CN1752216A

CN1752216A - Vestibule water catheter enlarging related gene mutation and detecting method thereof

Info

Publication number: CN1752216A
Application number: CN 200510087749
Authority: CN
Inventors: 王秋菊; 沈岩; 翟所强; 赵亚丽
Original assignee: Chinese PLA General Hospital; Sinogenomax Co Ltd
Current assignee: Chinese PLA General Hospital; Sinogenomax Co Ltd
Priority date: 2005-08-08
Filing date: 2005-08-08
Publication date: 2006-03-29
Anticipated expiration: 2025-08-08
Also published as: CN100368561C

Abstract

A method for diagnosing the vestibular aqueduct expansion (deafness) by detecting if there is SLC26A4 gene mutation in the specimen to be detected, its reagent kit for detecting said gene mutation, and its application for diagnosing and treating the vestibular aqueduct expansion are disclosed.

Description

Gene mutation related with LVA (large vesti-bular aqueduct) and detection method thereof

Technical field

The present invention relates to use the detection method that SLC26A4 mutator gene diagnosis aquaductus vestibuli enlarges relative disease.

The invention still further relates to the test kit that is used to detect the SLC26A4 mutator gene.

The invention still further relates to the application of SLC26A4 mutator gene in diagnosis and/or treatment aquaductus vestibuli expansion relative disease.

Background technology

The SLC26A4 gene is positioned at 7q31, it is by location such as Everett at first, and find that this transgenation can cause a kind of recessive hereditary disease: Pendred syndrome, clinical manifestation is a thyrocele, and merges the phonosensitive nerve deafness of aquaductus vestibuli expansion or Mondini deformity (aquaductus vestibuli enlarges merging cochlea underdevelopment).Afterwards, Usami etc. showed to the examination result that the simple aquaductus vestibuli of 6 examples enlarges the SLC26A4 gene of family that the sudden change of this gene can also cause simple aquaductus vestibuli to enlarge, and its hereditary pattern also is a recessive inheritance.This shows that the SLC26A4 transgenation can cause aquaductus vestibuli to enlarge---simple property aquaductus vestibuli enlarges or merges cochlea odd-shaped aquaductus vestibuli and enlarges.It is the modal deformity of inner ear that aquaductus vestibuli enlarges, and it accounts for 1～8% in hereditary hearing impairment.Mainly showing as high frequency hearing loss clinically is main phonosensitive nerve deafness, and the deafness multilist now is that severe or utmost point severe are deaf.Morbidity is many in the Childhood, and its premorbid often has flu, fever, wound etc. to make the inducement of intracranial hypertension.

Enlarge relevant SLC26A4 gene mRNA total length 4930bp with aquaductus vestibuli, contain 21 exons, open reading frame 2343bp runs through exon 2 and exon 21.The albumen of this genes encoding---Pendrin is that a molecular weight is 86kD, contains 780 amino acid whose transmembrane proteins, belongs to ion transport Zijia family.Scott and Bidart etc. discover, Pendrin mainly is expressed in the teleblem of thyroid follicular cells and the chief cell of ductus endolymphaticus and endolymphytic sac, the transhipment of mediation iodide ion and chlorion plays a role keeping on parathyroid tissue and the endolymphatic ionic equilibrium.At Tiroidina, when the Pendrin dysfunction, it can not in time be transported to the folliculus colloid to iodide ion, makes iodide ion in the follicular cells inner accumulated, thereby can not effectively organise and combine, thereby cause generation strumous in the Pendred syndrome with thyroglobulin.Qvortrup etc. think, the similar thyroid follicle of the endolymphytic sac of rat has balance gelatinoid filling blister cavities, and the function class of endolymphytic sac chief cell is similar to thyroid follicular cells, can synthesize, secretion, absorption, digesting protein.The chlorion transit barrier changes the composition of endolymph fluid, thereby the damage sensory epithelial cell causes phonosensitive nerve deafness.And because the toxic mechanism that osmotic pressure changes and composition changes causes the membranous labyrinth structural modification.Because just stasi when ductus endolymphaticus and endolymphytic sac to 4 year old, so their expansion can cause the change of surrounding bone structure, for example change of aquaductus vestibuli or cochlea structure.

Enlarge the examination of patient SLC26A4 transgenation for aquaductus vestibuli, abroad extensively carry out, the pathogenic mutation site of report surpasses 100, comprises missense mutation, the sudden change of frameing shift, nonsense mutation and splice site sudden change, is distributed in each exon.The SLC26A4 gene has tangible heterogeneity, and not agnate its mutant form is incomplete same.Its encoded protein can not accurately navigate on the cytolemma after this transgenation, but is present in endoplasmic reticulum or the golgi body, influences ion transport.

Aquaductus vestibuli enlarges the discovery of patient SLC26A4 transgenation, not only can promote the development of the fundamental researchs such as pathogeny that this is sick, also will promote the gene diagnosis of aquaductus vestibuli expansion and carrying out of treatment greatly.General examination by antenatal diagnosis examination and newborn infant SLC26A4 transgenation, can reduce the natality that aquaductus vestibuli enlarges infant, and realize diagnosing before the disease, thereby instruct the behavior of the infant that carries Disease-causing gene, prophylactic generation, the quantity that this will significantly reduce deafness patient alleviates social pressures.The research that the end is come will be devoted to the gene therapy aspect, can predict, and gene therapy brings qualitative leap will for the treatment that comprises deaf various heredopathias.

Summary of the invention

One object of the present invention is to provide the diagnosis method that aquaductus vestibuli enlarges, whether it by detecting from existing the SLC26A4 mutator gene with mutant form of the present invention to judge that patient's aquaductus vestibuli enlarges the generation and the type of disease in patient's the sample to be tested, and then provide reference for clinical diagnosis and treatment.

Another object of the present invention is to be provided for detecting the test kit that whether has the SLC26A4 transgenation from the sample of individuality to be measured.

A further object of the present invention is to provide the application of SLC26A4 mutator gene in diagnosis and/or treatment aquaductus vestibuli expansion relative disease.

According to an aspect of the present invention, the invention provides detection method a kind of and aquaductus vestibuli expansion disease related gene.This detection method is by detecting from whether there being the SLC26A4 transgenation in the sample of individuality to be measured, and diagnose aquaductus vestibuli in this individuality to be measured to enlarge the generation and the type of disease, wherein said SLC26A4 transgenation is the 349delC heterozygous mutant that is positioned at SLC26A4 gene extron 4, be positioned at the 916917insG heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 230A＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1673A＞T heterozygous mutant of SLC26A4 gene extron 15, be positioned at the 1336C＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 1594A＞C heterozygous mutant of SLC26A4 gene extron 14, be positioned at the 1343C＞A heterozygous mutant of SLC26A4 gene extron 12, be positioned at the 1318A＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 946G＞T heterozygous mutant of SLC26A4 gene extron 8, be positioned at the 1975G＞C heterozygous mutant of SLC26A4 gene extron 17 or be positioned at the 626G of SLC26A4 gene extron 6＞A sudden change.

Described detection method can comprise the steps:

1) blood, body fluid or the tissue samples of collection individuality to be measured extract DNA;

2) be template with this DNA, carry out the PCR reaction, obtain the PCR reaction product with PCR primer at described each the mutational site design of SLC26A4 gene;

3) the PCR product that obtains is carried out direct sequencing analysis, the sequence of resulting sequence and SLC26A4 normal gene is compared, determine whether to exist the described mutational site of SLC26A4 gene;

4) judge that according to above result the aquaductus vestibuli whether individuality to be measured causes for the SLC26A4 transgenation enlarges.

Described detection method can further comprise the steps:

5) translate to determine whether to exist following amino acid mutation site by the normal reading frame: X125, X329, K77I, N558I, Q446X, S532R, S448X, K440X, G316X, V659L or G209E.

Below, specify the detection method of the above-mentioned 11 kinds of sudden changes of SLC26A4 gene respectively:

(1) about the detection method of SLC26A4 gene 349delC heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 85 normal good hearings carries out examination, find infant that aquaductus vestibuli enlarges and father thereof have the new mutational site of SLC26A4 gene (349delC, X125).Do not detect this sudden change in 85 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.This mutational site is positioned at second of Pendrin and strides the film district, and it makes aminoacid sequence frame shift since 117, and back premature termination is in 125 amino acids.

Fig. 1 is the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and sudden change is positioned at the 349th bit base, and corresponding to the 117th amino acids, after the 349 bit bases disappearance, the frameshit of base generation thereafter causes amino acid translation premature termination in 125 amino acids.The structural representation of Pendrin is seen Fig. 2.

In the present invention, can adopt the ordinary method of the check point sudden change of this area to detect the mutational site of SLC26A4 gene, for example PCR (polymerase chain reaction) sequencing, adopt the SLC26A4 gene DNA probe hybridization method of mark or with the restriction fragment length polymorphism method etc., for some specific mutational site, also can adopt the digestion with restriction enzyme reaction.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 349delC heterozygous mutant in the sample of individuality to be measured, comprise the steps:

4.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

4.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS4-F：5’-TAATCACTTTGCATGTGCTTT-3’

PDS4-R：5’-GCCAAAACACTTTAAACATGA-3’

4.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 349delC mutational site of SLC26A4 gene; Or

4.4) adopt the Eco31I restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have the 349delC mutational site of SLC26A4 gene;

4.5) judge according to above result whether this individuality to be measured exists SLC26A4 gene 349delC sudden change.

Further, this method can optionally comprise the steps:

4.6) translate to determine whether to exist X125 amino acid mutation site by the normal reading frame.

The PCR primer of Shi Yonging can be according to known nucleotide sequence in the present invention, for example design according to the sequence of document descriptions such as Coyle, be generally 15-30 base, GC content is about 45-50%, combine with terminal specificity under suitable temperature, it can utilize special computer programming.In the present invention, the standard sequence of SLC26A4 gene can reference example such as Genbank GeneID:5172.

(2) about the detection method of SLC26A4 gene 916 917insG heterozygous mutants

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 87 normal good hearings carries out examination, the infant of finding an aquaductus vestibuli expansion have the new mutational site of SLC26A4 gene (916_917insG, X329).This mutational site is positioned at the seven-transmembrane district of Pendrin, and it makes aminoacid sequence frame shift since 306, and back premature termination is in 329 amino acids.All do not find this sudden change in 87 normal controls, it is closely related to illustrate that this mutational site and aquaductus vestibuli enlarge.

Fig. 7 is the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, sudden change between the 916th and 917 bit bases, corresponding to the 306th amino acids, insert bases G between 916917 after, thereafter base generation frameshit causes amino acid translation premature termination in 329 amino acids.The structural representation of Pendrin is seen Fig. 8.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 9169 17insG heterozygous mutants in the sample of individuality to be measured, comprise the steps:

5.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

5.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS7-F：5’-CATGGTTTTTCATGTGGGAAGATTC-3’

PDS7-R：5’-AATGGCAGTAGCAATTATCG-3’

5.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 916_917insG mutational site of SLC26A4 gene;

5.4) judge according to above result whether this individuality to be measured exists SLC26A4 gene 916_917insG sudden change.

Further, this method can optionally comprise the steps:

5.5) translate to determine whether the existing 916_917insG sudden change to cause the X329 mutational site by the normal reading frame.

(3) about the detection method of SLC26A4 gene 230A＞T heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 70 normal good hearings carries out examination, find infant that aquaductus vestibuli enlarges and mother thereof have the new mutational site of SLC26A4 gene (230A＞T, K77I).This mutational site is positioned at the N-terminal of Pendrin, makes the Methionin of 77 alkalescence become nonpolar hydrophobic Isoleucine.The Study on Evolution result of its aminoacid sequence and rat mouse homologous sequence shows that this mutational site has high conservative (Figure 17).Do not detect this sudden change in 70 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.

Figure 12 is the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and sudden change is positioned at the 230th bit base, becomes Isoleucine corresponding to the 77th amino acid by Methionin.The structural representation of Pendrin is seen Figure 13, and the 77th amino acids is positioned at the C-terminal of Pendrin.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 230A＞T heterozygous mutant in the sample of individuality to be measured, comprise the steps:

6.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

6.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS3-F：5’-GGCAAAAGCATGGTAAGCAC-3’

PDS3-R：5’-AGGGTAAGCAACCATCTGTCA-3’

6.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 230A＞T mutational site of SLC26A4 gene;

6.4) judge according to above result whether this individuality to be measured exists SLC26A4 gene 230A＞T sudden change.

Further, this method can optionally comprise the steps:

6.5) translate to determine whether to exist K77I amino acid mutation site by the normal reading frame.

(4) about the detection method of SLC26A4 gene 1673A＞T heterozygous mutant

Carry out examination by the SLC26A4 gene that 70 routine aquaductus vestibulis is enlarged the control group member of family member and 87 normal good hearings, find infant that aquaductus vestibuli enlarges and mother thereof have the new mutational site of SLC26A4 gene (1673A＞T, N558I).This mutational site is positioned at the N-terminal of Pendrin, makes 558 the neutral N of polarity become nonpolar hydrophobic Isoleucine.The Study on Evolution result of homologous amino acid sequence shows that this mutational site has high conservative (Figure 24).Do not detect this sudden change in 87 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.

Figure 18 is the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and sudden change is positioned at the 1673rd bit base, becomes Isoleucine corresponding to the 558th amino acid by N.The structural representation of Pendrin is seen Figure 19, and the 558th amino acids is positioned at the carboxyl terminal of Pendrin.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 1673A＞T heterozygous mutant in the sample of individuality to be measured, comprise the steps:

7.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

7.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS15-F：5’-GCTCCTCTGAGCAACTGTGA-3’

PDS15-R：5’-GGGTCTAGGGCCTATTCCTTG-3’

7.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 1673A＞T mutational site of SLC26A4 gene; Or

7.4) adopt BseM I restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have 1673A＞T mutational site;

7.5) judge according to above result whether this individuality to be measured exists SLC26A4 gene 1673A＞T sudden change.

In another embodiment of the invention, adopt the endonuclease reaction of BseM I restriction enzyme to detect mutator gene, after above-mentioned steps 7.2 resulting PCR reaction product are carried out endonuclease reaction with BseM I, agarose electrophoresis detects, normal gene can be cut by BseM I, and mutator gene can not be cut by BseM I enzyme, determines whether to exist the mutational site thus; Judge according to detected result whether individuality to be measured is that the aquaductus vestibuli that SLC26A4 gene 1336C＞the T sudden change causes enlarges.

(5) about the detection method of SLC26A4 gene 1336C＞T heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 70 normal good hearings carries out examination, find infant that aquaductus vestibuli enlarges and father thereof have the new mutational site of SLC26A4 gene (1336C＞T, Q446X).This sudden change causes the translation premature termination of Pendrin in the 446th amino acids, lacks 335 amino acid than normal aminoacid sequence.All do not find this sudden change in 70 normal controls, it is closely related to illustrate that this mutational site and aquaductus vestibuli enlarge.Figure 25 provides the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and with red fluorescence the position, mutational site is shown, and stops since 446 amino acid whose codings.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 1336C＞T heterozygous mutant in the sample of individuality to be measured, comprise the steps:

8.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

8.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’

8.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 1336C＞T mutational site of SLC26A4 gene;

8.4) judge according to above result whether this individuality to be measured exists SLC26A4 gene 1336C＞T sudden change.

Further, this method can optionally comprise the steps:

8.5) translate to determine whether to exist Q446X amino acid mutation site by the normal reading frame.

(6) about the detection method of SLC26A4 gene 1594A＞C heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 87 normal good hearings carries out examination, find one merge infant that the undergrown aquaductus vestibuli of cochlea enlarges and mother thereof have the new mutational site of SLC26A4 gene (1594A＞C, S532R).This mutational site is positioned at the C-terminal of Pendrin, makes 532 neutral Serine become alkaline arginine.The Study on Evolution result of its aminoacid sequence and rat mouse homologous sequence shows that this mutational site has high conservative (Figure 35).Do not detect this sudden change in 87 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.

Figure 30 is the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and sudden change is positioned at the 1594th bit base, becomes arginine corresponding to the 532nd amino acid by Serine.The structural representation of Pendrin is seen Figure 31, and the 532nd amino acids is positioned at the C-terminal of Pendrin.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 1594A＞C heterozygous mutant in the sample of individuality to be measured, comprise the steps:

9.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

9.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS14-F：5’-CAAAATACGGCTGTTCCAAA-3’

PDS14-R：5’-AATGGAGCTGCTGAAACTTC-3’

9.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 1594A＞C mutational site of SLC26A4 gene;

9.4) judge according to above result whether this individuality to be measured exists SLC26A4 gene 1594A＞C sudden change.

Further, this method can optionally comprise the steps:

9.5) translate to determine whether to exist S532R amino acid mutation site by the normal reading frame.

(7) about the detection method of SLC26A4 gene 1343C＞A heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 70 normal good hearings carries out examination, find infant that aquaductus vestibuli enlarges and father thereof have the new mutational site of SLC26A4 gene (1343C＞A, S448X).This sudden change causes the translation premature termination of Pendnn in the 448th amino acids, lacks 333 amino acid than normal aminoacid sequence.All do not find this sudden change in 70 normal controls, it is closely related to illustrate that this mutational site and aquaductus vestibuli enlarge.Figure 36 provides the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and with red fluorescence the position, mutational site is shown, and stops since 448 amino acid whose codings.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 1343C＞A heterozygous mutant in the sample of individuality to be measured, comprise the steps:

10.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

10.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’

10.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 1343C＞A mutational site of SLC26A4 gene;

10.4) judge according to above result whether this individuality to be measured exists SLC26A4 gene 1343C＞A sudden change.

Further, this method can optionally comprise the steps:

10.5) translate to determine whether to exist S448X amino acid mutation site by the normal reading frame.

(8) about the detection method of SLC26A4 gene 1318A＞T heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 70 normal good hearings carries out examination, find infant that aquaductus vestibuli enlarges and mother thereof have the new mutational site of SLC26A4 gene (1318A＞T, K440X).This sudden change causes the translation premature termination of Pendrin in the 440th amino acids, lacks 341 amino acid than normal aminoacid sequence.All do not find this sudden change in 70 normal controls, it is closely related to illustrate that this mutational site and aquaductus vestibuli enlarge.Figure 41 provides the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and with red fluorescence the position, mutational site is shown, and stops since 440 amino acid whose codings.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 1318A＞T heterozygous mutant in the sample of individuality to be measured, comprise the steps:

11.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

11.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’

11.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 1318A＞T mutational site of SLC26A4 gene; Or

11.4) adopt Hind III restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have 1318A＞T mutational site;

11.5) judge according to above result whether this individuality to be measured exists SLC26A4 gene 1318A＞T sudden change.

In another embodiment of the invention, adopt the endonuclease reaction of Hind III restriction enzyme to detect mutator gene, after above-mentioned steps 11.2 resulting PCR reaction product are carried out endonuclease reaction with Hind III, agarose electrophoresis detects, normal gene can be cut by Hind III, and mutator gene can not be cut by Hind III enzyme, determines whether to exist the mutational site thus; Judge according to detected result whether individuality to be measured is that the aquaductus vestibuli that SLC26A4 gene 1318A＞the T sudden change causes enlarges (Figure 46).

(9) about the detection method of SLC26A4 gene 946G＞T heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 80 normal good hearings carries out examination, finds an aquaductus vestibuli expansion patient have the new mutational site of SLC26A4 gene (946G＞T, G316X).This sudden change causes the translation premature termination of Pendrin in the 316th amino acids, lacks 465 amino acid than normal aminoacid sequence.All do not find this sudden change in 80 normal controls, it is closely related to illustrate that this mutational site and aquaductus vestibuli enlarge.Figure 47 provides the contrast figure of SLC26A4 coding region amino acid and nucleic acid base, and with red fluorescence the position, mutational site is shown, and ends at 316 from amino acid whose coding.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 946G＞T heterozygous mutant in the sample of individuality to be measured, comprise the steps:

12.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

12.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS8-F：5’-AAGTTCAGCATTATTTGGTTGACA-3’

PDS8-R：5’-TGGTTGTTTCTTCCAGATCACA-3’

12.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 946G＞T mutational site of SLC26A4 gene; Or

12.4) adopt Nde I restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have 946G＞T mutational site;

12.5) judge according to above result whether this individuality to be measured exists SLC26A4 gene 946G＞T sudden change.

In another embodiment of the invention, adopt the endonuclease reaction of Nde I restriction enzyme to detect mutator gene, after above-mentioned steps 12.2 resulting PCR reaction product are carried out endonuclease reaction with Nde I, agarose electrophoresis detects, normal gene can be cut by Nde I, and mutator gene can not be cut by Nde I enzyme, determines whether to exist the mutational site thus; Judge according to detected result whether individuality to be measured is that the aquaductus vestibuli that SLC26A4 gene 946G＞the T sudden change causes enlarges (Figure 52).

(10) about the detection method of SLC26A4 gene 1975G＞C heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 80 normal good hearings carries out examination, find aquaductus vestibuli enlarge patient and mother thereof have the new mutational site of SLC26A4 gene (1975G＞C, V659L).This mutational site is positioned at the C-terminal of Pendrin, makes 659 Xie Ansuan become leucine.The Study on Evolution result of its aminoacid sequence and rat mouse homologous sequence shows that this mutational site has high conservative (Figure 58).Do not detect this sudden change in 80 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.

In a specific embodiment of the present invention, detect the method that whether has SLC26A4 gene 1975G＞C heterozygous mutant in the sample of individuality to be measured, comprise the steps:

13.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

13.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS17-F：5’-TCTTCGTTTAGAATGGCATCA-3’

PDS17-R：5’-ATTGCCAAAGCTCCAAATGT-3’

13.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 1975G＞C mutational site of SLC26A4 gene;

13.4) judge according to above result whether this individuality to be measured exists SLC26A4 gene 1975G＞C sudden change.

Further, this method can optionally comprise the steps:

13.5) translate to determine whether to exist V659L amino acid mutation site by the normal reading frame.

(11) are about the detection method of SLC26A4 gene 626G＞A heterozygous mutant

The SLC26A4 gene of control group member by family member that 70 routine aquaductus vestibulis are enlarged and 80 normal good hearings carries out examination, find aquaductus vestibuli enlarge patient and elder brother thereof have the new mutational site of SLC26A4 gene (626G＞A, V659L).This mutational site is positioned at second cell ring loop of Pendrin, makes 209 nonpolar hydrophobic glycine become acid L-glutamic acid.The Study on Evolution result of its aminoacid sequence and rat mouse homologous sequence shows that this mutational site has high conservative (Figure 65).Do not detect this sudden change in 80 normal peoples' the examination, illustrate that this mutational site enlarges relevant with aquaductus vestibuli.

In a specific embodiment of the present invention, detect the method that whether exists SLC26A4 gene 626G＞A to suddenly change in the sample of individuality to be measured, comprise the steps:

14.1) gather blood, body fluid or the tissue samples of individuality to be measured, extract DNA;

14.2) be template with this DNA, carry out the PCR reaction with following PCR primer, obtain the PCR reaction product;

PDS6-F：5’-GGTTTCTATCTCAGGCAAACAT-3’

PDS6-R：5’-ATTGTTTCTGGAATGAACAGTGACC-3’

14.3) the PCR reaction product that obtains is directly checked order, and the sequence of sequencing result and SLC26A4 normal gene is compared, determine whether to exist the 626G＞A mutational site of SLC26A4 gene; Or

14.4) adopt EcoR I restriction enzyme to carry out endonuclease reaction the PCR reaction product that obtains, agarose electrophoresis detects, and determines whether to have 626G＞A mutational site;

14.5) judge according to above result whether this individuality to be measured exists SLC26A4 gene 626G＞A sudden change.

In another embodiment of the invention, adopt the endonuclease reaction of EcoR I restriction enzyme to detect mutator gene, after above-mentioned steps 14.2 resulting PCR reaction product are carried out endonuclease reaction with EcoR I, agarose electrophoresis detects, mutator gene can be cut by EcoR I, and normal gene can not be cut by the EcoRI enzyme, determines whether to exist the mutational site thus; Judge according to detected result whether individuality to be measured is that the aquaductus vestibuli that SLC26A4 gene 946G＞the T sudden change causes enlarges (Figure 64).

In above-mentioned detection method at each mutational site of SLC26A4 gene, resulting PCR reaction product can also detect with hybridization probe, used hybridization probe can be and normal SLC26A4 nucleotide sequence hybridization, or with the nucleotide sequence hybridization of sudden change, or with their probe of complementary sequence hybridization.These probes can be used radio isotope, chromonic material or fluorescent substance mark, especially can utilize the allele specific probe, whether have the sudden change that has been determined with examination.

According to a further aspect in the invention, the invention provides and be used to detect the test kit that whether has the SLC26A4 transgenation from the sample of individuality to be measured, can comprise one or more combination of following several reagent in the test kit:

From testing sample, extract the reagent of DNA;

The PCR primer and the corresponding PCR reaction reagent that are used for the following mutational site of amplified sample DNA SLC26A4 gene:

Be positioned at the 349delC heterozygous mutant of SLC26A4 gene extron 4, be positioned at the 916917insG heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 230A＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1673A＞T heterozygous mutant of SLC26A4 gene extron 15, be positioned at the 1336C＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 1594A＞C heterozygous mutant of SLC26A4 gene extron 14, be positioned at the 1343C＞A heterozygous mutant of SLC26A4 gene extron 12, be positioned at the 1318A＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 946G＞T heterozygous mutant of SLC26A4 gene extron 8, be positioned at the 1975G＞C heterozygous mutant of SLC26A4 gene extron 17 or be positioned at the 626G of SLC26A4 gene extron 6＞A sudden change;

Pcr amplification product is carried out the reagent of digestion with restriction enzyme reaction; And/or

The reagent that pcr amplification product is checked order.

For example, a test kit that detects the described sudden change of SLC26A4 gene is provided in one embodiment of the invention, be equipped with in the container in order to detect the composition of the described sudden change of SLC26A4 gene, what provide simultaneously with it can be through manufacturing, use and the marketing information of the audit of medication management mechanism of government, relevant medicine or biological products.For example, after adopting pcr amplification, the direct test kit in the described mutational site of SLC26A4 gene in the test sample can contain amplimer, dNTP, be used for one or more of the archaeal dna polymerase of PCR reaction and damping fluid, endonuclease reaction and/or the required reagent of sequencing reaction etc.It is known to those skilled in the art that above component only is that schematically for example, described primer can adopt above-mentioned a pair of PDS-F and PDS-R primer, the archaeal dna polymerase of the described PCR of being used for reaction is the enzyme that can be used in pcr amplification.

The using method of described test kit mainly comprises the steps:

(1) DNA of extraction blood sample to be measured utilizes above-mentioned a pair of PDS-F and PDS-R primer, carries out pcr amplification reaction;

(2) the endonuclease reaction detection that suddenlys change; And/or

(3) directly order-checking behind the PCR reaction product purifying is with the sequence of gained and the existence in the relatively more definite mutational site of the standard sequence among the Genbank;

(4) translate to determine whether to exist described amino acid mutation site by the normal reading frame.

This test kit can detect the described mutational site of SLC26A4 gene quickly and easily, thereby is applied to sudden change detection and diagnoses and treatment thereof that aquaductus vestibuli enlarges case.

In accordance with a further aspect of the present invention, the invention provides the application of SLC26A4 mutator gene in diagnosis and/or the expansion of treatment aquaductus vestibuli, by detecting deaf occurrence cause and the type of judging this patient from the described sudden change that whether has the SLC26A4 gene in patient's the sample to be tested, and then provide reference for clinical diagnosis and treatment; In addition; aspect further clinical treatment, detected result be the described sudden change of SLC26A4 gene has taken place after, normal gene can be imported the cell that carry mutator gene and express therein; it can be recombinated with the endogenous mutator gene, thereby can carry out gene therapy.

The present invention provides the described 11 kinds of sudden changes that have the SLC26A4 gene in Chinese population first, and illustrates that this mutator gene enlarges relevant with aquaductus vestibuli.Simultaneously, whether the present invention proposes by detecting exists the SLC26A4 transgenation to judge the deaf reason that takes place and the detection method of type among the patient, this will help carrying out clinically the SLC26A4 Mutation Screening work of deafness patient, for deafness patient provides diagnosis and treatment service.

Below in conjunction with accompanying drawing,, describe in detail but do not limit the present invention by explanation to better embodiment of the present invention.

Brief description of drawings

Fig. 1～6 are the accompanying drawing about SLC26A4 gene 349delC heterozygous mutant:

Fig. 1 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 349th bit base, and corresponding to the 117th amino acids, after the 349 bit bases disappearance, the frameshit of base generation thereafter causes amino acid translation premature termination in 125 amino acids; 117～124 amino acids among Fig. 1 (amino acid of grey mark part) change, and 125 become termination signal (*), thereafter aminoacid deletion (amino acid that has underscore);

Fig. 2 is the structural representation of Pendrin, and top asterisk illustrates the position at the 117th amino acids place, and following asterisk illustrates the position that this sudden change causes translation termination, the sequential amino acid deletion after this position;

Fig. 3 shows temperature of reaction and time for PCR reaction process synoptic diagram in the SLC26A4 gene 349delC heterozygous mutant detection method of the present invention;

Fig. 4 carries out the quantitative agarose gel electrophoretogram of electrophoresis in the SLC26A4 gene 349delC heterozygous mutant detection method of the present invention to the PCR product, there is shown the fragment position of quantitative Marker;

Fig. 5 is in the inventive method, the part backward sequencing result of SLC26A4 gene extron 4, the top is the normal control sequence, the below is a mutant nucleotide sequence, the arrow indication be the mutational site (349delC, X125);

Fig. 6 is mutational site restriction enzyme digestion and electrophoresis figure in the SLC26A4 gene 349delC heterozygous mutant detection method of the present invention; among the figure from left to right: first swimming lane is that molecular weight standard, second swimming lane are that restriction enzyme mapping, the 3rd swimming lane of normal gene is the restriction enzyme mapping of heterozygous mutant gene

Restriction enzyme Eco31I can cut at 72bp and 73bp bit base, and make normal people PCR product fragment be cut into 72bp and two fragments of 117bp by original 189bp, and this restriction enzyme site disappears after the sudden change, can not cut, because this example is a heterozygous mutant in this group, so be shown as 3 bands on the running gel figure;

Fig. 7～11 are the accompanying drawing about SLC26A4 gene 916_917insG heterozygous mutant:

Fig. 7 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is between the 916th and 917 bit bases, corresponding to the 306th amino acids, after the G base was inserted in the 916_917 position, the frameshit of base generation thereafter caused amino acid translation premature termination in 329 amino acids; 306～329 amino acids among Fig. 7 (amino acid of grey mark part) change, and 329 become termination signal (*), thereafter aminoacid deletion (amino acid that has underscore);

Fig. 8 is the structural representation of Pendrin, and red asterisk illustrates the position at the 306th amino acids place, and green asterisk illustrates the position that this sudden change causes translation termination, the sequential amino acid deletion after this position;

Fig. 9 shows temperature of reaction and time for PCR reaction process synoptic diagram in the SLC26A4 gene 916_917insG heterozygous mutant detection method of the present invention;

Figure 10 carries out the quantitative agarose gel electrophoretogram of electrophoresis in the SLC26A4 gene 916_917insG heterozygous mutant detection method of the present invention to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 11 is the part sequencing result of SLC26A4 gene extron 7 in the inventive method, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (916_917insG, X329);

Figure 12～17 are the accompanying drawing about SLC26A4 gene 230A＞T heterozygous mutant:

Figure 12 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 230th bit base, becomes Isoleucine (grey mark part be base and the amino acid that suddenlys change) corresponding to the 77th amino acid by Methionin;

Figure 13 is the structural representation of Pendrin, and asterisk illustrates the 77th amino acids position, and it is positioned at the C-terminal of Pendrin.

Figure 14 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 230A of the present invention＞T heterozygous mutant detection method;

Figure 15 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 230A of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 16 is the part sequencing result of the inventive method SLC26A4 gene extron 3, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (230A＞T, K77I);

Figure 17 is a different plant species Pendrin aminoacid sequence Study on Evolution,

By the different plant species aminoacid sequence is compared (the arrow indication is the K77I site), the Pendrin of visible different plant species is Methionin (K) in this site, illustrates that the K77I sudden change is positioned at conservative region;

Figure 18～24 are the accompanying drawing about SLC26A4 gene 1673A＞T heterozygous mutant:

Figure 18 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 1673rd bit base, becomes Isoleucine (grey mark part be base and the amino acid that suddenlys change) corresponding to the 558th amino acid by N;

Figure 19 is the structural representation of Pendrin, and asterisk illustrates the 558th amino acids position, and it is positioned at the carboxyl terminal of Pendrin;

Figure 20 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 1673A of the present invention＞T heterozygous mutant detection method;

Figure 21 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 1673A of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 22 is the part sequencing result of the inventive method SLC26A4 gene extron 15, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (1673A＞T, N558I);

Figure 23 is mutational site restriction enzyme digestion and electrophoresis figure in the N558I heterozygous mutant detection method in the inventive method, among the figure from left to right: first swimming lane be molecular weight standard, second for the restriction enzyme mapping of normal gene, the 3rd swimming lane are the restriction enzyme mapping of heterozygous mutant gene,

Restriction enzyme BseM I can cut at 1677 and 1678 bit bases, and make normal people PCR product fragment be cut into 170bp and two fragments of 132bp by original 302bp, and this restriction enzyme site disappears after the sudden change, can not cut, because this organizes this example for heterozygous mutant, so be shown as 3 bands on the running gel figure;

Figure 24 is the homologous amino acid sequence Study on Evolution,

Different plant species aminoacid sequence comparison (the arrow indication is the N558I site), the aminoacid sequence of the different plant species of visible pendrinz is N (N) in this site, illustrate that the N558I sudden change is positioned at conservative region;

Figure 25～29 are the accompanying drawing about SLC26A4 gene 1336C＞T heterozygous mutant:

Figure 25 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 1336th base, corresponding to the 446th amino acid become the translation termination signal (the grey mark part be the sudden change base and amino acid), thereafter aminoacid sequence underscore mark, this section sequential amino acid deletion.

Figure 26 is the structural representation of Pendrin, and asterisk illustrates the 446th amino acids position, the Q446X sudden change takes place after, sequential amino acid deletion thereafter;

Figure 27 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 1336C of the present invention＞T heterozygous mutant detection method;

Figure 28 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 1336C of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 29 is the part backward sequencing result of the inventive method SLC26A4 gene extron 11, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (1336C＞T, Q446X);

Figure 30～35 are the accompanying drawing about SLC26A4 gene 1594A＞C heterozygous mutant:

Figure 30 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 1594th bit base, becomes arginine (grey mark part be base and the amino acid that suddenlys change) corresponding to the 532nd amino acid by Serine;

Figure 31 is the structural representation of Pendrin, and asterisk illustrates the 532nd amino acids position, and it is positioned at the C-terminal of Pendrin;

Figure 32 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 1594A of the present invention＞C heterozygous mutant detection method;

Figure 33 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 1594A of the present invention＞C heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 34 is the part sequencing result of the inventive method SLC26A4 gene extron 14, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (1594A＞C, S532R);

Figure 35 is the Study on Evolution of different plant species Pendrin aminoacid sequence,

By the different plant species aminoacid sequence is compared (the arrow indication is the S532R site), the Pendrin of visible different plant species is Serine (S) in this site, illustrates that the S532R sudden change is positioned at conservative region.

Figure 36～40 are the accompanying drawing about SLC26A4 gene 1343C＞A heterozygous mutant:

Figure 36 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 1343rd base, corresponding to the 448th amino acid become the translation termination signal (the grey mark part be the sudden change base and amino acid), thereafter aminoacid sequence underscore mark, this section sequential amino acid deletion.

Figure 37 is the structural representation of Pendrin, and asterisk illustrates the 448th amino acids position, the S448X sudden change takes place after, sequential amino acid deletion thereafter;

Figure 38 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 1343C of the present invention＞A heterozygous mutant detection method;

Figure 39 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 1343C of the present invention＞A heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 40 is the part backward sequencing result of the inventive method SLC26A4 gene extron 12, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (1343C＞A, S446X);

Figure 41～46 are the accompanying drawing about SLC26A4 gene 1318A＞T heterozygous mutant:

Figure 41 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 1318th base, corresponding to the 440th amino acid become the translation termination signal (the grey mark part be the sudden change base and amino acid), thereafter aminoacid sequence underscore mark, this section sequential amino acid deletion.

Figure 42 is the structural representation of Pendrin, and asterisk illustrates the 440th amino acids position, the K440X sudden change takes place after, sequential amino acid deletion thereafter;

Figure 43 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 1318A of the present invention＞T heterozygous mutant detection method;

Figure 44 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 1318A of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 45 is the part backward sequencing result of the inventive method SLC26A4 gene extron 12, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (1318A＞T, K440X);

Figure 46 is mutational site restriction enzyme digestion and electrophoresis figure in 1318A in the inventive method＞T heterozygous mutant detection method, among the figure from left to right: first swimming lane be molecular weight standard, second for the restriction enzyme mapping of normal gene, the 3rd swimming lane are the restriction enzyme mapping of heterozygous mutant gene,

Restriction enzyme Hind III can cut at 1318 and 1319 bit bases, and make normal people PCR product fragment be cut into 126bp and two fragments of 277bp by original 403bp, and this restriction enzyme site disappears after the sudden change, can not cut, because this organizes this example for heterozygous mutant, so be shown as 3 bands on the running gel figure;

Figure 47～52 are the accompanying drawing about SLC26A4 gene 946G＞T heterozygous mutant:

Figure 47 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 946th base, corresponding to the 316th amino acid become the translation termination signal (the grey mark part be the sudden change base and amino acid), thereafter aminoacid sequence underscore mark, this section sequential amino acid deletion.

Figure 48 is the structural representation of Pendrin, and asterisk illustrates the 316th amino acids position, the G316X sudden change takes place after, sequential amino acid deletion thereafter;

Figure 49 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 946G of the present invention＞T heterozygous mutant detection method;

Figure 50 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 946G of the present invention＞T heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 51 is the part sequencing result of the inventive method SLC26A4 gene extron 8, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (946G＞T, G316X);

Figure 52 is mutational site restriction enzyme digestion and electrophoresis figure in 946G in the inventive method＞T heterozygous mutant detection method, among the figure from left to right: first swimming lane be molecular weight standard, second for the restriction enzyme mapping of normal gene, the 3rd swimming lane are the restriction enzyme mapping of heterozygous mutant gene,

Restriction enzyme Nde I can cut at 942 and 943 bit bases, and make normal people PCR product fragment be cut into 102bp and two fragments of 204bp by original 306bp, and this restriction enzyme site disappears after the sudden change, can not cut, because this organizes this example for heterozygous mutant, so be shown as 3 bands on the running gel figure;

Figure 53～58 are the accompanying drawing about SLC26A4 gene 1975G＞C heterozygous mutant:

Figure 53 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 1975th bit base, becomes leucine (grey mark part be base and the amino acid that suddenlys change) corresponding to the 659th amino acid by Xie Ansuan;

Figure 54 is the structural representation of Pendrin, and asterisk illustrates the 659th amino acids position, and it is positioned at the carboxyl terminal of Pendrin;

Figure 55 shows temperature of reaction and time for PCR reaction process synoptic diagram in SLC26A4 gene 1975G of the present invention＞C heterozygous mutant detection method;

Figure 56 carries out the quantitative agarose gel electrophoretogram of electrophoresis in SLC26A4 gene 1975G of the present invention＞C heterozygous mutant detection method to the PCR product, there is shown the fragment position of quantitative Marker;

Figure 57 is the part sequencing result of the inventive method SLC26A4 gene extron 17, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (1975G＞C, V659L);

Figure 58 is the homologous amino acid sequence Study on Evolution,

Different plant species aminoacid sequence comparison (the arrow indication is the V659L site), the Pendrin aminoacid sequence of visible different plant species is Xie Ansuan (V) in this site, illustrate that the V659L sudden change is positioned at conservative region;

Figure 59～65 are the accompanying drawing about SLC26A4 gene 626G＞A sudden change:

Figure 59 is the contrast of SLC26A4 coding region amino acid and nucleic acid base: sudden change is positioned at the 626th bit base, becomes L-glutamic acid (grey mark part be base and the amino acid that suddenlys change) corresponding to the 209th amino acid by glycine;

Figure 60 is the structural representation of Pendrin, and asterisk illustrates the 209th amino acids position, and it is positioned at second cell ring loop of Pendrin;

Figure 61 is a PCR reaction process synoptic diagram in SLC26A4 gene 626G of the present invention＞A sudden change detection method, shows temperature of reaction and time;

Figure 62 is in SLC26A4 gene 626G of the present invention＞A sudden change detection method, and the PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis, there is shown the fragment position of quantitative Marker;

Figure 63 is the part backward sequencing result of the inventive method SLC26A4 gene extron 6, and the top is the normal control sequence, and the below is a mutant nucleotide sequence, the arrow indication be the mutational site (626G＞A, G209E);

Figure 64 is mutational site restriction enzyme digestion and electrophoresis figure in 626G in the inventive method＞A sudden change detection method; among the figure from left to right: first swimming lane is that molecular weight standard, second swimming lane are that the restriction enzyme mapping of homozygous mutation gene, restriction enzyme mapping, the 4th swimming lane that the 3rd swimming lane is the heterozygous mutant gene are the restriction enzyme mapping of normal gene

Restriction enzyme EcoR I can dna sequence dna 625 and 626 incisions, and make the original 270bp of mutator gene be cut into 77bp and two fragments of 193bp, and normal gene does not have this restriction enzyme site, can not cut, this is organized this example and is homozygous mutation, its father is the carrier of this sudden change, is shown as 2 bands and 3 bands on running gel figure respectively;

Figure 65 is the homologous amino acid sequence Study on Evolution,

Different plant species aminoacid sequence comparison (the arrow indication is the G209E site), the aminoacid sequence of the Pendrin of visible different plant species is glycine (G) in this site, illustrate that the G209E sudden change is positioned at conservative region;

The embodiment of invention

The used test materials of the present invention if no special instructions, is commercially available purchase product.

Research process of the present invention: collect aquaductus vestibuli by deaf sick outpatient service and enlarge the patient, under the voluntary prerequisite of patient and family members thereof, the 5-10ml blood sample left and taken in signature informed consent postscript, set up the patient medical history database, incidence and contact method in detail record conditions of patients, the family.Then, use phenol chloroform method for extracting extraction genomic dna, quantitatively put in storage the back ,-20 ℃ of preservations, and every part of DNA sample is all accurately corresponding to the patient clinical data of registering.Then, describe the design primer, comprise SLC26A4 gene coding region and flanking sequence thereof, carry out pcr amplification according to the document of Coyle etc.The PCR product is directly checked order: sequencing primer is identical with the pcr amplification primer, uses the ABI 3730DNA of company sequenator and carries out forward and reverse order-checking.The sequence and the standard sequence among the Genbank that obtain are compared, determine the SLC26A4 mutational site.At some mutational site of SLC26A4 gene, the PCR product is also optionally carried out endonuclease reaction: use specific restriction enzyme and normal people PCR product can be cut into two fragments, and after sudden change occurring, restriction enzyme site disappears, and can not cut.Translate to determine the mutational site of SLC26A4 gene by the normal reading frame.

The detection of SLC26A4 gene 349delC heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 85 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 4, have the new mutational site of SLC26A4 gene (349delC, X125).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.Mother of patient is the carrier of 349delC (X125) sudden change.This mutational site all is not found in 70 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 1] SLC26A4 gene 349delC heterozygous mutant

One, the preparation of object blood sample DNA to be measured

1, research object: patient's 70 examples that the aquaductus vestibuli that deaf sick outpatient service is collected enlarges, normal control 85 examples.To all its medical history of participator's probe and family history, and it is carried out a medical examination, the otology inspection comprises otoscopy, audiological evaluation.Everyone blood sample collection 5-10ml after the signature Informed Consent Form.

2, extracting genome DNA: adopt the phenol chloroform extraction method.

First day

1) anticoagulation is done 1 times of dilution with PBS.

2) the lymph parting liquid (18 ℃-28 ℃) of 2 times of volumes of adding in centrifuge tube is spread the blood that 1 times of volume of one deck has diluted, room temperature 1000xg, centrifugal 20 minutes above.

3) supernatant is abandoned in suction, and karyocyte layer in the middle of the careful sucking-off changes in the 5mlEp pipe, and 5000xg centrifugal 10 minutes, washes once with PBS then.5000xg, centrifugal 10 minutes.

4) cell is suspended from the 2mlTE damping fluid, adds 10% SDS to final concentration 0.5% (100 μ l), protein kinase k 100-200 μ g/ml (10mg/ml), 50 ℃ water-bath 3-5 hour.

5) use the phenol chloroform extraction.With isopyknic saturated phenol, add mixing, 5000xg, centrifugal 10 minutes.

6) suct clear liquid to new centrifuge tube, abandon lower sediment, add equal-volume phenol: chloroform mixture, mixing, 5000xg, centrifugal 10 minutes.

7) suct clear liquid to new centrifuge tube, abandon lower sediment, add the equal-volume chloroform: iso pentane alcohol mixture, mixing, 5000xg, centrifugal 10 minutes.

8) suct clear liquid to new centrifuge tube, abandon lower sediment, add the sodium acetate of 1/10 volume, mixing.

9) dehydrated alcohol of 2.5 times of volumes of adding.

10)-20 a ℃ deposit D NA spends the night.

Second day

11) high speed centrifugation, 10000xg, 10 minutes, 4 ℃.

12) abandon supernatant, add 75% ethanol 2ml, high speed centrifugation 10000xg, 5 minutes.

13) abandon supernatant, dry up.

14) with TE damping fluid dissolving (200 μ l TE/5ml whole bloods, 400 μ l TE/10ml whole bloods).

15) packing.1% agarose electrophoresis and spectrophotometer are quantitative.

Two, the pcr amplification of SLC26A4 gene extron 4

1, primer sequence

Upstream primer:

PDS4-F：5’-TAATCACTTTGCATGTGCTTT-3’(nt11480-nt11500)

Downstream primer:

PDS4-R：5’-GCCAAAACACTTTAAACATGA-3’(nt11648-11nt668)

Annotate: SLC26A4 gene order searching number: GeneID:5172

2, the foundation of PCR reaction system

The composition of the PCR reaction system of SLC26A4 gene sees Table 1.

The PCR reaction system of table 1 SLC26A4 gene

Title	Original liquid concentration	Application of sample amount (μ l)	The system final concentration
Title	Original liquid concentration	Application of sample amount (μ l)	The system final concentration	Damping fluid	10×	2.5	1×
dNTP	2.SmM	2.0	0.2mM	Damping fluid	10×	2.5	1×
dNTP	2.SmM	2.0	0.2mM	Primer	10μM
	1	0.4μM		Primer	10μM
	1	0.4μM	The Taq enzyme	5units/μl	0.5	0.1units/μl
Template (DNA that extracts in the step 1)	100ng/μl	1	The Taq enzyme	5units/μl	0.5	0.1units/μl	4ng/μl
Template (DNA that extracts in the step 1)	100ng/μl	1	The total reaction system	ddH ₂O polishing to 25 μ l			4ng/μl

Wherein damping fluid, dNTP, Taq enzyme are buied from precious biotech firm.Primer is given birth to worker company by Shanghai and is synthesized.Reaction conditions: PCR is reflected on ABI company 9700 thermal cyclers and carries out, and reaction process (comprising temperature and time) as shown in Figure 3.

Three, the purifying of PCR product

1, in 96 orifice plates that the PCR product is housed, adds 50 μ l sterilized waters, mixing.

2, it is transferred in the Millipore purifying plate, be put on the vacuum pump suction filtration about 3 minutes, see in the purifying plate not having water to get final product.

3, the deionized water that adds 50 μ l in the purifying plate once more continues suction filtration, in the purifying plate, do not have water till.

4, the purifying plate is taken off from vacuum pump, in plate, add the deionized water of 20 μ l, static 15 minutes, shook again 15 minutes, be drawn onto then in new 96 orifice plates.

5, be kept in-20 ℃ of refrigerators.

Four, electrophoresis is quantitative

1, sample is prepared

PCR product (2 μ l)+sample-loading buffer (6 μ l) is totally 8 μ l * 96

Get one 96 hole point templates, every hole adds sample damping fluid 6 μ l earlier, and from the chamber plate that the PCR product is housed, PCR product (2 μ l) is shifted out with the volley of rifle fire in every hole, transfers on the point template, mixing, centrifugal (chamber plate hole number corresponding one by one with 96 hole point templates).

2, flow process

1) joins glue (1.0% agarose): take by weighing the 3.0g agarose, be suspended in (500ml Erlenmeyer flask) among 300ml 0.5 * TBE.

2) colloidal sol: high fire is heated to and boils in the microwave oven, and constantly boiling number minute is noted not boiling, and takes out mixing therebetween.

3) cool glue: treat that glue dissolves fully, from microwave oven, take out that cool to about 60 ℃, (about 10 μ l 10mg/ml), shake up to add 1 EB.

4) shop glue: obturage with adhesive plaster in dull and stereotyped two ends, the 250ml glue is all poured into flat board, inserts comb scale.

5) gluing: flat board is put in the electrophoresis chamber that fills electrophoresis liquid (0.5 * TBE, liquid level apart from glue face 1 to 2mm), pulled up comb scale.

6) application of sample:, add the DNA standard substance with single rifle at last with volley of rifle fire form application of sample in accordance with regulations.

7) walk glue: cover the electrophoresis chamber lid, check positive and negative level, open electrophoresis apparatus, regulate electrophoretic voltage.

8) quantitative: as to walk from well 1.5 to 2cm places when tetrabromophenol sulfonphthalein, close electrophoresis apparatus, carefully get glue, put into pickup camera and take a picture, carry out quantitatively.Quantitatively marker is DL 2000, as shown in Figure 4, through behind the electrophoresis, 6 bands is arranged as seen, and fragment length is respectively 2000bp, 1000bp, and 750bp, 500bp, 250bp, 100bp, the total concn of DL2000 is 300ng/5 μ l.Get 5ul DL2000 during electrophoresis, therefore the content of every band is 50ng.Get 3 μ l (PCR product)+5 μ l (sample-loading buffer) during PCR product electrophoresis and carry out electrophoresis.The content of relatively judging the PCR product according to the gray-scale value of gray-scale value behind the PCR product electrophoresis and DL2000.

Five, the direct order-checking of SLC26A4 gene PCR amplified production

1, the purity of PCR product D NA template and consumption requirement

DNA purity: OD260/OD280=1.6～2.0.

DNA concentration: PCR product 10ng/ μ L.

The DNA consumption:

The PCR product

100-200bp 1-3ng

200-500bp 3-10ng

500-1000bp 5-20ng

1000-2000bp 10-40ng

＞2000bp 40-100ng

2, sequencing reaction

(1) the required reagent of sequencing reaction should be fresh preparation, need can use after autoclaved reagent must be sterilized.The required equipment of sequencing reaction (first-class as 384 orifice plates, tip) should be cleaning sterile equally.

(2) in order to guarantee the fresh of sample and reaction reagent that check order, should be during application of sample in operation on ice.

(3) present reaction system is 5 μ l, and all ingredients add-on sees Table 2.

The sequencing reaction system of table 2 SLC26A4 gene PCR amplified production

Template	The PCR product of purifying (preparation among the embodiment 2)	200-500bp	3-10ng
		200-500bp	3-10ng	500-1000bp	5-20ng
		1000-2000bp	40-100ng	500-1000bp	5-20ng
		1000-2000bp	40-100ng	BigDye v3.1 ^*		0.25μl
5 * damping fluid (Tris-HCL pH9.0, MgCl ₂)			0.875μl(Kit)	BigDye v3.1 ^*		0.25μl
5 * damping fluid (Tris-HCL pH9.0, MgCl ₂)			0.875μl(Kit)	Primer		3.2pmol
Mend to 5 μ l with sterilized water				Primer		3.2pmol

^*A kind of fluorescence dye that is used for sequencing reaction that BigDye 3.1 produces for u.s.a. applied biosystem company (ABI).

(4) sample is put on the PCR instrument, and the process of the reaction of doing sees Table 3.

The sequencing reaction process of table 3 SLC26A4 gene PCR amplified production

Step	Effect


	1	96℃，2min.
2	1	96℃，2min.	Repeat 30 circulations of following process: ● 96 ℃, 10sec; ● 50 ℃, 5sec; ● 60 ℃, 4min.
2	3	Remain on 4 ℃, up to purifying

(5) sample that has reacted will in time take off from the PCR instrument, and the sample that will carry out purifying in the short period of time is positioned in 4 ℃ of C refrigerators, surpass more than one day could purifying sample to be positioned over-20 ℃ of refrigerators freezing.

3, the purifying of sequencing reaction thing and order-checking

(1) in every hole, adds 20 μ l, 80% ethanol, the centrifugal 30min of 4000rpm; Sample panel is placed on the paper handkerchief of rolling well, gets rid of in whizzer, speed can not surpass 1000rpm when getting rid of;

(2) in every hole, add 30 μ l, 70% ethanol, 4, the centrifugal 10min of 000rpm gets rid of;

(3) repeat the 2nd operation that goes on foot;

(4) repeat the 2nd operation that goes on foot;

(5) sample panel is put in the clean drawer the dry 30min of lucifuge;

(6) add 5 μ l methane amides, the envelope film is in the centrifugal being placed on-20 ℃ refrigerator;

(7) go up the preceding 95 ℃ of sex change of machine 5 minutes, placed 2 minutes on ice, sample is gone up in centrifugal back.

Sequencer map is seen shown in Figure 5.

Six, the mutational site endonuclease reaction detects

The composition of endonuclease reaction system sees Table 4, and reaction conditions is 37 ℃ of water-baths 4 hours.Use 2% agarose gel electrophoresis and detect, method is quantitative referring to electrophoresis among the embodiment 2.The endonuclease reaction electrophorogram as shown in Figure 6.

Restriction analysis is to have found a restriction enzyme Eco31I restriction enzyme site in this mutational site, when not undergoing mutation, restriction enzyme Eco31I can cut at 352 and 353 bit bases, and make PCR product fragment be cut into 72bp and two fragments of 117bp by original 189bp, and this restriction enzyme site disappears after the sudden change, can not cut.Because this patient is a heterozygous mutant, so be shown as 3 bands on the running gel figure.

Table 4 endonuclease reaction system

Reagent

Sample size

Eco31I	1μl
Eco31I	1μl	10×L Buffer	2μl
The PCR product	6μl	10×L Buffer	2μl
The PCR product	6μl	ddH ₂O	Up to 20μl

Eco31I restriction enzyme and 10 * L Buffer purchase in precious biotech firm, and the PCR product is the pcr amplification product among the embodiment 1.

[embodiment 2] mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (349delC, X125).

Study on Evolution: 349delC sudden change is positioned at second of Pendrin and strides the film district, and it makes aminoacid sequence from 117 living frameing shift of starting, and back premature termination is in 125 amino acids.This sudden change must influence the 26S Proteasome Structure and Function of Pendrin.

The detection of SLC26A4 gene 916_917insG heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 87 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 7, have the new mutational site of SLC26A4 gene (916_917insG, X329).This sudden change causes aminoacid sequence from 306 living frameing shift of starting, and back premature termination is in 329 amino acids.Do not find this sudden change at 87 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 3] SLC26A4 gene 916_917insG heterozygous mutant

Basic skills and step are carried out pcr amplification at SLC26A4 gene extron 7 coding regions and flanking sequence thereof referring to embodiment 1 in concrete method, the PCR primer sequence of use is:

Upstream primer:

PDS7-F：5’-CATGGTTTTTCATGTGGGAAGATTC-3’(nt22454-nt22478)

Downstream primer:

PDS7-R：5’-AATGGCAGTAGCAATTATCG-3’(nt22822-nt22841)

PCR is reflected on ABI company 9700 thermal cyclers and carries out, and reaction process (comprising temperature and time) as shown in Figure 9.The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 10; Sequencer map is seen shown in Figure 11.

[embodiment 4] mutational site Study on Evolution

Mutation analysis: the SeqmanTM software of using in DNAStar5.0 (Lasergene inc.) software package carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (916_917insG, X329).

Study on Evolution: 916_917insG (X329) is positioned at the 7th of Pendrin and strides the film district, and this sudden change makes aminoacid sequence from 306 living frameing shift of starting, and back premature termination is in 329 amino acids.Thereby cause the change of Pendrin 26S Proteasome Structure and Function.

The detection of SLC26A4 gene 230A＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 70 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 3, have the new mutational site of SLC26A4 gene (230A＞T, K77I).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.Mother of patient is the carrier of 230A＞T (K77I) sudden change.This mutational site all is not found in 70 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 5] SLC26A4 gene 230A＞T heterozygous mutant

Basic skills and step are referring to embodiment 1, and wherein the pcr amplification at SLC26A4 gene extron 3 uses following primer:

Upstream primer:

PDS3-F：5’-GGCAAAAGCATGGTAAGCAC-3’(nt2497-nt2516)

Downstream primer:

PDS3-R：5’-AGGGTAAGCAACCATCTGTCA-3’(nt2876-nt2896)

The PCR reaction system as shown in figure 14; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 15; Sequencer map is seen Figure 16.

[embodiment 6] mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (230A＞T, K77I).

Study on Evolution: 230A＞T sudden change is positioned at the N-terminal of Pendrin, make the Methionin of alkalescence become nonpolar hydrophobic Isoleucine, human Pendrin sequence shows that with rat and mouse homologous amino acid sequence comparison result this sudden change is positioned at conserved regions (Figure 17).

The detection of SLC26A4 gene 1673A＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 87 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 15, have the new mutational site of SLC26A4 gene (1673A＞T, N558I).Mother of patient is the carrier of 1673A＞T (N558I) sudden change.This mutational site all is not found in 87 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 7] SLC26A4 gene 1673A＞T heterozygous mutant

Basic skills and step are referring to embodiment 1.Pcr amplification primer sequence at SLC26A4 gene extron 15 is:

Upstream primer:

PDS15-F：5’-GCTCCTCTGAGCAACTGTGA-3’(nt39342-nt39351)

Downstream primer:

PDS15-R：5’-GGGTCTAGGGCCTATTCCTG-3’(nt39624-nt39643)

The PCR reaction process as shown in figure 20; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 21; Sequencer map is seen Figure 22.

The mutational site endonuclease reaction detects

The composition of endonuclease reaction system sees Table 5, and reaction conditions is 55 ℃ of water-baths 4 hours.The application agarose electrophoresis detects, and method is quantitative referring to electrophoresis among the embodiment 2.The endonuclease reaction electrophorogram as shown in figure 23.

Restriction analysis is to have found a restriction enzyme BseMI restriction enzyme site in this mutational site, when not undergoing mutation, restriction enzyme BseMI can cut at 1677 and 1678 bit bases, and make PCR product fragment be cut into 170bp and two fragments of 132bp by original 302bp, and this restriction enzyme site disappears after the sudden change, can not cut.Because this example is a heterozygous mutant, so be shown as 3 bands on the running gel figure.

Table 5 endonuclease reaction system

Reagent	Sample size
Reagent	Sample size	BseMI	0.1μl
10×L Buffer	2μl	BseMI	0.1μl
10×L Buffer	2μl	The PCR product	1μl
ddH ₂O	Up to 20μl	The PCR product	1μl

BseMI restriction enzyme and 10 * L Buffer purchase in crystalline substance U.S. biotech firm.

[embodiment 8] mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (1673A＞T, N558I).

Study on Evolution: 1673A＞T sudden change is positioned at the C-terminal of Pendrin, makes polarity neutral N become nonpolar hydrophobic Isoleucine, and homologous gene aminoacid sequence evolution Journal of Sex Research result shows that this sudden change is positioned at high conservative region (Figure 24).

The detection of SLC26A4 gene 1336C＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 70 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 11, have the new mutational site of SLC26A4 gene (1336C＞T, Q446X).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.Patient's father is the carrier of 1336C＞T (Q446X) sudden change.This mutational site all is not found in 70 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection of [embodiment 9] SLC26A4 gene 1336C＞T heterozygous mutant

Basic skills and step are referring to embodiment 1.Pcr amplification primer sequence at SLC26A4 gene extron 11:

Upstream primer:

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’(nt33698-nt33717)

Downstream primer:

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’(nt34079-n t34100)

The PCR reaction process as shown in figure 27; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 28; Sequencer map is seen Figure 29.

[embodiment 10] mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (1336C＞T, Q446X).

Study on Evolution: 1336C＞T sudden change causes that the aminoacid sequence premature termination that makes the SLC26A4 genes encoding in 446 amino acids, forms jejune Pendrin, and this change must cause that the 26S Proteasome Structure and Function of Pendrin is unusual.

The detection of SLC26A4 gene 1594A＞C heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination, finds that one merges the undergrown aquaductus vestibuli expansion of cochlea patient have SLC26A4 gene 1594A＞C (S532R) heterozygous mutant on exon 14 to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 70 routine normal controls.Mother of patient is the carrier of 1594A＞C (S532R) sudden change.This mutational site all is not found in 87 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection method of [embodiment 11] SLC26A4 gene 1594A＞C heterozygous mutant

Method is referring to embodiment 1; Pcr amplification primer sequence at SLC26A4 gene extron 14:

Upstream primer:

PDS14-F：5’-CAAAATACGGCTGTTCCAAA-3’(nt37358-nt37377)

Downstream primer:

PDS14-R：5’-AATGGAGCTGCTGAAACTTC-3’(nt37524-nt37543)

The PCR reaction process shown in figure 32; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 33; Sequencer map is seen Figure 34.

[embodiment 12] mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (1594A＞C, S532R).

Study on Evolution: 1594A＞C sudden change is positioned at the C-terminal of Pendrin, makes neutral Serine change the arginine of alkalescence into, shows that with rat and mouse homologous amino acid sequence comparison result this sudden change is positioned at conserved regions (Figure 35).

The detection of SLC26A4 gene 1343C＞A heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by should the disease core families to 70 examples and the examination of each exon of coding region of the SLC26A4 gene of 70 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 12, have the new mutational site of SLC26A4 gene (1343C＞A, S448X).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.Patient's father is the carrier of 1343C＞A (S448X) sudden change.This mutational site all is not found in 70 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection of [embodiment 13] SLC26A4 gene 1343C＞A heterozygous mutant

Upstream primer:

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’(nt33698-nt33717)

Downstream primer:

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’(nt34079-n t34100)

The PCR reaction process as shown in figure 38; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 39; Sequencer map is seen Figure 40.

[embodiment 14] mutational site Study on Evolution

Mutation analysis: use the Seqman in DNAStar5.0 (Lasergene inc.) software package ^TMSoftware carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (1343C＞A, S448X).

Study on Evolution: 1343C＞A sudden change causes that the aminoacid sequence premature termination that makes the SLC26A4 genes encoding in 448 amino acids, forms jejune Pendrin, and this change must cause that the 26S Proteasome Structure and Function of Pendrin is unusual.

The detection of SLC26A4 gene 1318A＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 70 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 11, have the new mutational site of SLC26A4 gene (1318A＞T, K440X).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.Mother of patient is the carrier of 1318A＞T (K440X) sudden change.This mutational site all is not found in 70 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection of [embodiment 15] SLC26A4 gene 1318A＞T heterozygous mutant

Upstream primer:

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’(nt33698-nt33717)

Downstream primer:

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’(nt34079-nt34100)

The PCR reaction process as shown in figure 43; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 44; Sequencer map is seen Figure 45.

The mutational site endonuclease reaction detects

The composition of endonuclease reaction system sees Table 6, and reaction conditions is 37 ℃ of water-baths 2 hours.The application agarose electrophoresis detects, and method is quantitative referring to electrophoresis among the embodiment 2.The endonuclease reaction electrophorogram as shown in figure 46.

Restriction analysis is to have found a restriction enzyme Hind III restriction enzyme site in this mutational site, when not undergoing mutation, restriction enzyme Hind III can cut at 1318 and 1319 bit bases, and make PCR product fragment be cut into 126bp and two fragments of 277bp by original 403bp, and this restriction enzyme site disappears after the sudden change, can not cut.Because this example is a heterozygous mutant, so be shown as 3 bands on the running gel figure.

Table 6 endonuclease reaction system

Reagent	Sample size
Reagent	Sample size	Hind III	1μ1
10×L Buffer	2μl	Hind III	1μ1
10×L Buffer	2μl	The PCR product	4μl
ddH ₂O	Up to 20μl	The PCR product	4μl

^*Hind III restriction enzyme and 10 * L Buffer purchase in precious biotinylated biomolecule Engineering Co., Ltd.

[embodiment 16] mutational site Study on Evolution

Mutation analysis: the SeqmanTM software of using in DNAStar5.0 (Lasergene inc.) software package carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, has found mutational site 1318A＞T (K440X).

Study on Evolution: 1318A＞T sudden change causes that the aminoacid sequence premature termination that makes the SLC26A4 genes encoding in 440 amino acids, forms jejune Pendrin, and this change must cause that the 26S Proteasome Structure and Function of Pendrin is unusual.

The detection of SLC26A4 gene 946G＞T heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 80 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 8, have the new mutational site of SLC26A4 gene (946G＞T, G316X).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.This mutational site all is not found in 80 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection of [embodiment 17] SLC26A4 gene 946G＞T heterozygous mutant

Basic skills and step are referring to embodiment 1.Pcr amplification primer sequence at SLC26A4 gene extron 8:

Upstream primer:

PDS8-F：5’-AAGTTCAGCATTATTTGGTTGACA-3’(nt22743-nt22766)

Downstream primer:

PDS8-R：5’-TGGTTGTTTCTTCCAGATCACA-3’(nt23144-nt23167)

The PCR reaction process as shown in figure 49; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 50; Sequencer map is seen Figure 51.

The mutational site endonuclease reaction detects

The composition of endonuclease reaction system sees Table 8, and reaction conditions is 37 ℃ of water-baths 2 hours.The application agarose electrophoresis detects, and method is quantitative referring to electrophoresis among the embodiment 2.The endonuclease reaction electrophorogram is shown in Figure 52.

Restriction analysis is to have found a restriction enzyme Nde I restriction enzyme site in this mutational site, when not undergoing mutation, restriction enzyme Nde I can be 942 and 943 incisions, PCR product fragment is cut into 102bp and two fragments of 204bp by original 306bp, and this restriction enzyme site disappears after the sudden change, can not cut.Because this example is a heterozygous mutant, so be shown as 3 bands on the running gel figure.

Table 7 endonuclease reaction system

Reagent	Sample size
Reagent	Sample size	Nde I	1μl
10×L Buffer	2μl	Nde I	1μl
10×L Buffer	2μl	The PCR product	4μl
ddH ₂O	Up to 20μl	The PCR product	4μl

^*Nde I restriction enzyme and 10 * L Buffer purchase in precious biotinylated biomolecule Engineering Co., Ltd.

[embodiment 18] mutational site Study on Evolution

Mutation analysis: the SeqmanTM software of using in DNAStar5.0 (Lasergene inc.) software package carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (946G＞T, G316X).

Study on Evolution: 946G＞T sudden change causes that the aminoacid sequence premature termination that makes the SLC26A4 genes encoding in 316 amino acids, forms jejune Pendrin, and this change must cause that the 26S Proteasome Structure and Function of Pendrin is unusual.

The detection of SLC26A4 gene 1975G＞C heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 80 routine normal controls, find aquaductus vestibuli enlarge the patient on exon 17, have the new mutational site of SLC26A4 gene (1975G＞C, V659L).This patient is a compound heterozygote, meets the pattern of autosomal recessive inheritance, and there is another sudden change in the allelotrope of its SLC26A4.Mother of patient is the carrier of 1975G＞C (V659L) sudden change.This mutational site all is not found in 80 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection of [embodiment 19] SLC26A4 gene 1975G＞C heterozygous mutant

Basic skills and step are referring to embodiment 1.Pcr amplification primer sequence at SLC26A4 gene extron 17:

Upstream primer:

PDS17-F：5’-TCTTCGTTTAGAATGGCATCA-3’(nt41182-nt41202)

Downstream primer:

PDS17-R：5’-ATTGCCAAAGCTCCAAATGT-3’(nt41449-nt41468)

The PCR reaction process is shown in Figure 55; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 56; Sequencer map is seen Figure 57.

[embodiment 20] mutational site Study on Evolution

Mutation analysis: the SeqmanTM software of using in DNAStar5.0 (Lasergene inc.) software package carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, has found mutational site 1975G＞C (V659L).

Study on Evolution: 1975G＞C sudden change makes 659 figured silk fabrics amino acid of SLC26A4 genes encoding become leucine, this sudden change is positioned at the C-terminal of Pendrin, show that with rat and mouse homologous amino acid sequence comparison result this sudden change is positioned at conserved regions (Figure 58).

The detection of SLC26A4 gene 626G＞A heterozygous mutant

The patient who enlarges with aquaductus vestibuli is as research object, by examination to each exon of coding region of the SLC26A4 gene of 70 example these patient and his family set members and 80 routine normal controls, two aquaductus vestibulis finding a family enlarge patients on exon 6, have the new mutational site of SLC26A4 gene (626G＞A, G209E).These two patients are homozygote.Patient's parents are the carrier of 626G＞A (G209E) sudden change.This mutational site all is not found in 80 control groups, illustrate this mutational site and aquaductus vestibuli enlarge be divided into from.

The detection of [embodiment 21] SLC26A4 gene 626G＞A heterozygous mutant

Basic skills and step are referring to embodiment 1.Pcr amplification primer sequence at SLC26A4 gene extron 6:

Upstream primer:

PDS6-F：5’-GGTTTCTATCTCAGGCAAACAT-3’(nt14259-nt14280)

Downstream primer:

PDS6-R：5’-ATTGTTTCTGGAATGAACAGTGACC-3’(nt14504-nt14528)

The PCR reaction process is shown in Figure 61; The PCR product is carried out the quantitative agarose gel electrophoretogram of electrophoresis see Figure 62; Sequencer map is seen Figure 63.

The mutational site endonuclease reaction detects

The composition of endonuclease reaction system sees Table 8, and reaction conditions is 37 ℃ of water-baths 2 hours.The application agarose electrophoresis detects, and method is quantitative referring to electrophoresis among the embodiment 2.The endonuclease reaction electrophorogram is shown in Figure 64.

Restriction analysis is to have found a restriction enzyme EcoR I restriction enzyme site in this mutational site, when undergoing mutation, restriction enzyme EcoR I can be in 625 and 626 incisions of dna sequence dna, PCR product fragment is cut into 77bp and two fragments of 193bp by original 270bp, and normal sequence does not have this restriction enzyme site, can not cut.Because this example is a homozygous mutation, so be shown as 2 bands on the running gel figure.

Table 8 endonuclease reaction system

Reagent	Sample size
Reagent	Sample size	EcoR I	1μl
10×L Buffer	2μl	EcoR I	1μl
10×L Buffer	2μl	The PCR product	4μl
ddH ₂O	Up to 20μl	The PCR product	4μl

^*EcoR I restriction enzyme and 10 * L Buffer purchase in precious biotinylated biomolecule Engineering Co., Ltd.

[embodiment 22] mutational site Study on Evolution

Mutation analysis: the SeqmanTM software of using in DNAStar5.0 (Lasergene inc.) software package carries out the sequence comparative analysis.The standard sequence that sequence that order-checking is obtained and NCBI retrieve is compared, and finds out mutant nucleotide sequence, found the mutational site (626G＞A, G209E).

Study on Evolution: 626G＞A sudden change makes 209 nonpolar hydrophobic glycine of SLC26A4 genes encoding become tart L-glutamic acid, and this sudden change occurs in ring loop in second cell.Show that with rat and mouse homologous amino acid sequence comparison result this sudden change is positioned at conserved regions (Figure 65).

Detect test kit and application thereof that aquaductus vestibuli enlarges relevant SLC26A4 transgenation

[embodiment 23]

The test kit that detects the SLC26A4 transgenation can be prepared into the independent test kit that detects each single mutational site as required, also can be prepared into the test kit that detects a plurality of or all SLC26A4 gene mutation sites, be that example describes with the test kit that detects 11 mutational sites of the present invention below:

1, test kit contains:

(1) amplification primers

Aforementioned is 9 pairs of primers that detect 11 mutational sites designs of SLC26A4 gene, perhaps other spendable primers at these 11 mutator genes designs;

Annotate: SLC26A4 gene order searching number: GeneID:5172

Other reagent that is included in alternatively in the test kit comprises following reagent:

(2) pcr amplification Taq enzyme 5U/ μ l

(3) 10 * damping fluids (contain 15ml MgCl ₂)

(4)dNTP 2.5mM

(5) Eco31 I restriction enzyme; BseM I restriction enzyme; Hind III restriction enzyme; Nde I restriction enzyme; Or EcoR I restriction enzyme

(6)10×L Buffer

(7)Big-Dye mix

2, using method

Mainly comprise the steps:

1) pcr amplification

Gather the sample (blood, body fluid, tissue etc.) of individuality to be measured, extract DNA, carry out pcr amplification respectively with above-mentioned primer at each mutator gene;

2) PCR product purification

The MultiScreen-PCR plate that will contain the PCR product vacuumizes, and adds deionized water, leaves standstill, and the MultiScreen-PCR plate is placed on the mixing tank shake subsequently, is transferred in 96 orifice plates of another cleaning after the PCR product behind the purifying dissolves again;

3) endonuclease reaction

Will be at the 349delC mutational site, at 1673A＞T mutational site, at 1318A＞T mutational site, carry out endonuclease reaction according to the endonuclease reaction system of

embodiment

1,7,15,17 and 21 respectively at 946G＞T mutational site or at the amplified production in 626G＞A mutational site, judge whether to exist above-mentioned five kinds of sudden changes with reference to Fig. 6, Figure 23, Figure 46, Figure 52 and Figure 64.

4) sequencing reaction and checking

The PCR product at each mutational site of above-mentioned amplification is carried out sequencing reaction with the PCR primer as sequencing primer, on the ABI9700 thermal cycler, carry out sequencing reaction.After reaction finished, extension products was splined on ABI PRISM 3730DNA sequenator.Resulting order-checking collection of illustrative plates is analyzed, relatively can be found the mutational site with the standard sequence among the Genbank;

4) translate to determine whether to exist corresponding amino acid mutation site by the normal reading frame.

Concrete grammar is referring to the detailed description among the embodiment of front.

Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.

Claims

1, a kind of detection method, by detecting from whether there being the SLC26A4 transgenation in the sample of individuality to be measured, and diagnose aquaductus vestibuli in this individuality to be measured to enlarge the generation and the type of disease, wherein said SLC26A4 transgenation is the 349delC heterozygous mutant that is positioned at SLC26A4 gene extron 4, be positioned at the 916_917insG heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 230A＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1673A＞T heterozygous mutant of SLC26A4 gene extron 15, be positioned at the 1336C＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 1594A＞C heterozygous mutant of SLC26A4 gene extron 14, be positioned at the 1343C＞A heterozygous mutant of SLC26A4 gene extron 12, be positioned at the 1318A＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 946G＞T heterozygous mutant of SLC26A4 gene extron 8, be positioned at the 1975G＞C heterozygous mutant of SLC26A4 gene extron 17 or be positioned at the 626G of SLC26A4 gene extron 6＞A sudden change.

2, the described detection method of claim 1 is characterized in that comprising the steps:

3, the described detection method of claim 2 is characterized in that described detection method further comprises the steps:

4, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 349delC heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS4-F：5’-TAATCACTTTGCATGTGCTTT-3’

PDS4-R：5’-GCCAAAACACTTTAAACATGA-3’

5, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 916_917insG heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS7-F：5’-CATGGTTTTTCATGTGGGAAGATTC-3’

PDS7-R：5’-AATGGCAGTAGCAATTATCG-3’

6, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 230A＞T heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS3-F：5’-GGCAAAAGCATGGTAAGCAC-3’

PDS3-R：5’-AGGGTAAGCAACCATCTGTCA-3’

7, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 1673A＞T heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS15-F：5’-GCTCCTCTGAGCAACTGTGA-3’

PDS15-R：5’-GGGTCTAGGGCCTATTCCTG-3’

8, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 1336C＞T heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’

9, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 1594A＞C heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS14-F：5’-CAAAATACGGCTGTTCCAAA-3’

PDS14-R：5’-AATGGAGCTGCTGAAACTTC-3’

10, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 1343C＞A heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’

11, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 1318A＞T heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS11-F：5’-ACACATCCAGTGAGCTGGAA-3’

PDS11-R：5’-AGGTGGTAGGTGACTTACAGCA-3’

12, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 946G＞T heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS8-F：5’-AAGTTCAGCATTATTTGGTTGACA-3’

PDS8-R：5’-TGGTTGTTTCTTCCAGATCACA-3’

13, the described detection method of claim 1 wherein detects the method that whether has SLC26A4 gene 1975G＞C heterozygous mutant in the sample of individuality to be measured, comprises the steps:

PDS17-F：5’-TCTTCGTTTAGAATGGCATCA-3’

PDS17-R：5’-ATTGCCAAAGCTCCAAATGT-3’

14, the described detection method of claim 1 wherein detects the method that whether exists SLC26A4 gene 626G＞A to suddenly change in the sample of individuality to be measured, comprises the steps:

PDS6-F：5’-GGTTTCTATCTCAGGCAAACAT-3’

PDS6-R：5’-ATTGTTTCTGGAATGAACAGTGACC-3’

15, a kind of detection kit, whether the sample that is used to detect from individuality to be measured exists the SLC26A4 transgenation, comprises following combination of agents:

From testing sample, extract the reagent of DNA;

Be positioned at the 349delC heterozygous mutant of SLC26A4 gene extron 4, be positioned at the 916_917insG heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 230A＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1673A＞T heterozygous mutant of SLC26A4 gene extron 15, be positioned at the 1336C＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 1594A＞C heterozygous mutant of SLC26A4 gene extron 14, be positioned at the 1343C＞A heterozygous mutant of SLC26A4 gene extron 12, be positioned at the 1318A＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 946G＞T heterozygous mutant of SLC26A4 gene extron 8, be positioned at the 1975G＞C heterozygous mutant of SLC26A4 gene extron 17 or be positioned at the 626G of SLC26A4 gene extron 6＞A sudden change;

The reagent that pcr amplification product is checked order.

16, the application of SLC26A4 transgenation in diagnosis and/or treatment and aquaductus vestibuli expansion relative disease, wherein said SLC26A4 transgenation is the 349delC heterozygous mutant that is positioned at SLC26A4 gene extron 4, be positioned at the 916_917insG heterozygous mutant of SLC26A4 gene extron 7, be positioned at the 230A＞T heterozygous mutant of SLC26A4 gene extron 3, be positioned at the 1673A＞T heterozygous mutant of SLC26A4 gene extron 15, be positioned at the 1336C＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 1594A＞C heterozygous mutant of SLC26A4 gene extron 14, be positioned at the 1343C＞A heterozygous mutant of SLC26A4 gene extron 12, be positioned at the 1318A＞T heterozygous mutant of SLC26A4 gene extron 11, be positioned at the 946G＞T heterozygous mutant of SLC26A4 gene extron 8, be positioned at the 1975G＞C heterozygous mutant of SLC26A4 gene extron 17 or be positioned at the 626G of SLC26A4 gene extron 6＞A sudden change.