479409. In another preferred embodiment, the determination is of the GG genotype of SNP rs2479409. Said kit may contain all those reagents necessary to analyze the genotype of the PCSK9 gene by means of any of the methods described hereinabove. The kit can also include, without any limitation, buffers, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out any of the methods of the invention.
Es también posible que el (los) oligonucleótido(s) o sondas estén inmovilizados en manchas sobre una superficie (preferiblemente sólida). En una de sus realizaciones, el kit comprende una micromatriz, o micromatriz de la invención. Una micromatriz de A N es una matriz sobre un sustrato sólido (normalmente un porta de vidrio o una celda de una película fina de silicio) que evalúa grandes cantidades de diferentes ARN que son detectables mediante sondas específicas inmovilizadas sobre manchas sobre un sustrato sólido. Cada mancha contiene una secuencia específica de ácido nucleico, que normalmente corresponde a una secuencia concreta de ADN. Aunque el número de manchas no está limitado de manera alguna, existe una realización preferida en la que la micromatriz se personaliza para los procedimientos de la invención. Por tanto, otro aspecto de la invención se refiere a un microarray, de ahora en adelante microarray de la invención, que comprende al menos una sonda para detectar el genotipo del gen PCSK9. It is also possible that the oligonucleotide (s) or probes are immobilized in spots on a (preferably solid) surface. In one of its embodiments, the kit comprises a microarray, or microarray of the invention. An A N microarray is a matrix on a solid substrate (usually a glass holder or a cell of a thin silicon film) that evaluates large amounts of different RNAs that are detectable by specific probes immobilized on spots on a solid substrate. Each stain contains a specific nucleic acid sequence, which normally corresponds to a specific DNA sequence. Although the number of spots is not limited in any way, there is a preferred embodiment in which the microarray is customized for the methods of the invention. Therefore, another aspect of the invention relates to a microarray, hereafter referred to as a microarray of the invention, comprising at least one probe for detecting the genotype of the PCSK9 gene.
Así, por ejemplo, las secuencias de oligonucleótidos son construidas en la superficie de un chip mediante el elongamiento secuencial de una cadena en crecimiento con un sólo nucleótido utilizando fotolitografía. Así, los oligonucleótidos son anclados por el extremo 3' mediante un método de activación selectiva de nucleótidos, protegidos por un reactivo fotolábil, mediante la incidencia selectiva de luz a través de una fotomáscara. La fotomáscara puede ser física o virtual. Así, las sondas oligonucleotidos pueden ser de entre 10 y 100 nucleotidos, más preferiblemente, de entre 20 y 70 nucleotidos, y aún más preferiblemente, de entre 24 y 30 nucleotidos. Para la cuantificación de la expresión génica, preferiblemente se emplean aproximadamente unos 40 oligonucleotidos por gen. La síntesis in situ sobre un soporte sólido (por ejemplo, vidrio), podría hacerse mediante tecnología chorro de tinta (ink-jet), lo que requiere sondas más largas. Los soportes podrían ser, pero sin limitarse, filtros o membranas de NC o nylon (cargadas), silicio, o Portas de vidrio para microscopios cubiertos con aminosilanos, polilisina, aldehidos o epoxy. La sonda es cada una de las muestras del chip. El target es la muestra a analizar: RNA mensajero, RNA total, un fragmento de PCR, etc. Otro aspecto de la invención se refiere al uso del kit o dispositivo, la micromatriz, o el microarray de la invención, para predecir o pronosticar la respuesta de un individuo al tratamiento del VHC con I FN-Peg más un análogo de guanosina. En una realización preferida, el análogo de la guanosina es la ribavirina (RBV). En otra realización preferida el VHC es del genotipo 3. Thus, for example, oligonucleotide sequences are constructed on the surface of a chip by sequential elongation of a growing chain with a single nucleotide using photolithography. Thus, the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask. The photomask can be physical or virtual. Thus, oligonucleotide probes can be between 10 and 100 nucleotides, more preferably, between 20 and 70 nucleotides, and even more preferably, between 24 and 30 nucleotides. For the quantification of gene expression, preferably about 40 oligonucleotides per gene are preferably used. Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy. The probe is each of the chip samples. The target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc. Another aspect of the invention relates to the use of the kit or device, the microarray, or the microarray of the invention, to predict or predict an individual's response to HCV treatment with I FN-Peg plus a guanosine analog. In a preferred embodiment, the guanosine analog is ribavirin (RBV). In another preferred embodiment the HCV is genotype 3.
Otro aspecto de la invención se refiere a un medio de almacenamiento legible por un ordenador que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos de cualquiera de los métodos de la invención (del primer o del segundo método de la invención). Another aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of any of the methods of the invention (of the first or second method of the invention ).
Los términos "polinucleótido" y "ácido nucleico" se usan aquí de manera intercambiable, refiriéndose a formas poliméricas de nucleotidos de cualquier longitud, tanto ribonucleótidos (ARN ó RNA) como desoxirribonucleótidos (ADN ó DNA). Los términos "secuencia aminoacídica", "péptido", "oligopéptido", "polipéptido" y "proteína" se usan aquí de manera intercambiable, y se refieren a una forma polimérica de aminoácidos de cualquier longitud, que pueden ser codificantes o no codificantes, química o bioquímicamente modificados. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA). The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, Chemically or biochemically modified.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Fig. 8. Representación gráfica del gen PSCK9 con las posiciones de los polimorfismos genotipados. Regiones no traducidas de los exones están representadas en gris. *Codificación SN Ps. Fig. 9. Posición genómica de los SNPs y los bloques de desequilibrio de ligamiento analizados. Fig. 8. Graphic representation of the PSCK9 gene with the positions of genotyped polymorphisms. Untranslated regions of exons are represented in gray. * Coding SN Ps. Fig. 9. Genomic position of the SNPs and the linkage imbalance blocks analyzed.
Fig. 10. Resultados del tratamiento conforme a los genotipos rs2479409. Las barras negras: rs2479409 AA/AG, barras grises: GG, RVR: respuesta virológica rápida; RVT: respuesta virológica temprana; NR: no respuesta; VB: progreso virológico; ETR: respuesta al fin de tratamiento; SVR: respuesta virológica sostenida. Fig. 10. Results of treatment according to rs2479409 genotypes. Black bars: rs2479409 AA / AG, gray bars: GG, RVR: rapid virological response; RVT: early virological response; NR: no response; VB: virological progress; ETR: response to end of treatment; SVR: sustained virological response.
EJEMPLOS DE LA INVENCIÓN EXAMPLES OF THE INVENTION
Población de estudio Study population
Los pacientes fueron seleccionados de una cohorte de pacientes infectados con VHC que fueron seguidos posteriormente por cuatro unidades de enfermedades infecciosas en España, entre Junio de 2000 y Diciembre de 2010. Los criterios de inclusión para esta cohorte fueron: i) Mayor de 18 años de edad. ii) Tratamiento suave previo. iii) El inicio de terapia dual con Peg-IFN más RBV. The patients were selected from a cohort of patients infected with HCV who were subsequently followed by four units of infectious diseases in Spain, between June 2000 and December 2010. The inclusion criteria for this cohort were: i) Over 18 years of age. age. ii) Prior gentle treatment. iii) The start of dual therapy with Peg-IFN plus RBV.
Todos los pacientes incluidos en esta cohorte, proporcionaron muestras de sangre total o de sangre periférica, para la determinación genética. Las muestras fueron criopreservadas a -80 grados centígrados. Aquellos pacientes infectados con VHC genotipo 3 fueron seleccionados de este estudio.All patients included in this cohort provided whole blood or peripheral blood samples for genetic determination. The samples were cryopreserved at -80 degrees Celsius. Those patients infected with HCV genotype 3 were selected from this study.
Las respuestas fueron determinadas en tratamiento, esto es, aquellos que interrumpieron el tratamiento debido a efectos adversos, o quiénes voluntariamente abandonaron, fueron excluidos.The responses were determined in treatment, that is, those who discontinued treatment due to adverse effects, or who voluntarily dropped out, were excluded.
Después de una visita en condiciones básales, los sujetos fueron visitados cada 4 semanas durante el tratamiento, incluyendo una visita 24 semanas después de la suspensión de la terapia para evaluar la respuesta virológica sostenida (RVS). After a visit in baseline conditions, subjects were visited every 4 weeks during treatment, including a visit 24 weeks after discontinuation of therapy to assess sustained virological response (SVR).
Estrategias de tratamiento Treatment strategies
Peg-IFN alfa 2a o alfa 2b fue administrado a dosis de 180 μg o 1,5 μg/kg una vez por semana, respectivamente, en combinación con dosis diarias de 800-1200 mg ribavirina (RBV). La duración del tratamiento en pacientes VHC monoinfectados fue de 12 semanas en aquellos que presentaron una carga de ARN de VHC basal <600000 lU/mL, ausencia de cirrosis y respuesta virológica rápida (RVR), y 24 semanas el resto. Pacientes co-infectados con VHC y VIH fueron tratados de 24 a 48 semanas, de acuerdo a si alcanzaron la RVR o no. Las reglas de suspensión fueron aplicadas siguiendo la pauta internacional vigente en el momento del tratamiento. Definición de respuesta Peg-IFN alpha 2a or alpha 2b was administered at doses of 180 μg or 1.5 μg / kg once a week, respectively, in combination with daily doses of 800-1200 mg ribavirin (RBV). The duration of treatment in monoinfected HCV patients was 12 weeks in those who presented a baseline HCV RNA load <600,000 lU / mL, absence of cirrhosis and rapid virological response (RVR), and 24 weeks the rest. Patients co-infected with HCV and HIV were treated for 24 to 48 weeks, depending on whether they achieved RVR or not. The suspension rules were applied following the international guidelines in force at the time of treatment. Answer definition
RVS fue definida como ARN de VHC indetectable en plasma, 24 semanas después de la terminación del tratamiento. RVR fue considerada como la indetectabilidad de ARN de VHC a la cuarta semana del tratamiento. Respuesta virológica temprana (RVT) fue definida tanto como la indetectabilidad o una disminución >2 loglO de ARN de VHC en la semana 12. ARN de VHC detectable en plasma durante el tratamiento después de haber alcanzado la indetectabilidad fue definido como progreso virologico. ARN de VHC detectable después de haber presentado una disminución >2 loglO a las 12 semanas, o la falta de RVT fue definido como no-respuesta. La respuesta a final de tratamiento (RFT) fue considerada cuando el ARN de VHC podría no ser detectado al final de terapia programada. La recaída fue definida como RFT sin lograr RVS. SVR was defined as undetectable HCV RNA in plasma, 24 weeks after treatment termination. RVR was considered as the undetectability of HCV RNA at the fourth week of treatment. Early virological response (RVT) was defined as either undetectability or a decrease> 2 log of HCV RNA at week 12. HCV RNA detectable in plasma during treatment after reaching undetectability was defined as virological progress. HCV RNA detectable after presenting a decrease> 2 logs at 12 weeks, or the lack of RVT was defined as non-response. The end-of-treatment response (RFT) was considered when HCV RNA may not be detected at the end of scheduled therapy. Relapse was defined as RFT without achieving SVR.
Selección y genotipo do de SNPs y cuantificación de ARN VHC. Selection and genotype of SNPs and quantification of HCV RNA.
ADN genómico fue aislado de la muestra de sangre general o de sangre periférica aislada de células mononucleares usando el método de extracción automatizado MagNa Puré DNA (Roche Diagnostic Corporation, Indianapolis, I N, USA) o el kit de extracción Quick Puré Blood DNA (Macherey-Nagel, Düren, Alemania). Los SNPs de PCSK9 fueron identificados como etiquetas SNPs, usando el software Haploview's V4.1, opción etiquetador (https://www.broad.harvard.edu/haploview/haploview) y los datos genotípicos de la población Hapmap-CEU (Tabla 1). Genomic DNA was isolated from the general blood sample or from peripheral blood isolated from mononuclear cells using the MagNa Puré DNA automated extraction method (Roche Diagnostic Corporation, Indianapolis, IN, USA) or the Quick Puré Blood DNA extraction kit (Macherey- Nagel, Düren, Germany). PCSK9 SNPs were identified as SNPs tags, using Haploview's V4.1 software, labeling option (https://www.broad.harvard.edu/haploview/haploview) and genotype data of the Hapmap-CEU population (Table 1 ).
Tabla 1: Polimorfismo de un solo nucleótido analizado en genes PCSK9. Table 1: Single nucleotide polymorphism analyzed in PCSK9 genes.
SNP1 BBP on Locus position Rate of MAF Alíeles chromosome l2 genotyping (%) SNP 1 BBP on Locus position Rate of MAF Ally them chromosome l 2 genotyping (%)
HWE rsl7111503 55276036 5' upstream 0.266 100 0.295 A:G rs2479408* 55276776 5' upstream 0.631 100 0.205 G:C rs2479409 55277238 5' upstream 0.954 99.2 0.336 A:G rsll591147* 55505647 Coding 1 100 0.019 C:A rsl0888896 55281801 Intron 0.91 99.2 0.275 G:C rs4927193* 55282460 Intron 0.631 100 0.205 A:G rs499718 55285137 Intron 0.211 100 0.25 G:A rsl0888897 55285649 Intron 0.843 100 0.462 G:A rs676297* 55514611 Intron 0.535 99.2 0.229 T:A rs572512 55289932 Intron 0.541 100 0.348 G:A rs2495477 55291055 Intron 0.066 100 0.364 A:G rs2479413 55291270 Intron 0.499 100 0.33 G:A rs7552841 55291340 Intron 1 100 0.477 G:A rs557435* 55293452 Intron 1 99.2 0.195 G:A rs483462 55297988 Intron 0.66 100 0.284 A:G rs603247* 55298456 Intron 1 99.2 0.164 A:G rsl0465832* 55301395 Intron 1 100 0.064 G:C HWE rsl7111503 55276036 5 ' upstream 0.266 100 0.295 A: G rs2479408 * 55276776 5 ' upstream 0.631 100 0.205 G: C rs2479409 55277238 5 ' upstream 0.954 99.2 0.336 A: G rsll591147 * 55505647 Coding 1 100 0.0191 C88196 0.881 C889696 C88196 0.881 C96 A96 0.275 G: C rs4927193 * 55282460 Intron 0.631 100 0.205 A: G rs499718 55285137 Intron 0.211 100 0.25 G: A rsl0888897 55285649 Intron 0.843 100 0.462 G: A rs676297 * 55514611 Intron 0.535 99.2 0.229 T: A rs572512 55289932 Intron 0.541 100 0.348 G: A rs2495477 55291024 Intr134999292929292929292924296296296296296296296296296296296296296296296296296296296296296296296296296296296296296296296296296296296126296296126 100 0.33 G: A rs7552841 55291340 Intron 1 100 0.477 G: A rs557435 * 55293452 Intron 1 99.2 0.195 G: A rs483462 55297988 Intron 0.66 100 0.284 A: G rs603247 * 55298456 Intron 1 99.2 0.164 A: G rsl0465832 Intr. G: C
^505151* 55529187 Coding 1 99.4 0.041 A:G rs662145!t 55302416 3' downstream 1 100 0.22 A:G ^ 505151 * 55529187 Coding 1 99.4 0.041 A: G rs662145 ! T 55302416 3 ' downstream 1 100 0.22 A: G
*SNPs que fueron descartados debido a los criterios de exclusión. ISNP: polimorfismo de un 5 solo nucleótido; 2BBP: posición de base par; de acuerdo al navegador NCBI36/hgl8 (http://genome.ucsc.edu/cgi-bin/hgGateway?hgsid=309991419&clade= * SNPs that were discarded due to exclusion criteria. ISNP: single nucleotide polymorphism; 2BBP: even base position; according to browser NCBI36 / hgl8 (http://genome.ucsc.edu/cgi-bin/hgGateway?hgsid=309991419&clade=
mammal&org=Human&db=hgl8) y dbSNP build 130 (http://www.ncbi.nlm.nih.gov/projects/SNP/); 3HWE: equilibrio de Hardy-Weinberg; 4MAF: frecuencia alélica menor. mammal & org = Human & db = hgl8) and dbSNP build 130 (http://www.ncbi.nlm.nih.gov/projects/SNP/); 3HWE: Hardy-Weinberg equilibrium; 4MAF: lower allelic frequency.
El gen PCSK9 se muestra en la figura 1. Todos los SNPs fueron genotipados usando un 5 ensayo de genotipado Golden Gate Veracode (lllumina, San Diego, CA, USA), de acuerdo a las instrucciones del fabricante. Aquellos SNPs que mostraron los siguientes criterios de exclusión fueron descartados: i) Menos frecuencia alélica de <0.25. ii) Tasa de genotipado <80% iii) P<0.05 para el equilibrio de Hardy-Weinberg. Finalmente, 10 SNPs de los 19 seleccionados previamente fueron analizados, El genotipado de IL28B por PC y la determinación de ARN-VHC en plasma se llevaron a cabo como se describió en otra parte. The PCSK9 gene is shown in Figure 1. All SNPs were genotyped using a Golden Gate Veracode genotyping assay (lllumina, San Diego, CA, USA), according to the manufacturer's instructions. Those SNPs that showed the following exclusion criteria were discarded: i) Less allele frequency of <0.25. ii) Genotyping rate <80% iii) P <0.05 for Hardy-Weinberg equilibrium. Finally, 10 SNPs of the 19 previously selected were analyzed, IL28B genotyping by PC and plasma HCV-RNA determination were carried out as described elsewhere.
Análisis estadístico Statistic analysis
El equilibrio Hardy-Weinberg fue calculado para los SNPs usando el recurso online del Instituto para la Genética Humana de la Universidad técnica de Munich, Alemania (http://ihg.gsf.de). Un primer análisis de asociaciones alélicas estándar de casos y controles (1 grado de libertad), así como de diferentes modelos genéticos, fue desarrollado usando el software PLINK (http://pngu.mgh.harvard.edu/~purcell/plink/gplink.shtml). Aquellos SNPs que mostraron una asociación con RVS en este estudio fueron seleccionados para entrar en análisis más profundos. El umbral para el valor p fue calculado dividiendo un p=0.05 por la suma del número de bloques de desequilibrio de ligamiento (DL) y el número de SNPs fuera de los bloques DL. Las variables continuas son presentadas como mediana (rango intercuantil, IQR) y comparadas por medio de la prueba de la t de student o la prueba U de Mann-Whitney, cuando sea aplicable. El test χ2 (chi-cuadrado) y el test exacto de Fischer, fueron usados para analizar las variables categóricas en tablas 2x2. El test χ2 para tablas RxC fue aplicado para variables clasificadas en más de 2 categorías, usando residual estandarizado (ASR) para la interpretación de la significancia. Las variables categóricas son expresadas como números (porcentajes). El resultado primario variable fue RVS. La asociación de los SNPs seleccionados con RVR, como una variable secundaria, fue también analizada. Además, la relación entre el genotipo PCSK9 y los diferentes resultados al tratamiento, así como la cinética viral, fue evaluada. Para identificar los factores independientemente asociados con RVS y RVR, un análisis de regresión logística multivariante, ajustado por edad, sexo, así como aquellos factores que estaban asociados con cualquiera de los puntos finales anteriores con un valor de significación estadística p<0.2 en un análisis univariante. Análisis descriptivos e inferenciales fueron llevados a cabo, usando el paquete de software SPSS estadístico 19.0 (IBM Corporation, Somers, NY, USA). Cuestiones éticas The Hardy-Weinberg equilibrium was calculated for SNPs using the online resource of the Institute for Human Genetics of the Technical University of Munich, Germany (http://ihg.gsf.de). A first analysis of standard allelic associations of cases and controls (1 degree of freedom), as well as of different genetic models, was developed using the PLINK software (http://pngu.mgh.harvard.edu/~purcell/plink/gplink .shtml). Those SNPs that showed an association with SVR in this study were selected to enter deeper analyzes. The threshold for the p-value was calculated by dividing a p = 0.05 by the sum of the number of linkage imbalance blocks (DL) and the number of SNPs outside the DL blocks. Continuous variables are presented as median (interquartile range, IQR) and compared by means of the student's t test or the Mann-Whitney U test, when applicable. The χ2 test (chi-square) and the exact Fischer test were used to analyze the categorical variables in 2x2 tables. The χ2 test for RxC tables was applied for variables classified in more than 2 categories, using standardized residual (ASR) for the interpretation of significance. Categorical variables are expressed as numbers (percentages). The primary variable outcome was RVS. The association of selected SNPs with RVR, as a secondary variable, was also analyzed. In addition, the relationship between the PCSK9 genotype and the different treatment results, as well as viral kinetics, was evaluated. To identify the factors independently associated with RVS and RVR, a multivariate logistic regression analysis, adjusted for age, sex, as well as those factors that were associated with any of the previous endpoints with a statistical significance value p <0.2 in an analysis univariate. Descriptive and inferential analyzes were carried out, using the statistical SPSS software package 19.0 (IBM Corporation, Somers, NY, USA). Ethical issues
El estudio fue diseñado y desarrollado de acuerdo a la declaración de Helsinki, y fue aprobado por el comité ético del Hospital Universitario de Valme (Sevilla). The study was designed and developed according to the declaration of Helsinki, and was approved by the ethical committee of the University Hospital of Valme (Seville).
RESULTADOS RESULTS
Características de la población de estudio. 132 individuos fueron seleccionados para el estudio. La media de la edad (rango intercuartil) fue de 41.5 (37.6-45.1) años y 99 (el 75%) eran hombres. Fibrosis avanzada fue diagnosticada en 44 (41.1%) de los 107 pacientes en los que la determinación de las etapas de la fibrosis por medio de una biopsia hepática (F>3 de acuerdo al índice de Scheuer) o elastometría transitoria (valor de rigidez hepática >11 kPa) fue posible. La carga viral basal media fue 5.94 (5.42-6.49) loglO U I/mL. 99 (75%) individuos fueron coinfectados con VI H, presentando un recuento medio de células CD4 de 470 (350-680) células/μί. Characteristics of the study population. 132 individuals were selected for the study. The mean age (interquartile range) was 41.5 (37.6-45.1) years and 99 (75%) were men. Advanced fibrosis was diagnosed in 44 (41.1%) of the 107 patients in whom the determination of the stages of fibrosis by means of a liver biopsy (F> 3 according to the Scheuer index) or transient elastometry (liver stiffness value > 11 kPa) was possible. The mean baseline viral load was 5.94 (5.42-6.49) log IU / mL. 99 (75%) individuals were coinfected with VI H, presenting an average CD4 cell count of 470 (350-680) cells / μί.
Genotipos PCSK9 PCSK9 genotypes
Tres bloques DL fueron identificados y 2 polimorfismos de un solo nucleótido (SN Ps) estaban fuera de los bloques (figura 2). Así, un valor p umbral de 0.01 fue aplicado para seleccionar SNP2 para estudios posteriores. En el análisis estándar de casos y controles de asociaciones alélicas, solo el SN P rs2479409 localizado en la región 5' proximal del gen cumplió los criterios para los estudios estadísticos posteriores (p=0.002). La distribución genotípica estaba conforme con el principio de equilibrio de Hardy-Weinberg (p=0.954). La distribución de los genotipos rs2479409 fue: AA fue encontrado en 57 (43.2%) pacientes, AG en 60 (45.4%) y GG en los 15 restantes (11.4%). Las características básicas de la población de estudio de acuerdo a rs2479409 PCSK9 se presentan en la tabla 2. Three DL blocks were identified and 2 single nucleotide polymorphisms (SN Ps) were outside the blocks (Figure 2). Thus, a p-threshold value of 0.01 was applied to select SNP2 for further studies. In the standard analysis of cases and controls of allelic associations, only SN P rs2479409 located in the proximal 5 'region of the gene met the criteria for subsequent statistical studies (p = 0.002). The genotypic distribution was in accordance with the Hardy-Weinberg equilibrium principle (p = 0.954). The distribution of the rs2479409 genotypes was: AA was found in 57 (43.2%) patients, AG in 60 (45.4%) and GG in the remaining 15 (11.4%). The basic characteristics of the study population according to rs2479409 PCSK9 are presented in Table 2.
Table 2: Características básales de la población de estudio de acuerdo con el rs2479409. Table 2: Baseline characteristics of the study population according to rs2479409.
Parameter rs2479409 Parameter rs2479409
AA/AG GG n=117 n=15 AA / AG GG n = 117 n = 15
Age (years)" 41.5 (37.2-45.2) 43 (38.9-44.4) 0.251 Age (years) "41.5 (37.2-45.2) 43 (38.9-44.4) 0.251
Male gender, no. (%) 85 (72.6) 14 (93.3) 0.082 Male gender, no. (%) 85 (72.6) 14 (93.3) 0.082
Body mass índex (kg/m2) 24.1 (21.8-27.2) 24.3 (22.6-25.4) 0.968 Body mass index (kg / m 2 ) 24.1 (21.8-27.2) 24.3 (22.6-25.4) 0.968
IDU, no. [%Ϋ 101 (88.6) 16 (88.9) 0.971 IDU, no. [% Ϋ 101 (88.6) 16 (88.9) 0.971
HlV-infected , no. (%) 86 (73.5) 13 (86.7) 0.268 HlV-infected, no. (%) 86 (73.5) 13 (86.7) 0.268
HBsAg positive, no. (%)2 2 (2.3) 0.646 HBsAg positive, no. (%) 2 2 (2.3) 0.646
IL28B rsl2979860 CC, no. (%)3 65 (56) 5 (33.3) 0.097 Plasma HCV- NA (log10 IU/mL)* 6.01 (5.43-6.5) 5.83 (5.13-6.01) 0.113 IL28B rsl2979860 CC, no. (%) 3 65 (56) 5 (33.3) 0.097 HCV-NA Plasma (log 10 IU / mL) * 6.01 (5.43-6.5) 5.83 (5.13-6.01) 0.113
Advanced fibrosis, no. (%)4 35 (37.2) 9 (69.2) 0.028 Advanced fibrosis, no. (%) 4 35 (37.2) 9 (69.2) 0.028
ALT (U/L)*5 83 (54-135) 81 (61-180) 0.608 ALT (U / L) * 5 83 (54-135) 81 (61-180) 0.608
Total cholesterol (mg/dL)* 151 (129-183) 162 (139-172) 0.69 Total cholesterol (mg / dL) * 151 (129-183) 162 (139-172) 0.69
LDL cholesterol (mg/dL)*6 87 (64.5-109) 91 (67-129) 0.458 LDL cholesterol (mg / dL) * 6 87 (64.5-109) 91 (67-129) 0.458
Triglycerides (mg/dL)* 113 (66-185) 97 (77-125) 0.643 Triglycerides (mg / dL) * 113 (66-185) 97 (77-125) 0.643
Peg-IFN alfa 2a, no. (%)7 27 (23.1) 3 (20) 0.789 Peg-IFN alpha 2a, no. (%) 7 27 (23.1) 3 (20) 0.789
Daily dose of RBV (mg/kg)*8 14 (12.6-15.7) 14.6 (13.6-15.5) 0.439 Daily dose of RBV (mg / kg) * 8 14 (12.6-15.7) 14.6 (13.6-15.5) 0.439
Undetectable HIV RNA, no. (%)9 65 (77.4) 11 (84.6) 0.556 Undetectable HIV RNA, no. (%) 9 65 (77.4) 11 (84.6) 0.556
CD4 cell count (cells/μΙ.)* 463 (351-664) 564 (272-746) 0.634 CD4 cell count (cells / μΙ.) * 463 (351-664) 564 (272-746) 0.634
*mediana (rango intercuartil) ; 1IDU: usuario de drogas intravenosas; 2disponible en 96 pacientes; 3disponible en 131 pacientes; 4determinado por biopsia de hígado (F>3 conforme al índice Scheuer) o un valor de rigidez hepática > como se determinó por elastometríatransitoria; disponible en 107 pacientes; 5ALT: alanina-aminotransferasa; 6LDL: lipoproteína de baja densidad, disponible en 117 pacientes; 7Peg-IFN: interferón pegilado; 8RBV: ribavirina; 9disponible en 97 pacientes de aquellos co-infectados con VHC y VIH. * median (interquartile range); 1IDU: user of intravenous drugs; 2 available in 96 patients; 3d available in 131 patients; 4determined by liver biopsy (F> 3 according to the Scheuer index) or a liver stiffness value> as determined by transient elastometry; available in 107 patients; 5ALT: alanine aminotransferase; 6LDL: low density lipoprotein, available in 117 patients; 7Peg-IFN: pegylated interferon; 8RBV: ribavirin; 9Available in 97 patients of those co-infected with HCV and HIV.
Respuesta al tratamiento general. Response to general treatment.
La respuesta virológica sostenida (SVR) fue lograda en 108 pacientes (81.8%). La respuesta viral rápida (RVR) pudo ser evaluada en 78 (67.8%) de los 115 pacientes (87.1%).Cuatro individuos(3%) no respondieron a la terapia y 6 (4.5%) experimentaron progreso virológico. (10.6%) de los pacientes recayeron. Los detalles de las tasas de SVR y RVR de acuerdo a las características de los pacientes, se muestran en la tabla 3.
Figure imgf000037_0001
Figure imgf000038_0001
r-. The sustained virological response (SVR) was achieved in 108 patients (81.8%). The rapid viral response (RVR) could be evaluated in 78 (67.8%) of the 115 patients (87.1%) Four individuals (3%) did not respond to therapy and 6 (4.5%) experienced virological progress. (10.6%) of the patients relapsed. The details of the SVR and RVR rates according to the characteristics of the patients are shown in Table 3.
Figure imgf000037_0001
Figure imgf000038_0001
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Respuesta a la terapia, de acuerdo al genotipo PCSK9. CL <
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Response to therapy, according to the PCSK9 genotype.
45 (78.9%) pacientes con el genotipo PCSK9 rs2479409 AA, 55 (91.7%) de los que tienen genotipo AG y 8 (53.3%) con genotipo GG, mostraron SV (p=0.002; ASR para SVR y AA= 0.7, AG=2.7 y GG=-3). El número de pacientes que lograron la RVS entre los genotipos rs2479409 fueron 34 (69,4%) para AA, 38 (73.1%) para AG y 6 (42.9%) para GG (p=0.095; ASR para RVR y AA=0.3, AG=1.1 y GG=-2.1). Los diferentes resultados al tratamiento de los genotipos rs2479409 GG frente a no-GG se muestran en la figura 3. 45 (78.9%) patients with the PCSK9 genotype rs2479409 AA, 55 (91.7%) of those with AG genotype and 8 (53.3%) with GG genotype, showed SV (p = 0.002; ASR for SVR and AA = 0.7, AG = 2.7 and GG = -3). The number of patients who achieved SVR among the rs2479409 genotypes were 34 (69.4%) for AA, 38 (73.1%) for AG and 6 (42.9%) for GG (p = 0.095; ASR for RVR and AA = 0.3 , AG = 1.1 and GG = -2.1). The different results to the treatment of rs2479409 GG genotypes versus non-GG are shown in Figure 3.
En la subpoblación de portadores de rs2479409 no-GG, 27 (87.1%) de aquellos que fueron tratados con un régimen abreviado, esto es 12 semanas para monoinfectados por VHC y 24 semanas para pacientes co-infectados con VHC y VIH, frente a 73 (84.9%) de aquellos tratados con la terapia alargada, lograron SVR (p=0.764). In the subpopulation of rs2479409 non-GG carriers, 27 (87.1%) of those who were treated with an abbreviated regimen, this is 12 weeks for HCV monoinfected patients and 24 weeks for patients co-infected with HCV and HIV, compared to 73 (84.9%) of those treated with elongated therapy, achieved SVR (p = 0.764).
Otros factores asociados con la respuesta a la terapia. Other factors associated with the response to therapy.
Además del genotipo PCSK9 rs2479409, la ausencia de fibrosis avanzada, usando I FN pegilado alfa 2§ y edades más jóvenes fueron asociadas con RVR en el análisis univariante (véase tabla 3). En el estudio multivariante, solo los genotipos rs2479409 AA y AG fueron identificados como predictores independientes de SVR, mientras la ausencia de fibrosis avanzada fue asociada independientemente con RVR (tabla 3). In addition to the PCSK9 rs2479409 genotype, the absence of advanced fibrosis, using pegylated FN alpha 2 § and younger ages were associated with RVR in the univariate analysis (see table 3). In the multivariate study, only the rs2479409 AA and AG genotypes were identified as independent predictors of SVR, while the absence of advanced fibrosis was independently associated with RVR (Table 3).
CLAUSULAS CLAUSES
I. - El uso del gen PCSK9 para predecir o pronosticar la respuesta de un individuo al tratamiento del VHC, con IFN-Peg más un análogo de guanosina. 2.- El uso del gen PCSK9 según la reivindicación anterior, donde el análogo de guanosina es la ribavirina ( BV). I. - The use of the PCSK9 gene to predict or predict an individual's response to HCV treatment, with IFN-Peg plus a guanosine analog. 2. The use of the PCSK9 gene according to the preceding claim, wherein the guanosine analog is ribavirin (BV).
3. -El uso del gen PCSK9, según cualquiera de las reivindicaciones 1-2, donde el individuo está infectado también con VI H. 3. The use of the PCSK9 gene, according to any of claims 1-2, wherein the individual is also infected with VI H.
4. - El uso del gen PCSK9 según cualquiera de las reivindicaciones 1-3, donde el VHC es del genotipo 3. 5.- Un método de obtención de datos útiles para predecir o pronosticar la respuesta de un individuo al tratamiento de VHC, con I FN-Peg más un análogo de guanosina, que comprende: a) obtener una muestra biológica aislada de un individuo, y b) determinar el genotipo del gen PCSK9, en la muestra aislada de a). 4. - The use of the PCSK9 gene according to any of claims 1-3, wherein the HCV is of genotype 3. 5.- A method of obtaining useful data to predict or predict the response of an individual to the treatment of HCV, with I FN-Peg plus a guanosine analog, comprising: a) obtaining an isolated biological sample from an individual, and b) determining the genotype of the PCSK9 gene, in the isolated sample of a).
6. - Un método para predecir o pronosticar la respuesta de un individuo al tratamiento del VHC con I FN- peg más un análogo de guanosina, que comprende los pasos a) y b) de la reivindicación anterior, y además comprende: c) clasificar a los individuos según el genotipo de PCSK9 que presentan y su respuesta al tratamiento con IFN-peg más un análogo de guanosina. 6. - A method for predicting or predicting an individual's response to HCV treatment with I FN-peg plus a guanosine analogue, comprising steps a) and b) of the preceding claim, and further comprising: c) classifying a the individuals according to the genotype of PCSK9 that they present and their response to IFN-peg treatment plus a guanosine analog.
7. - El método según cualquiera de las reivindicaciones 5-6, donde el análogo de guanosina 30 es la ribavirina (RBV). 7. - The method according to any of claims 5-6, wherein the guanosine analogue is ribavirin (RBV).
8. - El método según cualquiera de las reivindicaciones 5-7, donde el individuo está también infectado por VI H. 8. - The method according to any of claims 5-7, wherein the individual is also infected by VI H.
9. -El método según cualquiera de las reivindicaciones 5-8, donde el VHC es del genotipo 3. 9. The method according to any of claims 5-8, wherein the HCV is of genotype 3.
10. - El método según cualquiera de las reivindicaciones 5-9, que además comprende la determinación del genotipo del gen PCSK9. 10. - The method according to any of claims 5-9, which further comprises determining the genotype of the PCSK9 gene.
II. -El método según cualquiera de las reivindicaciones 5-10, donde la determinación del genotipo de PCSK9 se realiza mediante PCR. 12. - El método según cualquiera de las reivindicaciones 10-11, donde la determinación del genotipo de PCSK9, se realiza analizando el SN P rs2479409. II. -The method according to any of claims 5-10, wherein the determination of the PCSK9 genotype is performed by PCR. 12. - The method according to any of claims 10-11, wherein the determination of the PCSK9 genotype is performed by analyzing SN P rs2479409.
13. - El método según cualquiera de las reivindicaciones 10-12, donde el genotipo AA del SNP rs2479409, es indicativo de buena respuesta al tratamiento. 14.- El método según cualquiera de las reivindicaciones 10-12, donde el genotipo AG del SNP rs2479409, es indicativo de buena respuesta al tratamiento. 13. - The method according to any of claims 10-12, wherein the SNP AA genotype rs2479409 is indicative of good response to treatment. 14. The method according to any of claims 10-12, wherein genotype AG of SNP rs2479409, is indicative of good response to treatment.
15. - El método según cualquiera de las reivindicaciones 10-12, donde el genotipo GG del SNP rs2479409 es indicativo de una respuesta menor que en el caso de las reivindicaciones 13-14. 15. - The method according to any of claims 10-12, wherein the GG genotype of SNP rs2479409 is indicative of a lower response than in the case of claims 13-14.
16. - El método según cualquiera de las reivindicaciones 5-15, donde el análogo de la guanosina es la ribavirina ( BV). 16. - The method according to any of claims 5-15, wherein the guanosine analog is ribavirin (BV).
17. - El método según cualquiera de las reivindicaciones 5-16, donde la muestra biológica es o se obtiene de sangre periférica. 17. - The method according to any of claims 5-16, wherein the biological sample is or is obtained from peripheral blood.
18. - Un kit o dispositivo que comprende una o varias secuencias nucleotídicas necesarias para determinar el genotipo del gen PCKS9. 19.- Un kit o dispositivo según la reivindicación anterior, que comprende una o varias secuencias nucleotídicas necesarias para determinar el SN P rs2479409 del gen PCKS9. 18. - A kit or device comprising one or more nucleotide sequences necessary to determine the genotype of the PCKS9 gene. 19. A kit or device according to the preceding claim, comprising one or more nucleotide sequences necessary to determine the SN P rs2479409 of the PCKS9 gene.
20. - Un kit o dispositivo según las reivindicaciones 18-19, donde el genotipo AA del SN P rs2479409, es indicativo de buena respuesta al tratamiento. 20. - A kit or device according to claims 18-19, wherein the genotype AA of SN P rs2479409, is indicative of good response to treatment.
21. - Un kit o dispositivo según las reivindicaciones 18-19, donde el genotipo AG del SN P rs2479409, es indicativo de buena respuesta al tratamiento. 21. - A kit or device according to claims 18-19, wherein the genotype AG of SN P rs2479409, is indicative of good response to treatment.
22. - Un kit o dispositivo según las reivindicaciones 18-19, donde el genotipo GG del SN P rs2479409 es indicativo de una respuesta menor que en el caso de las reivindicaciones 20-21. 22. - A kit or device according to claims 18-19, wherein the GG genotype of SN P rs2479409 is indicative of a lower response than in the case of claims 20-21.
23. - El uso de un kit o dispositivo según las reivindicaciones 18-22, para predecir o pronosticar la respuesta de un individuo con VHC al tratamiento con IFN-Peg más un análogo de guanosina. 24.-EI uso de un kit o dispositivo según la reivindicación anterior, donde el análogo de la guanosina es la ribavirina (RBV). 23. - The use of a kit or device according to claims 18-22, to predict or predict the response of an individual with HCV to treatment with IFN-Peg plus a guanosine analog. 24.-The use of a kit or device according to the preceding claim, wherein the guanosine analog is ribavirin (RBV).
25.- El uso de un kit o dispositivo según las reivindicaciones 23-24, donde el VHC es del 15 genotipo 3. 25. The use of a kit or device according to claims 23-24, wherein the HCV is of genotype 3.

Claims

REIVINDICACIONES
1.- El uso de los polimorfismos genéticos: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 y rs6413504 del gen LDLR, para predecir o pronosticar la respuesta al tratamiento con interferón pegilado más ribavirina en pacientes infectados con VHC.  1.- The use of genetic polymorphisms: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504 of the LDLR gene, to predict or predict the response to treatment with pegylated interferon plus Vibavirin infected patients.
2.- El uso de los polimorfismos genéticos del gen LDLR según la reivindicación anterior, donde la respuesta al tratamiento es la respuesta virológica sostenida ( VS). 2. The use of the genetic polymorphisms of the LDLR gene according to the preceding claim, wherein the response to treatment is the sustained virological response (VS).
3.- El uso de los polimorfismos genéticos del gen LDLR según cualquiera de las reivindicaciones anteriores, donde el VHC es de los genotipos que se seleccionan de entre: genotipo 1, genotipo 2, genotipo 3, genotipo 4, o cualquiera de sus combinaciones. 3. The use of the genetic polymorphisms of the LDLR gene according to any of the preceding claims, wherein the HCV is one of the genotypes that are selected from: genotype 1, genotype 2, genotype 3, genotype 4, or any combination thereof.
4.- Un método de obtención de datos útiles para predecir o pronosticar la respuesta al tratamiento con interferón pegilado más ribavirina en pacientes infectados con VHC, que comprende: a) obtener una muestra biológica aislada de un individuo, y b) detectar los polimorfismos genéticos: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 y rs6413504 del gen LDLR, en la muestra biológica aislada de (a). 4.- A method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with HCV, which comprises: a) obtaining an isolated biological sample from an individual, and b) detecting genetic polymorphisms: rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504 of the LDLR gene, in the isolated biological sample of (a).
5.- El método de obtención de datos útiles según la reivindicación anterior, donde la respuesta al tratamiento es la respuesta virológica sostenida del VHC. 5. The method of obtaining useful data according to the preceding claim, wherein the response to treatment is the sustained virological response of HCV.
6. - El método de obtención de datos útiles según cualquiera de las reivindicaciones 4-5, que además comprende medir la carga viral del VHC en un individuo. 6. - The method of obtaining useful data according to any of claims 4-5, which further comprises measuring the viral load of HCV in an individual.
7. - El método de obtención de datos útiles según cualquiera de las reivindicaciones 4-6, que además comprende determinar los polimorfismo del gen IL28B. 7. - The method of obtaining useful data according to any of claims 4-6, which further comprises determining the polymorphism of the IL28B gene.
8. - El método de obtención de datos útiles según la reivindicación anterior, donde los polimorfismo del gen IL28B son el rsl2979860 y/o rs8099917. 8. - The method of obtaining useful data according to the preceding claim, wherein the polymorphism of the IL28B gene is rsl2979860 and / or rs8099917.
9. - El método según cualquiera de las reivindicaciones 5-8, donde el VHC es de los genotipos que se seleccionan de entre: genotipo 1, genotipo 2, genotipo 3, genotipo 4, o cualquiera de sus combinaciones. 9. - The method according to any of claims 5-8, wherein the HCV is one of the genotypes that are selected from: genotype 1, genotype 2, genotype 3, genotype 4, or any combination thereof.
10. - El método según cualquiera de las reivindicaciones 5-9, donde la determinación de los polimorfismos se realiza mediante PCR. 10. - The method according to any of claims 5-9, wherein the determination of the polymorphisms is carried out by PCR.
11. - Un kit o dispositivo que comprende una o varias secuencias nucleotídicas necesarias para detectar los polimorfismos del gen LDLR. 11. - A kit or device comprising one or several nucleotide sequences necessary to detect the polymorphisms of the LDLR gene.
12. - El kit o dispositivo según la reivindicación anterior, donde los polimorfismos del gen LDLR son rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 y rs6413504. 12. - The kit or device according to the preceding claim, wherein the LDLR gene polymorphisms are rs2738456, rs2738457, rs2569540, rs2738459, rs2738460, rs2116898, rsl433099 and rs6413504.
13.- El kit o dispositivo según la reivindicación anterior, que además comprende una o varias secuencias nucleotídicas necesarias para detectar los polimorfismos del gen IL28B. 13. The kit or device according to the preceding claim, which further comprises one or more nucleotide sequences necessary to detect polymorphisms of the IL28B gene.
14. - El kit o dispositivo según la reivindicación anterior donde los polimorfismos del gen IL28B son el rsl2979860 y el rs8099917 14. - The kit or device according to the preceding claim wherein the polymorphisms of the IL28B gene are rsl2979860 and rs8099917
15. - El uso de un kit o dispositivo según cualquiera de las reivindicaciones 11-14, para predecir o pronosticar la respuesta, en pacientes infectados con VHC, al tratamiento con interferon pegilado más ribavirina. 15. - The use of a kit or device according to any of claims 11-14, to predict or predict the response, in patients infected with HCV, to treatment with pegylated interferon plus ribavirin.
PCT/ES2014/070158 2013-03-01 2014-03-03 Combined genetic polymorphisms of ldlr and il28b for predicting response to treatment with pegylated interferon plus ribavirin in patients infected with the hepatitis c virus WO2014131934A1 (en)

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ES201330294A ES2490492B1 (en) 2013-03-01 2013-03-01 Combined genetic polymorphisms of LDLR and IL28B for prediction of the response to treatment with pegylated interferon plus ribavirin in patients infected with hepatitis C virus
ES201330292A ES2502017B1 (en) 2013-03-01 2013-03-01 Method of obtaining useful data to predict or predict the response to treatment with pegylated interferon plus ribavirin in patients infected with the hepatitis C virus genotype 3.

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