CN110272986B - Targeted detection for XY dysplastic disease - Google Patents
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Abstract
The invention provides a targeted detection for XY dysplasia diseases, in particular to an application of a gene related to sexual dysplasia or a locus thereof and/or a detection reagent thereof in preparing a reagent or a kit, wherein the reagent or the kit is used for detecting susceptibility of a detected object to the sexual dysplasia, and the gene related to the sexual dysplasia is selected from the following groups: SRD5A2, AR and NR5A1.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to targeted detection of XY dysplasia diseases.
Background
Sexual development diseases (DSD) are a group of heterogeneous genetic diseases with abnormal sex determination and sex differentiation, are diseases caused by chromosome aberration or monogenic mutation, and have the incidence rate of 1/4500 in the population. It is currently believed that sex is composed of several levels of chromosomal sex, gonadal sex, phenotypic sex and psychosocial sex, and when the sex is inconsistent, the disease causes dysplasia. Sex differentiation and sex determination in embryonic development, involving complex gene and molecular network regulation; two hormones secreted by the testes, testosterone and mullerian tube inhibitor, play important roles in the sexual differentiation process.
The research on molecular etiology of various DSD patients greatly promotes people to know the physiological process of sex differentiation, and the diagnosis and treatment of the diseases are gradually standardized and reasonable. Since the patients who found 46,XX phenotype males have Y-to-X chromosomal ectopy, they have been prompted to gradually find the testis Determining gene, sex Determining Region Y (SRY gene), on the Y chromosome; and to recognize its key regulatory role in sex determination. It is considered that the differentiation of the original gonad into testicular tissue is initiated by the downstream gene SOX9 and the like of the SRY gene on the Y chromosome, and that the sexual inversion is caused by gene mutation of SRY, SOX9, WT-1 and the like. Accessory middle renal tubular inhibitory factor (MIF), secreted by the supporting cells of the fetal testis, degenerates the accessory middle renal tubular structure (developing into the upper 1/3 of uterus, oviduct and vagina); androgen (mainly testosterone) secreted by the testicular interstitial cells causes the structure of the middle renal duct to become masculine and develop into an output blood vessel, a seminal duct, an epididymis and a seminal vesicle; in the target cells, testosterone is converted to dihydrotestosterone by 5 α -reductase 2, which makes the prostate and external genitalia masculinizing. However, there are many unknown molecules involved in sex determination and differentiation, and there are many unknown areas of molecular etiology for such diseases.
Among the DSD diseases, the most troublesome is 46,xy DSD, involving the correct and timely diagnosis of the disease, the selection of gender, and the timing and selection of external genitalia reconstruction surgical protocols. A 46,xy fetus can result in 46,xy DSD if there is insufficient androgen secretion from the testes during the embryonic period (e.g., deficiency in various enzymes that promote testosterone production), impaired androgen functioning (androgen receptor insensitivity), or impaired conversion of testosterone to dihydrotestosterone in the target cells. The clinical phenotype of such patients can be similar to that of the small penis of a normal male, with varying degrees of hypospadias and cryptorchidism to near-normal external female genitalia, and the molecular causes of the diseases are usually caused by mutations in androgen receptor gene (AR), 5 alpha-reductase 2 (SRD 5A 2) gene, 17-hydroxylase gene and the like. Through the analysis of DSD patient genes and animal model research, it is found that the genetic mutation of SF-1, WT-1, GATA-4, etc. can lead to the phenotype of testicular hypoplasia, suggesting that these genes are also involved in the differentiation of primitive gonad into testis. In addition, recent studies have found that female ovarian development is not a "default" process, and that there are molecular and material effects (e.g., FOXL2, etc.) that act against testicular differentiation. There are 80-90 genes that are currently known to cause DSD, and only a small fraction of diseases can be diagnosed by clinical phenotypic characteristics and hormone levels. The disease is used as a difficult and complicated disease, a plurality of patients with the same clinical phenotype can have distinct molecular causes, and the diagnostic value of endocrine hormone determination is limited; currently, the clinical gene diagnosis is less than 20 percent; moreover, the diagnosis and treatment usually require cooperation among different departments, and patients often run to different hospitals and departments and cannot be diagnosed for a long time, even misdiagnosed and treated by mistake, and suffer from serious physical and psychological trauma.
In the past, molecular diagnosis of such diseases often adopts a method of sequencing candidate genes Sanger according to clinical phenotypes, which is time-consuming and labor-consuming, and sometimes can not obtain clear results.
Therefore, the establishment of the targeted gene sequencing based on the next generation sequencing technology has important value and significance for the diagnosis of DSD diseases, and is expected to greatly improve the prognosis of patients. Therefore, there is an urgent need in the art to develop a gene chip for targeted second-generation sequencing of 46,xy dysplastic diseases.
Disclosure of Invention
The invention aims to provide a gene chip for targeted second-generation sequencing of 46,XY dysplasia.
Another objective of the invention is to provide a detection method for the next generation sequencing of targeted genes of 46,XY DSD patients.
The invention provides a usage of a gene related to sexual dysplasia or its locus, and/or a detection reagent thereof, for preparing a reagent or a kit, wherein the reagent or the kit is used for detecting susceptibility of a detected object to sexual dysplasia, and the gene related to sexual dysplasia is selected from the following group: SRD5A2, AR and NR5A1.
In another preferred example, the detected object includes: human or non-human mammals (e.g., domestic animals, laboratory animals, etc.).
In another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: EGF, ESR1.
In another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: AMH, BMP4, CBX2 and MAP3K1.
In another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: GJA4, MYH6, PTCH1.
In another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: SOX3, HSD17B3, POR, WNT4, WT1, LHCGR, MAMLD1, RXFP2, LHX9, MSX1, BMP7, DGKK, CST9, FGF10.
In another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: CYP17A1, FGF8, GATA4, RSPO1, SRY, ARNT2, BMP2, BNC2, EPHB2, ESR2, FGFR2, FKBP4, GSTM1, TESC.
In another preferred embodiment, the related genes include 5 to 80 genes, preferably 8 to 60 genes, and more preferably 20 to 40 genes.
In another preferred embodiment, the reagent or kit comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: SRD5A2: and (2) chr: 31754395, SRD5A2: and (2) chr: 31805954, SRD5A2: and (2) chr: 31754392, SRD5A2: and (2) chr: 31754418, SRD5A2: and (2) chr: 31805786, SRD5A2: and (2) chr: 31751294, SRD5A2: and (2) chr: 31805775, SRD5A2: and (2) chr: 31758693, SRD5A2: and (2) chr: 31754418, SRD5A2: and (2) chr: 31754497, NR5A1: and (chr 9): 127262605, NR5A1: and (chr 9): 127265467, NR5A1: and (9) chr: 127262390, NR5A1: and (9) chr: 127265589, NR5A1: and (chr 9): 127262797, NR5A1: and (chr 9): 127262973, NR5A1: and (chr 9): 127265498, NR5A1: and (9) chr: 127253389, NR5A1: and (chr 9): 127253398, NR5A1: and (9) chr: 1272459and AR: and (4) chrX:66942740, AR: and (4) chrX:66765516, AR: and (4) chrX:66766163, AR: and (4) chrX:66863249, AR: and (4) chrX:66943556, AR: and (4) chrX:66765872.
in another preferred embodiment, the reagent or kit comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: SRD5A2: and (2) chr: 31805775, SRD5A2: and (2) chr: 31758693, SRD5A2: and (2) chr: 31754418, SRD5A2: and (2) chr: 31754497, NR5A1: and (9) chr: 127265467, NR5A1: and (9) chr: 127262390, NR5A1: and (chr 9): 127265589, NR5A1: and (chr 9): 127262797, NR5A1: and (9) chr: 127262973, NR5A1: and (9) chr: 127265498, NR5A1: and (chr 9): 127253389, NR5A1: and (chr 9): 127253398, NR5A1: and (9) chr: 1272459and AR: and (4) chrX:66863249, AR: and (4) chrX:66943556, AR: and (4) chrX:66765872.
in another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: EGF: and (chr 9): 110920947, EGF: and (9) chr: 110884370, EGF: and (chr 9): 110932389, EGF: and (chr 9): 110902074, EGF: and (chr 9): 110834507, ESR1: and (2) chr6:152129480, ESR1: and (2) chr6:152129484.
in another preferred embodiment, the reagent or the kit further comprises a detection reagent for detecting one or more sites of genes related to sexual dysplasia selected from the group consisting of: AMH: and (2) chr19:2249647, AMH: and (chr 19): 2249450, AMH: and (2) chr19:2251466, BMP4: and (chr 14): 54417171, BMP4: and (chr 14): 54417226, CBX2: and (chr 17): 77758182, CBX2: and (2) chr17:77757651, CBX2: and (2) chr17:77755866, MAP3K1: and (2) chr5:56177692.
in another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: GJA4: and (2) chr1:35260541, GJA4: and (2) chr1:35260779, MYH6: and (chr 14): 23876318, MYH6: and (2) chr14:23865496, MYH6: and (chr 14): 23857492, PTCH1: and (9) chr: 98240468, PTCH1: and (9) chr: 98241383, PTCH1: and (9) chr: 98224163.
in another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: SOX3: and (4) chrX:139587069, HSD17B3: and (9) chr: 99060720, HSD17B3: and (9) chr: 98997823, POR: and (chr 7): 75615537, POR: and (2) chr7:75608886, WNT4: and (2) chr1:22456135, WNT4: and (2) chr1:22446752, WT1: and (chr 11): 32456873, WT1: and (9) chr11:32417943, LHCGR: and (2) chr: 48982782, LHCGR: and (2) chr: 48915950, MAMLD1: and (4) chrX:149639324, MAMLD1: and (4) chrX:149639544, RXFP2: and (chr 13): 32366083, RXFP2: and (2) chr13:32355815, LHX9: and (2) chr1: 197886989, LHX9: and (2) chr1:197890684, MSX1: and (2) chr4:4864775, MSX1: and (2) chr4:4862087, BMP7: and (chr 20): 55840949, BMP7: and (2) chr20:55840785, DGKK: and (4) chrX:50127741, DGKK: and (4) chrX:50213362, CST9: and (2) chr20:23584368, FGF10: and (chr 5): 44388594, FGF10: and (chr 5): 44305114.
in another preferred embodiment, the reagent or kit further comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: CYP17A1: and (2) chr10:104592955, FGF8: and (2) chr10:103531266, GATA4: and (chr 8): 11614521, RSPO1: and (2) chr1:38095564, SRY: and (2) chrY:2655418, ARNT2: and (2) chr15:80762743, BMP2: and (2) chr20:6751089, BNC2: and (9) chr: 16437095, EPHB2: and (2) chr1:23208869, ESR2: and (2) chr14:64701857, FGFR2: and (2) chr10:123353315, FKBP4: and (2) chr12:2910316, GSTM1: and (2) chr1:110233137, TESC: and (2) chr12:117494679.
in another preferred embodiment, the sites of the gene associated with sexual dysplasia comprise 10-500 sites, preferably 20-200 sites, more preferably 50-150 sites.
In another preferred embodiment, the reagent or the kit further comprises a detection reagent for detecting mutation sites of one or more sexual dysplasia-related genes selected from the group consisting of genes shown in table 1:
TABLE 1
In another preferred embodiment, the gene mutation site of the present invention is a biallelic mutation site.
In another preferred embodiment, the biallelic mutation site is selected from the group consisting of:
SRD5A2 chr2：31754395 C→T
SRD5A2 chr2：31754392 G→A
AR chrX：66942740 C→T
AR chrX：66765516 C→A
AR chrX：66766163 C→G
AR chrX：66943556 T→G
AR chrX：66765872 T→C
SOX3 chrX：139587069 C→G
LHCGR chr2：48982782 A→G
MAMLD1 chrX：149639544 C→T
DGKK chrX：50213362 C→T
SRY chrY：2655418 C→A
GSTM1 chr1：110233137 A→G。
in another preferred example, the detected object includes: human or non-human mammals (e.g., domestic animals, laboratory animals, etc.).
In another preferred embodiment, the detected objects include Asian population (preferably Chinese).
In another preferred embodiment, the reagent comprises a primer, a probe, a chip, or an antibody.
In another preferred embodiment, the kit contains one or more reagents selected from the group consisting of:
(A) Specific primers for gene detection;
(B) Specific probes for gene detection;
(C) A chip for gene detection;
(D) And the specific antibody is used for detecting the amino acid mutation corresponding to the mutant gene.
In another preferred embodiment, the reagent or kit is used for real-time fluorescent quantitative PCR detection.
In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature is between 60 and 67 ℃, and the length of the PCR amplification product is between 80 and 300bp.
In another preferred example, in the real-time fluorescent quantitative PCR, the annealing temperature of the fluorescent probe is between 60 and 70 ℃.
In another preferred example, the two ends of the probe are modified with chemical groups, the 5' end is modified with a fluorescence excitation group, and the 3 end is modified with a fluorescence quenching group.
In another preferred example, the detection is an auxiliary detection.
In a second aspect, the present invention provides a kit comprising a detection reagent for a gene associated with sexual dysplasia, wherein the gene associated with sexual dysplasia is selected from the group consisting of: SRD5A2, AR and NR5A1.
In another preferred embodiment, the kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: EGF, ESR1.
In another preferred embodiment, the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: AMH, BMP4, CBX2 and MAP3K1.
In another preferred embodiment, the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: GJA4, MYH6, PTCH1.
In another preferred embodiment, the kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: SOX3, HSD17B3, POR, WNT4, WT1, LHCGR, MAMLD1, RXFP2, LHX9, MSX1, BMP7, DGKK, CST9, FGF10.
In another preferred embodiment, the kit further comprises a detection reagent for detecting one or more sexual dysplasia-related genes selected from the group consisting of: CYP17A1, FGF8, GATA4, RSPO1, SRY, ARNT2, BMP2, BNC2, EPHB2, ESR2, FGFR2, FKBP4, GSTM1, TESC.
In another preferred embodiment, the related genes include 5 to 80 genes, preferably 8 to 60 genes, and more preferably 20 to 40 genes.
In another preferred embodiment, the kit comprises a detection reagent for one or more of the sexual dysplasia-associated gene loci selected from the group consisting of:
SRD5A2：chr2：31805775、SRD5A2：chr2：31758693、SRD5A2：chr2：31754418、SRD5A2：chr2：31754497、NR5A1：chr9：127265467、NR5A1：chr9：127262390、NR5A1：chr9：127265589、NR5A1：chr9：127262797、NR5A1：chr9：127262973、NR5A1：chr9：127265498、NR5A1：chr9：127253389、NR5A1：chr9：127253398、NR5A1：chr9：127245134、AR：chrX：66863249、AR：chrX：66943556、AR：chrX：66765872。
in another preferred embodiment, the reagent or kit further comprises a detection reagent for one or more genetic loci selected from the group consisting of:
EGF：chr9：110920947、EGF：chr9：110884370、EGF：chr9：110932389、EGF：chr9：110902074、EGF：chr9：110834507、ESR1：chr6：152129480、ESR1：chr6：152129484。
in another preferred embodiment, the reagent or kit further comprises a detection reagent for one or more genetic loci selected from the group consisting of:
AMH：chr19：2249647、AMH：chr19：2249450、AMH：chr19：2251466、BMP4：chr14：54417171、BMP4：chr14：54417226、CBX2：chr17：77758182、CBX2：chr17：77757651、CBX2：chr17：77755866、MAP3K1：chr5：56177692。
in another preferred embodiment, the reagent or kit further comprises a detection reagent further comprising one or more genetic loci selected from the group consisting of:
GJA4：chr1：35260541、GJA4：chr1：35260779、MYH6：chr14：23876318、MYH6：chr14：23865496、MYH6：chr14：23857492、PTCH1：chr9：98240468、PTCH1：chr9：98241383、PTCH1：chr9：98224163。
in another preferred embodiment, the kit further comprises a detection reagent for one or more gene loci selected from the group consisting of:
SOX3：chrX：139587069、HSD17B3：chr9：99060720、HSD17B3：chr9：98997823、POR：chr7：75615537、POR：chr7：75608886、WNT4：chr1：22456135、WNT4：chr1：22446752、WT1：chr11：32456873、WT1：chr11：32417943、LHCGR：chr2：48982782、LHCGR：chr2：48915950、MAMLD1：chrX：149639324、MAMLD1：chrX：149639544、RXFP2：chr13：32366083、RXFP2：chr13：32355815、LHX9：chr1：197886988、LHX9：chr1：197890684、MSX1：chr4：4864775、MSX1：chr4：4862087、BMP7：chr20：55840949、BMP7：chr20：55840785、DGKK：chrX：50127741、DGKK：chrX：50213362、CST9：chr20：23584368、FGF10：chr5：44388594、FGF10：chr5：44305114。
in another preferred embodiment, the kit further comprises a detection reagent for one or more gene loci selected from the group consisting of:
CYP17A1：chr10：104592955、FGF8：chr10：103531266、GATA4：chr8：11614521、RSPO1：chr1：38095564、SRY：chrY：2655418、ARNT2：chr15：80762743、BMP2：chr20：6751089、BNC2：chr9：16437095、EPHB2：chr1：23208869、ESR2：chr14：64701857、FGFR2：chr10：123353315、FKBP4：chr12：2910316、GSTM1：chr1：110233137、TESC：chr12：117494679。
in another preferred embodiment, the reagent or the kit further comprises a detection reagent for detecting mutation sites of one or more sexual dysplasia-related genes selected from the group consisting of those shown in table 1.
In another preferred embodiment, the detection reagent is:
(a) Specific primers for gene detection;
(b) Specific probes for gene detection;
(c) A chip for gene detection;
(d) And a specific antibody for detecting the mutation of the amino acid corresponding to the mutated gene.
In a third aspect, the present invention provides a method for in vitro detecting the presence of a gene mutation in a sample, comprising the steps of:
(a) Amplifying the polynucleotide of the sample by using a specific primer to obtain an amplification product; and
(b) And detecting whether the amplified products have mutation of the genes SRD5A2, AR and NR5A related to the dysplasia.
In another preferred embodiment, the amplification product is tested for the presence of one or more of the following sexual dysplasia-associated genetic loci: SRD5A2: and (2) chr: 31805775, SRD5A2: and (2) chr: 31758693, SRD5A2: and (2) chr: 31754418, SRD5A2: and (2) chr: 31754497, NR5A1: and (9) chr: 127265467, NR5A1: and (9) chr: 127262390, NR5A1: and (9) chr: 127265589, NR5A1: and (9) chr: 127262797, NR5A1: and (9) chr: 127262973, NR5A1: and (9) chr: 127265498, NR5A1: and (9) chr: 127253389, NR5A1: and (chr 9): 127253398, NR5A1: and (chr 9): 1272459and AR: and (4) chrX:66863249, AR: and (4) chrX:66943556, AR: and (4) chrX:66765872.
in another preferred embodiment, the method further comprises (c) detecting whether the amplification product further contains one or more of the following sexual dysplasia-related gene mutation sites:
SRD5A2：chr2：31754395 C→T
SRD5A2：chr2：31805954 G→A
AR：chrX：66942740 C→T；
AR chrX：66765516 C→A。
in another preferred embodiment, the method further comprises (d) detecting whether the amplification product further contains one or more of the following mutation sites of the sexual dysplasia-related gene:
EGF chr4：110920947 G→A
ESR1 chr6：152129480 G→A
ESR1 chr6：152129484 C→A。
in another preferred embodiment, the method further comprises the step (e) of detecting whether one or more of the following sexual dysplasia-associated gene mutation sites are also present in the amplification product:
AMH chr19：2249647 G→A
BMP4 chr14：54417171 C→T
MAP3K1 chr5：56177692 G→C。
in another preferred embodiment, the method comprises the step (f) of detecting whether the following mutation sites of the sexual dysplasia-related gene are also present in the amplification product:
GJA4 chr1：35260541 C→T。
in another preferred embodiment, the method comprises the step (g) of detecting whether the amplification product further contains one or more of the following mutation sites of the sexual dysplasia-related gene:
SOX3 chrX：139587069 C→G
CST9 chr20：23584368 G→A。
in another preferred embodiment, the method further comprises the step (h) of detecting whether one or more sexual dysplasia-related gene mutations shown in table 1 are also present in the amplification product.
In another preferred embodiment, the assay is non-diagnostic and non-therapeutic.
In another preferred embodiment, the sample is from a human.
In a fourth aspect, the invention provides a method of detecting susceptibility to sexual dysplasia in an individual, comprising the steps of:
(i) Detecting the SRD5A2, AR and NR5A1 genes of the individual; and
(ii) The difference between the gene locus and the corresponding normal gene locus indicates that the susceptibility of the individual to sexual dysplasia is higher than that of the normal population.
In another preferred embodiment, the method further comprises detecting a gene associated with sexual dysplasia selected from the group consisting of: EGF, ESR1.
In another preferred embodiment, the method further comprises detecting a sexual dysplasia-associated gene selected from the group consisting of: AMH, BMP4, CBX2 and MAP3K1.
In another preferred embodiment, the method further comprises detecting a gene associated with sexual dysplasia selected from the group consisting of: GJA4, MYH6, PTCH1.
In another preferred embodiment, the method further comprises detecting a sexual dysplasia-associated gene selected from the group consisting of: SOX3, HSD17B3, POR, WNT4, WT1, LHCGR, MAMLD1, RXFP2, LHX9, MSX1, BMP7, DGKK, CST9, FGF10.
In another preferred embodiment, the method further comprises detecting a sexual dysplasia-associated gene selected from the group consisting of: CYP17A1, FGF8, GATA4, RSPO1, SRY, ARNT2, BMP2, BNC2, EPHB2, ESR2, FGFR2, FKBP4, GSTM1, TESC.
In another preferred embodiment, said detecting SRD5A2, AR and NR5A1 genes comprises detecting one or more nucleotide variations of table 1.
In another preferred embodiment, the detecting SRD5A2, AR and NR5A1 genes comprises detecting nucleotide variations of one or more of table 2 below:
TABLE 2
| Gene | Gene mutation | Mutation site |
| SRD5A2 | chr2：31805775 | C→T |
| SRD5A2 | chr2：31758693 | G→A |
| SRD5A2 | chr2：31754418 | GA→G |
| SRD5A2 | chr2：31754497 | T→A |
| NR5A1 | chr9：127265467 | CTTG→C |
| NR5A1 | chr9：127262390 | G→T |
| NR5A1 | chr9：127265589 | G→T |
| NR5A1 | chr9：127262797 | TC→T |
| NR5A1 | chr9：127262973 | CT→C |
| NR5A1 | chr9：127265498 | G→T |
| NR5A1 | chr9：127253389 | G→A |
| NR5A1 | chr9：127253398 | A→G |
| NR5A1 | chr9：127245134 | G→T |
| AR | chrX：66863249 | G→C |
| AR | chrX：66943556 | T→G |
| AR： | chrX：66765872 | T→C。 |
In another preferred embodiment, the difference is selected from one or more of the nucleotide variations of table 1 or table 2.
In another preferred embodiment, the individual is a human.
In a fifth aspect, the present invention provides an isolated gene polynucleotide sequence derived from a fragment of a gene selected from the group consisting of: SRD5A2 gene, AR gene and NR5A1 gene, and the nucleotide sequences respectively have one or more gene mutation sites selected from the group shown in Table 2.
In another preferred embodiment, the nucleotide sequence is further derived from a fragment of a gene selected from the group consisting of: EGF, ESR1 or a combination thereof, and the nucleotide sequences respectively have one or more gene mutation sites selected from the genes shown in Table 1.
In another preferred embodiment, the nucleotide sequence is further derived from a fragment of a gene selected from the group consisting of: AMH, BMP4, CBX2, MAP3K1, or a combination thereof, and the nucleotide sequences respectively have one or more gene mutation sites selected from those shown in Table 1.
In another preferred embodiment, the nucleotide sequence is further derived from a fragment of a gene selected from the group consisting of: GJA4, MYH6, PTCH1, or a combination thereof, and the nucleotide sequences respectively have one or more gene mutation sites selected from those shown in Table 1.
In another preferred embodiment, the nucleotide sequence is further derived from a fragment of a gene selected from the group consisting of: SOX3, HSD17B3, POR, WNT4, WT1, LHCGR, MAMLD1, RXFP2, LHX9, MSX1, BMP7, DGKK, CST9, FGF10, or a combination thereof, and the nucleotide sequences respectively have one or more gene mutation sites selected from those shown in Table 1.
In another preferred embodiment, the nucleotide sequence is further derived from a fragment of a gene selected from the group consisting of: CYP17A1, FGF8, GATA4, RSPO1, SRY, ARNT2, BMP2, BNC2, EPHB2, ESR2, FGFR2, FKBP4, GSTM1, TESC, or a combination thereof, and the nucleotide sequences respectively have one or more gene mutation sites selected from those shown in Table 1.
In another preferred embodiment, the polynucleotide sequence is 30-1000bp in length; preferably 50-500bp, more preferably 200-250bp.
The invention also provides the use of a polynucleotide sequence as described in the fifth aspect of the invention as a positive control (standard).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.
Drawings
FIG. 1 shows the results of targeted gene secondary sequencing of 70 46,XY DSD patients.
The red marking gene is a DSD candidate gene, and the blue marking gene is a potential pathogenic gene of DSD. Potential disease genes refer to genes found in animal studies to be associated with sexual development but not found in DSD patients.
Detailed Description
Through extensive and intensive research and a large amount of screening, a group of highly relevant target genes and relevant mutation sites thereof related to the specific dysplastic diseases are developed for the first time. The experimental result of the invention shows that based on the target gene of the sexual dysplasia disease and the related mutation site thereof, the corresponding diagnostic reagent and the kit can be developed, thereby being used for early diagnosis or auxiliary diagnosis of whether the object is susceptible to the sexual dysplasia disease. The present invention has been completed based on this finding.
Term(s) for
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. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Target gene combination
In the target gene combination provided by the invention, the core target genes comprise SRD5A2, AR and NR5A1. On this basis, one or more additional target genes may also be included.
In a preferred example, the inventors found that the mutation was verified to be 113/127=89% by preliminary secondary sequencing, data analysis and Sanger sequencing of 70 46,xy DSD patients. Mutations were detected in 40 of 80 genes, including 8 homozygous mutations in the AR gene, 2 homozygous mutations in the SOX3 gene, 1 compound heterozygous mutation in the ESR1 gene, 2 heterozygous mutations in the WT1 gene, one homozygous mutation in the SRY gene, 3 heterozygous mutations in the BMP4 gene, 1 homozygous mutation in the LHCGR gene, 1 homozygous mutation in the MAMLD1 gene, 10 heterozygous mutations in the NR5A1 gene, 3 homozygous mutations in the SRD5A2 gene, and 4 compound heterozygous mutations. The results of the analysis suggest that in humans (particularly the asian population), the most central causative genes in 46,xy DSD patients are: SRD5A2, AR and NR5A1.
SRD5A2 gene
The SRD5A2 gene is a 5 alpha-reductase type II gene and is mainly expressed on external genital organs and prostate to promote the conversion of testosterone into dihydrotestosterone.
In a preferred embodiment of the present invention, the primer pairs for detecting the mutation sites at Arg227Gln, gln6, ala228Val, F219fs, ala62Glu, arg246Gln, gly66Arg, thr142Ile and N193I positions of the corresponding amino acid sequences in the SRD5A2 gene are as follows:
c.680C → T, p.Arg227Gln heterozygous mutation
SRD5A2 chr2：31754395 C→T
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:1)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:2)
c.16G → A, p.Gln6 + heterozygous mutation
SRD5A2 chr2：31805954 G→A
F:AAGGCAGCTCCTGCAGGAAC(SEQ ID NO.:3)
R:TCTCTTCTGGGAGGGCAGCG(SEQ ID NO.:4)
c.683G → A, p.Ala228Val homozygous mutation
SRD5A2 chr2：31754392 G→A
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:5)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:6)
c.656delT, p.F219fs heterozygous mutation
SRD5A2 chr2：31754418 GA→G
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:7)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:8)
c.185G → T, p.Ala62Glu heterozygous mutation
SRD5A2 chr2：31805786G→T
F:CCGCAAGGGAAAAACGCTAC(SEQ ID NO.:9)
R:TTGTACGTCGCGAAGCCTC(SEQ ID NO.:10)
c.737C → T, p.Arg246Gln heterozygous mutation
SRD5A2 chr2：31751294 C→T
F:TTCATCAGCATTGTGGGAGC(SEQ ID NO.:11)
R:GACCATCGAAATAGTCAGGC(SEQ ID NO.:12)
c.196C → T, p.Gly66Arg heterozygous mutation
SRD5A2 chr2：31805775 C→T
F:CCGCAAGGGAAAAACGCTAC(SEQ ID NO.:13)
R:TTGTACGTCGCGAAGCCTC(SEQ ID NO.:14)
c.425G → A, p.Thr142Ile heterozygous mutation
SRD5A2 chr2：31758693 G→A
F:TGGGAAGTAGGTGAGAAGTG(SEQ ID NO.:15)
R:TAACCTTTCCTCCCTGTGTG(SEQ ID NO.:16)
T578A, p.Asn193Ile heterozygous mutation
SRD5A2 chr2：31754497 T→A
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:17)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:18)
AR gene
The coding product of AR, also called androgen receptor gene, belongs to nuclear receptor family, mainly expresses in penis and urethra, and combines with testosterone and dihydrotestosterone compound to affect the expression of key genes in downstream differentiation process.
In a preferred embodiment of the present invention, the primer pairs for detecting the mutation sites at Arg841Cys, S176R, pro392Arg, gly590Arg, phe879Cys and Leu295Pro of the corresponding amino acid sequence of the AR gene are as follows:
c.2521C → T, p.Arg841Cys homozygous mutation
AR chrX：66942740 C→T
F：TGTCTAATGCTCCTTCGTGG(SEQ ID NO.:19)
R：ACCCTCCATCGTTTGCTTAC(SEQ ID NO.:20)
c.C528A, p.S176R homozygous mutation
AR chrX：66765516 C→A
F：CGCTCAGGATGTCTTTAAGG(SEQ ID NO.:21)
R：AGGAACAGCAACCTTCACAG(SEQ ID NO.:22)
c.1175C → G, p.Pro392Arg homozygous mutation
AR chrX：66766163 C→G
F：AAGTCCGGAGCACTGGACGA(SEQ ID NO.:23)
R：TGAGGGTGACCCAGAACCG(SEQ ID NO.:24)
c.1768G → C, p.Gly590Arg heterozygous mutation
AR chrX：66863249 G→C
F：GCATGTGCAAGACCCTTTAC(SEQ ID NO.:25)
R：CTGCCATTCAGTGACATGTG(SEQ ID NO.:26)
c.2636T → G, p.Phe879Cys homozygous mutation
AR chrX：66943556 T→G
F：GTCAACCCTGTTTTTCTCCC(SEQ ID NO.:27)
R：GGGAAATAGGGTTTCCAATG(SEQ ID NO.:28)
c.884T → C, p.Leu295Pro homozygous mutation
AR chrX：66765872 T→C
F：CAGCAGTATCTTCAGTGCTC(SEQ ID NO.:29)
R：TTTCTGACAACGCCAAGGAG(SEQ ID NO.:30)
NR5A1 gene
NR5A1, also known as steroidogenic factor 1, is a member of the nuclear receptor superfamily, regulating transcription of a number of downstream genes known to be involved in gonadal development, adrenal development, steroidogenesis and reproduction.
In a preferred embodiment of the present invention, the primer pairs for detecting the mutation sites at positions Gly212Ser, p.n44del, cys283, E148fsX295, R89fsX105, G35V, C370Y, E367G, S430I and Thr29Lys of the amino acid sequence corresponding to the NR5A1 gene are as follows:
c.634C → T, p.Gly212Ser heterozygous mutation
NR5A1 chr9：127262605 C→T
F：TCCTGCAGGCAGCCCAAGAT(SEQ ID NO.:31)
R：ACTGGCTGGCTACCTCTAC(SEQ ID NO.:32)
c.135delTTG, p.N44del heterozygous mutations
NR5A1 chr9：127265467 CTTG→C
F：CCTACCCCCTCAGGCTGTG(SEQ ID NO.:33)
R：CCTCGCTGACTCTCAGCTC(SEQ ID NO.:34)
c.849C → A, p.Cys283 heterozygous mutation
NR5A1 chr9：127262390 G→T
F：GAGGAGAGACTCACCTCCA(SEQ ID NO.:35)
R：AACGTGCCTGAGCTCATCCT(SEQ ID NO.:36)
c.86G → T, p.Thr29Lys hybrid mutations
NR5A1 chr9：127265589 G→T
F：AGTGCTTGTTGTTCTGCACC(SEQ ID NO.:37)
R：ACGCCGCGGGCATGGACTAT(SEQ ID NO.:38)
(this site repeats with the last mutation site)
c.443delC, p.E148fsX295 heterozygous mutation
NR5A1 chr9：127262797 TC→T
F：TACTCAGACTTGATGGCACG(SEQ ID NO.:39)
R：CTTCAAGCTGGAGACAGGG(SEQ ID NO.:40)
c.265delT, p.R89fsX105 heterozygous mutation
NR5A1 chr9：127262973 CT→C
F：TGGGAGGCAGCACGTAGTC(SEQ ID NO.:41)
R：GCTTAGAGAGGGTGAGTCTG(SEQ ID NO.:42)
c.G104T, p.G35V heterozygous mutation
NR5A1 chr9：127265498 G→T
F：AGTGCTTGTTGTTCTGCACC(SEQ ID NO.:43)
R：ACGCCGCGGGCATGGACTAT(SEQ ID NO.:44)
c.G1109A, p.C370Y heterozygous mutation
NR5A1 chr9：127253389 G→A
F：GACCCACGTCCTCTGACTGT(SEQ ID NO.:45)
R：CTGGCTGTCTCCACCTCTCT(SEQ ID NO.:46)
c.A1100G, p.E367G heterozygous mutation
NR5A1 chr9：127253398 A→G
F：GACCCACGTCCTCTGACTGT(SEQ ID NO.:47)
R：CTGGCTGTCTCCACCTCTCT(SEQ ID NO.:48)
c.G1289T, p.S430I heterozygous mutation
NR5A1 chr9：127245134 G→T
F：ATTCCAGCAGCTGCTGCTGT(SEQ ID NO.:49)
R：AATGAACCATGCGGAGCCAG(SEQ ID NO.:50)
c.86G → T, p.Thr29Lys hybrid mutation
NR5A1 chr9：127265589 G→T
F：AGTGCTTGTTGTTCTGCACC(SEQ ID NO.:51)
R：ACGCCGCGGGCATGGACTAT(SEQ ID NO.:52)
Combination of target sites
In the target site combination provided by the invention, the core target site package is as follows: SRD5A2: and (2) chr: 31805775, SRD5A2: and (2) chr: 31758693, SRD5A2: and (2) chr: 31754418, SRD5A2: and (2) chr: 31754497, NR5A1: and (chr 9): 127265467, NR5A1: and (chr 9): 127262390, NR5A1: and (chr 9): 127265589, NR5A1: and (chr 9): 127262797, NR5A1: and (chr 9): 127262973, NR5A1: and (9) chr: 127265498, NR5A1: and (9) chr: 127253389, NR5A1: and (9) chr: 127253398, NR5A1: and (chr 9): 1272459and AR: and (4) chrX:66863249, AR: and (4) chrX:66943556, AR: and (4) chrX:66765872. on this basis, one or more additional target sites may also be included.
Detection method of target gene aiming at 46,XY DSD patient
The invention provides a detection method of a target gene aiming at a 46,XY DSD patient. Preferably, the method is based on second generation sequencing, comprising the steps of:
step (1): it is clear that the etiology that is currently known to cause 46,xy DSD;
step (2): deriving gene sequences and coding information through UCSC and NCBI databases;
and (3): AA chip primer Design is carried out on the genes by using MassARRAY Assay Design software;
and (4): AA chip experiment (can be divided into 5 steps);
(1) loading: primers and templates in 96-well plates were pipetted onto 48.48Access Array IFC chips.
(2) Loading: the chip is put into a Pre-PCR IFC Controller AX instrument, and the primers and the template are automatically mixed.
(3) Thermal cycling: PCR amplification was performed using FC1Cycler instrument.
(4) Harvesting: PCR product collection and harvesting were performed using a Post-PCR IFC Controller AX instrument.
(5) And (3) recovering: and (3) sucking out the PCR product on the sample loading position of the chip template by using a line gun.
And (5): performing sequencing analysis by using an Illumina sequencer;
and (6): sequencing results were analyzed using BWA, SAMTOOLS and GATK software.
And (7): all SNVs measured as well as INDELs were filtered through public databases (ExAC, 1000 genomes, dbSNP and ClinVar) and ANNOVAR (http:// anovar. Openbioinformatics. Org /) functionally annotated.
And (8): the method meets the requirements that (1) double-hole SNV, alldepth is more than 20, vaf is more than 0.3; (2) single pore SNV, alldepth >1000, vaf >0.4; (3) double-well SNV, poor for single-well assay, good for double-well assay (alldepth > 1000) standard SNV was confirmed by Sanger sequencing.
And (9): meets (1) double diplopore INDEL; (2) single-well INDEL, alldepth >25, vaf > =0.4 Standard INDEL was confirmed by Sanger sequencing.
Preferably, the 80 genes involved in the onset of 46,XY DSD, and 857 primer pairs are assigned to both panels.
Preferably, the 40 genes of the first Panel are:
SRD5A2,NR5A1,AR,EGF,ESR1,AMH,BMP4,CBX2,MAP3K1,GJA4,MYH6,PTCH1,SOX3,HSD17B3,POR,WNT4,WT1,LHCGR,MAMLD1,RXFP2,LHX9,MSX1,BMP7,DGKK,CST9,FGF10,SRY,CYP17A1,RSPO1,FGF8,GATA4,GSTM1,TESC,BMP2,FGFR2,ARNT2,BNC2,EPHB2,ESR2,FKBP4
preferably, the second Panel has 40 genes:
SOX9、NR0B1、BMP15、DHH、ARX、STAR、AMHR2、CYP11A1、HSD3B2、CYB5A、HOXA13、LHB、HSD11B1、FDX1、FDXR、MAP3K11、EMX2、LHX1、CTNNB1、FOXL2、FST、SHOX、DMRT1、SOX8、TSPYL1、SMAD5、KIAA1310、MID1、ATF3、SHH、WNT5A、HOXD13、BMPR1A、MAFB、PIP、CYR61、CTGF、GADD45A、HOXA4、HOXB6。
preferably, the criteria for screening SNV are (1) double-well SNV, alldepth >20, vaf >0.3; (2) single pore SNV, alldepth >1000, vaf >0.4; (3) double-well SNV, poor for single-well assay, good for double-well assay (alldepth > 1000).
Preferably, the criteria for screening INDEL are (1) diplopore INDEL; (2) single hole INDEL, alldepth >25, vaf > =0.4.
The invention is suitable for the definition of molecular etiology of 46,XY dysplasia patients, makes up the defects and deletion in the field, and has the principle that 80 genes which are known to possibly cause 46,XY DSD at present are found through case accumulation observation and literature retrieval, including candidate genes which are known at present and certain genes which are shown to be related to gonadal development in animal models but have not been reported to be mutated in human bodies. Passing Access Array TM The System establishes 2 panels for next generation sequencing of targeted genes.
According to 2015 American society of molecular pathology and Genetics (American College of Medical Genetics and Genomics; ACMG)), the nucleotide variations detected were divided into 5 types: (1) pathogenic, (2) potentially curative, (3) clinically insignificant Variation (VUS), (4) potentially benign (5) and (5) potentially benign. The types of mutations that have been reported in the literature in the past, which have similar manifestations, are considered pathogenic. For new mutation types, three software were used for prediction, including SIFT, polyPhen2, and mutationmaster, with more than two predictions being pathogenic and associated with clinical phenotypes, classified as likely pathogenic variants; variants predicted to be benign (non-loss of function) or loss of function but not associated with the phenotype are of unknown clinical significance (see table 2 for details). After summing the pathogenic and possibly pathogenic variations, the diagnostic rate of 46,XY DSD was found to be 42.86% (30/70) higher than in the same study.
More significantly, the method firstly shows that a plurality of pathogenic genes in a plurality of patients have mutation, the genetic pattern is not random in DSD patients (P = 0.024) through Fisher test with 144 normal human whole exon sequencing data, the illustrative differentiated molecular control network has great complexity and is related to the phenotypic heterogeneity of the patients, and the necessity of accurate medical diagnosis for the difficult and complicated diseases is further proved. The invention is suitable for the patient who is clinically planned to be diagnosed as 46,XY DSD, can carry out high-throughput detection and clinical popularization, is beneficial to comprehensively selecting the treatment scheme of the patient, fully makes up the defects and shortcomings in the field, and has practical value.
The advantages of the invention mainly include:
(1) The invention screens a group of genes related to sexual dysplasia, related sites and related mutation sites thereof for the first time, and the invention combines the genes related to sexual dysplasia, the related sites and the related mutation sites thereof (such as the combination of SRD5A2, AR and NR5A 1), so that the diagnosis accuracy of the sexual development diseases can be obviously improved to 90 percent (the calculation mode is that the number of true positives/(the number of true positives + the number of false negatives) is multiplied by 100 percent).
(2) The diagnostic rate of the method of the invention for 46,XY DSD was 42.86% (30/70);
the calculating method comprises the following steps: according to ACGM criteria, (patients with pathogenic nucleotide variation + with potentially pathogenic nucleotide variation)/total patients.
(3) The method of the invention firstly shows the condition that a plurality of pathogenic genes have mutation in a plurality of patients, the Fisher test is carried out on the sequencing data of all exons of 144 normal persons, the genetic pattern is not randomly generated in DSD patients (P = 0.024), the molecular control network of the descriptive differentiation has great complexity and is related to the phenotypic heterogeneity of the patients, and the necessity of precise medical diagnosis on the difficult and complicated diseases is further proved. The invention is suitable for the patient who is clinically planned to be diagnosed as 46,XY DSD, can carry out high-throughput detection and clinical popularization, is beneficial to comprehensively selecting the treatment scheme of the patient, fully makes up the defects and shortcomings in the field, and has practical value.
(4) DSD diseases have large differences in clinical phenotypes and are intricate. For 46,XY DSD, which is atypical for the clinical phenotype, molecular diagnostics can be performed using the present invention without clinical triage. The timely and effective molecular diagnosis is helpful for making a long-term treatment scheme aiming at the difficult and complicated diseases, providing sex selection information and carrying out genetic consultation on families of patients, thereby comprehensively improving the prognosis of the patients.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 screening and identification of Gene loci involved in XY DSD
The method comprises the following specific implementation steps:
step (1): 80 pathogenic genes which are known to cause 46,XY DSD at present are selected, and the chromosomal position, the coding, the gene size, the pseudogene condition and the like of the genes are determined according to the gene sequence in GRCh37/hg19 in UCSC Genome Browser.
TABLE 3.80 known potential causative genes for 46,XY DSD
Step (2): according to the Access Array TM And (3) designing a primer and adding a tag sequence according to the requirement of System amplification (the size of a target sequence is 240bp and overlapping exists).
Take SRD5A2 gene as an example.
For example, SRD5A2 chr2:31805954 the primers are as follows:
F:AAGGCAGCTCCTGCAGGAAC(SEQ ID NO.:3)
R:TCTCTTCTGGGAGGGCAGCG(SEQ ID NO.:4)；
detection of SRD5A2 chr2:31754392 the primers are as follows:
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:5)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:6)
detection of SRD5A2 chr2:31754418 the primers are as follows:
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:7)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:8)
detection of SRD5A2 chr2:31805786 the primers are as follows:
F:CCGCAAGGGAAAAACGCTAC(SEQ ID NO.:9)
R:TTGTACGTCGCGAAGCCTC(SEQ ID NO.:10)
detection of SRD5A2 chr2:31751294 the primers are as follows:
F:TTCATCAGCATTGTGGGAGC(SEQ ID NO.:11)
R:GACCATCGAAATAGTCAGGC(SEQ ID NO.:12)
detection of SRD5A2 chr2:31805775 the primers are as follows:
F:CCGCAAGGGAAAAACGCTAC(SEQ ID NO.:13)
R:TTGTACGTCGCGAAGCCTC(SEQ ID NO.:14)
detection of SRD5A2 chr2:31758693 the primers are as follows:
F:TGGGAAGTAGGTGAGAAGTG(SEQ ID NO.:15)
R:TAACCTTTCCTCCCTGTGTG(SEQ ID NO.:16)
detection of SRD5A2 chr2:31754497 the primers are as follows:
F:AAAGCTACGTGAATGCTGCC(SEQ ID NO.:17)
R:TGACCTTCCGATTCTTCTGC(SEQ ID NO.:18)
and (3): after primer synthesis, dilution is performed according to the Access Array TM System amplification requires the preparation of primer mixtures and distribution in 96-well plates.
And (4): preparation of amplification template, quantification to 50ng/ul (less than 50ng/ul in 50 ng/ul), template mixture preparation, and 96-well plate.
And (5): primers and templates in 96-well plates were pipetted onto 48.48Access Array IFC chips.
And (6): the chip is put into a Pre-PCR IFC Controller AX instrument, the primers and the template are automatically mixed, and the PCR amplification reaction is carried out by utilizing an FC1Cycler instrument.
And (7): PCR product collection and harvest using Post-PCR IFC Controller AX instrument (Note that room replacement is required for harvest to prevent contamination)
And (8): preparation of a barcode mixture 1:100 dilution of the product, PCR reaction.
And (9): the product was purified by magnetic beads, run gel to confirm barcode addition, and prepared for sequencing on machine.
Step (10): hiseq 2500 machine sequencing was performed and the off-machine data was analyzed using BWA, SAMTOOLS and GATK software.
Step (11): the SNV and INDEL measured were filtered through public databases (ExAC, 1000 genomes, dbSNP and ClinVar) and ANNOVAR ((R))http://annovar.openbioinformatics.org/) And (6) functional annotation.
Step (12): the double-hole SNV, the alldepth is more than 20, and vaf is more than 0.3; (2) single pore SNV, alldepth >1000, vaf >0.4; (3) double-well SNV, poor for single-well assay, good for double-well assay (alldepth > 1000) standard SNV was confirmed by Sanger sequencing.
Step (13): the double-hole INDEL meets the requirements of (1), and SNP is removed; (2) single-well INDELs, alldepth >25, vaf > =0.4, with SNP criteria removed and INDELs confirmed by Sanger sequencing.
The invention is suitable for the detection of the known gene mutation of 46,XY dysplastic diseases, makes up the defects and deletion in the field, and has the principle that the invention designs primers through Mass ARRAY Assay Design software in 80 genes which are known to possibly cause 46,XY DSD at present, performs second-generation sequencing analysis after performing targeted gene exon multiple PCR amplification on 48.48Access ARRAY IFC, and makes the gene diagnosis of high flux and larger coverage for the diseases possible.
Through the previous 70 patients' study, it was found that mutations were detected in 40 of the 80 genes (table 4), and the most common causative genes in 46,xy DSD patients were: SRD5A2, AR and NR5A1, and the like. Furthermore, common genes in Chinese people can be gathered in one diagnosis panel, and gene detection of a large number of samples can be performed more efficiently and rapidly. The invention is very suitable for Chinese 46, XY DSD patients, can make up the deficiency and deletion in the field, and has practical value.
TABLE 4
The mutation frequency of the normal population is shown in Table 5.
TABLE 5
Example 2 preparation of kit and Effect verification
This example provides a kit for detecting 46,XY DSD.
The kit can detect gene mutation of the mutation sites in the following table 3:
the kit comprises the following main reagents:
(1) Polymorphic site amplification primers
An upstream primer (F) and a downstream primer (R) (designed by Mass ARRAY Assay Design software);
(2) Polymorphic site sequencing primer
Sequencing the primer (S) (designed according to methods conventional in the art);
(3) PCR Main reagents: pfu Hi-Fi enzyme, 10 XPCR Buffer, dNTPMixtur, ddH 2 O；
(4) Pyrosequencing major reagents: 70% ethanol solution, magnetic beads, denaturation buffer, annealing buffer, binding buffer, washing buffer, substrate (ASP, luciferin), enzyme mixture solution (DNA polymerase, luciferase, adenosine triphosphate sulfurylase, adenosine triphosphate diphosphatase), A/T/C/G base.
The kit provided by the embodiment is used for detecting 300 patients, the specific method refers to the embodiment 1, 229 patients carrying the gene mutation of the invention are detected, and finally, 46 XY DSD patients are confirmed by a clinical routine method to be 218 patients, the detection rate is up to more than 95%, and the specific detection results are shown in Table 6.
TABLE 6 results of second-generation sequencing of 300 patient-targeted genes
Example 3 identification of Gene loci involved in diseases associated with sexual dysplasia
In this example, the second population sample was tested in the same manner as in example 1. The number of samples in this example was 110 patients with dysplasia-related disease and 200 normal persons.
As a result:
among 110 patients with sexual dysplasia-related diseases, 51 patients were detected to have gene mutations in three genes of SRD5A2, NR5A1 and AR (some patients had SRD5A2 biallelic gene mutations), that is, one of the three genes was a causative gene of sexual dysplasia-related diseases in 51 patients. In 200 normal persons, however, the mutations of the above three genes were not detected.
Specifically, with respect to the single gene, 21 of the 110 patients with the dysplasia-related disease (19.09%, 21/110) carried SRD5A2 mutation, the mutation rate of NR5A1 was 10.91% (12/110), and the mutation rate of AR was 16.36% (18/110).
In addition, mutations were also detected in EGF, ESR1, BMP4, and LHX9 genes in 21 cases (19.1%).
Example 3 identification of Gene loci involved in diseases associated with sexual dysplasia
In this example, the third population sample was tested in the same manner as in example 1. The number of samples in this example was 200 patients with a sexual dysplasia-related disease and 200 normal persons.
As a result:
the newly found SNVs shown in table 2, which were detected in 12 (about 70%) of the patients with the sexual dysplasia-related disease in the third population and were not detected in normal persons, suggest that the newly found SNVs shown in table 2 have a certain frequency of occurrence in the patients with the sexual dysplasia-related disease and can be targets for auxiliary detection or early diagnosis of the sexual dysplasia-related disease.
Discussion of the related Art
There is significant heterogeneity in the clinical phenotypes of 46,xy DSD patients, which is very difficult to treat accurately in a timely manner, while diagnosis is very important for their sex determination, treatment strategies, and long-term genetic counseling at home. Given that the majority of DSD patients are mendelian genetic disease, traditional Sanger sequencing has been successfully used in some patients with phenotypically implicated Complete Androgen Insensitivity Syndrome (CAIS) and Congenital Adrenal Hyperplasia (CAH). In any event, sanger sequencing has a low diagnostic rate, particularly in patients with hypogonadism, due to the complex molecular etiology of such patients and the lack of clinically relevant molecular markers. Here, we used a targeted next-generation sequencing method, and found that a total of 113 mutations were detected in 52 patients, which were mainly distributed in 40 genes. The diagnostic rate was up to 42.86% (30/70), higher than that of the previous 2 studies, probably due to better mean sequencing depth and targeted gene region coverage.
Most patients present with varying degrees of penile, hypospadias and cryptorchidism. Patients with the classic complete androgen insensitivity syndrome and 17 alpha hydroxylase/17,20 lyase deficiency were excluded. Meanwhile, patients who have obvious virilization after puberty and cause social sex transformation are also excluded. Similar to the results of previous studies, the 3 genes with the highest mutation frequency in our study were SRD5A2 (15.04%, 17/113), NR5A1 (8.84%, 10/113) and AR (7.96%, 9/113), respectively, suggesting that they are the most prominent causes of 46,XY DSD. Of all the mutations detected, 15 were reported from SRD5A2, NR5A1, AR, LHCGR, HSD17B3, BMP4, ESR1 and MSX1 genes, and 34 previously unreported mutations were predicted to be potentially pathogenic.
In addition, 37 mutations occurred in 19 genes potentially causing DSD, which were found to be related to 46,xy DSD as first reported, mainly including EGF, LHX9, CST9, and the like. Patients DSD0037 and DSD0094 carried the NR5A1 gene mutations p.c283 and p.e367g, respectively, while they both detected a nonsense mutation p.r87 in the CST9 gene. Previous studies have shown that CST9 plays an important role in early testicular development, although no mutations in this gene were found in hypogonadal patients. LHX9 is used as a transcription factor, directly regulates the activity of NR5A1 gene with the help of WT1 and is very important for the formation of mouse gonad. However, ottolenghi et al did not find mutations in the LHX9 gene screening of 30 46,XY gonadal dysplastic patients, possibly due to insufficient sample size. In our study, patients DSD0044 and DSD0101 were both found to carry LHX9 gene heterozygous mutations, p.g201s and p.s12 ×, respectively. Patient DSD0044 was mainly characterized by 46,xy DSD with testicular degeneration, while patient DSD0101 found a cleft in the penis and hypo-urethral at birth. In addition, DSD0044 also carried 2 heterozygous mutations for the SRD5A2 gene (p.r227q and p.a62e), whereas DSD0101 only carried 1 heterozygous mutation for SRD5A2 (p.n 193i). Thus, we hypothesized that the LHX9 gene in combination with the SRD5A2 gene deficiency may lead to an atypical 5. Alpha. Reductase type 2 deficiency.
In our patients, the frequency of mutations in the NR5A1 gene is second (10/70). The NR5A1 gene is a member of the nuclear receptor superfamily, regulating the expression of downstream genes involved in adrenal and gonadal development, steroid hormone synthesis and reproduction. Different heterozygous mutations in the NR5A1 gene can lead to different clinical manifestations ranging from mild to severe hypogonadism, or to male infertility due to insufficient virilization or lack of adrenal insufficiency. In addition, the NR5A1 mutation was associated with primary premature ovarian failure as well as testicular or oodidymal DSD in 46,XX patients. We found 9 new NR5A1 gene mutations, mainly distributed in the DNA binding region and the ligand binding region. Wang et al found that a phospholipid molecule binds to the LBD region, and that mutations occurring in this region may block the binding of phospholipids, resulting in a decrease in transcriptional activity. Phosphorylation of serine at hinge region 203 can regulate recruitment of co-activators, while ubiquitination of lysines at positions 119 and 194 can result in inhibition of transcriptional activity. A previously reported mutation in p.gly212ser, located near serine 203, was found in the study and may affect its phosphorylation. The 2 frameshift mutations p.148fsxg295, p.r 89fsxg105 and the nonsense mutation p.cys283 may produce truncated proteins, disrupt the ligand binding region or affect NR5A1 phosphorylation. In functional studies, we found that 10 mutants had reduced transcriptional activity on downstream target genes CYP17A1 and CYP19A1, while nuclear localization was unaffected. The mutations p.T29K and p.N44del exhibit an abnormal phenomenon of nuclear aggregation compared to the wild type, and their transcriptional activity is also relatively low.
Notably, our study found 47.14% (33/70) of patients to find multiple gene mutations. Among 10 patients carrying the NR5A1 gene mutation, 8 were mutated at up to 80% (8/10) of the other genes. DSD0011 is a phenotype of almost complete women, manifested as complete gonadal dysplasia, juvenile vulva and primary amenorrhea; no testicular and ovarian structures were found by ultrasound examination. The gene screening result shows that the patient carries a new mutation point (p.R76L) of the SRY gene and a reported point (p.G212S) of the NR5A1 gene. The mutation of SRY gene is associated with 46,xy reversal or gonadal dysplasia, while the mutation p.g212s has been previously reported to be associated with a vietnam patient, which has a lighter phenotype and is mainly manifested as spermatorrhea disorder and infertility. The more severe phenotype of DSD0011 suggests that the SRY gene incorporating NR5A1 gene mutation may lead to the development of complete gonadal dysplasia, leading to a more complex clinical presentation. In addition, DSD0011 also carries FGF10 gene mutation (p.m 204v). Although previous studies have shown that FGF10 plays a role in the development of early genital nodules, it is unclear whether the mutant p.m204v is involved in its pathogenic process. DSD0049 patients show virilization (penile, perineal sub-urethral fissure, cryptorchidism and scrotal fissure), hormone findings indicating elevated FSH, LH and relatively low testosterone levels. Sequencing analysis found it to carry a mutation p.l295p on exon 1 of the AR gene, which was previously reported to be associated with Androgen Insensitive Syndrome (AIS). In addition to this, DSD0049 carries the NR5A1 gene mutation p.t29k, which may lead to its atypical androgen-insensitive syndrome phenotype, in particular low testosterone levels. Patient DSD0096 carried both missense mutation of NR5A1 gene p.s430i and mutation of WT1 gene p.t7p, manifested as gonadal dysplasia and relatively normal testosterone levels. The research shows that the WT1 gene plays a role in the development of bidirectional differentiation potential in the early gonad stage and directly regulates the activity of the NR5A1 gene. Thus, simultaneous mutation of the NR5A1 gene and the WT1 gene may result in overlapping clinical phenotypes. Patient DSD0092 shows that the patient DSD0092 is anogenital and gonadal dysgenesis, and the research result shows that the patient DSD0092 carries NR5A1 gene mutation p.C370Y and SOX3 gene mutation p.V53L. The SOX3 gene is expressed in mouse embryonic gonadal tissue, is not essential for gonadal formation, but is involved in testicular differentiation and spermatogenesis. The above results may partially explain the heterogeneity of clinical phenotypes of patients carrying the NR5A1 gene, ranging from severe hypogonadism, male infertility to male infertility alone.
To date, more and more NR5A1 gene heterozygous mutations, frameshift mutations, and missense mutations have been reported, and most of them are not inherited from parents. We found by mutation analysis of the parents of 3 patients that the mutations p.g35v and p.c370y were not inherited, whereas the mutation p.n44del was inherited from the father of the patient, whose father phenotype was normal. Lin et al found that the mother carried the same NR5A1 gene mutation as the patient, but showed normal sexual differentiation and normal fertility, probably due to a limited dominant genetic pattern. Based on the above results, we speculate that the NR5A1 gene mutation combined with other pathogenic mutations may cause a complex clinical phenotype in some, if not all, 46,xy DSD patients (such as DSD 0032). Patients carrying the same mutation exhibit different clinical phenotypes, which may be caused by an imbalance in allele expression, incomplete penetrance or different causative mutations that are not detected by targeted gene sequencing.
The SRD5A2 gene mutations p.r227q, p.q6 x, p.r246q and p.a228v are recurrent mutations. Consistent with previous results using Sanger sequencing, only a single heterozygous mutation in the SRD5A2 gene was found in 6 patients in our study to fail an autosomal recessive mode of inheritance for disease. Of these 6 patients, 5 were also accompanied by other gene mutations. Patients DSD0056 and DSD0073 both harbor mutations in the BMP4 gene, while they both harbor a single heterozygous mutation in the SRD5A2 gene. DSD0073 presents as a severe male insufficiency, almost as a female external genitalia, with the scrotum split. In addition, DSD0073 also underwent WNT4 gene mutation (p.p 96l). WNT4 gene antagonizes testicular development and plays an important role in the development of female external genitalia and in hindering testicular formation, while Chen et al found that BMP4 gene is involved in hypospadias. In combination with the above results, we found that in some patients there was a "secondary hit" phenomenon, indicating that DSD may not be as indicated in previous studies as a monogenic disease. It may present a genetic pattern of a double or oligogenic disease in at least some patients.
In addition, some rare DSD gene mutations were also found. The DSD0042 of a patient carries homozygous mutation p.P542S of the MAMLD1 gene and is mainly expressed as the small penis, perineum type hypospadias and cryptorchidism. Mutations in the MAMLD1 gene can cause X-linked hypospadias and 46,xy DSD. The CBX2 gene mutations p.R137C and p.G185E occurred in patients DSD0010 and DSD0017, respectively, and were first reported by Biason-Lauber et al to occur in 46,XY patients with female phenotype. The DSD0013 of the patient shows that the patient has serious cryptorchidism and penis without hypospadias and has poor reaction in an hCG test, and the patient is found to carry homozygous mutation p.L10P of the LHCGR gene by sequencing. The mutant p.L10P is positioned in a luteinizing hormone receptor signal peptide region and can influence Leydig cell development and receptor synthesis 47 . In addition, mutations in the BMP4 and ESR1 genes, which have been reported to be associated with hypospadias, have also been discovered.
Overall, the method of targeted next-generation sequencing can diagnose 42.86% of patients. This study revealed genetic characteristics of 46,xy DSD patients and expanded the candidate gene expression profile for the disease. The presence of multiple gene mutations in patients indicates that DSD may not be a simple monogenic genetic disease, and a potential bi-or oligogenic genetic pattern exists in some patients with hypogonadism. In the future, research on multi-gene mutation will emerge continuously, and important guidance will be provided for sex determination or genetic counseling of patients.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Shanghai university of traffic medical college affiliated ninth people hospital
<120> Targeted detection for XY dysplastic diseases
<130> P2017-0366
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Claims (7)
1. Use of a detection reagent for a sexual dysplasia-related gene selected from the group consisting of: SRD5A2, AR and NR5A1, wherein the detected object is a Chinese, and the kit comprises a detection reagent of the gene locus related to the sexual dysplasia, which is selected from the group consisting of:
SRD5A2：chr2：31805775、SRD5A2：chr2：31758693、SRD5A2：chr2：31754418、
SRD5A2: and (2) chr: 31754497, NR5A1: and (chr 9): 127265467, NR5A1: and (9) chr: 127262390, NR5A1: and (9) chr: 127265589, NR5A1: and (9) chr: 127262797, NR5A1: and (9) chr: 127262973, NR5A1: and (chr 9): 127265498, NR5A1: and (9) chr: 127253389, NR5A1: and (9) chr: 127253398, NR5A1: and (9) chr: 1272459and AR: and (4) chrX:66863249, AR: and (4) chrX:66943556, AR: and (4) chrX:66765872, the sexual dysplasia is 46,XY sexual dysplasia.
2. The use of claim 1, wherein the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: EGF, ESR1.
3. The use of claim 1, wherein the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: AMH, BMP4, CBX2 and MAP3K1.
4. The use of claim 1, wherein the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: GJA4, MYH6, PTCH1.
5. The use of claim 1, wherein the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: SOX3, HSD17B3, POR, WNT4, WT1, LHCGR, MAMLD1, RXFP2, LHX9, MSX1, BMP7, DGKK, CST9, FGF10.
6. The use of claim 1, wherein the kit further comprises a detection reagent for detecting one or more genes associated with sexual dysplasia selected from the group consisting of: CYP17A1, FGF8, GATA4, RSPO1, SRY, ARNT2, BMP2, BNC2, EPHB2, ESR2, FGFR2, FKBP4, GSTM1, TESC.
7. The use of claim 1, wherein the kit further comprises a detection reagent for detecting one or more sites of genes associated with sexual dysplasia selected from the group consisting of: EGF: and (9) chr: 110920947, EGF: and (9) chr: 110884370, EGF: and (9) chr: 110932389, EGF: and (9) chr: 110902074, EGF: and (9) chr: 110834507, ESR1: and (2) chr6:152129480, ESR1: and (2) chr6:152129484.
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