CN112695082B - Gene mutation combination as marker of MRKH syndrome and application thereof - Google Patents

Abstract

The invention discloses a gene mutation combination as a marker of MRKH syndrome and application thereof, and relates to the fields of biotechnology and medical diagnosis. The research of the invention utilizes whole exome sequencing to find the following mutation sites on the PAX8 gene of patients with MRKH syndrome rare disease worldwide for the first time: the experiment proves that the mutant PAX8 gene carrying the mutant site can be used as a diagnostic marker of MRKH syndrome and is used for diagnosing whether a patient has the MRKH syndrome and pre-pregnancy early warning, and a brand-new idea is provided for the research of pathogenesis of the MRKH syndrome.

Description

Gene mutation combination as marker of MRKH syndrome and application thereof

Technical Field

The invention belongs to the field of biotechnology and medical diagnosis, and particularly relates to a group of pathogenic gene mutation sites related to MRKH syndrome, and application of pathogenic genes carrying the gene mutation sites in preparation of a reagent or a kit for diagnosing the MRKH syndrome.

Background

MRKH (Mayer-Rokitansky-Kuster-Hauser) syndrome, also known as congenital vaginal-free syndrome, is a structural defect of female congenital reproductive system, and is characterized in that the clinical features of patients are that the uterus and 2/3 segments of the vagina are completely deficient (occasionally are primordial uterus), the functions of ovary reproduction and endocrine are normal, the development of secondary sexual characteristics is normal, the karyotype is 46,XX, and the incidence rate is about 1/4500-5000 newborn baby girls. MRKH syndrome can be classified into two types according to whether it incorporates malformations of other systems outside the genital tract, MRKH syndrome type I being a simple genital tract malformation, 44% in patients with MRKH syndrome, MRKH syndrome type II incorporating abnormal development of the renal and/or skeletal system, 56% in patients with MRKH syndrome (ChanYY, jayaprakasn K, zamora J, et al. The prediction of joint materials and organisms in unselected and high-rise positions: a systematic review [ J ]. Human Reproduction Update,2011,17 (6): 761-771.). The most common deformities observed in 40% to 60% of patients with MRKH syndrome type II are unilateral renal loss and/or renal ectopy.

Both type I and type II MRKH syndromes rarely occur simultaneously in multiple family members, suggesting that the cause of MRKH syndrome is unlikely to be a single, highly overt gene. The recurrence of the progeny of patients with MRKH syndrome is extremely rare in the biological progeny of patients with MRKH syndrome who are pregnant by assisted reproductive techniques, further suggesting that the cause of the progeny is unlikely to be a single, highly exogenic gene. On the contrary, the prevalence rate of the disease is relatively high in the general population (1/4500-5000 newborn baby girls), which suggests that the disease conforms to a polygene/multifactorial genetic model. Thus, the etiology of the disease may be a combined effect epigenetic of several genetic variations, and environmental factors may be modifiers of its different phenotypes. The genetic studies strongly motivate the understanding of MRKH syndrome pathogenesis in humans, and the phenotype of the mouse models of loss-of-function variants of many genes, suggest that these genes play a broad role in the development stage of the muller's canal, such as PAX8, HOX, WNT family genes, etc., and thus, become candidate pathogenic genes for MRKH syndrome (Pizzo a, langan A S, sturlese E, et al.

Currently, some candidate disease-causing genes have been preliminarily detected in MRKH syndrome by means of Sanger sequencing, in which PAX8, the most important disease-related gene, encodes a transcription factor critical to the normal development of thyroid and female reproductive systems, PAX8 ^-/- The mice reproduced the human MRKH syndrome phenotype: normal ovarian/fallopian tube and uterus are missing (Mittag, J., winterhager, E., bauer, K., and Grummer, R. (2007). Congenic hypothyroid animal pesticide thod hormone replacement therapy 148, 719-725.). However, as a rare disease, because the number of the case samples is small, the case samples are difficult to collect and the like, and patients cannot breed, families with good research value are rare, and the research on possible pathogenic gene mutation of the MRKH syndrome is still lacked, based on the fact that the invention firstly discovers the following mutation sites on the PAX8 gene by carrying out whole exon sequencing analysis on 442 MRKH tested patients and 941 female controls: c.236C>G、c.156_157dupCG、c.25+1G>T、c.195delC、c.322C>T and c.542C>T, the mutation site is proved to cause the loss of the function of the protein corresponding to the PAX8, thereby causing the MRKH syndrome.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to make up the blank of the research on related pathogenic gene mutation sites in the field of MRKH syndrome research, provides a group of pathogenic gene mutation sites related to MRKH syndrome, and the application of pathogenic genes carrying the mutation sites in the preparation of reagents or kits for diagnosing MRKH syndrome.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to the invention, a group of mutation sites are found on the PAX8 gene (Genebank: NM-003466.3) for the first time by sequencing the whole exon of a large sample of patients with MRKH syndrome, and the mutation sites can cause the function loss of the protein corresponding to the PAX8 and further cause the MRKH syndrome through verification, so that the group of PAX8 genes carrying the mutation sites can be used as markers for diagnosing the MRKH syndrome.

Specifically, the invention provides a group of diagnostic markers for MRKH syndrome, comprising PAX8 genes carrying the mutation sites c.236C > G (p.Ser79Cys), c.156-157 dupCG (p.Val53AlafsTer24), c.25+1G >T (Splice donor), c.195delC (p.Tyr66ThrfsTer10), c.322C > T (p.Arg108Ter), c.542C > T (p.Ser181Phe), respectively.

In a first aspect of the invention, a panel of diagnostic markers for MRKH syndrome is provided.

Further, the diagnostic marker comprises a mutant PAX8 gene and a mutant PAX8 protein.

Further, the mutant PAX8 gene comprises: PAX8 genes with gene mutation sites of c.236C > G, c.156-157 dupCG, c.25+1G >.

Further, the mutant PAX8 protein comprises: PAX8 proteins with protein mutation sites p.Ser79Cys, p.Val53AlafsTer24, splice donor, p.Tyr66ThrfsTer10, p.Arg108Ter, p.Ser181Phe, respectively.

In a second aspect, the invention provides a use of the mutant PAX8 gene of the first aspect in preparing a reagent or a kit for diagnosing MRKH syndrome.

In a third aspect, the invention provides the use of the mutant PAX8 protein of the first aspect in the preparation of an agent or a kit for diagnosing MRKH syndrome.

A fourth aspect of the present invention provides an agent for diagnosing MRKH syndrome.

Further, the reagent comprises specific amplification primers aiming at 6 mutation sites on the PAX8 gene;

preferably, the 6 mutation sites are c.236C > G, c.156_157dupCG, c.25+1G > -T, c.195delC, c.322C > T, c.542C > T located on the PAX8 gene.

Further, the reagents include reagents conventional in PCR amplification reactions, and/or reagents used in DNA extraction processes, and/or reagents used in DNA sequencing processes.

Further, other conventional reagents in the PCR amplification reaction include (but are not limited to): dNTP, PCR buffer, magnesium ions, tap polymerase and the like.

Further, the above 6 gene mutation sites can be detected by various methods including (but not limited to): 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 reagent of the invention comprises a reagent used in the method for detecting the gene mutation site.

Further, one skilled in the art can design primers for specific amplification or probes for specific detection according to the sequences upstream and downstream of the gene mutation site.

Further, the design methods of primers for specific amplification and probes for specific detection are routine in the art.

Further, the reagent comprises a specific antibody for detecting the mutant PAX8 protein;

preferably, the mutation sites of the mutant PAX8 protein include: p.ser79cys, p.val53alafster24, helice donor, p.tyr66thrfster10, p.arg108ter, p.ser181phe.

Further, the present invention provides a specific antibody which specifically binds to a mutant PAX8 protein encoded by a mutant PAX8 gene and does not act on a protein encoded by a wild-type PAX8 gene.

A fifth aspect of the present invention provides a kit for diagnosing MRKH syndrome.

Further, the kit comprises the following components:

(1) An effective amount of a reagent for detecting 6 mutation sites on the PAX8 gene and/or 6 mutation sites on the mutant PAX8 protein;

(2) One or more selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent;

preferably, the 6 mutation sites on the mutant PAX8 gene are c.236C > G, c.156-157 dupCG, c.25+1G >T, c.195delC, c.322C > T and c.542C > T;

preferably, the 6 mutation sites on the mutant PAX8 protein are p.Ser79Cys, p.Val53AlafsTer24, splice donor, p.Tyr66ThrfsTer10, p.Arg108Ter, p.Ser181Phe.

Further, the sample source detected by the diagnostic kit is blood.

The kit of the invention is accompanied with instructions for using the kit, and the instructions describe how to use the kit for detection and how to judge whether the subject has MRKH syndrome and/or the risk of the subject by using the detection result of the kit.

Using the kit of the present invention, the above 6 gene mutation sites can be detected by various methods selected from the group consisting of (but not limited to): 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.

Further, the diagnostic kit is used for diagnosing whether the individual of the subject has the MRKH syndrome by detecting whether the 6 mutation sites on the mutant PAX8 gene and/or the 6 mutation sites on the mutant PAX8 protein exist in the sample of the subject.

Further, the reagent may be any reagent capable of detecting 6 gene mutation sites. Including (but not limited to): reagents used in DNA sequencing, restriction Fragment Length Polymorphism (RFLP), single Strand Conformation Polymorphism (SSCP), allele-specific oligonucleotide hybridization (ASO).

In addition, the present invention provides a method for detecting the presence or absence of a gene mutation in the PAX8 gene, the method comprising the steps of:

(1) Extracting the genome DNA of a sample of a test subject;

(2) Amplifying the gene sequence of the PAX8 gene;

(3) Carrying out DNA sequencing;

(4) Comparing the DNA sequencing result of the sample of the tested subject with the DNA sequence of a normal human genome, and judging that the PAX8 gene has mutation if the C.236C > G, C.156-157 dupCG, c.25+1G >T, c.195delC, c.322C > T and c.542C > T sites exist on the PAX8 gene.

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. Further, some terms are explained as follows:

the term "marker" as used in the context of the present application refers to a molecular indicator with specific biological properties, biochemical characteristics, which can be used to determine the presence or absence of a particular disease or condition and/or the severity of a particular disease or condition.

The term "diagnosis" as used in the context of the present application includes the risk prediction of the MRKH syndrome disease, as well as the diagnosis of the presence or absence of the onset of the MRKH syndrome disease.

The term "primer" as used in the context of the present application refers to 7 to 50 nucleic acid sequences capable of forming a base pair complementary to a template strand (Basepair) and serving as a starting point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.

The term "probe", as used in the context of the present application, refers to a nucleic acid fragment such as RNA or DNA as short as a few to as long as a few hundred bases that can establish specific binding to mRNA and can determine the presence of a particular mRNA by the Labeling effect. The probe can be prepared in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like.

The term "specifically amplify" as used in the context of the present application means that the primers are capable of amplifying the gene of interest by a PCR reaction without amplifying other genes. In the present embodiment, specifically amplifying the PAX8 gene means that the primers only amplify the PAX8 gene and not other genes in the PCR reaction, and the design of such primers is well known to those skilled in the art.

The term "LGD variant" as used in the context of the present application, refers to gene interference variants, defined in the context of the present application as mutational sites predicted to result in loss of function (LoF) of the corresponding gene, including nonsense, frameshift and typical splice site mutational sites.

The term "D-mis variation" as used in the context of the present application refers to a missense variation, in the context of the present application, which indicates a mutation in which a substitution of a base pair changes a codon of mRNA to a codon encoding another amino acid, which mutation may cause structural and functional abnormalities of a certain protein or enzyme in the body, thereby causing disease.

The term "CH" used in the context of the present application refers to congenital hypothyroidism, which is the most common disorder of endocrine metabolism in newborn infants, and has various clinical manifestations, including hypomnesis, hyporesponsiveness, and dry skin, which may lead to severe developmental delay and growth retardation of nervous system if it cannot be diagnosed and treated in time.

The inventors of the present application found a group of mutation sites on the PAX8 gene (Genebank: NM-003466.3) for the first time by whole exon sequencing of large samples of patients with MRKH syndrome rare disease including 442 patients with MRKH syndrome, 941 normal female controls, and 150 patients with MRKH syndrome of mixed ethnic groups from North America, south America, and Europe, respectively: c.236C > G, c.156-157 dupCG, c.25+1G > -T, c.195delC, c.322C > T and c.542C > T, and the PAX8 gene carrying the mutation site can be used as a group of brand-new diagnostic markers for diagnosing MRKH syndrome, is used for diagnosing whether a patient has MRKH syndrome and pre-pregnancy early warning, indicates that progeny of a mutant PAX8 gene carrier have the risk of MRKH syndrome, and provides a brand-new thought for researching pathogenesis of MRKH syndrome.

The invention has the following advantages and beneficial effects:

(1) The invention utilizes whole exome sequencing to discover the following mutation sites on the PAX8 gene of the patient with MRKH syndrome rare disease for the first time in the world: c.236C > G, c.156-157 dupCG, c.25+1G >.

(2) The group of PAX8 genes which are found and verified to carry mutation sites of c.236C > G, c.156-157 dupCG, c.25+1G >T, c.195delC, c.322C > T and c.542C > T can be used as a group of brand-new diagnostic markers of MRKH syndrome and used for diagnosing whether a patient has the MRKH syndrome.

(3) The mutation sites c.236C > G, c.156-157 dupCG, c.25+1G >T, c.195delC, c.322C > T and c.542C > T on a group of PAX8 genes discovered and verified by the invention provide a brand-new idea for further researching the pathogenesis of MRKH syndrome.

(4) The invention also discovers that the mutation site on the PAX8 gene is inherited from father and has strong pathogenicity, so that the birth reduction of children suffering from the MRKH syndrome can be avoided as early as possible and the birth reduction rate of the birth defect infants is reduced by screening whether men carry the mutation site before pregnancy or marriage, so that the incidence rate of the MRKH syndrome is reduced.

Drawings

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

FIG. 1 is a drawing of the extension process of the Muller/Walf tube;

fig. 2 is a genealogical analysis plot and Sanger sequencing results plot of 3 PAX8 variant families in the study cohort and 1 PAX8 variant family in the replicate cohort, wherein, panel a: MRK49, panel B: MRK467, panel C: MRK330, graph D: BH9080;

FIG. 3 is a graph showing the results of luciferase assay for a mutation site on the disease-causing gene PAX8, in which WT: wild type, positive: a known deleterious variant R31C positive control, n =5, # p <0.05;

FIG. 4 is a mutation profile of LGD mutants and deleterious missense mutants in PAX 8.

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.

EXAMPLES identification and verification of mutation sites of pathogenic genes

1. Research population

Study cohort: 442 patients with MRKH syndrome from Beijing coordination hospital and Shenzhen Luo lake hospital were recruited together, among which 196 patients from Beijing coordination hospital, 246 patients from Shenzhen Luo lake hospital, 330 patients with type I MRKH syndrome (74.7%), and 112 patients with type II MRKH syndrome (25.3%). Patients with MRKH syndrome were diagnosed by gynecological ultrasound, pelvic MRI, karyotyping and medical records collected. Each subject signed an informed consent, which was approved by the ethical committee of the Beijing collaborating Hospital and Shenzhen Luo lake Hospital.

Control group: in the present invention, 941 healthy women were recruited as controls, and all subjects signed informed consent.

And (3) verifying the queue: the present invention recruited a total of 150 patients with MRKH syndrome from a mixed ethnic group in north america, south america and europe.

2. Research method

2.1 exome sequencing of all subjects

Peripheral blood DNA was extracted from each subject separately and exome sequencing was performed on all subjects. Preparing an Illumina paired-end library from a DNA sample, capturing exons, and then sequencing on an Illumina HiSeq 4000 platform; variants were called and filtered using an in-house developed data analysis platform (PUMP).

2.2 screening and testing of mutation load in Gene

Mutation load of 19 candidate genes was compared among 442 MRKHS cases and 941 female controls and to identify rare mutations, we screened mutation sites with smaller allele frequency (MAF) <0.001 using internal (case and control population) and external (ExAC and gnomAD) sequencing data. Rare non-synonymous variants (deletions, unintentional, frame shifts, in-frame insertions deletions, classical splice site mutations) that are likely to be pathogenic are then screened. After preparing the data, two models were used for mutation burden screening: a model of possible gene interference (LGD) and a model of LGD + deleterious missense mutations (D-mis). The mutation load of rare mutations was analyzed using the SNP-set (sequence) kernel association optimization test (SKAT-O) test to determine the association of the mutation load in 19 genes.

2.3 screening of potential interfering Gene loci in validation cohorts

The present invention recruits 150 patients with MRKH syndrome from a mixed ethnicity in north america, south america and europe as a validation cohort. Similar screening of 19 candidate genes was performed in the validation cohort, with possible interfering gene sites, D-mis and Copy Number Variation (CNV) in the validation cohort being screened. Finally, 11 mutation sites of c.236C > G, c.156-157 dupCG, c.25+1G >T, c.195delC, c.322C > T, c.542C > T, c.136G > A, c.266T > C, c.619C > T, c.727C > G and c.68G > T of the PAX8 gene are obtained by screening, 2 mutation sites of c.275-287 pTGGAGGAGGGCGG and c.621+1G A of the TBX6 gene, 3 mutation sites of c.367G > T, C-132-1G >A/-, c.766C > T of the BMP4 gene, c.1036-2A >G of the BMP7 gene, c.621+1G A of the TBX6 gene and c.979C > T of the B gene are obtained by screening.

2.4 verification of the selected mutation sites by Sanger sequencing

The screened LGD and D-mis mutation is verified to be true mutation by Sanger sequencing, the sequencing result is shown in figure 2, and mutation sites are in a box; the sequences of the primers used for Sanger sequencing are shown in Table 1.

TABLE 1 primer sequences used for Sanger sequencing

2.5 Dual fluorescein report experiments verify the influence of the screened mutant sites on the transcriptional activity of PAX

In order to further verify the screened mutation sites, the invention further verifies the influence of the mutation sites on PAX transcription activity by using a double fluorescein reporter experiment (Luciferase assay). The specific verification method comprises the following steps:

(1) HeLa cells were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum, 2mM L-glutamine and 50U/mL penicillin and 50. Mu.g/mL streptomycin. Digesting the cells with good growth condition, inoculating into 24-well cell culture plate, inoculating 3 multiple wells of each plasmid, adding 2.5 × 10 cells into each well after counting ⁵ After each digested cell, supplemented to 500. Mu.L per well volume by adding DMEM medium, 24-well cell culture plates were placed in a cell incubator at 37 ℃ for 24 hours and transfected with empty vector (pEGFP), PAX8 wild-type or mutant plasmid and pRL-SV40 Renilla control plasmid. The pGL4-pTG1084 plasmid contains the cloned human thyroglobulin promoter (-5' flanking region from-1043 bp to +41 bp) inserted into the pGL4.14 plasmid using KpnI and HindIII restriction sites. Transfection was performed using Lipofectamine 3000.

(2) Typical binding sequences for PAX8 were amplified using the following primers:

forward primer 5'-GCGGGGTACCACCTGGCTGGATTGGTCTTC-3' (SEQ ID NO. 11)

Reverse primer 5'-GGCGAAGCTTTTTCCTGGCCCTTCCTGGG-3' (SEQ ID NO. 12)

(3) The pRL-SV40 Renilla control plasmid contains the SV40 promoter and the Renilla Renilformis luciferase gene. The fluorescence of renilla luciferase was used to normalize the efficiency of PAX8 plasmid transfection. Independent assays were repeated 5 times. Luciferase activity was measured using the DualGlo luciferase assay measurement system.

(4) And (3) detecting the transcription activity:

a) After 24 hours of transfection, the 24-well cell culture plate was removed and the supernatant was discarded by pipette;

b) Adding 200 mu L PBS into each hole, washing for 2 times, and removing the PBS by using a pipette;

c) Adding 100 mu L of 1X Passive Lysis Buffer lysate which is diluted by PBS in advance into each well to perform Lysis on cells, and placing a 24-well plate on a shaking table to perform Lysis for 30 minutes at room temperature;

d) The lysed cells were collected into 1.5mL EP tubes; after centrifugation at 3000rpm for 5 minutes, 20. Mu.L of supernatant from each EP tube was added to the white plate;

e) Loading a Dual Luciferase reporter system detection reagent, firstly adding 50 mu L of luminescence reagent (Luciferase Assay Substrate) into each hole, and detecting immunofluorescence luminescence intensity (Firefly Luciferase concentrations, LUC) under an enzyme-linked immunosorbent Assay (450 nm);

f) Taking out the white board, respectively adding 50 mu L of inactivation reagent (Stop & Glo Buffer) into each hole, and detecting the inactivated luminous intensity (Renilla Luciferase counts, RLUC) under an enzyme-linked immunosorbent assay (450 nm);

g) Recording the LUC value and the RLUC value, and calculating the ratio of the LUC/RLUC as the transcription activity intensity;

h) And (6) analyzing and counting data.

2.6 comparative genomic hybridization microarray (aCGH)

The ES-identified pathogenic CNVs were validated using aCGH. Genomic DNA and gender-matched reference DNA were obtained from samples extracted from peripheral blood of each subject and fragmented using the AluI and RsaI enzymes. The Agilent SureTag DNA labeling kit is used for DNA labeling. Cy5-dUTP was used for dye labeling of each DNA, and Cy3-dUTP was used as a reference DNA. The labeled test DNA and reference DNA were hybridized to an Agilent Sureprint G3 Custom CGH Array. DNA processing, microarray processing and data analysis were performed according to the agilent oligonucleotide CGH protocol.

2.7 digital droplet PCR (ddPCR)

To verify the chimerism of the BMP4 variant c.367g > T (p.glu123ter), two TaqMan probes were designed for detection of the wild type and mutant sequences. For the simultaneous detection of CNVs identified in the validation column, two TaqMan probes designed with unique fluorescent tags are used, which are activated and detected when the corresponding binding region TBX6 (FAM probe) or housekeeping gene TERT (HEX probe) is amplified by PCR. Standard protocol systems and standard thermocyclers for TaqMan reactions were performed using Bio-Rad QX200 AutoDG ddPCR. To increase the efficiency of the reaction, hindIII restriction endonuclease was used to aid DNA fragmentation without cutting the region to be amplified by PCR.

3. Results of the experiment

Cohort demographic information and individual phenotypes are shown in table 1, and 12 LGD mutation sites were identified among 7 candidate genes, including PAX8 (MIM: 167415), BMP4 (MIM: 112262), BMP7 (MIM: 112267), TBX6 (MIM: 602427), HOXA10 (MIM: 142957), EMX2 (MIM: 600035), and WNT9B (MIM: 602864) (see table 3 and fig. 1), whereas no LGD variation was detected in any of the candidate genes from the genome of 941 female control samples (P =1.2E-06, skat-O). Of the 12 LGD mutant sites, 8 were truncated by the protein and 4 affected the standard splice sites. All truncation mutation sites, except one in WNT9B and BMP4, were predicted to result in unstable mutant RNAs that were susceptible to nonsense-mediated mRNA degradation by NMDEscPredictor 15.

The results show that 4 LGD mutation sites were identified on the disease-causing gene PAX 8: c.156_157dupCG (p.Val53AlafsTer24), c.25+1G >T (Splice doror), c.195delC (p.Tyr 66ThrfsTer10), c.322C > T (p.Arg108Ter) (see tables 3 and 4), all 4 patients harboring a mutation site of PAX8 heterozygous LGD exhibited MRKH syndrome type I at the time of initial visit, no phenotype of hypothyroidism (CH) was clinically observed, and thyroid hormone levels of MRK49 (c.25 +1G > T) and MRK467 (c.322C > T) were within normal ranges. Furthermore, sequencing the parental DNA of both individuals revealed that both PAX8 LGD mutants were paternally inherited (see fig. 2), consistent with the dominant disease profile of sex-related penetrance. 3D-misPAX 8 mutants were identified from 3 cases in the study cohort (c.542C > T [ p.Ser181Phe ], c.266T > C [ p.Val89Ala ], c.236C > G [ p.Ser79Cys ]) (see Table 4). The results of the luciferase assay showed that c.236C > G resulted in a reduced transactivation potential and localization of PAX8 protein on its consensus binding sequence (see FIG. 3), demonstrating that it is a LoF allele. The study of MRK330 (c.236C > G) in this individual revealed that this mutant was also inherited from its father (see FIG. 2). Both MRK330 and its father suffer from CH, demonstrating the genetic pleiotropic nature of PAX 8. Nonsense mutants c.619C > T (p.Arg207Ter) were also identified in PAX8, and two heterozygous D-mis mutants c.136G > A (p.Asp46Asn), c.727C > G (p.Gln243Glu) on the PAX8 gene were identified in 3 patients with type I MRKHS (see Table 4). The results of the luciferase assay showed that the c.136g > a variant, which is also paternally inherited, is responsible for the LoF of PAX8 (see fig. 3), is consistent with an autosomal dominant mode of inheritance, and appears to be restrictively inherited (see fig. 2). The LGD and D-mis variants in PAX8 are enriched in the DNA-binding PAX domain of this protein (see fig. 4), suggesting that the DNA-binding function of PAX8 plays an important role in the pathogenesis of MRKHS. To further investigate the genetic pleiotropic effects of PAX8, 5 female individuals carrying the CH pathogenic PAX8 mutant were studied and the results showed that one (CH 123) of them did not show uterine hypoplasia before the visit period, suggesting a clinical diagnosis of MRKHS. The PAX8 variation in this individual was c.68g > T (p.gly23val), verified in vivo as the LoF allele (see figure 3). The phenotype of the individual is consistent with the symptoms of the syndrome, namely congenital hypothyroidism-MRKH syndrome (CH-MRKH syndrome).

The results show that hybrid termination gain mutation c.367G > T (p.Glu123Ter) and hybrid Splice acceptor mutation c. -132-1G > -A (Splice accepter) in BMP4 (Genebank: NM-001202.3) were identified in MRK644 and MRK166, respectively. MRK644 inherits the BMP4 allele of the c.367g > T mutant from the mother chimera. ES detected the BMP4 variant c.367g > T in proband as 26/55 (47%) mutants/total read, presumably heterozygous mutants. In contrast, the blood DNA of their mothers, as detected by ES, gave Vr/Tr =13/60 (22%) consistent with the chimeric variant allele. The digital droplet PCR (ddPCR) further verifies the chimeric phenomenon, and the result shows that the mutation rate of proband is 50% and the mutation rate of mother is 10%. For the second MRKH166 mutant with BMP4 LGD, the splice acceptor mutant was inherited from the parental line, exhibiting restricted inheritance similar to PAX 8. In a replication cohort of ES studies, another LGD variation in BMP4, c.766c > T (p.arg256ter), was found in individuals with type I MRKHS, further suggesting that BMP4 variation is associated with MRKHS.

The results showed that in BMP7 (Genebank: NM-001719.2), the Splice acceptor mutant c.1036-2A > (Splice donor) from MRK444 and the frameshift mutant c.275-287 dupTGGAGGAGGGCGGG (p.Pro98GlyfsTer 31) from MRK342 were identified. Both people show MRKH syndrome type I.

The results showed that TBX6 (Genebank: NM-004608.3) Splice donor variant c.621+1G > -A (Splice donor) was identified in individual MRK639 of type II MRKH syndrome. In addition to the aetiosis of the mullerian tube, MRK639 also suffers from congenital scoliosis. Sanger sequencing results show that the individual carries the c.621+1G >. In the replication cohort, another frame-shift mutant c.856_859delAATG (p.His286CysfsTer28) was identified in TBX6 of Mul23, and this individual was diagnosed with type II MRKH syndrome.

The results showed that missense variation in WNT9B (Genebank: NM-001320458.2) was first found in a cohort of 42 individuals with Chinese MRKH syndrome. In this study, we identified a heterozygous nonsense variant c.976c > T (p.gln 326ter) in MRK57, which was diagnosed as type I MRKH syndrome (see table 3).

Table 2 cohort demographic information and individual phenotypes

⁺ Of the 150 samples in the replicate cohort, the data on the classification of MRKH syndrome applies to all individuals, the age of follow-up and the unionData relating to the complications originated from 96 patients, and the percentage in this column was calculated based on the individuals who had the available information.

TABLE 3 analysis of mutation load of 19 candidate genes in 442 cases and 941 female controls

Rare variants were analyzed using SNP-set (Sequence) Kernel Association test-optimized (SKAT-O) test to determine the relevance of the 19 gene mutation loads. Abbreviations: pLI, probability of intolerance of loss of function from the ExAC database (http:// ExAC. Broadproperty. Org /); FDR, false discovery rate; OR, ratio of ratios.

TABLE 4 analysis of mutation load of 19 candidate genes in 442 cases and 941 female controls

Abbreviations: gnomAD, genome aggregation database; exAC, exome integration database; AF, allele frequency; vR, variant reads; tR, total read; VAF, allelic variation frequency; -, not applicable; loF, loss of function; CADD, in conjunction with annotation, relies on depletion.

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> gene mutation combination as marker of MRKH syndrome and application thereof

<141> 2020-12-30

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cctgagcaaa ctgctctcgt 20

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cagtccccag ctcaactgta a 21

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tccctgcctg attgttcagc 20

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagggaggct ctggctaaat c 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agatgccctc tttggtccat c 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agtgggtttg gagaatggct g 21

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgatggagca caggctcatt t 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtgagtcag cttggagtca g 21

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gctctcgaga tccaaccacc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcctcctact cctggcagac 20

<210> 11

<211> 30

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 11

gcggggtacc acctggctgg attggtcttc 30

<210> 12

<211> 29

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 12

ggcgaagctt tttcctggcc cttcctggg 29

Claims (4)

1. The application of the reagent for detecting the gene mutation site or the protein mutation site in a sample in preparing a kit for diagnosing MRKH syndrome is characterized in that the gene mutation site is the mutation site c.236C > G on the PAX8 gene;

the protein mutation site is a mutation site p.Ser79Cys on the PAX8 protein.

2. The use according to claim 1, wherein the reagents comprise specific amplification primers for the mutation site in the PAX8 gene.

3. Use according to claim 2, wherein the reagents comprise reagents conventional in PCR amplification reactions, and/or reagents used in DNA extraction processes, and/or reagents used in DNA sequencing processes.

4. The use according to claim 3, wherein the reagent further comprises a specific antibody for detecting the mutation site of the protein.

Images