Abstract:
A purified polynucleotide having a chain of nucleotides corresponding to a mutated sequence, which in a wild form encodes a polypeptide implicated in hereditary sensory defect, wherein said mutated purified polynucleotide presents a mutation responsible for prelingual non-syndromic deafness selected from the group consisting of a specific deletion of at least one nucleotide.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    The present invention concerns a mutation responsible for autosomal prelingual non-syndromic deafness and a method for the detection of this hereditary sensory defect for homozygous and heterozygous individuals. The invention concerns more particularly a specific deletion of at least one nucleotide in the connexin  26  (Cx 26) gene and especially in a guanosine rich region, notably between the nucleotides 27 and 32. The invention is also directed to the use of polynucleotide, or fragments thereof, for example as tools useful for the in vitro detection of a mutation of a gene belonging to the Cx26 gene family.  
           [0002]    Profound or severe prelingual deafness affects one child in a thousand in developed countries (Morton N E. Genetic epidemiology of hearing impairment. In  Genetics of hearing impairment.  (The New York Acad Sci, New York 1991; 630:16-31). It is a major handicap as it impedes language acquisition.  
           [0003]    According to studies performed in a U.S. population of children with non-syndromic (isolated) prelingual deafness and in whom an obvious environmental cause has been excluded, it is estimated that up to two-thirds of the cases have a genetic basis (Marazita M L, Ploughman L M, Rawlings B, Remington E, Arnos K S, Nance W E. Genetic epidemiological studies of early-onset deafness in the U.S. school-age population.  Am J Med Genet  1993; 46:486-91). These forms are mainly sensorineural and are almost exclusively monogenic. The major mode of inheritance is autosomal recessive (DFNB), involving 72% to 85% of cases, this fraction increasing to 90% when only profound deafness is taken into account.  
           [0004]    Autosomal recessive prelingual deafness is known to be genetically highly heterogeneous. Estimates of the number of DFNB loci vary from thirty to one hundred (Petit C. Autosomal recessive non-syndromal hearing loss. In  Genetics and Hearing Impairment.  Martini A, Read A P, Stephens D, eds (Whurr, London) 1996; 197-212), for a review), of which fourteen have so far been mapped to the human chromosomes (Petit C. Genes responsible for human hereditary deafness:  symphony of a thousand. Nature Genet  1996; 14:385-91) for review, (Verhoeven K, Van Camp G, Govaerts P J, et al. A gene for autosomal dominant non-syndromic hearing loss (DFNA12) maps to chromosome 11q22-24.  Am J Hum Genet  1997; 60:1168-74 and Campbell D A, McHale D P, Brown K A, et al. A new locus for non-syndromal autosomal recessive sensorineural hearing loss (DFNB16) maps to human chromosome 15q21-q22.  J Med Genet  1997; in press).  
           [0005]    A majority of the families attending genetic counseling clinics consist of normal hearing parents with a single deaf child who wish to know the risk of recurrence of the defect. In most cases, given the major role of environmental causes of prelingual deafness, it is not usually possible even to recognize whether the hearing loss is of genetic origin. Genetic counseling in such families would be greatly improved by an ability to detect DFNB mutations. In this respect, the high genetic heterogeneity of the condition represents a major obstacle.  
           [0006]    After the initial identification of the DFNB1 locus on 13q11 in a large consanguineous Tunisian family (Guilford P, Ben Arab S, Blanchard S, et al. A non-syndromic form of neurosensory, recessive deafness maps to the pericentromeric region of chromosome 13q.  Nature Genet  1994; 6:24-8), two studies performed on New Zealand/Australian families (Maw M A, Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locus to neurosensory deafness in a Caucasian population.  Am J Hum Genet  1995; 57:629-35), and on Italian/Spanish families (Gasparini P, Estivill X, Volpini V, et al. Linkage of DFNB1 to non-syndromic neurosensory autosomal-recessive deafness in Mediterranean families.  Eur J Hum Genet  1997; 5:83-8) suggested that this locus might be a major contributor to prelingual deafness in these populations, although individual lod scores obtained in these families were not significant owing to the small size of these families.  
           [0007]    Recently, the Cx26 gene, which encodes a gap junction protein, connexin 26, has been shown to underlie DFNB1 deafness. Two different G-&gt;A substitutions resulting in premature stop codons in three DFNB1 linked consanguineous Pakistani families have been reported (Kelsell D P, Dunlop J, Stevens H P, et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness.  Nature  1997; 387:80-3). These two substitutions were identified, respectively, at codon 77 and at codon 24. This result has offered the opportunity directly to assess this hypothesis.  
           [0008]    The difficulties encountered in genetic counseling for prelingual non-syndromic deafness due to the inability to distinguish genetic and non-genetic deafness in the families presenting a single deaf child was one of the reasons that led the inventors to undertake a characterization of the spectrum and prevalence of mutations present in the Cx26 gene in 35 families from several parts of the world with autosomal recessive prelingual deafness.  
         SUMMARY OF THE INVENTION  
         [0009]    The determination of a mutation in the Cx26 gene has notably rendered possible the use of a detection probe as a tool for the identification of a specific form of autosomal prelingual non-syndromic deafness, and more particularly the useful role of a newly identified 30delG (a G deletion at position 30; position 1 being the first base of the initiator codon) mutation in such families. This invention establishes that the contribution of the DFNB1 locus predominantly results essentially from the 30delG mutation. It is now believed that the 30delG accounts for about three-quarters of all recessive DFNB1 mutations.  
           [0010]    The invention is thus intended to provide a purified polynucleotide having a chain of nucleotides corresponding to a mutated sequence, which in a wild form encodes a polypeptide implicated in hereditary sensory defect. The mutated purified polynucleotide presents a mutation responsible for prelingual non-syndromic deafness.  
           [0011]    The invention also provides oligonucleotides comprising of 15 to 50 consecutive nucleotides of the-mutated purified polynucleotide that are useful as primers or as probes.  
           [0012]    In addition, the invention aims to supply a method and a kit for the detection of the hereditary sensory defect for homozygous as heterozygous individuals.  
           [0013]    According to the invention, the purified polynucleotide having a chain of nucleotides corresponding to a mutated sequence, which encodes in a wild form a polypeptide implicated in hereditary sensory defect, presents a mutation responsible for prelingual non-syndromic deafness selected from the group consisting of a specific deletion of at least one nucleotide.  
           [0014]    By mutation, according to the invention it means a specific deletion of at least one nucleotide. Thus, a mutated sequence means a polynucleotide sequence comprising at least a mutation.  
           [0015]    A chain of nucleotides, according to the invention, means a polynucleotide, which encodes not necessarily a polypeptide, but which presents between 27 and 2311 nucleotides linked together.  
           [0016]    The invention particularly concerns a purified polynucleotide wherein, the specific mutation is a deletion located in a region encoding connexin 26 of chromosome 13q11-12, preferably located in a guanosine rich region starting at nucleotide 27 preferably at nucleotide 30, and extending to nucleotide 32 or nucleotide 35, all the recited nucleotides being inclusive. More particularly according to the invention, the specific deleted purified polynucleotide encodes for a truncated polypeptide.  
           [0017]    By truncated polypeptide, according to the invention it means a fragment of the polypeptide, which does not present the properties of the wild form of the polypeptide either in length, in amino acid composition, or in functional properties.  
           [0018]    A preferred embodiment of a specific deletion is a guanosine deletion at position 30, also called “30delG mutation”. Another preferred embodiment of the specific deletion is a 38 bp deletion beginning at position 30.  
           [0019]    The invention also includes a purified polynucleotide, which hybridizes specifically with any one of the polynucleotides as defined above under the following stringent conditions: at low temperatures between 23° C. and 37° C., in the presence of 4×SSC buffer, 5×Denhardt&#39;s solution, 0.05% SDS, and 100 μg/ml of salmon sperm DNA. (1×SSC corresponds to 0.15 M NaCl and 0.05 M sodium citrate; 1×Denhardt&#39;s solution corresponds to 0.02% Ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin).  
           [0020]    The invention also concerns an oligonucleotide useful as a primer or as a probe comprising 15 to 50 consecutive nucleotides of the polynucleotide according to any one of the polynucleotides as defined above. The oligonucleotide sequence is selected from the following group:  
           [0021]    A first couple:  
                                           5′-TCTTTTCCAGAGCAAACCGCC   (SEQ ID No. 1)-3′                           5′-TGAGCACGGGTTGCCTCATC.   (SEQ ID No. 2)-3′          
 
           [0022]    The length of the PCR product has been obtained from 285 bp in length;  
           [0023]    A second couple allowing to explore the other part of the reading frame:  
                                           5′-GACACGAAGATCAGCTGCAG   (SEQ ID No. 3)-3′                           5′-CCAGGCTGCAAGAACGTGTG   (SEQ ID No. 4)-3′          
 
           [0024]    A third couple:  
                               5′-CTAGTGATTCCTGTGTTGTGTGC; and   (SEQ ID No. 9)-3′                   5′ATAATGCGAAAAATGAAGAGGA and   (SEQ ID No. 10)-3′          
 
           [0025]    A fourth couple:  
                               5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCTAGTGATTCCTGTGTTGTGTGC; and   (SEQ ID No. 14)-3′                   5′ ATAATGCGAAAAATGAAGAGGA.   (SEQ ID No. 10)-3′          
 
           [0026]    Another oligonucleotide useful as a probe is selected from the following group:  
                               5′-AGACGATCCTGGGGGTGTGAACAAA   (SEQ ID No. 5)-3′                   5′-ATCCTGGGGGTGTGA   (SEQ ID No. 6)-3′               5′-AGACGATCCTGGGGGCTCACCGTCCTC.   (SEQ ID No. 7)-3′          
 
           [0027]    In addition, the invention concerns a method for the detection of an hereditary sensory defect, namely autosomal prelingual non-syndromic deafness, for homozygous as heterozygous individuals in a biological sample containing DNA, comprising the steps of:  
           [0028]    a) bringing the biological sample into contact with a oligonucleotide primers as defined above, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample;  
           [0029]    b) amplifying the DNA;  
           [0030]    c) revealing the amplification products;  
           [0031]    d) detecting the mutation.  
           [0032]    Step d) of the above-described method may comprise a Single-Strand Conformation Polymorphism (SSCP), a Denaturing Gradient Gel Electrophoresis (DGGE) sequencing (Smith, L. M., Sanders, J. Z., Kaiser, R. J., Fluorescence detection in automated DNA sequence analysis.  Nature  1986; 321:674-9); a molecular hybridization capture probe or a temperature gradient gel electrophoresis (TGGE).  
           [0033]    Step c) of the above-described method may comprise the detection of the amplified products with an oligonucleotide probe as defined above.  
           [0034]    According to the invention, a biological sample can be a blood sample extracted from people suffering from any kind of deafness with any criteria as follows: neurosensorial or mixed isolated deafness, advanced or not, at any degree of severity, concerning familial or sporadic case, or individuals exposed to noise, or individuals suffering from a low acoustic, or individuals susceptible to carry an anomaly in the gene, or from an embryo for antenatal diagnostic.  
           [0035]    Another aim of the invention comprises a method for the detection of an hereditary sensory defect, the autosomal prelingual non-syndromic deafness, for homozygous and heterozygous individuals in a biological sample containing DNA, comprising the steps of:  
           [0036]    a) bringing the biological sample into contact with an oligonucleotide probe according to the invention, the DNA contained in the sample having been optionally made available to hybridization and under conditions permitting a hybridization of the primers with the DNA contained in the biological sample; and  
           [0037]    b) detecting the hybrid formed between . the oligonucleotide probe and the DNA contained in the biological sample.  
           [0038]    Step b) of the above-described method may consist in a single-strand conformation. Polymorphism (SSCP), a denaturing gradient gel electrophoresis (DGGE) or amplification and sequencing.  
           [0039]    The invention also includes a kit for the detection of an hereditary sensory defect, the autosomal prelingual non-syndromic deafness, for homozygous as heterozygous individuals, said kit comprising:  
           [0040]    a) oligonucleotides according to the invention;  
           [0041]    b) the reagents necessary for carrying out DNA amplification; and  
           [0042]    c) a component that makes it possible to determine the length of the amplified fragments or to detect a mutation. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0043]    This invention will be more described in greater detail by reference to the drawings in which:  
         [0044]    FIG. l depicts the results of temperature gradient gel electrophoresis for detection of mutants in which:  
         [0045]    Lanes 1 and 2: DNA from normal patients.  
         [0046]    Lanes 3 and 4: DNA from homozygous patients with 30delG mutation.  
         [0047]    Lanes 5 and 6: DNA from heterozygous patients.  
         [0048]    Lane 7: PCR control without DNA.  
         [0049]    Lane 8: PCR fragment amplified from a normal DNA and hybridized with a standard DNA fragment harboring the 30delG mutation.  
         [0050]    Lane 9: PCR fragment amplified from a mutant homozygous DNA and hybridized with a normal standard DNA fragment harboring the guanine 30. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0051]    Prelingual non-syndromic (isolated) deafness is the most frequent hereditary sensory defect in children. The inheritance in most is autosomal recessive. Several dozens of genes might be involved, only two of which, DFNB1 and DFNB2, have so far been identified (Kelsell, D. P., et al., Connexin 26 mutations in hereditary non-syndromic sensorineural deafness.  Nature  1997; 387:80-3; Liu, X-Z, et al., Mutations in the myosin VIIA gene cause non-syndromic recessive deafness,  Nature Genet  1997; 16:188-90; and Weil, D., et al., The autosomal recessive isolated deafness, DFNB2, and the Usher 1B syndrome are allelic defects of the myosin-VIIA.  Nature Genet  1997; 16:191-3). A search was made searched for mutations in the gene encoding connexin 26, Cx26, which has recently been shown to be responsible for DFNB1. Mutation analysis of Cx26 was performed by PCR amplification on genomic DNA and sequencing of the single coding exon.  
       EXAMPLE 1  
     Patients  
       [0052]    Thirty-five affected families from various geographical regions, mainly France, New Zealand and Australia, Tunisia and Lebanon, were studied. They could be classified into three categories: (1) consanguineous families each having a significant linkage to the DFNB1 locus; (2) small non-consanguineous families in which linkage analysis was compatible with the involvement of DFNB1; and (3) small families in which no linkage analysis had been undertaken.  
         [0053]    The first category consists of six large families living in geographically isolated regions. Five were from Tunisia, two from the north and three from the south. Linkage to the DFNB1 locus in the two families from northern Tunisia (families 20 and 60) had previously been reported (Guilford P, Ben Arab S, Blanchard S, et al., A non-syndromic form of neurosensory, recessive deafness maps to the pericentromeric region of chromosome 13q.  Nature Genet  1994; 6:24-8); the three families from southern Tunisia (S15, S19 and ST) and the family from Lebanon (LH) comprise total of three, five, two, and five deaf children, respectively, the deafness being of severe or profound degree. The marriages were between first cousins (S15, ST and LH) and between first and second cousins (S19). Linkage analysis of these six families resulted in individual lod scores ranging from 2.5 to 10 with polymorphic markers from the DFNB1 region (D13S175, D13S141, D13S143 and D13S115).  
         [0054]    The second category of patients comprises seven New Zealand families with at least two deaf siblings (families 51, 1160, 1548, 1608, 1773, 1873, 1877) and one Australian (9670) family. Family 1608 was atypical in that four siblings sharing the same DFNB1 marker haplotypes had a mild to moderate deafness (severe at high frequency), with the child of one of them being profoundly deaf. In family 1873, the unrelated parents (individuals II.2 and II.3) were deaf as well as their two children, and we have therefore considered this as two families, bringing to nine the total of independent families. Apart from families 1608 and 1873, no parent acknowledged any hearing impairment. These nine families showed cosegregation between deafness and polymorphic markers of the DFNB1 region with maximum individual lod scores ranging from 0.6 to 1.2. Ten other families in the original study of Maw et al. (Maw M A, Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locus to neurosensory deafness in a Caucasian population.  Am J Hum Genet  1995; 57:629-35) had shown no cosegregation and one other cosegregating family was not tested for Cx26 mutations. The New Zealand families were all of Caucasian origin with no known Polynesian admixture. According to the antecedent family names, the ancestral proportion among the families reflected that of the general Caucasian New Zealand population with the great predominance being of Anglo-Celtic patrimony and a small fraction due to migration from continental Europe. Neither parental consanguinity, nor links between any of the families were recognized. In the Australian case, the father was from Northern Ireland and the mother from Yorkshire, England.  
         [0055]    The third category is composed of nineteen families living in France and two in New Zealand, each with at least two children having a severe to profound deafness. No parent acknowledged any hearing impairment, except for the mother in family P16 and the father. in family P17 who had moderate and progressive high-frequency hearing loss. Five of these families had foreign ancestors from Lebanon (family P3), Turkey (family P4), Portugal (family P9), Algeria (family P14) and Poland (father in family P16). In two of the families (P7 and P14), the parents were distantly related.  
       EXAMPLE 2  
       [0056]    Amplification of the coding exon of Cx26 PCRs were carried out on genomic DNA using a set-of primers that allowed the amplification of the entire coding sequence of the Cx26 gene, which consists of a single coding exon (Kelsell D P, Dunlop J, Stevens H P, et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness.  Nature  1997; 387: 80-3). Primer sequences were as follows:  
                               5′-TCTTTTCCAGAGCAAACCGCC and   (SEQ ID No. 1)-3′                   5′-TGAGCACGGGTTGCCTCATC.   (SEQ ID No. 2)-3′          
 
         [0057]    PCR conditions were: 35 cycles of 95° C., 1 min; 58° C., 1 min; 72° C., 2 min. The PCR product obtained was 777 bp in length.  
       EXAMPLE 3  
     DNA sequencing  
       [0058]    Sequencing of the PCR products was performed as previously described (Smith L M, Sanders J Z, Kaiser R J, et al., Fluorescence detection in automated DNA sequence analysis,  Nature  1986; 321:674-9) using the dideoxy chain terminator method on an Applied Biosystems DNA sequencer ABI373 with fluorescent dideoxynucleotides. The primers used were the same as those for the PCR amplification plus two internal primers  
                                           5′-GACACGAAGATCAGCTGCAG and   (SEQ ID No. 3)-3′                           5′-CCAGGCTGCAAGAACGTGTG.   (SEQ ID No. 4)-3′          
 
       EXAMPLE 4  
     Mutations in Consanguineous Tunisian and Lebanese DFNB1 Families  
       [0059]    In these families the involvement of the DFNB1 locus could be demonstrated by linkage analysis. In four of the five families from Tunisia (S15, S19, 20, and 60) and in the Lebanese family (LH), the same mutation was detected in all affected children on both Cx26 alleles, namely, a deletion of a guanosine (G) in a sequence of six G extending from position 30 to 35 (position 1 being the first base of the initiator codon) (Table 1). This mutation is hereafter referred to as 30delG mutation according to the nomenclature proposed by Beaudet and Tsui ((Beaudet A L, Tsui L-C. A suggested nomenclature for designating mutations,  Hum Mutation  1993; 2: 245-8)). It creates a frameshift, which results in a premature stop codon at nucleotide position 38. The mutation segregating in the fifth family from Tunisia (ST) was identified as a G to T transversion at nucleotide position G39 creating a premature stop codon (GAG-&gt;TAG) at codon 47, and was designated E47X. In each family, normal hearing parents were found to be heterozygous for the corresponding mutation.  
       EXAMPLE 5  
     Mutations in Small Nonconsanguineous New Zealand and Australian Families Consistent with DFNB1 Linkage  
       [0060]    In these families, segregation analysis has previously been reported as compatible with the involvement of the DFNB1 locus (Maw M A, Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locus to neurosensory deafness in a Caucasian population.  Am J Hum Genet  1995; 57: 629-35). The deaf individuals from five of the nine families (51, 1160, 1608 (III.20), 1873 (II.3) and 1877) were homozygous for the 30delG mutation. The deaf children from family 1773 were heterozygous for 30delG. Deaf individual II.2 from family 1873 (see “subjects” and Table 1) was heterozygous for a deletion of 38 bp beginning at nucleotide position G30, designated 30de138. No other mutation was detected in the deaf children of family 1773 and the deaf individual (II.2) in family 1873. Nevertheless, in this last individual, a deletion of the polymorphic marker immediately proximal to the Cx26 gene (locus D13S175) had previously been observed (Maw M A, Allen-Powell D R, Goodey R J, et al. The contribution of the DFNB1 locus to neurosensory deafness in a Caucasian population.  Am J Hum Genet  1995; 57: 629-35), which may indicate that a DNA rearrangement has impaired the functioning of the other Cx26 allele of the gene in cis. In family 9670, compound heterozygosity for a missense mutation (R184P) and an in frame single-codon deletion (delE138) was observed in affected siblings. In only one family (1548) was no Cx26 mutation detected. Results are summarized in Table 1.  
       EXAMPLE 6  
     Mutations in Small Families Uncharacterized for DFNB1 Linkage Living in France and New Zealand  
       [0061]    Nineteen families (P1 to 17, L14190 and L13131) living in France and two in New Zealand (families 1885 and 2254) were studied. In these families, cosegregation of the deafness with polymorphic markers had not been analysed. Deaf children from six of the twenty-one families (P1, P3, P5, P9, P10, and P16) were found to be homozygous for the mutation 30delG. In five additional families (P6, P11, P14, P17, and 1885), deaf children were heterozygous for this mutation; no other mutation was detected in these families. In the ten remaining families, no mutation in the Cx26 gene was found.  
       EXAMPLE 7  
     Molecular Hybridization Using Allele-Specific Capture Probes  
       [0062]    Molecular hybridization capture probe (see, e.g., D. Chevrier et al. PCR product quantification by non-radioactive hybridization procedures using an oligonucleotide covalently bound to microwells. Molecular and Cellular Probes 1993; 7: 187-197 and D Chevrier et al. Rapid detection of Salmonella subspecies I by PCR combined with non-radioactive hybridization using covalently immobilized oligonucleotide on a microplate. FEMS Immunology and Medical Microbiology 1995; 10: 245-252 each of which is incorporated by reference herein) permit specific detection of the 30delG mutation. The technique has been adapted to permit rapid diagnosis of prelingual non-syndromic deafness caused by the 30delG mutation. The technique provides certain advantages in a clinical setting because it uses stable, nonradioactive molecules, it can be easily automated, and. it is well adapted to large scale analysis.  
         [0063]    Using primers designed for PCR amplification, the region of interest in the Cx26 gene is amplified from genomic DNA samples. The primer sequences are as follows:  
         [0064]    CONN3: 5′-CTAGTGATTCCTGTGTTGTGTGC(SEQ ID No. 9)-3′ 
         [0065]    CONN4: 5′ ATAATGCGAAAAATGAAGAGGA(SEQ ID No. 10)-3′ 
         [0066]    PCR is performed with the CONN3 (SEQ ID No. 9) and CONN4 (SEQ ID No. 10) primers (1 μM each), an aliquot of the DNA to be analyzed (2 μl, 100-300 ng), 1.5 mM MgCl 2 , 200 μM dNTP, and Taq polymerase. The amplification program consists of the following steps: 1) 95° C., 5 min; 2) addition of enzyme, 95° C., 1 min; 3) 60° C., 1 min (ramp rate=0.25° C./s); 4) 72° C., 1 min; 5) repeat steps 2 to 4 for 40 cycles; and 6) 72° C., 10 min. PCR products are verified by a rapid gel electrophoresis.  
         [0067]    The amplified PCR product contains either the normal or the mutant Cx26 sequence. To distinguish between the normal and mutant sequence, two capture probes are designed. The sequences of these two capture probes are as follows:  
         [0068]    For detection of normal sequence:  
         [0069]    CONN6: 5′-AAAAAAAATCCTGGGGGGTGTG(SEQ ID No. 11)-3′ 
         [0070]    For detection of mutant sequence:  
         [0071]    CONN7: 5′-AAAAAAAATCCTGGGGGTGTGA(SEQ ID No. 12)-3′ 
         [0072]    Each capture probe must be 22 nucleotides long. Furthermore, to be efficient, the capture probe must include an A 7  spacer at its 5′ end and a hybridization region of 15 bases. Such a capture probe is able to specifically differentiate the mutant sequence from the normal sequence. Thus, CONN6 (SEQ ID No. 11) is designed to specifically hybridize with the normal sequence, whereas CONN7 (SEQ ID No. 12) is designed to specifically hybridize with the mutant sequence.  
         [0073]    Before attaching the capture probes to a microtiter plate, they are phosphorylated at their 5′ ends. The phosphorylation is carried out for 1 hour at 37° C. in presence of 20 nmoles of CONN6 (SEQ ID No. 11) or CONN7 (SEQ ID No. 12) oligonucleotides, 100 μM ATP, 10 units T4 polynucleotide kinase in 200 μl of buffer (50 mM Tris-HCl pH 7.4; 10 mM MgCl 2 ; 5 mM dithiothreitol; and 1 mM spermidine). The mixture is heated for 10 min. at 68° C. to inactivate the T4 polynucleotide kinase, then the oligonucleotide is precipitated by adding 145 μl of 10 M CH 3 COONH 4 , 15 μl H2O, and 800 μl iced ethanol. After a 30 min. incubation in ice, the mixture is centrifuged for 20 min. at  12,000×g  at 4° C. The resulting pellet is washed with 500 μl iced ethanol (70%) and dissolved in 800 μl of TE buffer. The phosphorylated oligonucleotide concentration is determined by optical density at 260 nm.  
         [0074]    Before attaching the phosphorylated oligonucleotides to microplates, they are denatured by heating at 95° C. for 10 min . and rapidly cooled in ice to avoid the formation of secondary structure. 500 ng of phosphorylated CONN6 (SEQ ID No. 11) or CONN7 (SEQ ID No. 12) and 1 μl of 1 M 1-methylimidazole, pH 7, is added to each well of a microplate, which is kept on ice. The total volume of each well is adjusted to 70 μl with distilled water, before adding 30 μl of a cold, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide solution (167 mM). The microplate is covered and incubated for 5 hours at 50° C. in an incubator (Thermomix® from Labsystems). After the 5-hour incubation, the microplate is washed three times with a warm solution (50° C.) of 0.4 N NaOH containing 0.25% SDS. The microplate is incubated for 5 min. with the same warm solution and washed again with warm NaOH/SDS (50° C.). Finally, the microplate is washed five times with TE buffer. The coated microplate can be kept several months at 4° C., if the wells are filled with TE buffer.  
         [0075]    The amplified sequences from the genomic DNA samples are incubated with a biotinylated detection probe in the coated microplates. Unlike the capture probes, which are allele specific, the detection probe can hybridize with both the normal and mutant sequences. The sequence of the detection probe is:  
         [0076]    CONN12: 5′-CAGCATTGGAAAGATCTGGCTCA(SEQ ID No. 13)-3′. The amplified sequences and the detection probe, which is biotinylated at its 5′ end, are denatured directly in the microplates by successively adding to each well: 95 μl of water, 5 μl of PCR reaction, 40 μl of biotinylated probe (SEQ ID No. 13) at 22 nM diluted in water, and 14 μl 1 N NaOH. After 10 min., 21 μl of 1 M NaH 2 PO 4  and 1% Sarkosyl is added to each well to bring the total volume to 175 μl per well. The final concentration of the detection probe is 5 nM. The microplate is covered and incubated overnight at 40° C. in an incubator (Thermomix® from Labsystems) and then extensively washed (5 times) with TBS-Tween to remove the excess biotinylated probe (SEQ ID No. 13).  
         [0077]    An immunoenzymatic method is used to detect the hybridized probe. Each well receives 100 μl of the conjugate (Extravidine-alkaline phosphatase, Sigma E-2636) diluted {fraction (1/4000)} in TBS-BSA-Tween. The microplate is covered and incubated for 1 hour at 25° C. Following the incubation, the microplate is washed 5 times with TBS-Tween. Then 200 μl of preheated (37° C.) substrate (7.5 mg para-nitro-phenyl-phosphate in 20 ml of the following buffer: 1 M diethanolamine pH 9.8 containing 1 mM MgCl 2 ) are added to each well. The microplate is covered and incubated for 3 hours at 37° C. The absorbance is measured at 405 nm to determine the specific signal and at 630 nm to determine the background noise.  
         [0078]    The hybridization ratio (R) between the signal obtained with CONN6 (SEQ ID No. 11) probe (normal sequence) and that obtained with CONN7 (SEQ ID No. 12) probe mutant sequence) is calculated. The calculated R values are used to determine the genotypes of the sample DNA as follows: homozygous for the normal Cx26 sequence (R≧2), heterozygous for the 30delG mutation (0.5&lt;R&lt;2), and homozygous for the 30delG mutation (R≦0.5). The range of the hybridization ratio (R) can be slightly modified when the number of samples increases. The following table represents an example of results obtained with 39 samples.  
                                                                                   Hybridization ratio (R)            Genotype:   Normal   Homozygous 30delG   Heterozygous                        5.96   0.48   1.33           5.43   0.17   1.13           3.39   0.21   0.73           4.14   0.16   0.63           4.09   0.28   1.4           2.76   0.13   0.73           2.2   0.21   0.76           3.97   0.4   0.73           4.07       1.06           3           2.76           3.66           3.87           3.92           3.26           5.17           2.74           4.51           6.3           3.49           4.05           3.17       Number   22   8   9       Mean value   3.91   0.26   0.94       Standard   1.06   0.12   0.29       deviation       Range   (6.3-2.2)   (0.48-0.13)   (1.4-0.63)                  
 
       EXAMPLE 8  
     Temperature Gradient Gel Electrophoresis  
       [0079]    Temperature gradient gel electrophoresis (TGGE) permits the detection of any type of mutation, including deletions, insertions, and substitutions, which is within a desired region of a gene. (See, e.g. D. Reiner et al. Temperature-gradient gel electrophoresis of nucleic acids: Analysis of conformational transitions, sequence variations and protein-nucleic acid interactions. Electrophoresis 1989; 10: 377-389; E. P. Lessa and G. Applebaum Screening techniques for detecting allelic variation in DNA sequences. Molecular Ecology 1993; 2: 119-129 and A. L. Börresen-Dale et al. Temporal Temperature Gradient Gel Electrophoresis on the D code™ System. Bio-Rad US/EG Bulletin 2133; the entire disclosure of each publication is incorporated by reference herein.) However, TGGE does not permit one to determine precisely the type of mutation and its location.  
         [0080]    As in the previously described molecular hybridization technique, the region of interest in the Cx26 gene is first amplified from genomic DNA samples by PCR. The primer sequences are as follows:  
                                   CONN2:   5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCT   (SEQ ID No. 14)-3′                  AGTGATTCCTGTGTTGTGTGC               CONN4:   5′ATAATGCGAAAAATGAAGAGGA   (SEQ ID No. 10)-3′          
 
         [0081]    PCR is performed with 1 μM of the CONN2 (SEQ ID No. 14) primer, which has a GC clamp at its 5′ end, and 1 μM of the CONN4 (SEQ ID No. 10) primer, an aliquot of the DNA to be analyzed (2 μl, 100-300ng), 1.5 mM MgCl 2 , 200 μM dNTP, and Taq polymerase. The amplification program consists of the following steps: 1) 95° C., 5 min; 2) addition of enzyme, 95° C., 1 min; 3) 60° C., 1 min (ramp rate=0.25° C./s); 4) 72° C., 1 min; 5) repeat steps 2 to 4 for 40 cycles; and 6) 72° C., 10 min.  
         [0082]    Analyzing these PCR amplification fragments by TGGE can differentiate between homozygous (normal or mutant) samples, which produce a single band on a gel, and heterozygous samples, which produce three bands. However, differentiating between genomic samples that are homozygous for the normal sequence and genomic samples that are homozygous for the 30delG mutants requires an additional step.  
         [0083]    To differentiate normal homozygous versus mutant homozygous samples, an aliquot of the amplified PCR product is mixed with either a known, normal homozygous sample or a known, 30delG mutant homozygous sample and analyzed for heteroduplex formation. If the amplified PCR product derives from a normal, homozygous sample, it will form a heteroduplex with the known, 30delG mutant homozygous sample. On the other hand, if the amplified PCR product derives from a mutant, homozygous sample, it will form a heteroduplex with the known, normal homozygous sample. To promote heteroduplex formation in these mixtures, they are denatured at 95° C. for 5 min, followed by a renaturation step at 60° C. for 45 min.  
         [0084]    The PCR fragments from the initial amplification and those that are subjected to the additional heating steps to permit heteroduplex formation are analyzed on a 10% polyacrylamide gel containing 7 M urea. By way of example, a 30 ml gel is prepared by combining the following ingredients:  
         [0085]    12.6 g urea  
         [0086]    0.75 ml SOX TAE  
         [0087]    7.5 ml acrylamide:bisacrylamide (37.5:1) at 40%  
         [0088]    water to bring volume to 30 ml  
         [0089]    30 μl Temed (added extemporaneously)  
         [0090]    300 μl 10% ammonium persulfate (added extemporaneously).  
         [0091]    After adding the Temed and ammonium persulfate, the gel is poured between two glass plates (Dcode Universal Mutation Detection System® from BIORAD) and allowed to polymerize for 1 hour.  
         [0092]    An aliquot (7.5 μl ) of the PCR mixture is mixed with 7.5 μl of 2X sample solution (2 mM EDTA pH 8; 70% glycerol; 0.05% xylene cyanol; 0.05% bromophenol blue), and introduced into a gel well. Electrophoresis is performed for 4-5 hours at 150V in 1.25X TAE buffer with a temperature gradient ranging from 61° C. to 62° C. at a rate of 0.2° C. per hour. Following electrophoresis, the gel is incubated for 6 min. in 1.25X TAE containing 25 μg/ml ethidium bromide. Excess ethidium bromide is removed by a 20 min. wash in 1.25X TAE, and the DNA fragments are visualized with a UV transilluminator.  
         [0093]    A typical TGGE result is represented in FIG. 1. The. amplified DNA from homozygous patients (normal or mutant) produces only one band. The amplified DNA from heterozygous patients results in three different fragments in the polyacrylamide gel. The more intense band, which migrates more rapidly, corresponds to both homoduplexes, which cannot be separated in this gel. The other two bands, which migrate more slowly, correspond to both kinds of heteroduplexes.  
         [0094]    The DNA of normal homozygous patients can be differentiated from the DNA of mutant homozygous patients by analyzing the PCR fragments that were subjected to the conditions that permitted heteroduplex formation. Heteroduplexes form when the PCR amplified fragment from a normal homozygous genome is mixed with sequences from a known, mutant homozygous genome, or when the PCR. amplified fragment from a mutant homozygous genome is mixed with sequences from a known, normal homozygous genome. These heteroduplexes are visible by TGGE analysis. Consequently, the DNA of normal and mutant homozygous patients can be easily differentiated by this technique using the primers described in the present study.  
         [0095]    In all the known DFNB1 families (6/6), in all but one (8/9) of the putatively DFNB1-linked families, and in about half (11/21) of the families not tested for DFNB1 linkage, a mutation in Cx26 was detected. Furthermore, of the 44 chromosomes reckoned to be independent upon which a Cx26 mutant allele was identified or inferred, 33(75%) were found to carry the same deletion of a guanosine, G, at position 30 (30delG).  
         [0096]    Cx26 mutations represent a major cause of recessively inherited prelingual deafness and would be implicated in about half of cases in the examined populations. In addition, one specific mutation, 30delG, accounts for the majority (about three-quarters in our series) of the Cx26 mutant alleles.  
         [0097]    The wild type connexin 26 gene published in LEE S. W. et al. (1992)J. Cell Biol. 118: 1213-1221 has the following sequence:  
                               1   GATTTAATCC TATGACAAAC TAAGTTGGTT CTGTCTTCAC CTGTTTTGGT                   51   GAGGTTGTGT AAGAGTTGGT GTTTGCTCAG GAAGAGATTT AAGCATGCTT               101   GCTTACCCAG ACTCAGAGAA GTCTCCCTGT TCTGTCCTAG CTATGTTCCT               151   GTGTTGTGTG CATTCGTCTT TTCCAGAGCA AACCGCCCAG AGTAGAAG AT                 201     G GATTGGGGC ACGCTGCAGA CGATCCTGGG GGGTGTGAAC AAACACTCCA               251   CCAGCATTGG AAAGATCTGG CTCACCGTCC TCTTCATTTT TCGCATTATG               301   ATCCTCGTTG TGGCTGCAAA GGAGGTGTGG GGAGATGAGC AGGCCGACTT               351   TGTCTGCAAC ACCCTGCAGC CAGGCTGCAA GAACGTGTGC TACGATCACT               401   ACTTCCCCAT CTCCCACATC CGGCTATGGG CCCTGCAGCT GATCTTCGTG               451   TCCAGCCCAG CGCTCCTAGT GGCCATGCAC GTGGCCTACC GGAGACATGA               501   GAAGAAGAGG AAGTTCATCA AGGGGGAGAT AAAGAGTGAA TTTAAGGACA               551   TCGAGGAGAT CAAAACCCAG AAGGTCCGCA TCGAAGGCTC CCTGTGGTGG               601   ACCTACACAA GCAGCATCTT CTTCCGGGTC ATCTTCGAAG CCGCCTTCAT               651   GTACGTCTTC TATGTCATGT ACGACGGCTT CTCCATGCAG CGGCTGGTGA               701   AGTGCAACGC CTGGCCTTGT CCCAACACTG TGGACTGCTT TGTGTCCCGG               751   CCCACGGAGA AGACTGTCTT TCACAGTGTT CATGATTGCA GTGTCTGGAA               801   TTTGCATCCT GCTGAATGTC ACTGAATTGT GTTATTTGCT AATTAGATAT               851   TGTTCTGGGA AGTCAAAAAA GCCAGTTTAA CGCATTGCCC AGTTGTTAGA               901   TTAAGAAATA GACAGCATGA GAGGGATGAG GCAACCCGTG CTCAGCTGTC               951   AAGGCTCAGT CGCCAGCATT TCCCAACACA AAGATTCTGA CCTTAAATGC               1001   AACCATTTGA AACCCCTGTA GGCCTCAGGT GAAACTCCAG ATGCCACAAT               1051   GAGCTCTGCT CCCCTAAAGC CTCAAAACAA AGGCCTAATT CTATGCCTGT               1101   CTTAATTTTC TTTCACTTAA GTTAGTTCCA CTGAGACCCC AGGCTGTTAG               1151   GGGTTATTGG TGTAAGGTAC TTTCATATTT TAAACAGAGG ATATCGGCAT               1201   TTGTTTCTTT CTCTGAGGAC AAGAGAAAAA AGCCAGGTTC CACAGAGGAC               1251   ACAGAGAAGG TTTGGGTGTC CTCCTGGGGT TCTTTTTGCC AACTTTCCCC               1301   ACGTTAAAGG TGAACATTGG TTCTTTCATT TGCTTTGGAA GTTTTAATCT               1351   CTAACAGTGG ACAAAGTTAC CAGTGCCTTA AACTCTGTTA CACTTTTTGG               1401   AAGTGAAAAC TTTGTAGTAT GATAGGTTAT TTTGATGTAA AGATGTTCTG               1451   GATACCATTA TATGTTCCCC CTGTTTCAGA GGCTCAGATT GTAATATGTA               1501   AATGGTATGT CATTCGCTAC TATGATTTAA TTTGAAATAT GGTCTTTTGG               1551   TTATGAATAC TTTGCAGCAC AGCTGAGAGA GGCTGTCTGT TGTATTCATT               1601   GTGGTCATAG CACCTAACAA CATTGTAGCC TCAATCGAGT GAGACAGACT               1651   AGAAGTTCCT AGTTGGCTTA TGATAGCAAA TGGCCTCATG TCAAATATTA               1701   GATGTAATTT TGTGTAAGAA ATACAGACTG GATGTACCAC CAACTACTAC               1751   CTGTAATGAC AGGCCTGTCC AACACATCTC CCTTTTCCAT GCTGTGGTAG               1801   CCAGCATCGG AAAGAACGCT GATTTAAAGA GGTGAGCTTG GGAATTTTAT               1851   TGACACAGTA CCATTTAATG GGGAGACAAA AATGGGGGCC AGGGGAGGGA               1901   GAAGTTTCTG TCGTTAAAAA CGAGTTTGGA AAGACTGGAC TCTAAATTCT               1951   GTTGATTAAA GATGAGCTTT GTCTACCTTC AAAAGTTTGT TTGGCTTACC               2001   CCCTTCAGCC TCCAATTTTT TAAGTGAAAA TATAACTAAT AACATGTGAA               2051   AAGAATAGAA GCTAAGGTTT AGATAAATAT TGAGCAGATC TATAGGAAGA               2101   TTGAACCTGA ATATTGCCAT TATGCTTGAC ATGGTTTCCA AAAAATGGTA               2151   CTCCACATAG TTCAGTGAGG GTAAGTATTT TCCTGTTGTC AAGAATAGCA               2201   TTGTAAAAGC ATTTTGTAAT AATAAAGAAT AGCTTTAATG ATATGCTTGT               2251   AACTAAAATA ATTTTGTAAT GTATCAAATA CATTTAAAAC ATTAAAATAT               2301   AATCTCTATA AT          
 
         [0098]    The wild type connexin 26 gene published in Kiang, D. T. et al. (1997) Gene 199 (1-2): 165-171; has the following sequence:  
                               1   GATTTAATCC TATGACAAAC TAAGTTGGTT CTGTCTTCAC CTGTTTTGGT                   51   GAGGTTGTGT AAGAGTTGGT GTTTGCTCAG GAAGAGATTT AAGCATGCTT               101   GCTTACCCAG ACTCAGAGAA GTCTCCCTGT TCTGTCCTAG CTAGTGATTC               151   CTGTGTTGTG TGCATTCGTC TTTTCCAGAG CAAACCGCCC AGAGTAGAAG               201     ATG GATTGGG GCACGCTGCA GACGATCCTG GGGGGTGTGA ACAAACACTC               251   CACCAGCATT GGAAAGATCT GGCTCACCGT CCTCTTCATT TTTCGCATTA               301   TGATCCTCGT TGTGGCTGCA AAGGAGGTGT GGGGAGATGA GCAGGCCGAC               351   TTTGTCTGCA ACACCCTGCA GCCAGGCTGC AAGAACGTGT GCTACGATCA               401   CTACTTCCCC ATCTCCCACA TCCGGCTATG GGCCCTGCAG CTGATCTTCG               451   TGTCCACGCC AGCGCTCCTA GTGGCCATGC ACGTGGCCTA CCGGAGACAT               501   GAGAAGAAGA GGAAGTTCAT CAAGGGGGAG ATAAAGAGTG AATTTAAGGA               551   CATCGAGGAG ATCAAAACCC AGAAGGTCCG CATCGAAGGC TCCCTGTGGT               601   GGACCTACAC AAGCAGCATC TTCTTCCGGG TCATCTTCGA AGCCGCCTTC               651   ATGTACGTCT TCTATGTCAT GTACGACGGC TTCTCCATGC AGCGGCTGGT               701   GAAGTGCAAC GCCTGGCCTT GTCCCAACAC TGTGGACTGC TTTGTGTCCC               751   GGCCCACGGA GAAGACTGTC TTTCACAGTG TTCATGATTG CAGTGTCTGG               801   AATTTGCATC CTGCTGAATG TCACTGAATT GTGTTATTTG CTAATTAGAT               851   ATTGTTCTGG GAAGTCAAAA AAGCCAGTTT AACGCATTGC CCAGTTGTTA               901   GATTAAGAAA TAGACAGCAT CAGAGGGATG AGGCAACCCG TGCTCAGCTG               951   TCAAGGCTCA GTCGCCAGCA TTTCCCAACA CAAAGATTCT GACCTTAAAT               1001   GCAACCATTT GAAACCCCTG TAGGCCTCAG GTGAAACTCC AGATGCCACA               1051   ATGAGCTCTG CTCCCCTAAA GCCTCAAAAC AAAGGCCTAA TTCTATGCCT               1101   GTCTTAATTT TCTTTCACTT AAGTTAGTTC CACTGAGACC CCAGGCTGTT               1151   AGGGGTTATT GGTGTAAGGT ACTTTCATAT TTTAAACAGA GGATATCGGC               1201   ATTTGTTTCT TTCTCTGAGG ACAAGAGAAA AAAGCCAGGT TCCACAGAGG               1251   ACACAGAGAA GGTTTGGGTG TCCTCCTGGG GTTCTTTTTG CCAACTTTCC               1301   CCACGTTAAA GGTGAACATT GGTTCTTTCA TTTGCTTTGG AAGTTTTAAT               1351   CTCTAACAGT GGACAAAGTT ACCAGTGCCT TAAACTCTGT TACACTTTTT               1401   GGAAGTGAAA ACTTTGTAGT ATGATAGGTT ATTTTGATGT AAAGATGTTC               1451   TGGATACCAT TATATGTTCC CCCTGTTTCA GAGGCTCAGA TTGTAATATG               1501   TAAATGGTAT GTCATTCGCT ACTATGATTT AATTTGAAAT ATGGTCTTTT               1551   GGTTATGAAT ACTTTGCAGC ACAGCTGAGA GAGGCTGTCT GTTGTATTCA               1601   TTGTGGTCAT AGCACCTAAC AACATTGTAG CCTCAATCGA GTGAGACAGA               1651   CTAGAAGTTC CTAGTTGGCT TATGATAGCA AATGGCCTCA TGTCAAATAT               1701   TAGATGTAAT TTTGTGTAAG AAATACAGAC TGGATGTACC ACCAACTACT               1751   ACCTGTAATG ACAGGCCTGT CCAACACATC TCCCTTTTCC ATGCTGTGGT               1801   AGCCAGCATC GGAAAGAACG CTGATTTAAA GAGGTGAGCT TGGGAATTTT               1851   ATTGACACAG TACCATTTAA TGGGGAGACA AAAATGGGGG CCAGGGGAGG               1901   GAGAAGTTTC TGTCGTTAAA AACGAGTTTG GAAAGACTGG ACTCTAAATT               1951   CTGTTGATTA AAGATGAGCT TTGTCTACCT TCAAAAGTTT GTTTGGCTTA               2001   CCCCCTTCAG CCTCCAATTT TTTAAGTGAA AATATAACTA ATAACATGTG               2051   AAAAGAATAG AAGCTAAGGT TTAGATAAAT ATTGAGCAGA TCTATAGGAA               2101   GATTGAACCT GAATATTGCC ATTATGCTTG ACATGGTTTC CAAAAAATGG               2151   TACTCCACAT ACTTCAGTGA GGGTAAGTAT TTTCCTGTTG TCAAGAATAG               2201   CATTGTAAAA GCATTTTGTA ATAATAAAGA ATAGCTTTAA TGATATGCTT               2251   GTAACTAAAA TAATTTTGTA ATGTATCAAA TACATTTAAA ACATTAAAAT               2301   ATAATCTCTA TAAT          
 
         [0099]    SEQ ID No. 8). The ATG underlined in the sequences corresponds to the start codon. The guanine residue “G”, which is in bold print, marks the end of the guanosine rich region between nucleotides 27 and 32, inclusive.  
                                                                                                                                 TABLE 1                           Mutations in the Cx26 coding exon in individuals       affected with familial forms of prelingual       deafness            Family   30delG   Other           (geographical origin)   mutation   mutation   Deafness                    DENB1-linked families                S15       homozygous   —   profound           (sTu)       S19       homozygous   —   profound           (sTu)       ST       —   homozygous   profound           (sTu)       E47X        20       homozygous   —   profound           (nTu)        60       homozygous   —   profound           (nTu)       LH       homozygous   —   severe-profound           (Leb)            Families consistent with           DFNB1 linkage             51       homozygous   —   severe-profound           (NZ)       1160       homozygous   —   moderate-severe*           (NZ)       1548       —   —   profound           (NZ)       1608       homozygous   —   profound**           (NZ)       1773       heterozygous   —   profound           (NZ)       1873       homozygous   —   moderate       individual       II.3           (NZ)       1873       —   heterozygous   profound       individual       II.2           (NZ)       30del38       1877       homozygous   —   profound           (NZ)       9670           delE118/R14   moderate-severe           (Aust)       8P            Families uncharacterized           for DFNB1 linkage            P1       homozygous   —   severe-profound           (Fr)       P2       —   —   profound           (Fr)       P3       homozygous   —   severe-profound           (Leb)       P4       —   —   severe-profound           (Tur)       p5       homozygous   —   profound           (Fr)       P6       heterozygous   —   severe-profound           (Fr)       p7       —   —   moderate           (Fr)       P8       —   —   moderate           (Fr)       L13131       —   —   profound           (Fr)       L14190       —   —   mild-moderate           (Fr)       P9       homozygous   —   severe-profound           (Por)       P10       homozygous   —   severe-profound           (Fr)       P11       heterozygous   —   moderate-severe           (Fr)       P12       —   —   severe-profound           (Fr)       P13       —   —   profound           (Fr)       P14       heterozygous   —   moderate-severe           (Alg)       P15       —   —   severe-profound           (Fr)       P16   (mother/Fr,   homozygous   —severe**           father/Pol)       P17       heterozygous   —   severe***           (Fr)       1885       heterozygous   —   profound           (NZ)       2254       —   —   moderate-severe           (NZ)                                                          
 
         [0100]    [0100] 
     
       
       
         1 
         
           
             15  
           
           
             1  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            1 

tcttttccag agcaaaccgc c                                               21 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            2 

tgagcacggg ttgcctcatc                                                 20 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            3 

gacacgaaga tcagctgcag                                                 20 

 
           
             4  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            4 

ccaggctgca agaacgtgtg                                                 20 

 
           
             5  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            5 

agacgatcct gggggtgtga acaaa                                           25 

 
           
             6  
             15  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            6 

atcctggggg tgtga                                                      15 

 
           
             7  
             27  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            7 

agacgatcct gggggctcac cgtcctc                                         27 

 
           
             8  
             2314  
             DNA  
             Homo sapiens  
           
            8 

gatttaatcc tatgacaaac taagttggtt ctgtcttcac ctgttttggt gaggttgtgt     60 

aagagttggt gtttgctcag gaagagattt aagcatgctt gcttacccag actcagagaa    120 

gtctccctgt tctgtcctag ctagtgattc ctgtgttgtg tgcattcgtc ttttccagag    180 

caaaccgccc agagtagaag atggattggg gcacgctgca gacgatcctg gggggtgtga    240 

acaaacactc caccagcatt ggaaagatct ggctcaccgt cctcttcatt tttcgcatta    300 

tgatcctcgt tgtggctgca aaggaggtgt ggggagatga gcaggccgac tttgtctgca    360 

acaccctgca gccaggctgc aagaacgtgt gctacgatca ctacttcccc atctcccaca    420 

tccggctatg ggccctgcag ctgatcttcg tgtccacgcc agcgctccta gtggccatgc    480 

acgtggccta ccggagacat gagaagaaga ggaagttcat caagggggag ataaagagtg    540 

aatttaagga catcgaggag atcaaaaccc agaaggtccg catcgaaggc tccctgtggt    600 

ggacctacac aagcagcatc ttcttccggg tcatcttcga agccgccttc atgtacgtct    660 

tctatgtcat gtacgacggc ttctccatgc agcggctggt gaagtgcaac gcctggcctt    720 

gtcccaacac tgtggactgc tttgtgtccc ggcccacgga gaagactgtc tttcacagtg    780 

ttcatgattg cagtgtctgg aatttgcatc ctgctgaatg tcactgaatt gtgttatttg    840 

ctaattagat attgttctgg gaagtcaaaa aagccagttt aacgcattgc ccagttgtta    900 

gattaagaaa tagacagcat gagagggatg aggcaacccg tgctcagctg tcaaggctca    960 

gtcgccagca tttcccaaca caaagattct gaccttaaat gcaaccattt gaaacccctg   1020 

taggcctcag gtgaaactcc agatgccaca atgagctctg ctcccctaaa gcctcaaaac   1080 

aaaggcctaa ttctatgcct gtcttaattt tctttcactt aagttagttc cactgagacc   1140 

ccaggctgtt aggggttatt ggtgtaaggt actttcatat tttaaacaga ggatatcggc   1200 

atttgtttct ttctctgagg acaagagaaa aaagccaggt tccacagagg acacagagaa   1260 

ggtttgggtg tcctcctggg gttctttttg ccaactttcc ccacgttaaa ggtgaacatt   1320 

ggttctttca tttgctttgg aagttttaat ctctaacagt ggacaaagtt accagtgcct   1380 

taaactctgt tacacttttt ggaagtgaaa actttgtagt atgataggtt attttgatgt   1440 

aaagatgttc tggataccat tatatgttcc ccctgtttca gaggctcaga ttgtaatatg   1500 

taaatggtat gtcattcgct actatgattt aatttgaaat atggtctttt ggttatgaat   1560 

actttgcagc acagctgaga gaggctgtct gttgtattca ttgtggtcat agcacctaac   1620 

aacattgtag cctcaatcga gtgagacaga ctagaagttc ctagttggct tatgatagca   1680 

aatggcctca tgtcaaatat tagatgtaat tttgtgtaag aaatacagac tggatgtacc   1740 

accaactact acctgtaatg acaggcctgt ccaacacatc tcccttttcc atgctgtggt   1800 

agccagcatc ggaaagaacg ctgatttaaa gaggtgagct tgggaatttt attgacacag   1860 

taccatttaa tggggagaca aaaatggggg ccaggggagg gagaagtttc tgtcgttaaa   1920 

aacgagtttg gaaagactgg actctaaatt ctgttgatta aagatgagct ttgtctacct   1980 

tcaaaagttt gtttggctta cccccttcag cctccaattt tttaagtgaa aatataacta   2040 

ataacatgtg aaaagaatag aagctaaggt ttagataaat attgagcaga tctataggaa   2100 

gattgaacct gaatattgcc attatgcttg acatggtttc caaaaaatgg tactccacat   2160 

acttcagtga gggtaagtat tttcctgttg tcaagaatag cattgtaaaa gcattttgta   2220 

ataataaaga atagctttaa tgatatgctt gtaactaaaa taattttgta atgtatcaaa   2280 

tacatttaaa acattaaaat ataatctcta taat                               2314 

 
           
             9  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            9 

ctagtgattc ctgtgttgtg tgc                                             23 

 
           
             10  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            10 

ataatgcgaa aaatgaagag ga                                              22 

 
           
             11  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            11 

aaaaaaaatc ctggggggtg tg                                              22 

 
           
             12  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            12 

aaaaaaaatc ctgggggtgt ga                                              22 

 
           
             13  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            13 

cagcattgga aagatctggc tca                                             23 

 
           
             14  
             63  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      Oligonucleotide  
             
           
            14 

cgcccgccgc gccccgcgcc cggcccgccg cccccgcccc ctagtgattc ctgtgttgtg     60 

tgc                                                                   63 

 
           
             15  
             2312  
             DNA  
             Homo sapiens  
           
            15 

gatttaatcc tatgacaaac taagttggtt ctgtcttcac ctgttttggt gaggttgtgt     60 

aagagttggt gtttgctcag gaagagattt aagcatgctt gcttacccag actcagagaa    120 

gtctccctgt tctgtcctag ctatgttcct gtgttgtgtg cattcgtctt ttccagagca    180 

aaccgcccag agtagaagat ggattggggc acgctgcaga cgatcctggg gggtgtgaac    240 

aaacactcca ccagcattgg aaagatctgg ctcaccgtcc tcttcatttt tcgcattatg    300 

atcctcgttg tggctgcaaa ggaggtgtgg ggagatgagc aggccgactt tgtctgcaac    360 

accctgcagc caggctgcaa gaacgtgtgc tacgatcact acttccccat ctcccacatc    420 

cggctatggg ccctgcagct gatcttcgtg tccagcccag cgctcctagt ggccatgcac    480 

gtggcctacc ggagacatga gaagaagagg aagttcatca agggggagat aaagagtgaa    540 

tttaaggaca tcgaggagat caaaacccag aaggtccgca tcgaaggctc cctgtggtgg    600 

acctacacaa gcagcatctt cttccgggtc atcttcgaag ccgccttcat gtacgtcttc    660 

tatgtcatgt acgacggctt ctccatgcag cggctggtga agtgcaacgc ctggccttgt    720 

cccaacactg tggactgctt tgtgtcccgg cccacggaga agactgtctt tcacagtgtt    780 

catgattgca gtgtctggaa tttgcatcct gctgaatgtc actgaattgt gttatttgct    840 

aattagatat tgttctggga agtcaaaaaa gccagtttaa cgcattgccc agttgttaga    900 

ttaagaaata gacagcatga gagggatgag gcaacccgtg ctcagctgtc aaggctcagt    960 

cgccagcatt tcccaacaca aagattctga ccttaaatgc aaccatttga aacccctgta   1020 

ggcctcaggt gaaactccag atgccacaat gagctctgct cccctaaagc ctcaaaacaa   1080 

aggcctaatt ctatgcctgt cttaattttc tttcacttaa gttagttcca ctgagacccc   1140 

aggctgttag gggttattgg tgtaaggtac tttcatattt taaacagagg atatcggcat   1200 

ttgtttcttt ctctgaggac aagagaaaaa agccaggttc cacagaggac acagagaagg   1260 

tttgggtgtc ctcctggggt tctttttgcc aactttcccc acgttaaagg tgaacattgg   1320 

ttctttcatt tgctttggaa gttttaatct ctaacagtgg acaaagttac cagtgcctta   1380 

aactctgtta cactttttgg aagtgaaaac tttgtagtat gataggttat tttgatgtaa   1440 

agatgttctg gataccatta tatgttcccc ctgtttcaga ggctcagatt gtaatatgta   1500 

aatggtatgt cattcgctac tatgatttaa tttgaaatat ggtcttttgg ttatgaatac   1560 

tttgcagcac agctgagaga ggctgtctgt tgtattcatt gtggtcatag cacctaacaa   1620 

cattgtagcc tcaatcgagt gagacagact agaagttcct agttggctta tgatagcaaa   1680 

tggcctcatg tcaaatatta gatgtaattt tgtgtaagaa atacagactg gatgtaccac   1740 

caactactac ctgtaatgac aggcctgtcc aacacatctc ccttttccat gctgtggtag   1800 

ccagcatcgg aaagaacgct gatttaaaga ggtgagcttg ggaattttat tgacacagta   1860 

ccatttaatg gggagacaaa aatgggggcc aggggaggga gaagtttctg tcgttaaaaa   1920 

cgagtttgga aagactggac tctaaattct gttgattaaa gatgagcttt gtctaccttc   1980 

aaaagtttgt ttggcttacc cccttcagcc tccaattttt taagtgaaaa tataactaat   2040 

aacatgtgaa aagaatagaa gctaaggttt agataaatat tgagcagatc tataggaaga   2100 

ttgaacctga atattgccat tatgcttgac atggtttcca aaaaatggta ctccacatag   2160 

ttcagtgagg gtaagtattt tcctgttgtc aagaatagca ttgtaaaagc attttgtaat   2220 

aataaagaat agctttaatg atatgcttgt aactaaaata attttgtaat gtatcaaata   2280 

catttaaaac attaaaatat aatctctata at                                 2312