CN107475423B - New pathogenic gene of headless spermatozoon disease and application thereof - Google Patents
New pathogenic gene of headless spermatozoon disease and application thereof Download PDFInfo
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Abstract
A new pathogenic gene of headless spermatozoon disease and application thereof, relating to a marker of headless spermatozoon disease and application thereof. Through large-scale screening, a new pathogenic gene TSGA10 of the headless spermatozoon is discovered for the first time. Taking a plurality of diseased families and diseased individuals of headless spermatozoa as research objects, exome sequencing and comparison are carried out on the diseased individuals in the families, and different patients are found to carry TSGA10 gene mutation. These mutations can be used to detect headless spermatozoa.
Description
Technical Field
The invention relates to a pathogenic gene, in particular to a novel pathogenic gene of headless spermatozoon and application thereof.
Background
Headless spermatozoa (achalolic spermatozoa) is a recessive genetic teratospermia that causes male infertility, a rare genetic disease whose main clinical features are that the spermatozoa have no head or loose connections between head and tail. Early studies also reported that the disease was decapitated spermatozoia (decapitated spermatozoa) and large-headed acicular sperms (pinhead sperms). The SUN5 gene was mutated as found in the early international molecular pathogenesis study (Zhu F, Wang F, Yang X, Zhang J, Wu H, Zhang Z, He X, Zhou P, Wei Z, Gecz J and CaoY. binary SUN5Mutations used in mouse Autosomal-Reccess Acephalic SpermatozoaSyndome. am J Hum Genet.2016; 99(4): 942-949).
At present, the gene mutation spectrum of headless spermatozoa is not completely discovered, and the relationship between genotype and phenotype is not clear. At present, the research on single-gene diseases starts to adopt a large number of methods of whole-exome sequencing (whole-exome sequencing) and whole-genome sequencing (whole-genome sequencing), and the two methods are successfully applied to find the pathogenic genes of rare single-gene diseases. The whole exome sequencing and whole genome sequencing technology is proved to be a powerful and effective means for reducing rare single-gene disease candidate genes and even discovering pathogenic genes thereof. The success rate of screening for disease-related variations is greatly enhanced by sequencing only the whole exome or genome of a few rare individuals, including patients and normal controls.
Therefore, the research on the headless spermatozoon disease in the field is not clear, and the cause of the disease is not clear, so that the research on the pathogenic mechanism of the headless spermatozoon disease and the search of a new pathogenic gene and a mutation site are urgently needed in the field.
Disclosure of Invention
The first object of the present invention is to provide a biomarker for headless spermatozoa.
It is a second object of the present invention to provide a method for detecting headless spermatozoa.
The third objective of the invention is to provide a primer pair used in detecting TSGA10 gene or TSGA10 protein mutation by PCR.
It is a fourth object of the present invention to provide a nucleic acid probe complementary to the mutant TSGA10 gene.
The fifth object of the present invention is to provide a kit for detecting a mutant TSGA10 gene or TSGA10 protein.
The sixth purpose of the invention is to provide a kit for detecting a mutant TSGA10 gene.
The invention determines a new pathogenic gene of headless spermatozoon by exome sequencing.
The biomarker for headless spermatozoa is a mutated TSGA10 gene or TSGA10 protein, having a mutated TSGA10 gene or TSGA10 protein selected from:
frame shift mutations in exon 2 (c.211delG; p.A71Hfs 12);
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs 6).
The mutated TSGA10 gene has the following mutations in the sequence of SEQ ID NO: 1:
a frameshift mutation in exon 2 (c.211delG);
a nonsense mutation in exon 9 (c.473g > a);
a frameshift mutation in exon 16 (c.1446delC).
The mutated TSGA10 protein is a mutant protein with the following mutations in the sequence of SEQ ID NO: 2:
frameshift mutations in exon 2 (p.a71hfs 12);
a nonsense mutation in exon 9 (p.r 158h);
frame shift mutations in exon 16 (p.L483Yfs. multidot.6).
The mutant TSGA10 protein is a frame shift mutation in exon 2 in the sequence of SEQ ID NO:2 (p.A71Hfs 12); the sequence of the mutant TSGA10 protein is SEQ ID NO 9.
The mutant TSGA10 protein is a sequence of SEQ ID NO:2, and has a frame shift mutation in an exon 16 (p.L483Yfs 6); the sequence of the mutant TSGA10 protein is SEQ ID NO 10.
The method for detecting headless spermatozoa comprises detecting whether a mutation site exists in TSGA10 gene or TSGA10 protein of a subject, wherein if the mutation site exists, the subject is identified as having or being susceptible to headless spermatozoa, and the mutation site is selected from any one or combination of the following:
frame shift mutations in exon 2 (c.211delG; p.A71Hfs 12);
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs 6).
The TSGA10 protein is represented by a sequence shown as SEQ ID NO. 2.
The TSGA10 gene is represented by the sequence of SEQ ID NO: 1.
The method for detecting headless spermatozoa disease comprises the following steps of amplifying at least one group of primers:
3 and 4;
5 and 6 SEQ ID NO;
SEQ ID NO 7 and SEQ ID NO 8.
In the method for detecting headless spermatozoa, mutation sites are detected by a technique selected from the group consisting of: sequencing, electrophoresis, nucleic acid hybridization, in situ hybridization, PCR, reverse transcriptase chain reaction, and denaturing high performance liquid chromatography.
The primer pair used in the detection of TSGA10 gene or TSGA10 protein mutation by PCR, the mutation is selected from any one or combination of the following:
frame shift mutations in exon 2 (c.211delG; p.A71Hfs 12);
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs. multidot.6),
wherein the primer pairs are designed based on a position selected from the group consisting of: 211. 473 and 1446, numbering based on the cDNA sequence of TSGA 10.
The nucleic acid probe complementary to a mutant TSGA10 gene, wherein the mutation is selected from any one or a combination of the following:
frame shift mutations in exon 2 (c.211delG; p.A71Hfs 12);
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs. multidot.6),
the probe comprises a position selected from the group consisting of: 211. 473 and 1446, numbering based on the cDNA sequence of TSGA 10.
The kit for detecting the mutant TSGA10 gene or TSGA10 protein comprises at least one group of primer pairs, wherein the mutation is selected from any one or the combination of the following:
frame shift mutations in exon 2 (c.211delG; p.A71Hfs 12);
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs. multidot.6),
wherein the primer pairs are designed based on the following positions respectively, so that the amplification products cover the positions: 211. 473 and 1446, numbering based on the cDNA sequence of TSGA 10.
The kit for detecting the mutant TSGA10 gene or TSGA10 protein comprises at least one group of primers selected from the following group:
3 and 4;
5 and 6 SEQ ID NO;
SEQ ID NO 7 and SEQ ID NO 8.
The kit for detecting the mutant TSGA10 gene comprises at least one nucleic acid probe, wherein the mutation is selected from any one or the combination of the following:
frame shift mutations in exon 2 (c.211delG; p.A71Hfs 12);
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs. multidot.6),
the probe is complementary to a region of the mutant TSGA10 gene comprising a position selected from the group consisting of: 211. 473 and 1446, numbering based on the cDNA sequence of TSGA 10.
The invention lays an important foundation for researching pathogenesis of the headless spermatozoon and provides a brand new theoretical basis for treating patients with the headless spermatozoon.
Detailed Description
In the present invention, mutations are expressed using methods commonly used in the art. For example, in the mutation (c.473G > A; p.R158H), c represents cDNA, p represents protein, and a mutation at the DNA level corresponds to a mutation at the protein level; in the frameshift mutation (c.211delG; p.A71Hfs. 12), c.211delG indicates the deletion G at position 211 on the DNA level, p.A71Hfs. 12 indicates a frameshift of 11 amino acids from position 71, and the resulting mutein has only 81 amino acids as shown in SEQ ID NO: 9.
The cDNA sequence of the wild type TSGA10 gene is shown in SEQ ID NO. 1.
2, SEQ ID NO: amino acid sequence of wild-type TSGA10 protein.
For reference to gene sequences in the present invention, it will be understood by those skilled in the art that the actual inclusion of either or both of the complementary double strands is intended. For convenience, in the present invention, although one strand is given in most cases, the other strand complementary thereto is also disclosed in practice. For example, reference is made to the cDNA sequence of the TSGA10 gene, which actually includes this sequence as well as its complement. For example, reference to SEQ ID NO 1 actually includes the complementary sequence thereof. One skilled in the art will also appreciate that one strand may be used to detect the other strand and vice versa.
The gene sequence in the present invention includes a DNA form or an RNA form, and one is disclosed, meaning that the other is also disclosed. For example, reference is made to the cDNA sequence of the TSGA10 gene, and indeed to the corresponding RNA sequence.
Specific examples are given below.
Example 1 the causative gene of headless spermatozoa was determined.
14 headless spermatozoon cases are collected, and all patients show typical headless spermatozoon disease characteristics such as sterility, headless, pin-like head or loose head-tail connection.
All patients were subjected to whole exome sequencing, with the following specific steps:
sample preparation: collecting peripheral blood of the patient and a parent mother, extracting genomic DNA (QIAamp DNA Mini Kit 51304 Qiagen, USA) in peripheral blood leukocyte by using a Kit, measuring the concentration and purity of the DNA (Thermo Scientific, USA) by using NanoDrop 2000, and obtaining the OD260/OD280 of the genomic DNA of each sample, wherein the concentration is not less than 100 ng/mul, and the total amount is not less than 30 mul.
Then, exome sequences of the above 4 samples were sequenced. The sequencing platform was Illumina Hiseq2000, and sequencing was performed according to Illumina Standard library construction Specification (see http:// www.illumina.com /), briefly as follows:
1) a DNA Library was prepared using TruSeq DNA Library Prep Kit available from Illumina. The Agilent kit SureSelect Human All Exon V5 was used during Exon capture;
2) performing parallel sequencing on the captured exon regions by using an Illumina HiSeq2000 sequencing platform to read the length of 90bp, wherein the average sequencing depth of each sample is at least 100;
3) the sequencing results were aligned to the ginseng reference genome using a Burrows-Wheeler Aligner (http:// bio-bw. sourceforce. net /) (hg 19). Duplicate reads were removed using SAMtools, adjusted and calibrated by GATK (https:// www.broadinstitute.org/GATK /). SNVs and InDels were then mined using SAMtools (http:// www.samtools.sourceforge.net /) and named with ANNOVAR (http:// www.openbioinformatics.org/ANNOVAR /).
Mutation sites with allele frequencies greater than 1% were knocked out (reference database included dbSNP, 1000 genes, exterior Aggregation Consortium (ExAC)).
3 patients were found to have homozygous or heterozygous mutations in the gene TSGA 10: these sites were probably pathogenic by co-segregation of 1 patient and 2 normal SNP screens in 1 family.
The results are shown in Table 1.
TABLE 1
Therefore, it is considered that the TSGA10 gene is highly likely to be a causative gene of headless spermatozoa.
Example 2 the causative gene of the above headless spermatozoa was confirmed in other cases.
The inventor included all patients with headless spermatozoa, and verified that TSGA10 gene is the causative gene of headless spermatozoa. In the verification, the mutation of the TSGA10 gene is verified by Sanger sequencing, wherein the sample verification conditions such as family inside, family outside, distribution and the like are considered.
Genes were examined for 3 patients and 5 normal persons in the family (i.e., parents or brothers of 5 of them, all of whom did not develop disease) respectively.
The method comprises the following specific steps:
DNA extraction:
peripheral venous blood was extracted from 3 patients and 5 normal persons in the family, respectively, and genomic DNA was extracted and DNA content was measured according to the method of example 1.
2. Primer design and PCR reaction
Primer design was referenced to the human genome sequence database hg19, see table 2 for details.
a) The primer sequence is as follows:
TABLE 2
b) Reaction system:
PCR amplification System:
c) and (3) PCR reaction conditions:
denaturation at 94 ℃ for 5 min and then in each cycle, denaturation at 94 ℃ for 30s, annealing (typically 55 ℃ and set according to the annealing temperature of the different primers) for 30s, extension at 72 ℃ for 1min, for a total of 30 cycles. All cycles were completed, final extension at 72 ℃ for 10min, and PCR products were stored at 4 or-20 ℃.
3) The PCR-amplified products obtained in step 2 from 3 patients with headless spermatozoa, 5 normal persons in the family (i.e., parents or brothers of the 5 patients who had no disease) were directly subjected to DNA sequencing in the same manner as in example 1.
Significant mutation sites were detected in the TSGA10 gene in 3 out of 14 collected headless sperm cases. Meanwhile, the found mutation sites can not be detected in family normal people. The frequency of these mutant sites in the ExAC database was very low or none (see Table 1), suggesting that the detected sites are likely not SNPs. Therefore, the TSGA10 gene is considered to be a pathogenic gene of headless spermatozoa.
Description of sequence listing:
1, SEQ ID NO: cDNA sequence ATGATGCGAAGTAGGTCTAAAAGTCCAAGACGCCCATCACCAACTGCCCGGGGTGCAAACTGTGATGTAGAACTTTTGAAGACAACAACAAGAGATCGTGAAGAACTTAAATGCATGCTGGAAAAATATGAGCGCCATTTGGCAGAAATTCAGGGTAATGTCAAGGTTCTTAAATCTGAGAGAGACAAGATCTTCCTTCTTTATGAACAGGCACAGGAAGAAATTACCCGACTTCGACGAGAAATGATGAAAAGCTGTAAGAGTCCTAAATCAACAACGGCACATGCTATTCTCCGGCGAGTGGAGACTGAAAGAGATGTAGCCTTTACTGATTTACGAAGAATGACCACAGAACGAGATAGTCTAAGGGAGAGGCTAAAGATTGCTCAAGAGACAGCATTTAATGAGAAGGCTCACCTGGAACAAAGGATAGAGGAGCTGGAGTGTACAGTTCATAATCTTGATGATGAACGTATGGAGCAAATGTCAAATATGACTTTGATGAAGGAAACCATAAGCACTGTGGAAAAAGAAATGAAATCACTAGCAAGAAAGGCAATGGATACCGAAAGTGAACTTGGCAGACAAAAAGCAGAGAATAATTCTTTGAGACTTTTGTATGAAAACACAGAAAAAGATCTTTCTGATACTCAGCGACACCTTGCTAAGAAAAAATATGAGCTACAGCTTACTCAGGAGAAAATTATGTGCTTGGATGAAAAAATTGATAACTTTACAAGGCAAAATATTGCACAGCGAGAAGAAATCAGCATTCTTGGTGGAACCCTCAATGATCTGGCTAAAGAAAAGGAATGCCTGCAAGCATGTTTGGATAAAAAATCTGAGAATATTGCATCCCTTGGAGAGAGTTTGGCAATGAAAGAAAAGACCATTTCAGGCATGAAGAATATCATTGCTGAGATGGAACAGGCATCAAGACAGTGTACTGAGGCCCTAATTGTGTGTGAACAAGACGTTTCCAGAATGCGTCGGCAATTGGATGAGACAAATGATGAGCTGGCCCAGATCGCCAGGGAAAGAGATATCTTGGCTCATGACAATGACAATCTCCAGGAACAGTTTGCTAAAGCTAAACAAGAAAACCAGGCACTGTCCAAAAAATTGAATGACACTCATAATGAACTTAATGACATAAAACAGAAGGTTCAAGATACTAATTTGGAGGTTAACAAGCTGAAGAATATATTAAAGTCTGAAGAATCTGAGAACCGGCAAATGATGGAACAACTTCGAAAAGCCAATGAAGATGCTGAAAACTGGGAAAATAAAGCCCGTCAATCAGAGGCAGATAACAATACCCTCAAACTGGAACTTATCACTGCTGAGGCAGAGGGTAACAGATTAAAAGAAAAAGTAGATTCCCTCAACAGAGAGGTTGAGCAACACTTAAATGCAGAAAGGTCTTACAAGTCCCAGATTTCTACCTTACATAAATCTGTTGTAAAAATGGAAGAGGAGCTTCAGAAGGTTCAGTTTGAAAAAGTGTCCGCTCTTGCAGATTTGTCTTCTACTAGGGAACTCTGTATTAAACTTGACTCAAGCAAAGAACTTCTTAATCGACAGCTGGTTGCTAAAGATCAAGAAATAGAAATGAGGGAGAATGAGTTAGATTCTGCTCATTCTGAAATTGAACTCCTGAGGAGTCAGATGGCAAATGAGAGAATCTCCATGCAGAATCTAGAAGCTTTGCTGGTGGCCAATCGAGACAAAGAATATCAGTCTCAGATAGCACTTCAAGAAAAAGAATCTGAAATTCAGCTTCTTAAAGAACACCTTTGTTTGGCAGAAAATAAAATGGCCATCCAGAGTAGAGATGTGGCCCAGTTCAGAAATGTTGTCACACAATTGGAAGCTGATTTAGACATTACCAAAAGACAACTAGGAACAGAGCGCTTTGAAAGGGAGAGGGCCGTACAAGAACTTCGCCGCCAAAATTATTCAAGTAATGCTTATCATATGAGTTCTACAATGAAGCCAAATACAAAATGTCATTCACCAGAACGTGCTCACCATCGATCTCCTGACCGAGGCCTAGATCGATCATTAGAAGAGAATCTTTGCTACAGAGATTTCTGA of wild type TSGA10 gene
2, SEQ ID NO: amino acid sequence MMRSRSKSPRRPSPTARGANCDVELLKTTTRDREELKCMLEKYERHLAEIQGNVKVLKSERDKIFLLYEQAQEEITRLRREMMKSCKSPKSTTAHAILRRVETERDVAFTDLRRMTTERDSLRERLKIAQETAFNEKAHLEQRIEELECTVHNLDDERMEQMSNMTLMKETISTVEKEMKSLARKAMDTESELGRQKAENNSLRLLYENTEKDLSDTQRHLAKKKYELQLTQEKIMCLDEKIDNFTRQNIAQREEISILGGTLNDLAKEKECLQACLDKKSENIASLGESLAMKEKTISGMKNIIAEMEQASRQCTEALIVCEQDVSRMRRQLDETNDELAQIARERDILAHDNDNLQEQFAKAKQENQALSKKLNDTHNELNDIKQKVQDTNLEVNKLKNILKSEESENRQMMEQLRKANEDAENWENKARQSEADNNTLKLELITAEAEGNRLKEKVDSLNREVEQHLNAERSYKSQISTLHKSVVKMEEELQKVQFEKVSALADLSSTRELCIKLDSSKELLNRQLVAKDQEIEMRENELDSAHSEIELLRSQMANERISMQNLEALLVANRDKEYQSQIALQEKESEIQLLKEHLCLAENKMAIQSRDVAQFRNVVTQLEADLDITKRQLGTERFERERAVQELRRQNYSSNAYHMSSTMKPNTKCHSPERAHHRSPDRGLDRSLEENLCYRDF of wild-type TSGA10 protein
9 of SEQ ID NO: amino acid sequence MMRSRSKSPRRPSPTARGANCDVELLKTTTRDREELKCMLEKYERHLAEIQGNVKVLKSERDKIFLLYEQHRKKLPDFDEK of mutant TSGA10 protein (p.a71hfs 12)
10, SEQ ID NO: amino acid sequence MMRSRSKSPRRPSPTARGANCDVELLKTTTRDREELKCMLEKYERHLAEIQGNVKVLKSERDKIFLLYEQAQEEITRLRREMMKSCKSPKSTTAHAILRRVETERDVAFTDLRRMTTERDSLRERLKIAQETAFNEKAHLEQRIEELECTVHNLDDERMEQMSNMTLMKETISTVEKEMKSLARKAMDTESELGRQKAENNSLRLLYENTEKDLSDTQRHLAKKKYELQLTQEKIMCLDEKIDNFTRQNIAQREEISILGGTLNDLAKEKECLQACLDKKSENIASLGESLAMKEKTISGMKNIIAEMEQASRQCTEALIVCEQDVSRMRRQLDETNDELAQIARERDILAHDNDNLQEQFAKAKQENQALSKKLNDTHNELNDIKQKVQDTNLEVNKLKNILKSEESENRQMMEQLRKANEDAENWENKARQSEADNNTLKLELITAEAEGNRLKEKVDSLNREVEQHLNAERSYKSQISTYINLL of mutant TSGA10 protein (p.l483yfs x 6).
Sequence listing
<110> Xiamen city women and children health care hospital (Xiamen city family planning service center), the first hospital affiliated to Jinzhou medical university, the Beijing women's hospital affiliated to the capital medical university, the tobacco stand Yu Jiang hospital
<120> novel pathogenic gene of headless spermatozoon and application thereof
<130>2017
<160>10
<170>SIPOSequenceListing 1.0
<210>1
<211>2097
<212>DNA
<213>Homo Sapiens
<400>1
atgatgcgaa gtaggtctaa aagtccaaga cgcccatcac caactgcccg gggtgcaaac 60
tgtgatgtag aacttttgaa gacaacaaca agagatcgtg aagaacttaa atgcatgctg 120
gaaaaatatg agcgccattt ggcagaaatt cagggtaatg tcaaggttct taaatctgag 180
agagacaaga tcttccttct ttatgaacag gcacaggaag aaattacccg acttcgacga 240
gaaatgatga aaagctgtaa gagtcctaaa tcaacaacgg cacatgctat tctccggcga 300
gtggagactg aaagagatgt agcctttact gatttacgaa gaatgaccac agaacgagat 360
agtctaaggg agaggctaaa gattgctcaa gagacagcat ttaatgagaa ggctcacctg 420
gaacaaagga tagaggagct ggagtgtaca gttcataatc ttgatgatga acgtatggag 480
caaatgtcaa atatgacttt gatgaaggaa accataagca ctgtggaaaa agaaatgaaa 540
tcactagcaa gaaaggcaat ggataccgaa agtgaacttg gcagacaaaa agcagagaat 600
aattctttga gacttttgta tgaaaacaca gaaaaagatc tttctgatac tcagcgacac 660
cttgctaaga aaaaatatga gctacagctt actcaggaga aaattatgtg cttggatgaa 720
aaaattgata actttacaag gcaaaatatt gcacagcgag aagaaatcag cattcttggt 780
ggaaccctca atgatctggc taaagaaaag gaatgcctgc aagcatgttt ggataaaaaa 840
tctgagaata ttgcatccct tggagagagt ttggcaatga aagaaaagac catttcaggc 900
atgaagaata tcattgctga gatggaacag gcatcaagac agtgtactga ggccctaatt 960
gtgtgtgaac aagacgtttc cagaatgcgt cggcaattgg atgagacaaa tgatgagctg 1020
gcccagatcg ccagggaaag agatatcttg gctcatgaca atgacaatct ccaggaacag 1080
tttgctaaag ctaaacaaga aaaccaggca ctgtccaaaa aattgaatga cactcataat 1140
gaacttaatg acataaaaca gaaggttcaa gatactaatt tggaggttaa caagctgaag 1200
aatatattaa agtctgaaga atctgagaac cggcaaatga tggaacaact tcgaaaagcc 1260
aatgaagatg ctgaaaactg ggaaaataaa gcccgtcaat cagaggcaga taacaatacc 1320
ctcaaactgg aacttatcac tgctgaggca gagggtaaca gattaaaaga aaaagtagat 1380
tccctcaaca gagaggttga gcaacactta aatgcagaaa ggtcttacaa gtcccagatt 1440
tctaccttac ataaatctgt tgtaaaaatg gaagaggagc ttcagaaggt tcagtttgaa 1500
aaagtgtccg ctcttgcaga tttgtcttct actagggaac tctgtattaa acttgactca 1560
agcaaagaac ttcttaatcg acagctggtt gctaaagatc aagaaataga aatgagggag 1620
aatgagttag attctgctca ttctgaaatt gaactcctga ggagtcagat ggcaaatgag 1680
agaatctcca tgcagaatct agaagctttg ctggtggcca atcgagacaa agaatatcag 1740
tctcagatag cacttcaaga aaaagaatct gaaattcagc ttcttaaaga acacctttgt 1800
ttggcagaaa ataaaatggc catccagagt agagatgtgg cccagttcag aaatgttgtc 1860
acacaattgg aagctgattt agacattacc aaaagacaac taggaacaga gcgctttgaa 1920
agggagaggg ccgtacaaga acttcgccgc caaaattatt caagtaatgc ttatcatatg 1980
agttctacaa tgaagccaaa tacaaaatgt cattcaccag aacgtgctca ccatcgatct 2040
cctgaccgag gcctagatcg atcattagaa gagaatcttt gctacagaga tttctga 2097
<210>2
<211>698
<212>PRT
<213>Homo Spiens
<400>2
Met Met Arg Ser Arg Ser Lys Ser Pro Arg Arg Pro Ser Pro Thr Ala
1 5 10 15
Arg Gly Ala Asn Cys Asp Val Glu Leu Leu Lys Thr Thr Thr Arg Asp
20 25 30
Arg Glu Glu Leu Lys Cys Met Leu Glu Lys Tyr Glu Arg His Leu Ala
35 40 45
Glu Ile Gln Gly Asn Val Lys Val Leu Lys Ser Glu Arg Asp Lys Ile
50 55 60
Phe Leu Leu Tyr Glu Gln Ala Gln Glu Glu Ile Thr Arg Leu Arg Arg
65 70 75 80
Glu Met Met Lys Ser Cys Lys Ser Pro Lys Ser Thr Thr Ala His Ala
85 90 95
Ile Leu Arg Arg Val Glu Thr Glu Arg Asp Val Ala Phe Thr Asp Leu
100 105 110
Arg Arg Met Thr Thr Glu Arg Asp Ser Leu Arg Glu Arg Leu Lys Ile
115 120 125
Ala Gln Glu Thr Ala Phe Asn Glu Lys Ala His Leu Glu Gln Arg Ile
130 135 140
Glu Glu Leu Glu Cys Thr Val His Asn Leu Asp Asp Glu Arg Met Glu
145 150 155 160
Gln Met Ser Asn Met Thr Leu Met Lys Glu Thr Ile Ser Thr Val Glu
165 170 175
Lys Glu Met Lys Ser Leu Ala Arg Lys Ala Met Asp Thr Glu Ser Glu
180 185 190
Leu Gly Arg Gln Lys Ala Glu Asn Asn Ser Leu Arg Leu Leu Tyr Glu
195 200 205
Asn Thr Glu Lys Asp Leu Ser Asp Thr Gln Arg His Leu Ala Lys Lys
210 215 220
Lys Tyr Glu Leu Gln Leu Thr Gln Glu Lys Ile Met Cys Leu Asp Glu
225 230 235 240
Lys Ile Asp Asn Phe Thr Arg Gln Asn Ile Ala Gln Arg Glu Glu Ile
245 250 255
Ser Ile Leu Gly Gly Thr Leu Asn Asp Leu Ala Lys Glu Lys Glu Cys
260 265 270
Leu Gln Ala Cys Leu Asp Lys Lys Ser Glu Asn Ile Ala Ser Leu Gly
275 280 285
Glu Ser Leu Ala Met Lys Glu Lys Thr Ile Ser Gly Met Lys Asn Ile
290 295 300
Ile Ala Glu Met Glu Gln Ala Ser Arg Gln Cys Thr Glu Ala Leu Ile
305 310 315 320
Val Cys Glu Gln Asp Val Ser Arg Met Arg Arg Gln Leu Asp Glu Thr
325 330 335
Asn Asp Glu Leu Ala Gln Ile Ala Arg Glu Arg Asp Ile Leu Ala His
340 345 350
Asp Asn Asp Asn Leu Gln Glu Gln Phe Ala Lys Ala Lys Gln Glu Asn
355 360 365
Gln Ala Leu Ser Lys Lys Leu Asn Asp Thr His Asn Glu Leu Asn Asp
370 375 380
Ile Lys Gln Lys Val Gln Asp Thr Asn Leu Glu Val Asn Lys Leu Lys
385 390 395 400
Asn Ile Leu Lys Ser Glu Glu Ser Glu Asn Arg Gln Met Met Glu Gln
405 410 415
Leu Arg Lys Ala Asn Glu Asp Ala Glu Asn Trp Glu Asn Lys Ala Arg
420 425 430
Gln Ser Glu Ala Asp Asn Asn Thr Leu Lys Leu Glu Leu Ile Thr Ala
435 440 445
Glu Ala Glu Gly Asn Arg Leu Lys Glu Lys Val Asp Ser Leu Asn Arg
450 455 460
Glu Val Glu Gln His Leu Asn Ala Glu Arg Ser Tyr Lys Ser Gln Ile
465 470 475 480
Ser Thr Leu His Lys Ser Val Val Lys Met Glu Glu Glu Leu Gln Lys
485 490 495
Val Gln Phe Glu Lys Val Ser Ala Leu Ala Asp Leu Ser Ser Thr Arg
500 505 510
Glu Leu Cys Ile Lys Leu Asp Ser Ser Lys Glu Leu Leu Asn Arg Gln
515 520 525
Leu Val Ala Lys Asp Gln Glu Ile Glu Met Arg Glu Asn Glu Leu Asp
530 535 540
Ser Ala His Ser Glu Ile Glu Leu Leu Arg Ser Gln Met Ala Asn Glu
545 550 555 560
Arg Ile Ser Met Gln Asn Leu Glu Ala Leu Leu Val Ala Asn Arg Asp
565 570 575
Lys Glu Tyr Gln Ser Gln Ile Ala Leu Gln Glu Lys Glu Ser Glu Ile
580 585 590
Gln Leu Leu Lys Glu His Leu Cys Leu Ala Glu Asn Lys Met Ala Ile
595 600 605
Gln Ser Arg Asp Val Ala Gln Phe Arg Asn Val Val Thr Gln Leu Glu
610 615 620
Ala Asp Leu Asp Ile Thr Lys Arg Gln Leu Gly Thr Glu Arg Phe Glu
625 630 635 640
Arg Glu Arg Ala Val Gln Glu Leu Arg Arg Gln Asn Tyr Ser Ser Asn
645 650 655
Ala Tyr His Met Ser Ser Thr Met Lys Pro Asn Thr Lys Cys His Ser
660 665 670
Pro Glu Arg Ala His His Arg Ser Pro Asp Arg Gly Leu Asp Arg Ser
675 680 685
Leu Glu Glu Asn Leu Cys Tyr Arg Asp Phe
690 695
<210>3
<211>25
<212>DNA
<213> Artificial sequence (Homo sapiens)
<400>3
atttgttttg atttgctgtg aattg 25
<210>4
<211>25
<212>DNA
<213> Artificial sequence (Homo sapiens)
<400>4
cctaaagtga tgtgataagg tgtgg 25
<210>5
<211>25
<212>DNA
<213> Artificial sequence (Homo sapiens)
<400>5
tgtaatttat taatgttggg caaca 25
<210>6
<211>25
<212>DNA
<213> Artificial sequence (Homo sapiens)
<400>6
tattctgtac ccagaagagc tttgt 25
<210>7
<211>25
<212>DNA
<213> Artificial sequence (Homo sapiens)
<400>7
ccagtcaaga attcaaaaac agatg 25
<210>8
<211>25
<212>DNA
<213> Artificial sequence (Homo sapiens)
<400>8
aaataaacct caacctcatg gttct 25
<210>9
<211>81
<212>PRT
<213>Homo Spiens
<400>9
Met Met Arg Ser Arg Ser Lys Ser Pro Arg Arg Pro Ser Pro Thr Ala
1 5 10 15
Arg Gly Ala Asn Cys Asp Val Glu Leu Leu Lys Thr Thr Thr Arg Asp
20 25 30
Arg Glu Glu Leu Lys Cys Met Leu Glu Lys Tyr Glu Arg His Leu Ala
35 40 45
Glu Ile Gln Gly Asn Val Lys Val Leu Lys Ser Glu Arg Asp Lys Ile
50 55 60
Phe Leu Leu Tyr Glu Gln His Arg Lys Lys Leu Pro Asp Phe Asp Glu
65 70 75 80
Lys
<210>10
<211>487
<212>PRT
<213>Homo Sapiens
<400>10
Met Met Arg Ser Arg Ser Lys Ser Pro Arg Arg Pro Ser Pro Thr Ala
1 5 10 15
Arg Gly Ala Asn Cys Asp Val Glu Leu Leu Lys Thr Thr Thr Arg Asp
20 25 30
Arg Glu Glu Leu Lys Cys Met Leu Glu Lys Tyr Glu Arg His Leu Ala
35 40 45
Glu Ile Gln Gly Asn Val Lys Val Leu Lys Ser Glu Arg Asp Lys Ile
50 55 60
Phe Leu Leu Tyr Glu Gln Ala Gln Glu Glu Ile Thr Arg Leu Arg Arg
65 70 75 80
Glu Met Met Lys Ser Cys Lys Ser Pro Lys Ser Thr Thr Ala His Ala
85 90 95
Ile Leu Arg Arg Val Glu Thr Glu Arg Asp Val Ala Phe Thr Asp Leu
100 105 110
Arg Arg Met Thr Thr Glu Arg Asp Ser Leu Arg Glu Arg Leu Lys Ile
115120 125
Ala Gln Glu Thr Ala Phe Asn Glu Lys Ala His Leu Glu Gln Arg Ile
130 135 140
Glu Glu Leu Glu Cys Thr Val His Asn Leu Asp Asp Glu Arg Met Glu
145 150 155 160
Gln Met Ser Asn Met Thr Leu Met Lys Glu Thr Ile Ser Thr Val Glu
165 170 175
Lys Glu Met Lys Ser Leu Ala Arg Lys Ala Met Asp Thr Glu Ser Glu
180 185 190
Leu Gly Arg Gln Lys Ala Glu Asn Asn Ser Leu Arg Leu Leu Tyr Glu
195 200 205
Asn Thr Glu Lys Asp Leu Ser Asp Thr Gln Arg His Leu Ala Lys Lys
210 215 220
Lys Tyr Glu Leu Gln Leu Thr Gln Glu Lys Ile Met Cys Leu Asp Glu
225 230 235 240
Lys Ile Asp Asn Phe Thr Arg Gln Asn Ile Ala Gln Arg Glu Glu Ile
245 250 255
Ser Ile Leu Gly Gly Thr Leu Asn Asp Leu Ala Lys Glu Lys Glu Cys
260 265 270
Leu Gln Ala Cys Leu Asp Lys Lys Ser Glu Asn Ile Ala Ser Leu Gly
275280 285
Glu Ser Leu Ala Met Lys Glu Lys Thr Ile Ser Gly Met Lys Asn Ile
290 295 300
Ile Ala Glu Met Glu Gln Ala Ser Arg Gln Cys Thr Glu Ala Leu Ile
305 310 315 320
Val Cys Glu Gln Asp Val Ser Arg Met Arg Arg Gln Leu Asp Glu Thr
325 330 335
Asn Asp Glu Leu Ala Gln Ile Ala Arg Glu Arg Asp Ile Leu Ala His
340 345 350
Asp Asn Asp Asn Leu Gln Glu Gln Phe Ala Lys Ala Lys Gln Glu Asn
355 360 365
Gln Ala Leu Ser Lys Lys Leu Asn Asp Thr His Asn Glu Leu Asn Asp
370 375 380
Ile Lys Gln Lys Val Gln Asp Thr Asn Leu Glu Val Asn Lys Leu Lys
385 390 395 400
Asn Ile Leu Lys Ser Glu Glu Ser Glu Asn Arg Gln Met Met Glu Gln
405 410 415
Leu Arg Lys Ala Asn Glu Asp Ala Glu Asn Trp Glu Asn Lys Ala Arg
420 425 430
Gln Ser Glu Ala Asp Asn Asn Thr Leu Lys Leu Glu Leu Ile Thr Ala
435440 445
Glu Ala Glu Gly Asn Arg Leu Lys Glu Lys Val Asp Ser Leu Asn Arg
450 455 460
Glu Val Glu Gln His Leu Asn Ala Glu Arg Ser Tyr Lys Ser Gln Ile
465 470 475 480
Ser Thr Tyr Ile Asn Leu Leu
485
Claims (3)
1. A biomarker for headless spermatozoa, characterised by being a mutated TSGA10 gene or TSGA10 protein, the biomarker being a mutated TSGA10 gene or TSGA10 protein having any one of the following:
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs 6);
the mutated TSGA10 gene has any of the following mutations in the sequence of SEQ ID NO: 1:
a nonsense mutation in exon 9 (c.473g > a);
a frameshift mutation in exon 16 (c.1446delC);
the mutant TSGA10 protein is a mutant with any one of the following mutations in the sequence of SEQ ID NO: 2:
a nonsense mutation in exon 9 (p.r 158h);
frame shift mutations in exon 16 (p.L483Yfs. multidot.6).
2. A nucleic acid probe complementary to a mutant TSGA10 gene, characterized in that the mutation is selected from any one of the following:
a nonsense mutation in exon 9 (c.473G > A; p.R158H);
frame-shift mutations in exon 16 (c.1446delC; p.L483Yfs 6);
the cDNA sequence of the wild type TSGA10 gene is shown as SEQ ID NO:1 is shown.
3. Kit for the detection of a mutant TSGA10 gene or TSGA10 protein, characterized in that it comprises a nucleic acid probe according to claim 2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107164520A (en) * | 2017-06-26 | 2017-09-15 | 厦门市妇幼保健院（厦门市计划生育服务中心） | A kind of new Disease-causing gene of severe asthenospermia and its application |
| CN107176994A (en) * | 2017-06-26 | 2017-09-19 | 厦门市妇幼保健院（厦门市计划生育服务中心） | A kind of new Disease-causing genes of MMAF and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164520A (en) * | 2017-06-26 | 2017-09-15 | 厦门市妇幼保健院（厦门市计划生育服务中心） | A kind of new Disease-causing gene of severe asthenospermia and its application |
| CN107176994A (en) * | 2017-06-26 | 2017-09-19 | 厦门市妇幼保健院（厦门市计划生育服务中心） | A kind of new Disease-causing genes of MMAF and its application |
Non-Patent Citations (1)
| Title |
|---|
| TSGA10 is a novel candidate gene associated with acephalic spermatozoa;Yan-Wei Sha等;《CLINICAL GENETICS》;20170914;第93卷(第4期);摘要，第9页最后1行 * |
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