CN104073499B - TMC1 gene mutation body and its application - Google Patents
TMC1 gene mutation body and its application Download PDFInfo
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- CN104073499B CN104073499B CN201310100466.4A CN201310100466A CN104073499B CN 104073499 B CN104073499 B CN 104073499B CN 201310100466 A CN201310100466 A CN 201310100466A CN 104073499 B CN104073499 B CN 104073499B
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Abstract
TMC1 gene mutation body and its application.The present invention relates to the nucleic acid of detached coding TMC1, detached polypeptide, the method for screening the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease, the system for screening the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease and the kit for screening the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease.Wherein, the nucleic acid of detached coding TMC1 gene mutation body, with SEQ ID NO：1 compares, with selected from following at least one mutation：c.589G>A、c.1171C>T.Whether there is in biological sample by detecting the mutant, can effectively detect whether biological sample is susceptible to suffer from phonosensitive nerve deafness disease.
Description
Technical field
The present invention relates to technical field.Specifically, the present invention relates to TMC1 gene mutation body and its application.
Background technology
Research shows that 60% deafness patient is caused due to inherent cause, and in addition 40% deafness patient is relevant with environmental factor.With
Lack deep understanding etiologic etiological to deaf inheritance and diagnostic techniques toward due to people, it is impossible to which clearly deafness molecular disease is because more
Its generation cannot be prevented.Over nearly 30 years, with the fast development of deaf inheritance teiology and Protocols in Molecular Biology, existing so far
84 deaf genes are cloned.Some Common genes have obtained deep understanding, and deaf Molecular Etiology diagnosis is possibly realized.
Thus, at present the research of phonosensitive nerve deafness disease is still needed to be goed deep into.
Content of the invention
It is contemplated that at least solving one of above-mentioned technical problem to a certain extent.For this purpose, one object of the present invention
Be to propose a kind of can Effective selection be susceptible to suffer from phonosensitive nerve deafness disease biological sample method.
The present invention is completed based on the following work of inventor：Inventor is waited by high flux sequencing of extron group joint
The method of gene mutation checking is selected to determine the new Disease-causing gene mutational site of phonosensitive nerve deafness disease（TMC1 gene
c.589G>A、c.1171C>T is mutated）.
According to the first aspect of the invention, the present invention proposes a kind of nucleic acid of detached coding TMC1 gene mutation body.
Embodiments in accordance with the present invention, the nucleic acid and SEQ ID NO：1 compares, with selected from following at least one mutation：
c.589G>A、c.1171C>T.I.e. with respect to wild type TMC1 gene, the TMC1 gene of the present invention has a missense mutation
c.589G>A (p.G197R) or a nonsense mutation are c.1171C>At least one of T (p.Q391X).Reality according to the present invention
Example is applied, inventor determines the new mutant of TMC1 gene, the mutant and the close phase of the morbidity of phonosensitive nerve deafness disease
Closing, so as to whether there is in biological sample by detecting the new mutant, can effectively detect whether biological sample is susceptible to suffer from
Phonosensitive nerve deafness disease.
According to the second aspect of the invention, the present invention proposes a kind of detached polypeptide, the polypeptide and SEQ ID NO：2
Compare, with selected from following at least one mutation：P.G197R, p.Q391X, by detecting in biological sample whether express this
Polypeptide, can effectively detect whether biological sample is susceptible to suffer from phonosensitive nerve deafness disease.Embodiments in accordance with the present invention, this are many
Peptide be by above-mentioned nucleic acid coding.
According to three, the ground aspect of the present invention, the present invention proposes a kind of life that screens and be susceptible to suffer from phonosensitive nerve deafness disease
The method of thing sample, the method are comprised the following steps：From the extraction from biological material sample of nucleic acid；Determine the sample of nucleic acid
Nucleotide sequence；The nucleotide sequence of the sample of nucleic acid and SEQ ID NO：1 compares, with selected from c.589G>A or c.1171C>
At least one mutation of T is the instruction that the biological sample is susceptible to suffer from phonosensitive nerve deafness disease, optionally, the biological sample
It is that optionally, the sample of nucleic acid is complete genome DNA, optionally selected from blood of human body, skin, hypodermic at least one
Ground, the phonosensitive nerve deafness disease is autosomal recessive disease.Easy by screening according to embodiments of the present invention
The method of the biological sample of sense phonosensitive nerve deafness disease, can effectively screen the life of susceptible phonosensitive nerve deafness disease
Thing sample.
According to the fourth aspect of the invention, the present invention proposes a kind of biology for screening and being susceptible to suffer from phonosensitive nerve deafness disease
The system of sample, the system include：Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus are used for from the extraction from biological material nucleic acid
Sample；Nucleotide sequence determining device, the nucleotide sequence determining device are connected with the nucleic acid-extracting apparatus, for the core
Acid sample is analyzed, to determine the nucleotide sequence of the sample of nucleic acid；Judgment means, the judgment means and the nucleic acid
Sequence Determination Means are connected, so as to the nucleotide sequence based on the sample of nucleic acid and SEQ ID NO：1 compares, if having
c.589G>A、c.1171C>At least one mutation of T, judges whether the biological sample is susceptible to suffer from phonosensitive nerve deafness disease；
Optionally, the phonosensitive nerve deafness disease is autosomal recessive disease.Using the system, effectively can implement
The method of the aforementioned biological sample for screening susceptible phonosensitive nerve deafness disease, such that it is able to effectively screen susceptible phonosensitive nerve
Property deaf disease biological sample.
According to the fifth aspect of the invention, the present invention propose a kind of for screening be susceptible to suffer from phonosensitive nerve deafness disease
The kit of biological sample, the kit contain：The reagent of TMC1 gene mutation body is adapted to detect for, wherein with SEQ ID NO：1
Compare, the TMC1 gene mutation body has selected from following at least one mutation：c.589G>A、c.1171C>T, optionally,
The reagent is nucleic acid probe or primer, and optionally, the nucleic acid probe or primer have such as SEQ ID NO：Shown in 7-10
Nucleotide sequence, optionally, described is autosomal recessive disease.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from the description with reference to accompanying drawings below to embodiment
Substantially and easy to understand, wherein：
Fig. 1：Show the biological sample for screening and being susceptible to suffer from phonosensitive nerve deafness disease according to an embodiment of the invention
System and its part schematic diagram, wherein,
A is the system of the biological sample for being susceptible to suffer from phonosensitive nerve deafness Disease according to the screening of the embodiment of the present invention
Schematic diagram,
B is the schematic diagram of the nucleic acid-extracting apparatus according to the embodiment of the present invention,
C is the schematic diagram of the nucleotide sequence determining device according to the embodiment of the present invention.
Fig. 2：Show the family collection of illustrative plates of phonosensitive nerve deafness Disease according to an embodiment of the invention.
Fig. 3：Show the temporal bone CT picture of phonosensitive nerve deafness Disease according to an embodiment of the invention.
Fig. 4：Show the Sanger method sequencing result of TMC1 gene mutation body according to an embodiment of the invention.
Specific embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from start to finish
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and be not considered as limiting the invention.
TMC1 gene mutation body
According to the first aspect of the invention, the present invention proposes a kind of nucleic acid of detached coding TMC1 gene mutation body.
Embodiments in accordance with the present invention, the nucleic acid and SEQ ID NO：1 compares, with selected from following at least one mutation：
c.589G>A、c.1171C>T.I.e. with respect to wild type TMC1 gene, the TMC1 gene of the present invention has a missense mutation
c.589G>A (p.G197R) or a nonsense mutation are c.1171C>At least one of T (p.Q391X).Reality according to the present invention
Example is applied, inventor determines the new mutant of TMC1 gene, the mutant and the close phase of the morbidity of phonosensitive nerve deafness disease
Closing, so as to whether there is in biological sample by detecting the new mutant, can effectively detect whether biological sample is susceptible to suffer from
Phonosensitive nerve deafness disease.
The expression way " nucleic acid of coding TMC1 mutant " for herein being used, refers to and coding TMC1 mutant
The corresponding nucleic acid substances of gene, the i.e. type of nucleic acid are not particularly limited, and can be any volumes comprising with TMC1 mutant
The corresponding deoxyribonucleotide of code gene and/or the polymer of ribonucleotide, including but not limited to DNA, RNA or
cDNA.According to a specific example of the present invention, the nucleic acid of foregoing coding TMC1 mutant is DNA.According to the present invention
Embodiment, inventor determines the new mutant of TMC1 gene, and these new mutant are sent out with phonosensitive nerve deafness disease
Disease is closely related, so as to whether there is in biological sample by detecting the new mutant, can effectively detect biological sample
Whether phonosensitive nerve deafness disease is susceptible to suffer from, it is also possible to be whether there is in organism by detecting these mutant, Ke Yiyou
Whether prediction organism in effect ground is susceptible to suffer from phonosensitive nerve deafness disease.
The nucleic acid of coding TMC1 mutant, is that present inventor combines candidate by high flux sequencing of extron group
New mutation on the Disease-causing gene of the phonosensitive nerve deafness disease that the method for gene mutation checking determines.The mutational site is existing
Have in technology and be not mentioned.
Wherein, the cDNA of wild type TMC1 gene has nucleotide sequence as follows：
ATGTCACCCAAAAAAGTACAAATCAAAGTGGAGGAAAAAGAAGACGAGACTGAGGAAAGCTCAAGTGAAGAGGAAGA
GGAGGTGGAAGATAAGCTACCTCGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTTATCAATGAGG
ATGACCCAGAACCTGAACCAGAGGATGAAGAAACAAGGAAGGCAAGAGAAAAAGAGAGGAGGAGGAGGCTAAAGAGA
GGAGCAGAAGAAGAAGAAATTGATGAAGAGGAATTGGAAAGATTGAAGGCAGAGTTAGATGAGAAAAGACAAATAAT
TGCTACTGTCAAATGCAAACCATGGAAGATGGAGAAGAAAATTGAAGTTCTCAAGGAGGCAAAAAAATTTGTGAGTG
AAAATGAAGGGGCTCTTGGGAAAGGAAAAGGAAAACGGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCA
AAATTCCTCCGTGATTTTGAGAACTTCAAAGCTGCGTGTGTCCCATGGGAAAATAAAATCAAGGCTATTGAAAGTCA
GTTTGGCTCCTCAGTGGCCTCATACTTCCTCTTCTTGAGATGGATGTATGGAGTCAATATGGTTCTCTTTATCCTGA
CATTTAGCCTCATCATGTTGCCAGAGTACCTCTGGGGTTTGCCATATGGCAGTTTACCTAGGAAAACCGTTCCCAGA
GCCGAAGAGGCATCGGCAGCAAACTTTGGTGTGTTGTACGACTTCAATGGTTTGGCACAATATTCCGTTCTCTTTTA
TGGCTATTATGACAATAAACGAACAATTGGATGGATGAATTTCAGGTTGCCGCTCTCCTATTTTCTAGTGGGGATTA
TGTGCATTGGATACAGCTTTCTGGTTGTCCTCAAAGCAATGACCAAAAACATTGGTGATGATGGAGGTGGAGATGAC
AACACTTTCAATTTCAGCTGGAAGGTCTTTACCAGCTGGGACTACCTGATCGGCAATCCTGAAACAGCAGACAACAA
ATTTAATTCTATCACAATGAACTTTAAGGAAGCTATCACAGAAGAAAAAGCAGCCCAAGTAGAAGAAAACGTCCACT
TGATCAGATTCCTGAGGTTTCTGGCTAACTTCTTCGTGTTTCTAACACTTGGAGGGAGTGGATACCTCATCTTTTGG
GCTGTGAAGCGATCCCAGGAATTTGCACAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACAT
GGTTATGTCCCTCCTAGGGATGTTCTGTCCAACATTGTTTGACTTATTTGCTGAATTAGAAGACTACCATCCTCTCA
TCGCTTTGAAATGGCTACTGGGACGCATTTTTGCTCTTCTTTTAGGCAATTTATACGTATTTATTCTTGCATTAATG
GATGAGATTAACAACAAGATTGAAGAGGAGAAGCTAGTAAAGGCCAATATTACCCTTTGGGAAGCCAATATGATCAA
GGCCTACAATGCATCATTCTCTGAAAATAGCACTGGACCACCCTTTTTTGTTCACCCTGCAGATGTACCTCGAGGAC
CTTGCTGGGAAACAATGGTGGGACAGGAGTTTGTGAGGCTGACAGTCTCTGATGTTCTGACCACCTACGTCACAATC
CTCATTGGGGACTTTCTAAGGGCATGTTTTGTGAGGTTTTGCAATTATTGCTGGTGCTGGGACTTGGAGTATGGATA
TCCTTCATACACCGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCT
CCTTCTTTGCTCCCAGCCTCCCAGGCATCAATATCCTTCGACTCCATACATCCATGTACTTCCAGTGCTGGGCCGTT
ATGTGCTGCAATGTTCCTGAGGCCAGGGTCTTCAAAGCTTCCAGATCAAATAACTTCTACCTGGGCATGCTACTGCT
CATCCTCTTCCTGTCCACAATGCCTGTCTTGTACATGATCGTGTCCCTCCCACCATCTTTTGATTGTGGTCCATTCA
GTGGCAAAAATAGAATGTTTGAAGTCATTGGAGAGACCCTGGAGCACGATTTCCCAAGCTGGATGGCGAAGATCTTG
AGACAGCTTTCAAACCCTGGGCTGGTCATTGCTGTCATTTTGGTGATGGTTTTGGCCATCTATTATCTCAATGCTAC
TGCCAAGGGCCAGAAGGCAGCGAATCTGGATCTCAAAAAGAAGATGAAAATGCAAGCTTTGGAGAACAAAATGCGAA
ACAAGAAAATGGCAGCTGCACGAGCAGCTGCAGCTGCTGGTCGCCAGTAA（SEQ ID NO：1）,
The protein of its coding has amino acid sequence as follows：
MSPKKVQIKVEEKEDETEESSSEEEEEVEDKLPRRESLRPKRKRTRDVINEDDPEPEPEDEETRKAREKERRRRLKR
GAEEEEIDEEELERLKAELDEKRQIIATVKCKPWKMEKKIEVLKEAKKFVSENEGALGKGKGKRWFAFKMMMAKKWA
KFLRDFENFKAACVPWENKIKAIESQFGSSVASYFLFLRWMYGVNMVLFILTFSLIMLPEYLWGLPYGSLPRKTVPR
AEEASAANFGVLYDFNGLAQYSVLFYGYYDNKRTIGWMNFRLPLSYFLVGIMCIGYSFLVVLKAMTKNIGDDGGGDD
NTFNFSWKVFTSWDYLIGNPETADNKFNSITMNFKEAITEEKAAQVEENVHLIRFLRFLANFFVFLTLGGSGYLIFW
AVKRSQEFAQQDPDTLGWWEKNEMNMVMSLLGMFCPTLFDLFAELEDYHPLIALKWLLGRIFALLLGNLYVFILALM
DEINNKIEEEKLVKANITLWEANMIKAYNASFSENSTGPPFFVHPADVPRGPCWETMVGQEFVRLTVSDVLTTYVTI
LIGDFLRACFVRFCNYCWCWDLEYGYPSYTEFDISGNVLALIFNQGMIWMGSFFAPSLPGINILRLHTSMYFQCWAV
MCCNVPEARVFKASRSNNFYLGMLLLILFLSTMPVLYMIVSLPPSFDCGPFSGKNRMFEVIGETLEHDFPSWMAKIL
RQLSNPGLVIAVILVMVLAIYYLNATAKGQKAANLDLKKKMKMQALENKMRNKKMAAARAAAAAGRQ（SEQ ID
NO：2）.
Saltant type is c.589G>AcDNA has nucleotide sequence as follows：
ATGTCACCCAAAAAAGTACAAATCAAAGTGGAGGAAAAAGAAGACGAGACTGAGGAAAGCTCAAGTGAAGAGGAAGA
GGAGGTGGAAGATAAGCTACCTCGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTTATCAATGAGG
ATGACCCAGAACCTGAACCAGAGGATGAAGAAACAAGGAAGGCAAGAGAAAAAGAGAGGAGGAGGAGGCTAAAGAGA
GGAGCAGAAGAAGAAGAAATTGATGAAGAGGAATTGGAAAGATTGAAGGCAGAGTTAGATGAGAAAAGACAAATAAT
TGCTACTGTCAAATGCAAACCATGGAAGATGGAGAAGAAAATTGAAGTTCTCAAGGAGGCAAAAAAATTTGTGAGTG
AAAATGAAGGGGCTCTTGGGAAAGGAAAAGGAAAACGGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCA
AAATTCCTCCGTGATTTTGAGAACTTCAAAGCTGCGTGTGTCCCATGGGAAAATAAAATCAAGGCTATTGAAAGTCA
GTTTGGCTCCTCAGTGGCCTCATACTTCCTCTTCTTGAGATGGATGTATAGAGTCAATATGGTTCTCTTTATCCTGA
CATTTAGCCTCATCATGTTGCCAGAGTACCTCTGGGGTTTGCCATATGGCAGTTTACCTAGGAAAACCGTTCCCAGA
GCCGAAGAGGCATCGGCAGCAAACTTTGGTGTGTTGTACGACTTCAATGGTTTGGCACAATATTCCGTTCTCTTTTA
TGGCTATTATGACAATAAACGAACAATTGGATGGATGAATTTCAGGTTGCCGCTCTCCTATTTTCTAGTGGGGATTA
TGTGCATTGGATACAGCTTTCTGGTTGTCCTCAAAGCAATGACCAAAAACATTGGTGATGATGGAGGTGGAGATGAC
AACACTTTCAATTTCAGCTGGAAGGTCTTTACCAGCTGGGACTACCTGATCGGCAATCCTGAAACAGCAGACAACAA
ATTTAATTCTATCACAATGAACTTTAAGGAAGCTATCACAGAAGAAAAAGCAGCCCAAGTAGAAGAAAACGTCCACT
TGATCAGATTCCTGAGGTTTCTGGCTAACTTCTTCGTGTTTCTAACACTTGGAGGGAGTGGATACCTCATCTTTTGG
GCTGTGAAGCGATCCCAGGAATTTGCACAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACAT
GGTTATGTCCCTCCTAGGGATGTTCTGTCCAACATTGTTTGACTTATTTGCTGAATTAGAAGACTACCATCCTCTCA
TCGCTTTGAAATGGCTACTGGGACGCATTTTTGCTCTTCTTTTAGGCAATTTATACGTATTTATTCTTGCATTAATG
GATGAGATTAACAACAAGATTGAAGAGGAGAAGCTAGTAAAGGCCAATATTACCCTTTGGGAAGCCAATATGATCAA
GGCCTACAATGCATCATTCTCTGAAAATAGCACTGGACCACCCTTTTTTGTTCACCCTGCAGATGTACCTCGAGGAC
CTTGCTGGGAAACAATGGTGGGACAGGAGTTTGTGAGGCTGACAGTCTCTGATGTTCTGACCACCTACGTCACAATC
CTCATTGGGGACTTTCTAAGGGCATGTTTTGTGAGGTTTTGCAATTATTGCTGGTGCTGGGACTTGGAGTATGGATA
TCCTTCATACACCGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCT
CCTTCTTTGCTCCCAGCCTCCCAGGCATCAATATCCTTCGACTCCATACATCCATGTACTTCCAGTGCTGGGCCGTT
ATGTGCTGCAATGTTCCTGAGGCCAGGGTCTTCAAAGCTTCCAGATCAAATAACTTCTACCTGGGCATGCTACTGCT
CATCCTCTTCCTGTCCACAATGCCTGTCTTGTACATGATCGTGTCCCTCCCACCATCTTTTGATTGTGGTCCATTCA
GTGGCAAAAATAGAATGTTTGAAGTCATTGGAGAGACCCTGGAGCACGATTTCCCAAGCTGGATGGCGAAGATCTTG
AGACAGCTTTCAAACCCTGGGCTGGTCATTGCTGTCATTTTGGTGATGGTTTTGGCCATCTATTATCTCAATGCTAC
TGCCAAGGGCCAGAAGGCAGCGAATCTGGATCTCAAAAAGAAGATGAAAATGCAAGCTTTGGAGAACAAAATGCGAA
ACAAGAAAATGGCAGCTGCACGAGCAGCTGCAGCTGCTGGTCGCCAGTAA（SEQ ID NO：3）,
The protein of its coding has amino acid sequence as follows：
MSPKKVQIKVEEKEDETEESSSEEEEEVEDKLPRRESLRPKRKRTRDVINEDDPEPEPEDEETRKAREKERRRRLKR
GAEEEEIDEEELERLKAELDEKRQIIATVKCKPWKMEKKIEVLKEAKKFVSENEGALGKGKGKRWFAFKMMMAKKWA
KFLRDFENFKAACVPWENKIKAIESQFGSSVASYFLFLRWMYRVNMVLFILTFSLIMLPEYLWGLPYGSLPRKTVPR
AEEASAANFGVLYDFNGLAQYSVLFYGYYDNKRTIGWMNFRLPLSYFLVGIMCIGYSFLVVLKAMTKNIGDDGGGDD
NTFNFSWKVFTSWDYLIGNPETADNKFNSITMNFKEAITEEKAAQVEENVHLIRFLRFLANFFVFLTLGGSGYLIFW
AVKRSQEFAQQDPDTLGWWEKNEMNMVMSLLGMFCPTLFDLFAELEDYHPLIALKWLLGRIFALLLGNLYVFILALM
DEINNKIEEEKLVKANITLWEANMIKAYNASFSENSTGPPFFVHPADVPRGPCWETMVGQEFVRLTVSDVLTTYVTI
LIGDFLRACFVRFCNYCWCWDLEYGYPSYTEFDISGNVLALIFNQGMIWMGSFFAPSLPGINILRLHTSMYFQCWAV
MCCNVPEARVFKASRSNNFYLGMLLLILFLSTMPVLYMIVSLPPSFDCGPFSGKNRMFEVIGETLEHDFPSWMAKIL
RQLSNPGLVIAVILVMVLAIYYLNATAKGQKAANLDLKKKMKMQALENKMRNKKMAAARAAAAAGRQ（SEQ ID
NO：4）.
Saltant type is c.1171C>T cDNA has nucleotide sequence as follows：
ATGTCACCCAAAAAAGTACAAATCAAAGTGGAGGAAAAAGAAGACGAGACTGAGGAAAGCTCAAGTGAAGAGGAAGA
GGAGGTGGAAGATAAGCTACCTCGAAGAGAGAGCTTGAGACCAAAGAGGAAACGGACCAGAGATGTTATCAATGAGG
ATGACCCAGAACCTGAACCAGAGGATGAAGAAACAAGGAAGGCAAGAGAAAAAGAGAGGAGGAGGAGGCTAAAGAGA
GGAGCAGAAGAAGAAGAAATTGATGAAGAGGAATTGGAAAGATTGAAGGCAGAGTTAGATGAGAAAAGACAAATAAT
TGCTACTGTCAAATGCAAACCATGGAAGATGGAGAAGAAAATTGAAGTTCTCAAGGAGGCAAAAAAATTTGTGAGTG
AAAATGAAGGGGCTCTTGGGAAAGGAAAAGGAAAACGGTGGTTTGCATTTAAGATGATGATGGCCAAGAAATGGGCA
AAATTCCTCCGTGATTTTGAGAACTTCAAAGCTGCGTGTGTCCCATGGGAAAATAAAATCAAGGCTATTGAAAGTCA
GTTTGGCTCCTCAGTGGCCTCATACTTCCTCTTCTTGAGATGGATGTATGGAGTCAATATGGTTCTCTTTATCCTGA
CATTTAGCCTCATCATGTTGCCAGAGTACCTCTGGGGTTTGCCATATGGCAGTTTACCTAGGAAAACCGTTCCCAGA
GCCGAAGAGGCATCGGCAGCAAACTTTGGTGTGTTGTACGACTTCAATGGTTTGGCACAATATTCCGTTCTCTTTTA
TGGCTATTATGACAATAAACGAACAATTGGATGGATGAATTTCAGGTTGCCGCTCTCCTATTTTCTAGTGGGGATTA
TGTGCATTGGATACAGCTTTCTGGTTGTCCTCAAAGCAATGACCAAAAACATTGGTGATGATGGAGGTGGAGATGAC
AACACTTTCAATTTCAGCTGGAAGGTCTTTACCAGCTGGGACTACCTGATCGGCAATCCTGAAACAGCAGACAACAA
ATTTAATTCTATCACAATGAACTTTAAGGAAGCTATCACAGAAGAAAAAGCAGCCCAAGTAGAAGAAAACGTCCACT
TGATCAGATTCCTGAGGTTTCTGGCTAACTTCTTCGTGTTTCTAACACTTGGAGGGAGTGGATACCTCATCTTTTGG
GCTGTGAAGCGATCCTAGGAATTTGCACAGCAAGATCCTGACACCCTTGGGTGGTGGGAAAAAAATGAAATGAACAT
GGTTATGTCCCTCCTAGGGATGTTCTGTCCAACATTGTTTGACTTATTTGCTGAATTAGAAGACTACCATCCTCTCA
TCGCTTTGAAATGGCTACTGGGACGCATTTTTGCTCTTCTTTTAGGCAATTTATACGTATTTATTCTTGCATTAATG
GATGAGATTAACAACAAGATTGAAGAGGAGAAGCTAGTAAAGGCCAATATTACCCTTTGGGAAGCCAATATGATCAA
GGCCTACAATGCATCATTCTCTGAAAATAGCACTGGACCACCCTTTTTTGTTCACCCTGCAGATGTACCTCGAGGAC
CTTGCTGGGAAACAATGGTGGGACAGGAGTTTGTGAGGCTGACAGTCTCTGATGTTCTGACCACCTACGTCACAATC
CTCATTGGGGACTTTCTAAGGGCATGTTTTGTGAGGTTTTGCAATTATTGCTGGTGCTGGGACTTGGAGTATGGATA
TCCTTCATACACCGAATTCGACATCAGTGGCAACGTCCTCGCTCTGATCTTCAACCAAGGCATGATCTGGATGGGCT
CCTTCTTTGCTCCCAGCCTCCCAGGCATCAATATCCTTCGACTCCATACATCCATGTACTTCCAGTGCTGGGCCGTT
ATGTGCTGCAATGTTCCTGAGGCCAGGGTCTTCAAAGCTTCCAGATCAAATAACTTCTACCTGGGCATGCTACTGCT
CATCCTCTTCCTGTCCACAATGCCTGTCTTGTACATGATCGTGTCCCTCCCACCATCTTTTGATTGTGGTCCATTCA
GTGGCAAAAATAGAATGTTTGAAGTCATTGGAGAGACCCTGGAGCACGATTTCCCAAGCTGGATGGCGAAGATCTTG
AGACAGCTTTCAAACCCTGGGCTGGTCATTGCTGTCATTTTGGTGATGGTTTTGGCCATCTATTATCTCAATGCTAC
TGCCAAGGGCCAGAAGGCAGCGAATCTGGATCTCAAAAAGAAGATGAAAATGCAAGCTTTGGAGAACAAAATGCGAA
ACAAGAAAATGGCAGCTGCACGAGCAGCTGCAGCTGCTGGTCGCCAGTAA（SEQ ID NO：5）,
The protein of its coding has amino acid sequence as follows：
MSPKKVQIKVEEKEDETEESSSEEEEEVEDKLPRRESLRPKRKRTRDVINEDDPEPEPEDEETRKAREKERRRRLKR
GAEEEEIDEEELERLKAELDEKRQIIATVKCKPWKMEKKIEVLKEAKKFVSENEGALGKGKGKRWFAFKMMMAKKWA
KFLRDFENFKAACVPWENKIKAIESQFGSSVASYFLFLRWMYGVNMVLFILTFSLIMLPEYLWGLPYGSLPRKTVPR
AEEASAANFGVLYDFNGLAQYSVLFYGYYDNKRTIGWMNFRLPLSYFLVGIMCIGYSFLVVLKAMTKNIGDDGGGDD
NTFNFSWKVFTSWDYLIGNPETADNKFNSITMNFKEAITEEKAAQVEENVHLIRFLRFLANFFVFLTLGGSGYLIFW
AVKRS（SEQ ID NO：6）.
The TMC1 gene mutation body that inventor has found and SEQ ID NO：1 compares, and the TMC1 gene of the present invention has one
Missense mutation is c.589G>A (p.G197R) or a nonsense mutation are c.1171C>At least one of T (p.Q391X).Specifically,
With respect to wild type TMC1 gene, the 589th G of the cDNA of the TMC1 gene mutation body of the present invention sports A, and/or
The C of 1171 sports G.Thus, the albumen of its coding：At least there is one kind of p.G197R and p.Q391X.Need explanation
Be, in " p.Q391X " X represent termination translation, be by c.1171C>The mutation that T causes, mutant protein are 390 amino
Acid.
Further, inventor has found, when occurring p.G197R and p.Q391X mutation in TMC1 gene mutation body simultaneously,
The autosomal recessive inheritance type phonosensitive nerve deafness that detection biological sample is suffering from.There are 2 mutational sites by detection
TMC1 gene mutation body whether there is in biological sample, can accurately and effectively predict whether organism suffers from autosome hidden
Property genotype phonosensitive nerve deafness.
According to the second aspect of the invention, the present invention proposes a kind of detached polypeptide.Embodiments in accordance with the present invention,
With SEQ ID NO：2 compare, and the polypeptide has selected from following at least one mutation：p.G197R、p.Q391X.According to the present invention
Specific embodiment, the polypeptide be by the nucleic acid coding of aforementioned detached coding TMC1 gene mutation body.Biological by detecting
The polypeptide whether is expressed in sample, can effectively detect the whether susceptible phonosensitive nerve deafness disease of biological sample, it is also possible to
Whether there is in organism by detecting these polypeptides, can effectively predict the whether susceptible phonosensitive nerve deafness of organism
Disease.
The method that screening is susceptible to suffer from the biological sample of phonosensitive nerve deafness disease
According to the third aspect of the present invention, the present invention proposes a kind of life that screens and be susceptible to suffer from phonosensitive nerve deafness disease
The method of thing sample.Embodiments in accordance with the present invention, the method that the screening is susceptible to suffer from the biological sample of phonosensitive nerve deafness disease
May comprise steps of：
First, from the extraction from biological material sample of nucleic acid.Embodiments in accordance with the present invention, the type of biological sample is not
It is particularly limited, as long as sample of nucleic acid of the reflection biological sample TMC1 with the presence or absence of mutation can be extracted from the biological sample
?.Embodiments in accordance with the present invention, biological sample can be selected from blood of human body, skin, hypodermic at least one.By
This, can easily be sampled and detect such that it is able to improve the life that screening is susceptible to suffer from phonosensitive nerve deafness disease further
The efficiency of thing sample.Embodiments in accordance with the present invention, term " sample of nucleic acid " used herein above should be interpreted broadly, and which is permissible
It is any sample that can reflect TMC1 in biological sample with the presence or absence of mutation, for example, can is extracting directly from biological sample
Complete genome DNA, or the full-length genome in the part comprising TMC1 coded sequence, can be from biological sample
The total serum IgE of extraction, or the mRNA extracted from biological sample.According to one embodiment of present invention, the nucleic acid sample
This is complete genome DNA.Thus, it is possible to expand the source range that comes of biological sample, and can be while to the multiple of biological sample
Information is determined such that it is able to improve the efficiency that screening is susceptible to suffer from the biological sample of phonosensitive nerve deafness disease.In addition, according to
Embodiments of the invention, for using RNA as sample of nucleic acid, may further include from extraction from biological material sample of nucleic acid：
From extraction from biological material RNA sample, preferably RNA sample is mRNA；And based on obtained RNA sample, anti-by reverse transcription
Should, cDNA sample is obtained, obtained cDNA sample constitutes sample of nucleic acid.Thus, it is possible to be improved by the use of RNA as core further
Acid sample screening is susceptible to suffer from the efficiency of the biological sample of phonosensitive nerve deafness disease.
Next, after sample of nucleic acid is obtained, can be analyzed to sample of nucleic acid such that it is able to core obtained by determining
The nucleotide sequence of acid sample.Embodiments in accordance with the present invention, the method and apparatus of the nucleotide sequence of sample of nucleic acid obtained by determining
It is not particularly restricted.According to a particular embodiment of the invention, sequence measurement can be passed through, determines the nucleic acid sequence of sample of nucleic acid
Row.Embodiments in accordance with the present invention, the method and apparatus that can be used for being sequenced are not particularly restricted.According to the present invention's
Embodiment, can adopt second generation sequencing technologies, it would however also be possible to employ the sequencing technologies of the third generation and forth generation or more advanced.
Specific example according to the present invention, it is possible to use selected from least the one of Hiseq2000, SOLiD, 454 and single-molecule sequencing device
Plant and nucleotide sequence is sequenced.Thus, in conjunction with newest sequencing technologies, higher sequencing depth can be reached for Single locus
Degree, detection sensitivity and accuracy are greatly improved, it is thus possible to using the high flux of these sequencing devices, the spy of deep sequencing
Point, improves the efficiency tested and analyzed by sample of nucleic acid further.Thus, it is possible to improve subsequently be analyzed to sequencing data
When accuracy and the degree of accuracy.Thus, embodiments in accordance with the present invention, determine that the nucleotide sequence of sample of nucleic acid can be wrapped further
Include：First, for obtained sample of nucleic acid, nucleic acid sequencing library is built；And obtained nucleic acid sequencing library is carried out
Sequencing, to obtain the sequencing result being made up of multiple sequencing datas.According to some embodiments of the present invention, can adopt and be selected from
Hiseq2000, SOLiD, 454 and at least one of single-molecule sequencing device are sequenced to obtained nucleic acid sequencing library.
In addition, embodiments in accordance with the present invention, can screen to sample of nucleic acid, TMC1 extron is enriched with, the screening enrichment is permissible
Before sequencing library is built, during building sequencing library, or carry out after building sequencing library.According to the present invention one
Individual embodiment, for sample of nucleic acid, builds nucleic acid sequencing library and further includes：Drawn using TMC1 gene extron specificity
Thing, enters performing PCR amplification to sample of nucleic acid；And for obtained amplified production, build nucleic acid sequencing library.Thus, it is possible to
Expanded by PCR, be enriched with TMC1 gene extron such that it is able to improve the susceptible phonosensitive nerve deafness disease of screening further
The efficiency of biological sample.Embodiments in accordance with the present invention, the sequence of TMC1 gene extron specific primer are not particularly limited,
According to a preferred embodiment of the invention, these TMC1 gene extron specific primers have nucleotides sequence as shown in the table
Row, i.e. SEQ ID NO：Nucleotide sequence shown in 7-10.It is surprisingly found by the inventors that, by adopting these primers, Ke Yi
Significantly effectively complete to TMC1 extron especially c.589G in PCR reaction system>A、c.1171C>It is outer aobvious that T mutation is located
The amplification of subsequence.It should be noted that these SEQ ID NO：Nucleotide sequence shown in 7-10 is the present inventor
After arduous labor has been paid, unexpected acquisition.
According to a particular embodiment of the invention, above-mentioned phonosensitive nerve deafness disease is autosomal recessive inheritance disease
Disease.
With regard to sample of nucleic acid is directed to, method and the flow process of sequencing library are built, and those skilled in the art can be according to difference
Sequencing technologies suitably selected, with regard to the details of flow process, may refer to the such as Illumina company of manufacturer of instrument is sequenced
The code for being provided, for example, see Illumina company Multiplexing Sample Preparation Guide（Part#
1005361;Feb2010）Or Paired-End SamplePrep Guide（Part#1005063；Feb2010）, by referring to
It is incorporated into herein.Embodiments in accordance with the present invention, from the method and apparatus of extraction from biological material sample of nucleic acid, also by not especially
Limit, can be carried out using the nucleic acid extraction kit of commercialization.
It should be noted that the term " nucleotide sequence " for being used here should broadly understood, its can be to core
Acid sample carries out being sequenced the complete nucleic acid sequence information that after the sequencing data for obtaining assembled, obtains, or directly
Using the sequencing data obtained by by being sequenced to sample of nucleic acid（reads）As nucleotide sequence, as long as these nucleic acid sequences
Coded sequence containing corresponding TMC1 gene in row.
Finally, after the nucleotide sequence for determining sample of nucleic acid, by the nucleotide sequence of obtained sample of nucleic acid and SEQ
ID NO：1 sequence is mutually compared.If had c.589G in obtained nucleotide sequence>A、c.1171C>T is mutated, it indicates that
Biological sample is susceptible to suffer from phonosensitive nerve deafness disease.Thus, phonosensitive nerve is susceptible to suffer from by screening according to embodiments of the present invention
The method of the biological sample of deaf disease, can effectively screen the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease.According to
Embodiments of the invention, to nucleotide sequence and SEQ ID NO：1 method and apparatus that compares is not particularly restricted, permissible
Operated using the software of any conventional, according to the instantiation of the present invention, can be compared using SOAP software.
It should be noted that according to embodiments of the present invention, " screening is susceptible to suffer from the biological sample of phonosensitive nerve deafness disease
Method " purposes be not particularly limited, for example can serve as the screening technique of non-diagnostic purpose.
Screening is susceptible to suffer from the system of the biological sample of phonosensitive nerve deafness disease and kit
According to the fourth aspect of the present invention, the present invention proposes a kind of life that screens and be susceptible to suffer from phonosensitive nerve deafness disease
The system of thing sample.
With reference to Fig. 1, embodiments in accordance with the present invention, what the screening was susceptible to suffer from the biological sample of phonosensitive nerve deafness disease is
System 1000 includes nucleic acid-extracting apparatus 100, nucleotide sequence determining device 200 and judgment means 300.
Embodiments in accordance with the present invention, nucleic acid-extracting apparatus 100 are used for from extraction from biological material sample of nucleic acid.As front institute
State, embodiments in accordance with the present invention, the type of sample of nucleic acid is not particularly restricted, for using RNA as sample of nucleic acid, then
Nucleic acid-extracting apparatus further include RNA extraction unit 101 and reverse transcription unit 102, and wherein, extraction unit 101 is used for from life
Thing sample extraction RNA sample, reverse transcription unit 102 are connected with RNA extraction unit 101, anti-for carrying out reverse transcription to RNA sample
Should, to obtain cDNA sample, obtained cDNA sample constitutes sample of nucleic acid.
Embodiments in accordance with the present invention, nucleotide sequence determining device 200 are connected with nucleic acid-extracting apparatus 100, for core
Acid sample is analyzed, to determine the nucleotide sequence of sample of nucleic acid.As previously shown, nucleic acid can be determined using the method for sequencing
The nucleotide sequence of sample.Thus, according to one embodiment of present invention, the nucleotide sequence determining device 200 can be further
Including：Library construction unit 201 and sequencing unit 202.Library construction unit 201 is used for, for sample of nucleic acid, building nucleic acid
Sequencing library；Sequencing unit 202 is connected with library construction unit 201, for being sequenced to nucleic acid sequencing library, to obtain
The sequencing result being made up of multiple sequencing datas.As it was previously stated, can be expanded by PCR, TMC1 extron is enriched with, is carried further
High screening is susceptible to suffer from the efficiency of the biological sample of phonosensitive nerve deafness disease.Thus, library construction unit 201 can be wrapped further
Include PCR amplification module（In figure is not shown）, TMC1 extron specific primer is provided with the PCR amplification module, with facility
With TMC1 extron specific primer, enter performing PCR amplification, according to a particular embodiment of the invention, TMC1 to the sample of nucleic acid
Gene extron specific primer has such as SEQ ID NO：Nucleotide sequence shown in 7-10.Embodiments in accordance with the present invention,
Sequencing unit 202 can include at least one selected from HISEQ2000, SOLiD, 454 and single-molecule sequencing device.Thus, tie
Newest sequencing technologies are closed, higher sequencing depth can be reached for Single locus, detection sensitivity and accuracy are carried significantly
High, it is thus possible to using the high flux of these sequencing devices, the feature of deep sequencing, to improve further and sample of nucleic acid is examined
The efficiency of cls analysis.So as to improve accuracy when being subsequently analyzed and the degree of accuracy to sequencing data.
Embodiments in accordance with the present invention, judgment means 300 are connected with nucleotide sequence determining device 200, are suitable to nucleic acid sample
This nucleotide sequence is compared, so as to the nucleotide sequence based on sample of nucleic acid and SEQ ID NO：1 difference judges biological sample
Whether product are susceptible to suffer from phonosensitive nerve deafness disease.Specifically, the nucleotide sequence based on sample of nucleic acid and SEQ ID NO：1 compares,
Whether have c.589G>A、c.1171C>At least one mutation of T, judges whether biological sample is susceptible to suffer from phonosensitive nerve deafness disease
Disease.As it was previously stated, according to one embodiment of present invention, the nucleotide sequence of sample of nucleic acid and SEQ ID NO：1 compares, and has
c.589G>A、c.1171C>At least one of T is mutated, and is the instruction that biological sample is susceptible to suffer from phonosensitive nerve deafness disease.Such as front
Described, embodiments in accordance with the present invention, to nucleotide sequence and SEQ ID NO：1 equipment that compares is not particularly restricted,
Can be operated using the software of any conventional, according to the instantiation of the present invention, can be compared using SOAP software.
Thus, using the system, the aforementioned biological sample for screening and being susceptible to suffer from phonosensitive nerve deafness disease can effectively be implemented
The method of product, such that it is able to effectively screen the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease.
According to the fifth aspect of the invention, the present invention propose a kind of for screening be susceptible to suffer from phonosensitive nerve deafness disease
The kit of biological sample.Embodiments in accordance with the present invention, this are used for screening the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease
The kit of product includes：The reagent of TMC1 gene mutation body is adapted to detect for, wherein with SEQ ID NO：1 compares, the TMC1 gene
C.589G mutant has>A、c.1171C>At least one mutation of T.Using kit according to an embodiment of the invention, energy
Enough effectively screenings are susceptible to suffer from the biological sample of phonosensitive nerve deafness disease.Herein, the term for being used " is adapted to detect for
The reagent of TMC1 gene mutation body " should be interpreted broadly, you can be reagent, or the detection for detecting TMC1 encoding gene
The reagent of TMC1 mutant polypeptide, for example can be using the antibody of identification specific position.According to one embodiment of present invention,
The reagent is nucleic acid probe, thus, it is possible to efficiently screening is susceptible to suffer from the biological sample of phonosensitive nerve deafness disease.According to this
The specific embodiment of invention, above-mentioned phonosensitive nerve deafness disease are autosomal recessive disease.
It should be noted that being susceptible to suffer from the method part of the biological sample of phonosensitive nerve deafness disease in screening herein above
Described in feature and advantage, be equally applicable to screen the system of the biological sample for being susceptible to suffer from phonosensitive nerve deafness disease or
Kit, will not be described here.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.
If not specializing, the conventional hand that the technological means employed in embodiment is well known to those skilled in the art
Section, is referred to《Molecular Cloning:A Laboratory guide》The third edition or Related product are carried out, and the reagent for being adopted and product are also
Available commercial.The various processes not described in detail and method are conventional methods as known in the art, agents useful for same
Source, trade name and it is necessary its constituent person is listed, all indicates when occurring first, identical reagent used thereafter is for example nothing special
Different explanation, all identical with the content that indicates first.
Embodiment 1 determines the Disease-causing gene of phonosensitive nerve deafness disease
1st, sample collection：
Inventor collects the Chinese phonosensitive nerve deafness patient family in 2 generations, and 208 outside the family are just
The gene of ordinary person.Fig. 1 shows the family collection of illustrative plates of phonosensitive nerve deafness patient family 1.As shown in Fig. 2 wherein, just represents
The normal male sex, zero represents normal female, and ■ represents male patient, ● represent female patient.As shown in Figure 2, the family has 4 and becomes
Member, wherein second generation children are phonosensitive nerve deafness patient, and the father and mother of the first generation are normal member.
Additionally, inventor has all been carried out to all patients in the family, and temporal bone CT examination is normal, inner ear nothing is lopsided（See figure
2）, but nothing concurrency systemic disease, result above proves that the patient's illnesses in the family are phonosensitive nerve ear really
Deaf.
Inventor is flat using the full exon trapping of NimbleGen SeqCap EZ Human Exome Library v2.0
Two patients and its normal father and mother of phenotype, in conjunction with the high throughput sequencing technologies of Illumina Hiseq2000, are carried out by platform
Full extron group capture sequencing, detailed step are as follows：
The 1st, genomic DNA is broken into the fragment of 250-300bp or so at random, is subsequently connect on fragment two ends connect respectively
Head prepares Hybrid Library（Refer tohttp://www.illumina.com/The Illumina/Solexa standard of offer builds storehouse explanation
Book）.
2nd, library after purification is entered through the linear amplification of ligation-mediated PCR (LM-PCR) and capture agent
Row hybridization enrichment, carries out machine sequencing after carrying out the linear amplification of LM-PCR.Microarray dataset is Illumina Hiseq2000, reads
Length being taken for 90bp, it is 50 that the average sequencing depth of each sample is minimum ×.
3rd, the initial data obtained after being sequenced is used by Illumina basecalling Software1.7 process
SOAPaligner2.20 compares the reads after filtration to reference gene group, obtains comparing the Unique on genome
mapped reads.Using software SOAPsnp[3]Determine the genotype of target region.
After information analysis, case 1 finds 86101 SNPs（SNPs）With 6476 insertion/deletions
（Indels）, 88376 SNP and 6601 Indel of discovery of case 2.By result and dbSNP database (http:// hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp132.txt.gz), HapMap database
(ftp://ftp.ncbi.nlm.nih.gov/hapmap), thousand human genome databases (ftp:// ftp.1000genomes.ebi.ac.uk/vol1/ftp), Yan Di and Huang Di, two legendary rulers of remote antiquity's database (http://yh.genomics.org.cn/) etc.
Public database is compared, and filters out all known variations.
Phonosensitive nerve deafness belongs to often hidden and normal aobvious two kinds of hereditary patterns, and this research is made with reference to family actual conditions
Use recessive inheritance analysis of strategies.By the family result after analysis at present it has been reported that take friendship with deaf related list of genes
Collection, obtains the mutation of 10 knowns.Inventor's synthesis sequencing quality, the reference of information analysis data screening, in conjunction with recessive something lost
Arq mode：I.e. father and mother's heterozygous mutant, child's homozygous mutation or father and mother's same gene are respectively with a mutation, child while have two
Individual mutation（Compound heterozygosis）, it is found that the mutation on TMC1 is eligible.There are 2 to sport patient to have in TMC1：One missense
Mutation is c.589G>A (p.G197R), a nonsense mutation is c.1171C>T (p.Q391X), according to bioinformatics analysis, in this family
As father and mother, respectively band one is mutated, and the missense mutation in TMC1 and nonsense mutation can form compound heterozygous mutations.For determining analysis result,
Inventor carries out sanger sequence verification to two case and the normal father and mother of phenotype（It is shown in Table 3）, as a result find that two patients carry
Missense mutation on TMC1 is c.589G>A (p.G197R) and nonsense mutation are c.1171C>T (p.Q391X), mother only carry missense
Mutation, father only carry nonsense mutation, no this two kinds mutation in normal person.Because TMC1 gene is deaf Disease-causing gene, then can push away
Disconnected：Missense mutation is c.589G>A (p.G197R) and nonsense mutation are c.1171C>The compound heterozygous mutations that T (p.Q391X) is formed are
The family pathogenic mutation.
TMC1 includes 24 extrons for known deafness Disease-causing gene, the gene, and encoding proteins size is 87.78KDa.
TMC1 is transmembrane protein, participates in cochlear hair cell normal physiological function.Research shows that the mutation of the gene can cause autosome
Dominant and recessive is deaf.
TMC1 gene is just chain encoding.Wild type cDNA sequence such as SEQ ID NO：Shown in 1.
The Disease-causing gene of embodiment 2Sanger method sequence verification phonosensitive nerve deafness disease
Collection family Fig. 2 in four members peripheral blood, using QIAmp Blood kit (Qiagen, Hilden,
Germany) genomic DNA in extracting PBL, is surveyed using Qubit Fluorometer and agarose gel electrophoresis
The concentration and purity of amount DNA, each sample genomic DNA OD260/OD280 of gained are respectively positioned between 1.7-2.0, and concentration is not
Less than 50ng/ul, total amount is no less than 3 μ g.
Respectively to 2 patients（II in Fig. 2:1、Ⅱ:2）, normal person in 2 familys（I in Fig. 2:1、Ⅰ:2）And 208
Outside family, normal people's gene is detected, for the TMC1 gene compound heterozygous mutations site place primers, is passed through
PCR is expanded, product purification, and the method for sequencing obtains relevant sequence, belongs to saltant type or wild type according to sequencing results,
And whether mutation is in isolate to verify correlation with phenotype in family.Concrete grammar step is as follows：
1）DNA is extracted：
Take 2 family troubles persons respectively, in 2 familys normal person and the outer 208 Normal human peripheral's venous blood of family by
Method according to embodiment 1 is extracted genomic DNA, determines DNA content and purity.
2）Design of primers and PCR reaction
Design of primers refers to human gene data unit sequence storehouse GRCh37/hg19, is specifically shown in down.
A) primer sequence：
B) PCR reaction system：
| Distilled water | 15.3μl |
| 10X buffer solution | 2μl |
| Template | 1μl |
| Upstream medicine | 0.5μl |
| Downstream primer | 0.5μl |
| Deoxy-ribonucleoside triphosphate | 0.5μl |
| TransStart Taq polymerase | 0.2μl |
| Cumulative volume | 20μl |
C) PCR reaction condition：
| 95℃ | 5 minutes | |
| 95℃ | 45 seconds | 9 circulations；- 0.2/ circulation |
| 58℃ | 45 seconds | |
| 72℃ | 30 seconds | |
| 95℃ | 45 seconds | 34 circulations；- 0.1/ circulation |
| 55℃ | 45 seconds | |
| 72℃ | 30 seconds | |
| 72℃ | 5 minutes | |
| 10℃ | ∞ |
3）The pcr amplification product available from normal person in 2 patients, 2 familys obtained in step 2 is directly carried out DNA
Sequencing.
Mutation investigation is carried out to TMC1 gene mutation site place coded sequence and flanking sequence in patients' family member,
Patient II:1、Ⅱ:C.1171C 2 all have>T and c.589G>A compound heterozygous mutations, I:1、Ⅰ:2 are carrier, i.e., I:1 only has
c.1171C>T is mutated；Ⅰ:2 only c.589G>A（It is shown in Table 1 and Fig. 4）.
Table 1
New compound heterozygous mutations in the TMC1 gene of the present invention：Missense mutation is c.589G>A (p.G197R) and nonsense
Mutation is c.1171C>T (p.Q391X), can determine whether the ill possibility of the crowd that do not fall ill in this family, is simultaneously available for family
The assessment of offspring's illness probability and diagnosis, are that patient and family's offer genetic counselling and pre-natal diagnosis avoid carrying the deaf of the mutation
Youngster is born.
Research confirms that TMC1 gene mutation can cause autosomal dominant and recessive deafness.Our research indicate that：
TMC1c.589G>A and c.1171C>T is the mutation for causing recessive hereditary deaf.
The invention discloses deaf gene known to two kinds（TMC1）New mutation, confirm which is autosomal recessive first
The molecular disease of hereditary hearing impairment is because carrying out the examination in the site, being feminine gender in 208 Chinese normal-hearing people.Originally grind
Study carefully and the deaf gene spectrum of mutation is enriched, be to carry out hereditary hearing impairment molecular diagnosis to provide genetics foundation.
3 detection kit of embodiment
Detection kit is prepared, wherein containing detection mutation c.589G>A and c.1171C>The primer pair of T see the table below：
Person under test DNA is extracted according to the method described in embodiment 1, carried out with above-mentioned primer as template with the DNA for being extracted
PCR reacts, and PCR primer is purified according to this area conventional method, by the product sequencing of purifying.Sequence obtained by observation sequencing
Whether have c.589G>A and c.1171C>T is mutated.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy described with reference to the embodiment or example
Point is contained at least one embodiment or the example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example are necessarily referred to.And, the specific features of description, structure, material or feature can be any
One or more embodiments or example in combine in an appropriate manner.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is in the principle without departing from the present invention and objective
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
Claims (3)
1. a kind of nucleic acid of detached coding TMC1 gene mutation body, it is characterised in that with SEQ ID NO：1 compares, the core
Acid has selected from following at least one mutation：c.589G>A、c.1171C>T.
2. a kind of detached polypeptide, it is characterised in that with SEQ ID NO：2 compare, and the detached polypeptide has selected from following
At least one mutation：p.G197R、p.Q391X.
3. a kind of for screening be susceptible to suffer from phonosensitive nerve deafness disease biological sample kit, it is characterised in that contain：
The reagent of TMC1 gene mutation body is adapted to detect for, wherein with SEQ ID NO：1 compares, and the TMC1 gene mutation body has
Selected from following at least one mutation：c.589G>A、c.1171C>T,
The reagent is nucleic acid probe or primer, the nucleotide sequence such as SEQ ID NO of the nucleic acid probe or primer：7-10 institute
Show, the phonosensitive nerve deafness disease is autosomal recessive disease.
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| CN105925663A (en) * | 2016-03-30 | 2016-09-07 | 广州精科生物技术有限公司 | Kit and application thereof, and method and system for detecting area target variation |
| CN108823297A (en) * | 2018-06-13 | 2018-11-16 | 领星生物科技（上海）有限公司 | Transcript profile sequencing approach based on RT-WES |
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|---|---|---|---|---|
| CN102154509A (en) * | 2010-12-28 | 2011-08-17 | 深圳华大基因科技有限公司 | Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154509A (en) * | 2010-12-28 | 2011-08-17 | 深圳华大基因科技有限公司 | Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation |
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| Title |
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| Dominant and recessive deafness caused by mutations of a novel gene, TMC1,required for cochlear hair-cell function;Kiyoto Kurima等;《nature genetics》;20020219;第30卷;第277-283页 * |
| Four Novel TMC1 (DFNB7/DFNB11) Mutations in Turkish Patients With Congenital Autosomal Recessive Nonsyndromic Hearing Loss;E. Kalay1 等;《HUMAN MUTATION》;20051231;第1-7页 * |
| 非综合征型耳聋分子病因学研究;惠培林;《中国优秀硕士学位论文全文数据库》;20100115(第01期);第57-62页 * |
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