CN114836534A - SAMD9L gene mutation as marker for diagnosis of type I interferon diseases and application thereof - Google Patents

Abstract

The invention discloses SAMD9L gene mutation as a marker for I-type interferon disease diagnosis and application thereof, wherein the mutation is SAMD9L gene truncation mutation, the truncation mutation is located in a P-lopNTPase region of an SAMD9L gene, the SAMD9L gene mutation is c.2642-2729ins/delTCCTGT, and verification experiments prove that the SAMD9L gene truncation mutation is a pathogenic mutation and has higher accuracy and reliability in I-type interferon disease diagnosis and/or auxiliary diagnosis.

Description

SAMD9L gene mutation as marker for diagnosis of type I interferon diseases and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and relates to SAMD9L gene autosomal dominant genetic truncated mutation and diagnosis of I-type interferon diseases, in particular to a truncated mutation of a P-loop NTPase region of an SAMD9L gene, and more particularly to SAMD9L gene mutation as a marker for diagnosis of I-type interferon diseases and application thereof.

Background

SAMD9L, a sterile alpha motif domain 9-like gene, is located on human chromosome 7, q21.2, is linked head-to-tail to SAMD9, is expressed in various cells such as CD4 cells, NK cells, DC cells, monocytes, hematopoietic stem cells, and the like, is mainly localized to the endosome in the cells, is ubiquitously expressed in various tissues of the human body, and plays roles in regulating cell proliferation and inhibiting tumorigenesis. SAMD9L gene-related gain-of-function Mutations may lead to Ataxia Pancytopenia Syndrome (OMIM 611170), which was first discovered in 1987, the causative gene of which was found in 2016 (Chen DH, Below JE, Shimamura A, Keel SB, Matsushita M, Wolff J, et Al. Ataxia-Pankytopenia Syn Is used by my misssens Mutations in SAMD L. am J Hum 2016; 98(6): 1146) and in 2017 bone marrow failure-related Diseases listed by IUIS (Picard C, Bobby GaspearH, Al-Hertz W, Bousha Ima JL A, Casanova J L., Chaula T, Chaloa of International patent application J.7: primer J.96. origin of culture J.38). The disease is an autosomal dominant hereditary disease and is characterized by cerebellar ataxia, cytopenia, susceptibility to bone marrow failure, myelodysplasia and myeloid leukemia, sometimes accompanied by chromosome 7 monomeric syndrome, usually in childhood onset, severe damage to the blood system, similar to aplastic anemia or idiopathic thrombocytopenic purpura. Age of onset, severity and progression of neurological and hematological abnormalities (Tesi B, Davidsson J, Voss M, Rahikkala E, Holmes TD, Chiang SCC, et al. gain-of-function D9 SAM 9L constants house a syndrome of cytopenia, immunodeficiencies, MDS, and neurological symptoms. blood 2017; 129(16): 2266-.

At present, a signal path mechanism of SAMD9L gene mutation action is not clear, the invention reports that a neonate with SAMD9L insertional deletion newborn mutation causing protein translation termination in advance shows chilblain-like rash, intracranial calcification and ISGs level increase, the clinical expression of the neonate is different from ATXPC, the neonate accords with the characteristics of I-type interferon diseases, and clinical symptoms are obviously relieved after a JAK inhibitor is used for blocking a related signal path, so that the SAMD9L gene truncation mutation can be considered to cause the I-type interferon diseases. Type I interferon disease was first proposed in 2011 (Crow YJ. type I interferon diseases: a novel set of inborn errors of immunity. Ann N Y Acad Sci 2011; 1238:91-98.), and is a group of auto-inflammatory diseases associated with persistent activation of type I interferon signaling. According to the 2019 Union of International Union of Immunological Societies (IUIS) classification standard, different type I interferon diseases caused by 15 gene mutations have been found. The pattern recognition receptor secretes type I interferon after sensing exogenous or self-derived nucleic acid, and then acts on type I receptor (IFNAR1/2), by inducing a series of transcriptions of genes called Interferon Stimulated Genes (ISGs) through Janus kinase (JAK) signal transduction and transcriptional activation (STAT) pathways, any gene defect which causes increased nucleic acid load, enhanced receptor sensing function or loss of negative feedback system function can cause serious inflammatory response by up-regulating type I interferon signals, thereby causing type I interferon diseases, the related specific clinical characteristics comprise intracranial calcification, leukoencephalopathy, chilblain-like rash or panniculitis, interstitial pneumonia, systemic inflammatory reaction, increase of ISGs and the like, and the JAK inhibitor is a specific treatment drug for I-type interferon diseases at present and can inhibit inflammatory reaction by blocking a JAK-STAT signal channel, so that the treatment effect is achieved.

Early diagnosis and symptomatic treatment of type I interferon diseases are effective ways to reduce morbidity, mortality and disease severity, and at present, diagnosis of type I interferon diseases is mainly based on clinical symptoms, but clinical symptoms of type I interferon diseases are complex in manifestation and similar to clinical manifestations of various other diseases, and are easily confused with other diseases, so detection at a molecular level is of great significance to the diseases, and especially for infants with prenatal diagnosis and incompletely revealed clinical phenotypes, molecular biological diagnosis is the most accurate way to provide early diagnosis evidence. Some patients who are not diagnosed clearly often show only partial disease characteristics, the clinical manifestations can not meet the current diagnosis standard of the type I interferon disease, and the patients also need gene detection to assist the further diagnosis of the type I interferon disease so as to be treated early. Therefore, the mutation site c.2642-2729ins/delTCCTGT on SAMD9L gene is identified in one patient with type I interferon disease in China for the first time, the mutation site is the truncation mutation of the P-loop NTPase region of SAMD9L gene, and the truncation mutation is verified to have better diagnostic efficacy as a biomarker of type I interferon disease.

Disclosure of Invention

Aiming at the technical problem that the diagnosis of the I-type interferon diseases is difficult in the prior art, the invention aims to provide the SAMD9L gene truncated mutation serving as a marker for the diagnosis of the I-type interferon diseases and application thereof, wherein the truncated mutation is positioned in a P-loop NTPase region of an SAMD9L gene, the SAMD9L gene mutation is c.2642-2729ins/delTCCTGT, and experiments prove that the SAMD9L gene truncated mutation is a pathogenic mutation and has higher accuracy and reliability in the diagnosis and/or auxiliary diagnosis of the I-type interferon diseases.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect of the invention there is provided a gene mutation and/or a protein mutation for use in the diagnosis and/or diagnosis of type I interferon diseases.

Further, the gene mutation and/or the protein mutation is SAMD9L gene truncation mutation;

preferably, the type I interferon disease caused by the truncation mutation of the SAMD9L gene is an autosomal dominant inheritance pattern;

preferably, the gene mutation and/or protein mutation is a truncated mutation of the P-loop NTPase region of SAMD9L gene;

more preferably, the genetic mutation is c.2642-2729 ins/delTCCTGT;

more preferably, the protein is mutated to p.k881ifs 2.

Further, the Gene ID:219285 of SAMD9L, the detailed sequence information of which is available at https:// www.ncbi.nlm.nih.gov/Gene.

Further, c.2642-2729ins/delTCCTGT as used herein refers to a truncated mutation c.2642-2729delins TCCTGT occurring in the P-loop NTPase region of SAMD9L gene.

The second aspect of the invention provides the use of a reagent for detecting mutations in SAMD9L gene in any of the following aspects:

(1) preparing a product for diagnosing and/or assisting in diagnosing the type I interferon disease;

(2) preparing a product for assessing and/or aiding in the assessment of the risk of a subject to develop type I interferon disease;

(3) preparing a product for assessing and/or aiding in the assessment of the risk of developing type I interferon disease in offspring of a subject;

preferably, the product comprises a kit, a chip, test paper and a detection and analysis system;

preferably, the gene mutation is a SAMD9L gene truncation mutation;

more preferably, said SAMD9L gene truncation mutation is autosomal dominant inheritance;

more preferably, the gene mutation is a truncated mutation of the P-loop NTPase region of SAMD9L gene;

most preferably, the genetic mutation is c.2642-2729 ins/delTCCTGT.

The third aspect of the present invention provides the use of a reagent for detecting mutations in the SAMD9L protein in any one of the following aspects:

(1) preparing a product for diagnosing and/or assisting in diagnosing the type I interferon disease;

(2) preparing a product for assessing and/or aiding in the assessment of the risk of a subject to develop type I interferon disease;

(3) preparing a product for assessing and/or aiding in the assessment of the risk of developing type I interferon disease in offspring of a subject;

preferably, the product comprises a kit, a chip, test paper and a detection and analysis system;

preferably, the protein mutation is a SAMD9L gene truncation mutation;

more preferably, said SAMD9L gene truncation mutation is autosomal dominant inheritance;

more preferably, the protein is mutated to a truncated mutation of the P-loop NTPase region of the SAMD9L gene;

most preferably, the protein is mutated to p.k881ifs 2.

The fourth aspect of the present invention provides reagents for detecting mutations in SAMD9L gene and/or for detecting mutations in SAMD9L protein;

further, the reagents comprise primers for specifically amplifying the SAMD9L gene mutation, and/or probes for specifically recognizing the SAMD9L gene mutation, and/or antibodies or ligands for specifically binding to the SAMD9L protein mutation;

preferably, the gene mutation and the protein mutation are SAMD9L gene truncation mutations;

more preferably, said SAMD9L gene truncation mutation is autosomal dominant inheritance;

more preferably, the gene mutation and the protein mutation are truncated mutations of a P-loop NTPase region of the SAMD9L gene;

most preferably, the genetic mutation is c.2642-2729 ins/delTCCTGT;

most preferably, the protein is mutated to p.k881ifs 2;

most preferably, the sequence of the primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2;

most preferably, the primer may be modified by any one of a thio modification, an amino modification, a thiol modification, a phosphorylation modification, a biotin modification, a locked nucleotide modification, a 5-bromo-deoxyuracil modification, a 2' -O-methyl ribonucleic acid modification, a deoxyinosine modification, a 5-methyl deoxycytidine modification, a 2-aminopurine modification, a 2-fluoro ribonucleic acid modification, a reverse dT modification, a dideoxycytidine modification, and a spacer modification, or may be modified in a plurality of ways simultaneously.

Further, the reagent isComprises dNTPs, Taq enzyme and Mg ²⁺ And PCR reaction buffer.

A fifth aspect of the invention provides a kit for diagnosing and/or aiding in the diagnosis of type I interferon disease.

Further, the kit comprises:

(1) an effective amount of a reagent for detecting mutations in SAMD9L gene and/or for detecting mutations in SAMD9L protein;

(2) selecting the following group of substances: containers, positive controls, negative controls, buffers, adjuvants, solvents, solutions for suspending or immobilizing cells, detectable labels or labels, solutions for facilitating hybridization of nucleic acids, solutions for lysing cells, solutions for nucleic acid purification, instructions for use;

preferably, the reagent described in step (1) is a reagent according to the fourth aspect of the present invention.

Further, the kit is used for diagnosing and/or assisting in diagnosing whether a subject individual has the type I interferon disease by detecting the gene mutation and/or the protein mutation of the first aspect of the invention in a test sample of the subject;

preferably, the test sample comprises a tissue, body fluid or excreta of the subject;

more preferably, the bodily fluid comprises blood, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid, or aqueous humor;

most preferably, the test sample is derived from the blood of a subject.

Further, the kit is accompanied with instructions for using the kit, wherein the instructions describe how to use the kit for detection, how to use the detection result to judge whether the subject has the type I interferon disease, and how to use the detection result to select the treatment scheme of the subject.

Further, the kit also comprises other reagents clinically used for judging the type I interferon disease and selecting a treatment scheme so as to assist or verify the result obtained by detecting the mutation, and a person skilled in the art can perform routine selection according to specific needs.

Further, with the kit of the present invention, the above-mentioned mutation can be detected by various methods (including but not limited to) selected from the group consisting of: PCR (polymerase chain reaction) combined with one-generation sequencing, gene mutation DNA probe hybridization using a marker, restriction fragment length polymorphism method or sequence-specific primer method.

Furthermore, the kit of the invention can be used for diagnosing and/or assisting in diagnosing the type I interferon disease by adopting the following method:

(1) obtaining a test sample from a subject;

(2) contacting a sample to be detected with a reagent detected in the kit;

(3) detecting said mutation in said sample;

(4) and (3) carrying out diagnosis and/or auxiliary diagnosis of the type I interferon disease according to the detection result: and if the detection result shows that the sample to be detected of the subject carries the mutation, the tendency that the subject suffers from the type I interferon disease is suggested.

Further, the subject includes mammals, such as humans, monkeys, mice, rats, rabbits.

Further, the test sample comprises a tissue, a body fluid or an excrement of the subject;

preferably, the bodily fluid comprises blood, extracellular fluid, interstitial fluid, lymphatic fluid, cerebrospinal fluid, or aqueous humor;

more preferably, the test sample is derived from the blood of a subject.

According to a sixth aspect of the invention there is provided the use of a genetic mutation and/or a protein mutation as described in the first aspect of the invention in the preparation of a reagent as described in the fourth aspect of the invention.

The seventh aspect of the invention provides the use of a gene mutation and/or a protein mutation according to the first aspect of the invention in the preparation of a kit according to the fifth aspect of the invention.

An eighth aspect of the invention provides the use of a kit according to the fifth aspect of the invention in any one of the following aspects:

(1) diagnosing and/or aiding in the diagnosis of type I interferon disease;

(2) assessing and/or aiding in the assessment of the risk of a subject to develop type I interferon disease;

(3) assessing and/or aiding in the assessment of the risk of developing type I interferon disease in the offspring of the subject.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the specification of the present invention are only for describing specific embodiments and are not intended to limit the present invention, and furthermore, some terms are explained as follows.

The term "diagnosis" or "aided diagnosis" as used herein refers to the identification or classification of a molecular or pathological state, disease or condition. "diagnosis" or "diagnosis-aiding" may also refer to the classification of a particular subtype of disease, for example by molecular characterization (e.g., a subpopulation of patients characterized by nucleotide variations in a particular gene or nucleic acid region).

The term "probe" as used herein refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that binds to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing, the probe being capable of binding to the target polynucleotide to which the probe lacks complete sequence complementarity, the probe being directly or indirectly labeled, depending on the stringency of the hybridization conditions, and includes within its scope primers, exemplary probes including PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to a molecular barcode, and probes immobilized on beads, in a manner including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays, the specific method of probe design and synthesis is not limited and can be performed using any suitable method in the art, depending on the target nucleic acid sequence. For example, probes that are customized to the target nucleic acid sequence of interest can be ordered from commercial companies (including but not limited to Thermo Fisher Scientific, Agilent, Nimblegen, IDT, etc.), the length of the probes is not limited, it is variable, and is generally dependent on the experimental design, e.g., the average length of the probes can be about 20 to about 200 nucleotides.

The term "primer" as used herein refers to 7 to 50 nucleic acid sequences capable of forming a Base pair (Base pair) complementary to a template strand and serving as a starting point for copying the template strand, and the primer is generally synthesized, but a naturally occurring nucleic acid may be used, and the sequence of the primer is not necessarily completely identical to the sequence of the template, and may be mixed with additional features that do not alter the basic properties of the primer as long as it is sufficiently complementary to be able to hybridize with the template, and examples of the additional features that may be mixed include methylation, capping, substitution of one or more nucleic acids with a homolog, and modification between nucleic acids, but are not limited thereto.

The term "sample" as used herein refers to a biological material isolated from a subject, which biological sample may contain any biological material suitable for detecting the marker and may comprise cellular and/or non-cellular material of the subject, which sample may be from any suitable biological tissue or fluid such as liver tissue, blood, plasma, blood, urine, thoracic fluid, cerebrospinal fluid, and "subject" refers to any animal, preferably a mammal, such as a human, monkey, mouse, rat, rabbit.

The term "chip" as used herein includes gene chips, protein chips, gene chips refer to probes comprising solid phase carriers and probes immobilized on the solid phase carriers, said probes comprising probes for said markers for detecting said markers, and protein chips refer to probes comprising solid phase carriers and antibodies and/or ligands immobilized on the solid phase carriers which specifically bind to proteins encoded by said markers.

The invention has the advantages and beneficial effects that:

the invention identifies the mutation site c.2642-2729ins/delTCCTGT on SAMD9L gene in one patient with I-type interferon disease in China for the first time, the mutation site is the truncation mutation of the P-loop NTPase region of SAMD9L gene, the inventor proves that the truncation mutation is the pathogenic mutation through verification experiments, and the invention has higher accuracy and reliability in I-type interferon disease diagnosis and/or auxiliary diagnosis;

the discovery of SAMD9L gene truncated mutation lays a foundation for explaining the etiology of the type I interferon disease, and simultaneously, the mutation can be used as a biomarker of the type I interferon disease, is used for diagnosis and/or auxiliary diagnosis of the type I interferon disease, evaluation of the risk of a subject suffering from the type I interferon disease, pre-pregnancy early warning and selection of a treatment scheme, indicates the risk of the offspring of a mutation carrier suffering from the type I interferon disease, can provide guidance in genetics for the type I interferon disease patient, and achieves early discovery and early treatment.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows graphs showing the results of variation in the clinical phenotypic characteristics of individuals having the c.2642-2729ins/delTCCTGT mutation in SAMD9L gene, wherein, A is a graph: before treatment, panel B: treatment for 6 months, panel C: treating for 1 year;

FIG. 2 shows a graph of the results of cranial CT in individuals having the c.2642-2729ins/delTCCTGT mutation in SAMD9L gene, wherein, A is: before treatment, panel B: after 1 year of treatment;

FIG. 3 shows a map of the results of cranial MRI in individuals with the c.2642-2729ins/delTCCTGT mutation in SAMD9L gene, wherein, map A: before treatment, panel B: after 1 year of treatment;

FIG. 4 shows a graph of the results of sequencing the SAMD9L gene of a patient and his parent, in which, Panel A: patient, panel B: father, fig. C: mother;

FIG. 5 shows the results of electrophoretic analysis of PCR amplification products of gDNA and cDNA, in which HC: healthy control, M: mother, F: father, P: patient, Marker: standard substance comparison;

FIG. 6 shows a graph of the results of sequencing of the 530bp mutant band;

FIG. 7 is a statistical chart showing the results of SAMD9L expression before and after JAK inhibitor treatment;

FIG. 8 is a statistical chart showing the results of ISGs expression before and after JAK inhibitor treatment;

FIG. 9 shows graphs of the results of Wild Type (WT) and heterozygous knockout (Het) cell line identification;

FIG. 10 is a graph showing the results of a growth curve of a WT cell line under stimulation with different concentrations of IFN;

FIG. 11 shows the results of a growth curve of a cell line of type Het in the presence of different concentrations of IFN;

FIG. 12 shows the results of growth curves for WT and Het cell lines under stimulation with 0, 10IU/mL IFN;

FIG. 13 shows the results of growth curves for WT and Het cell lines under 100, 1000IU/mL IFN stimulation;

FIG. 14 is a graph showing the results of qPCR detection of SAMD9L full-length mRNA and total mRNA (full-length + truncated) expression.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 screening for SAMD9L Gene mutations associated with type I Interferon disease

1. Study object

The study object is a first example of the clinical manifestation of the first in China in Beijing coordination hospital (PUMCH) as a newborn with scattered facial and limb spots and papules, hepatosplenomegaly, blood three-system reduction, increased inflammation index, backward growth and development, intracranial calcification and leukoencephalopathy, the clinical data is collected, all exome gene capture high-flux sequencing is carried out, all exome data is analyzed, suspected candidate genes of an infant patient and parents are subjected to primary sequencing by DNA and cDNA after RNA reverse transcription extraction for verification, the informed consent of the patient and the parents is obtained, and the study is approved by the PUMCH ethical examination committee.

2. Whole exome detection and data analysis

The infant and parents underwent Trios full exome test, which was reported as negative by the detection company, beijing michinono medical laboratory. And obtaining data obtained after the second generation sequencing annotation of the patient is reanalyzed after the patient is informed of consent, filtering out variation sites with high frequency, out of functional regions and inconsistent genetic patterns, and screening candidate genes by combining pathogenicity conjecture.

3. Patient and parental candidate gene generation sequencing validation

Taking peripheral blood of children patients and parents, and extracting DNA. According to the results of the second generation sequencing, SAMD9L is suggested to have three sections of new deletion mutations: c.2642_2661del, p.k881ifs 10; c.2666_2712del, p.f889cfs 11; c.2715_2729 delCAGGAATATCCTAT CCTAAA, p.905_910 delVRNILkinSV. Primers were designed outside both ends of the deleted fragment using Primer 3, and PCR was performed using 13. mu.L, ddH, of 2 XTAQ PCR Master mix (Biomed Co.) ₂ Carrying out PCR on the DNA of the infant patient in a GeneAmp PCR System 9700 System (Applied Biosystems) under the conditions of pre-denaturation at 95 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 45s and final extension at 72 ℃ for 5min for 40 cycles, covering the whole mutation region by the Applied primers, carrying out agarose gel (2%) electrophoresis, cutting gel purification and Sanger sequencing, wherein the proportion of the primers is 1 μ L;

the primer sequences are as follows:

SAMD9L-F(5’-3’)：CTGGTCACCTATAGGGCAAA(SEQ ID NO:1)；

SAMD9L-R(5’-3’)：TCCCATATTCTGCAACTTCTG(SEQ ID NO:2)。

4. further validation of patient and parent SAMD9L insertion deletion mutant DNA and cDNA

The fragment of the infant is proved to be in a set peak by first-generation sequencing verification, and the mutation is SAMD9L heterozygous insertion deletion mutation by artificial sequence alignment: 2642-2729ins/delTCCTGT, which is a truncated SAMD9L gene, and the sequence after mutation is 82 bases less than the original sequence. In order to avoid the manual alignment error, the cDNA reverse transcribed after total RNA extraction from the DNA of the sick child and the parents and the peripheral blood is subjected to PCR again, 2 XTaq PCR Master mix (Biomed) is Applied to the cDNA of the sick child, the parents and the healthy person in a GeneAmp PCR System 9700 System (Applied Biosystems) under the conditions of pre-denaturation 95 ℃ for 10min, denaturation 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 45s, final extension at 72 ℃ for 5min, and 40 cycles, and the Applied primers cover the whole mutation region. Performing gel running for 1 hour by 2% agarose gel electrophoresis, and sequencing the cut gel of the mutant band after the mutant band and the wild band are obviously distinguished;

the primer sequences are as follows:

SAMD9L-F(5’-3’)：CTGGTCACCTATAGGGCAAA(SEQ ID NO:1)；

SAMD9L-R(5’-3’)：TCCCATATTCTGCAACTTCTG(SEQ ID NO:2)。

5. SAMD9L expression and ISGs assay

Adding Trizol into whole blood, extracting total RNA in peripheral blood of children, parents and normal control, evaluating RNA concentration with spectrophotometer, applying PrimeScript RT reagent Kit with DNA Eraser (Takara company), reverse transcribing RNA, reverse transcribing 300ng RNA into cDNA with ABI veriti96 PCR instrument, and performing fluorescence quantitative real-time PCR detection (two-step detection) with ABI ViiA7 qPCR instrument (Applied Biosystems) under the conditions of 37 ℃ for 15min, 85 ℃ for 5s, cooling to 4 ℃: pre-denaturation at 95 ℃ for 30sec, denaturation at 95 ℃ for 3sec, annealing and elongation at 60 ℃ for 30sec, 40 cycles. Wherein, the sequences of the SAMD9L primers are as follows:

SAMD9L-F(5’-3’)：GAAACAGGAGCACTCAATCTCA(SEQ ID NO:3)；

SAMD9L-R(5’-3’)：CAGCCTTACTGGTGATTTTCACA(SEQ ID NO:4)；

detecting Interferon Stimulating Genes (ISGs) in whole blood of a patient by taking healthy human controls and actin as an internal reference, wherein the detected interferon stimulating genes are as follows: IFI27, IFI44L, IFIT1, ISG15, RSAD2 and SIGLEC1, wherein the application primers are (F/R):

IFI27-F(5’-3’)：TGCTCTCACCTCATCAGCAGT(SEQ ID NO:5)；

IFI27-R(5’-3’)：CACAACTCCTCCAATCACAACT(SEQ ID NO:6)；

IFI44L-F(5’-3’)：TTGTGTGACACTATGGGGCTA(SEQ ID NO:7)；

IFI44L-R(5’-3’)：GAATGCTCAGGTGTAATTGGTTT(SEQ ID NO:8)；

IFIT1-F(5’-3’)：AGAAGCAGGCAATCACAGAAAA(SEQ ID NO:9)；

IFIT1-R(5’-3’)：CTGAAACCGACCATAGTGGAAAT(SEQ ID NO:10)；

ISG15-F(5’-3’)：GAGGCAGCGAACTCATCTTT(SEQ ID NO:11)；

ISG15-R(5’-3’)：AGCATCTTCACCGTCAGGTC(SEQ ID NO:12)；

RSAD2-F(5’-3’)：TGCTTTTGCTTAAGGAAGCTG(SEQ ID NO:13)；

RSAD2-R(5’-3’)：AGGTATTCTCCCCGGTCTTG(SEQ ID NO:14)；

SIGLEC1-F(5’-3’)：AGCTGAGGCCAACTCCCTGA(SEQ ID NO:15)；

SIGLEC1-R(5’-3’)：AGGCTCCTCGGACCTGGAAG(SEQ ID NO:16)；

ACTIN-F(5’-3’)：CCAACCGCGAGAAGATGA(SEQ ID NO:17)；

ACTIN-R(5’-3’)：CCAGAGGCGTACAGGGATAG(SEQ ID NO:18)。

6. results of the experiment

(1) Case analysis results

The study object is a female infant, the infant is born prematurely in 35 weeks, delivered by caesarean delivery, normal post-natal Alzheimer score and easy to irritate, the whole body is scattered at a bleeding point, the face and four limbs of the infant are scattered at maculopapule on the 4 th day after the birth, and gradually increase, fuse into pieces, break, and remove toe red swelling (see figure 1A), the result of skull CT shows double basal ganglia, thalamus, lateral ventricles and multiple spot platy calcification in the center of hemioval (see figure 2A), the result of skull MRI shows double basal ganglia and lateral ventricles fat sheet abnormal signals, bleeding is considered (see figure 3A), the diagnosis can be neonatal lupus, hormone is treated, platelets are recovered to be normal, the rash is improved, but the hormone dosage is not reduced smoothly, the hormone dosage is adjusted for multiple times, the rash is diagnosed repeatedly, 2019-10 treatment is carried out, and the clinical characteristics of chilblain-like rash, growth and development laggardt, epilepsy, cytopenia, intracranial hemorrhage and the like are considered, diagnosing suspected I-type interferon disease, continuously maintaining hormone, 1.25mg QD of 2019-11-08 household tofacitinib, obviously improving clinical symptoms, basically recovering normal rash, blood three systems and liver enzymes, recovering normal CRP and ESR, recovering normal body weight and head circumference, and displaying multiple symmetrical calcification of bilateral cerebellar hemispheres, basal ganglia and radial crowns and reduction of white matter density around bilateral ventricles (see figure 2B) by a head CT result, wherein the calcification range is reduced compared with that before treatment; the results of cranial MRI showed changes in white matter demyelination in the bilateral frontal parietal lobe, expansion of the ventricular system, thinner pituitary (see fig. 3B), facial and extremity results for children treated for 6 months and 1 year of treatment are shown in fig. 1B and 1C, and the auxiliary examinations are detailed in table 1.

(2) Results of Gene analysis

The results of secondary analysis of the whole exome Trios data indicate that three sections of new deletion mutations exist in SAMD 9L: c.2642_2661del, p.k881ifs 10; c.2666_2712del, p.f889cfs 11; 2715_2729 delCAGGAATATCCTAT CCTAAA, p.905_910 delVRNILkinSV; after first generation verification, the mutation is proved to be a new hybrid insertion-deletion mutation: c.2642-2729ins/delTCCTGT, p.K881Ifs 2, which is a truncated mutation of SAMD9L gene, and the sequence charts of the infant patient and parent generation are shown in FIGS. 4A-C (sequentially from top to bottom, infant patient, father and infant patient);

after the PCR is carried out again on the DNA and the cDNA of the sick child, the parent and the normal person, and 2% agarose gel electrophoresis is carried out for 1 hour, the cDNA and the DNA of the sick child have two bands of 530bp (mutant type) and 612bp (wild type), the cDNA and the DNA of the parent only have the wild type band, the normal control DNA only has the wild type band (shown in figure 5), the sequencing result of the 530bp mutant type band is shown in figure 6, and the mutation at the 530bp position is c.2642-2729 ins/delTCCTGT.

(3) Results of peripheral blood expression of SAMD9L before and after treatment

The results of examining the expression of SAMD9L in peripheral blood before and after treatment in a child patient showed that the expression of SAMD9L in the whole child before the JAK inhibitor treatment was elevated, and that SAMD9L expressed in the child patient expressed both wild type and truncated mutant type, in combination with the results of the above gene analysis (2); total SAMD9L in the peripheral blood of children patients returned to normal levels following treatment with JAK inhibitors (see figure 7).

(4) Results of ISGs expression before and after JAK inhibitor treatment

The expression level of ISGs in children is obviously increased by applying RT-PCR detection before treatment, the ISGs are rechecked to be normal after the JAK inhibitor is treated for 1 year (see figure 8), the expression of the ISGs before treatment is increased, and the ISGs are basically recovered to be normal after the JAK inhibitor is treated for 1 year (see table 1).

TABLE 1 comparison before and after treatment with JAK inhibitors

Example 2 truncation mutation of SAMD9L Gene causes type I Interferon disease

1. Experimental methods

(1) Construction of cell lines

Constructing a p-loop NTPase region heterozygous knockout U87MG cell line by using a virus method, and obtaining a p-loop NTPase region heterozygous knockout cell (Het type) through PCR detection and identification, wherein the identification result is shown in figure 9, and the wild type is WT; the heterozygous knockout type is Het; f1, R: the primer pair crossing the knockout region, WT has only 1615bp band; het has 1615bp bands and 558bp bands at the same time; f2, R: primer pairs with upstream within the knockout region and downstream outside the knockout region. Both WT and Het types had a 715bp band.

(2) Cell proliferation assay

Cells are inoculated on a 96-well plate, 10000 cells per well, and 0IU/mL, 10IU/mL, 100IU/mL and 1000IU/mL IFN-alpha 2 beta are respectively applied to treat WT and Het type cells. After adherent culture for 4h, adding CCK-810 mu L, collecting absorbance at the wavelength of 450nm every half hour after 40min, continuously monitoring for 25h, and drawing a cell growth curve.

(3) Comparison of IFN beta secretion in WT and Het type cells under IFN stimulation

The WT cells and the Het cells with the same cell amount are cultured for 24h or 48h under the stimulation of IFN-alpha 2 beta of 0IU/mL and 100IU/mL respectively, and are washed for 2 times by PBS after stimulation (exogenous IFN is removed), a fresh culture medium is added for continuous culture for 24h or 48h, ELISA detection is applied, and the concentration of the IFN beta secreted in the culture medium supernatants of the WT and Het cells with or without IFN stimulation is compared.

(4) Comparison of SAMD9L expression in WT and Het cells in the absence and presence of IFN stimulation

WT cells and Het cells with the same cell amount are cultured for 24h under the stimulation of 0 and 100IU/mL IFN-alpha 2 beta respectively, and SAMD9L full-length mRNA and total mRNA (full-length + truncated) expression are detected by qPCR.

2. Results of the experiment

The experimental results showed that WT type cells increased rapidly with increasing IFN-. alpha.2beta.concentration and still increased slowly for 25h, not reaching the plateau phase (see FIG. 10). Het type cells have basically similar growth trend when stimulated by IFN-alpha 2 beta at low concentration (100IU/mL and below), and grow faster when stimulated by IFN-alpha 2 beta at high concentration of 1000 IU/mL; and begins to fade after 10h (see fig. 11). WT increased less rapidly than Het before 18h stimulation with 0, 10IU/mL IFN-. alpha.2beta.and continued to increase beyond Het after 18h (see FIG. 12). WT increases at higher rates than Het with 100IU/mL and 1000IU/mL IFN-. alpha.2beta (see FIG. 13).

The supernatant IFN beta concentration of the Het group and the WT group has no significant difference (P is 0.602) under the condition of no IFN stimulation, and 21.38 +/-5.22 Vs.22.79 +/-3.74 pg/mL; after 24h or 48h of IFN stimulation, the secretion of IFN beta is remarkably increased in the Het group compared with that in the WT group (P is 0.030), and the secretion of IFN beta is 88.22 +/-14.12 Vs.71.48 +/-17.48 pg/mL; after 24h or 48h of washout after IFN stimulation, IFN β secretion was significantly reduced in the Het group compared to the WT group (P ═ 0.004), 34.51 ± 2.00vs.84.37 ± 4.25 pg/mL.

WT cells and Het cells with the same cell amount are cultured for 24h under 0 and 100IU/mL IFN-alpha 2 beta stimulation respectively, and SAMD9L full-length mRNA and total mRNA (full-length type + truncated type) expression conditions are detected by qPCR, and the results show that SAMD9L total mRNA is increased after IFN stimulation by WT and Het, and the Het full-length mRNA and Het truncated mRNA are expressed (see figure 14).

The above experimental results show that SAMD9L accelerates the cell cycle of Het type cells under the stimulation of interferon after the p-loop NTPase region is subjected to heterozygous knockout, the decline period appears in advance, and the growth speed difference between WT cells and Het cells is reversed at the concentration of more than 100 IU/mL. That is, IFN can promote the growth of WT and Het type U87MG cells, but the promotion effect on Het is lower than that of WT and the regression phase of Het appears in advance, which indicates that the truncation mutation generated by deletion of p-loop NTPase region leads to the change of response to IFN stimulation. The secretion of IFN β by WT and Het cells is not different in the absence of IFN stimulation, but after IFN stimulation, Het is secreted more than WT, indicating a sustained activation of the type I interferon pathway in Het cells under stimulation with type I IFN, i.e. this type of mutation leads to type I interferon disease. Het cells express both full-length and truncated mRNAs. The experiment result shows that the SAMD9L gene truncation mutation is pathogenic mutation and can cause I-type interferon related diseases, and further proves that the SAMD9L gene truncation mutation can be used as a marker for I-type interferon related disease diagnosis.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

<110> Beijing coordination hospital of Chinese academy of medical sciences

<120> SAMD9L gene mutation as marker for diagnosis of type I interferon diseases and application thereof

<141> 2022-06-06

<150> 2021106374639

<151> 2021-06-08

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Claims (10)

1. Gene mutation and/or protein mutation for the diagnosis and/or the aided diagnosis of type I interferon diseases, characterized in that the gene mutation and/or protein mutation is a SAMD9L gene truncation mutation;

preferably, the type I interferon disease caused by said truncation mutation of SAMD9L gene is an autosomal dominant inheritance pattern;

preferably, the gene mutation and/or protein mutation is a truncated mutation of the P-loop NTPase region of SAMD9L gene;

more preferably, the genetic mutation is c.2642-2729 ins/delTCCTGT;

more preferably, the protein is mutated to p.k881ifs 2.

2. The application of the reagent for detecting the mutation of the SAMD9L gene in any one of the following aspects:

(1) preparing a product for diagnosing and/or assisting in diagnosing the type I interferon disease;

(2) preparing a product for assessing and/or aiding in the assessment of the risk of a subject to develop type I interferon disease;

(3) preparing a product for assessing and/or aiding in the assessment of the risk of developing type I interferon disease in offspring of a subject;

preferably, the product comprises a kit, a chip, test paper and a detection and analysis system;

preferably, the gene mutation is a SAMD9L gene truncation mutation;

more preferably, the gene mutation is a truncated mutation of the P-loop NTPase region of SAMD9L gene;

most preferably, the genetic mutation is c.2642-2729 ins/delTCCTGT.

3. The application of the reagent for detecting the mutation of the SAMD9L protein in any one of the following aspects:

(1) preparing a product for diagnosing and/or assisting in diagnosing the type I interferon disease;

(2) preparing a product for assessing and/or aiding in the assessment of the risk of a subject to develop type I interferon disease;

(3) preparing a product for assessing and/or aiding in the assessment of the risk of developing type I interferon disease in offspring of a subject;

preferably, the product comprises a kit, a chip, test paper and a detection and analysis system;

preferably, the protein mutation is a SAMD9L gene truncation mutation;

more preferably, the protein is mutated to a truncated mutation of the P-loop NTPase region of the SAMD9L gene;

most preferably, the protein is mutated to p.k881ifs 2.

4. Reagent for the detection of SAMD9L gene mutations and/or for the detection of SAMD9L protein mutations, characterized in that said reagent comprises a primer specifically amplifying SAMD9L gene mutations, and/or a probe specifically recognizing SAMD9L gene mutations, and/or an antibody or ligand specifically binding SAMD9L protein mutations;

preferably, the gene mutation and the protein mutation are SAMD9L gene truncation mutations;

more preferably, the gene mutation and the protein mutation are truncated mutations of a P-loop NTPase region of the SAMD9L gene;

most preferably, the genetic mutation is c.2642-2729 ins/delTCCTGT;

most preferably, the protein is mutated to p.k881ifs 2;

most preferably, the sequence of the primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2;

most preferably, the primer may be modified by any one of a thio modification, an amino modification, a thiol modification, a phosphorylation modification, a biotin modification, a locked nucleotide modification, a 5-bromo-deoxyuracil modification, a 2' -O-methyl ribonucleic acid modification, a deoxyinosine modification, a 5-methyl deoxycytidine modification, a 2-aminopurine modification, a 2-fluoro ribonucleic acid modification, a reverse dT modification, a dideoxycytidine modification, and a spacer modification, or may be modified in a plurality of ways simultaneously.

5. The reagent of claim 4, wherein the reagent further comprises dNTPs, Taq enzyme, Mg ²⁺ And PCR reaction buffer.

6. A kit for diagnosing and/or aiding in the diagnosis of type I interferon disease, said kit comprising:

(1) an effective amount of a reagent for detecting mutations in SAMD9L gene and/or for detecting mutations in SAMD9L protein;

(2) selecting the following group of substances: containers, positive controls, negative controls, buffers, adjuvants, solvents, solutions for suspending or immobilizing cells, detectable labels or labels, solutions for facilitating hybridization of nucleic acids, solutions for lysing cells, solutions for nucleic acid purification, instructions for use;

preferably, the reagent described in step (1) is the reagent described in claim 4 or 5.

7. The kit of claim 6, wherein the kit is used for diagnosing and/or assisting in diagnosing whether a subject individual has the type I interferon disease by detecting the gene mutation and/or the protein mutation of claim 1 in a test sample of the subject;

preferably, the test sample is derived from the blood of a subject.

8. Use of the gene mutation and/or protein mutation of claim 1 for the preparation of the reagent of claim 4 or 5.

9. Use of the genetic mutation and/or protein mutation of claim 1 for the preparation of a kit of claim 6 or 7.

10. Use of the kit of claim 6 or 7 in any of the following aspects:

(1) diagnosing and/or aiding in the diagnosis of type I interferon disease;

(2) assessing and/or aiding in the assessment of the risk of a subject to develop type I interferon disease;

(3) assessing and/or aiding in the assessment of the risk of developing type I interferon disease in the offspring of the subject.

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