HUMAN MUTATION Mutation in Brief #773 (2005) Online

MUTATION IN BRIEF

Novel TMC1 Structural and Splice Variants Associated with Congenital Nonsyndromic Deafness in a Sudanese Pedigree
Christian G. Meyer1*, Nagla M. Gasmelseed2, Adil Mergani2, Mubarak M.A. Magzoub2,3, Birgit Muntau1, Thorsten Thye1, and Rolf D. Horstmann1
1 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany; 2Institute of Nuclear Medicine, Molecular Biology & Oncology, University of Gezira, Wad Medani, Sudan; 3Ministry of Higher Education and Research, Khartoum, Sudan

*Correspondence to: Christian G. Meyer, Dept. Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, 20359 Hamburg, Germany; Tel.: +49 40 42818-501; Fax +49 40 42818-512; E-mail: c.g.meyer@bni.uni-hamburg.de
Communicated by Henrick Dahl

Mutations of the transmembrane channel-like gene 1 (TMC1) have been shown to cause autosomal dominant and recessive forms of congenital nonsyndromic deafness linked to the loci DFNA36 and DFNB7/B11, respectively. In a Sudanese pedigree affected by an apparently recessive form of nonsyndromic deafness, we performed a linkage analysis using markers covering the deafness loci DFNB1 DFNB30. A two-point LOD score of 3.08 was obtained at marker position D9S1876, located within the first intron of the TMC1 gene at DFNB7/B11. By DNA sequencing of TMC1 exons 3-22, we identified the structural variant c.1165C>T in exon 13, leading to the stop codon p.Arg389X, and the splice-site variant c.19+5G>A, independently segregating with the deafness phenotype. The c.1165C>T [p.Arg389X] mutation was also observed in four out of 243 unrelated deaf Sudanese individuals, but none of the mutations was found among 292 normal hearing controls. The finding of TMC1 mutations contributing to deafness in Sudan confirms and extends previous reports on the role of TMC1 in recessive nonsyndromic deafness and shows that deafnesscausing TMC1 mutations may occur in various ethnic groups. 2004 Wiley-Liss, Inc.
KEY WORDS: transmembrane channel-like gene 1l; TMC1; autosomal recessive nonsyndromic deafness; DFNB7/11; mutation analysis; mass spectrometry; Sudan

INTRODUCTION

Hereditary deafness is one of the most prevalent sensory defects in humans with an incidence of approximately 1 in 2000 births. Dominant, recessive and X-linked forms of deafness are highly heterogeneous disorders involving a variety of loci designated DFNA, DFNB, and DFN, respectively (www.uia.ac.be/dnalab/hhh/). In recent years, enormous progress has been made in defining the underlying genes and their disease-causing variants. In Caucasian populations, nonsyndromic recessive deafness was mostly found to be of the DFNB1 type, which results from a great number of distinct mutations of the GJB2 gene encoding connexin 26 (Cx26) (www.crg.es/deafness/). In contrast, variants of this gene were virtually absent in a sample of 183 Sudanese individuals studied for congenital nonsyndromic deafness (Gasmelseed et al., 2004). We, therefore, performed a linkage analysis in a small affected and GJB2-wildtype consanguineous pedigree and, in addition, determined the frequency of novel TMC1 mutations (MIM# 606706) identified by DNA sequencing among deaf and normal hearing Sudanese

Received 8 July 2004; accepted revised manuscript 12 October 2004.

2004 WILEY-LISS, INC. DOI: 10.1002/humu.9302

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individuals by a biplex mass spectrometry assay.

MATERIAL AND METHODS

Study group. The study design was approved by the Ethics Committee of the Ministry of Higher Education and Research, Khartoum, Sudan. In addition to a small affected pedigree, 222 congenitally deaf and 21 individuals with a postnatal onset of deafness were recruited from Schools for the Deaf in the States of Khartoum and Gezira for a study on GJB2-associated deafness (Gasmelseed et al., 2004). Furthermore, blood samples from 292 normal hearing Sudanese individuals were available. All samples had been collected during a previous study on GJB2associated deafness with written consent obtained from all participating individuals. Profound deafness was assessed audiometrically, and no additional symptoms were found upon clinical examination. Linkage analysis. The twelve members of the pedigree comprising six congenitally affected and six normal hearing individuals were considered informative for linkage (Fig. 1). We tested for linkage to recessive deafness loci recognized to be responsible for nonsyndromic deafness with 81 microsatellite markers covering DFNB1 DFNB30 on chromosomes 14, 7, 911, 1315, 1719, and 2122. Genotypes were determined using the GenScan and GenoTyper software (Applied Biosystems, Foster City, CA). Inheritance of alleles was verified by the PedCheck program (OConnell & Weeks, 1998). Order and distances of markers were obtained from the Marshfield and deCODE gene maps (Broman et al., 1998; Kong et al., 2002) and verified by calculating the order with own marker data using the CRIMAP software (Lander & Green, 1987). Genotyping of microsatellite markers was done on an ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA), and the Genehunter software was applied for linkage analysis (Kruglyak et al., 1996). PCR and DNA sequencing. PCR assays were established to amplify the TMC1 structural exons 3-22 (GenBank accession # NM_138691.2). Amplicons were sequenced (ABI PRISM 3100 Genetic Analyzer, BigDye terminator cycle sequencing ready reaction kit; Applied Biosystems, Foster City, CA) according to the manufacturers instructions with primers identical to those of the primary PCR. Primers for the PCR/sequencing reactions are listed in Table 1. Mass spectometry. To assess the frequency of the two single nucleotide polymorphisms (SNPs) among deaf and normal hearing Sudanese individuals we designed a biplex matrix-assisted laser desorption-ionization time-of flight mass spectrometry (MALDI-TOF MS; Autoflex Bruker-Daltonics, Bremen, Germany) assay to identify mutations at nucleotide positions 1165 of exon 13 (c.1165C) and at c.19+5. The primary biplex PCR yielded 159 bp and 178 bp products the regions containing c.1165 (exon 13) and c.19+5, respectively, which subsequently were digested (shrimp alkaline phosphatase, SAP) to remove excess dNTPs (primers given in Table 1). After SAP digestion an oligonucleotide probe was annealed to the PCR product adjacent to the nucleotide under consideration, and a biplex primer extension reaction was initiated using 5- biotin-labelled primers containing a UV-light linker (photocleavable o-nitrobenzyl derivative) that is inserted to replace one nucleotide closely adjacent to the mutation under consideration (primers given in Table 1). The products were purified according to the manufacturers recommendations. Elongation of the probe depends on the known variability of the template (e.g. exerted through SNPs), which is reflected in the content and composition of stop nucleotides (ddNTPs) within the reaction mixture. Here, ddATP and ddGTP were used to identify the c.19+5 mutation in a sense and the exon 13 mutation in an antisense reaction. The reaction product was purified (Genesis Workstation 200; Tecan, Durham, NC) by binding of biotin-labelled elongated oligocucleotides to streptavidin-coated microplates (Genostrep; Bruker Daltonics), washing/desalting steps, and UV-photocleaving (360 nm wave length). One l of the elongated analyte was embedded into a 3hydroxypicolinic-acid matrix on anchor chips with 400 m hydrophilic anchors in a hydrophobic surface for subsequent desorption/ionization. Spectrometric data were generated by summing of several independent spectra obtained per spot and genotypes were analyzed by the Genotools software (Bruker Daltonics) and displayed according to their mass/charge ratio (m/z).

TMC1 Variants in Nonsydromic Deafness

Table 1. Primers for PCR/sequencing, Mass Spectometry and Extension Reactions

TMC1ex3 TMC1ex4 TMC1ex5 TMC1ex6 TMC1ex7 TMC1ex8 TMC1ex9 TMC1ex10 TMC1ex11 TMC1ex12 TMC1ex13 TMC1ex14/15 TMC1ex16 TMC1ex17 TMC1ex18 TMC1ex19 TMC1ex20/21 TMC1ex22

Primers used for PCR/sequencing of TMC1 exons 3-22 sense antisense 5-gagtactccgtcccttaaatgg-3 5-ggatgaatgatttcaccaactgg-3 5-atcgtgctcattttggcaag-3 5-gcccttatagtgccaactgg-3 5-gcttggtagtgggaggaagc-3 5-gaaaggagtgatcttcttcagc-3 5-atatctgatacctgccttcc-3 5-gatatacatgtttatgactgtgacc-3 5-acagtctaggttcacctgtg-3 5-gtgttcttcttagaggcaatatg-3 5-gtatttgctgccagagagac-3 5-ttggactaaggtagaacacgagag-3 5-gaccaatgcctcacaattaatgg-3 5-tcctatgactctaagacgtg-3 5-ctggatttagtagtcgattc-3 5-agacattcagcctgacccag-3 5-gctcatgtcagagctgtgacccaag-3 5-catttcatctcttacagactctg-3 5-cactcatgatcatgttttgttgc-3 5-tactgcactactggaccagatg-3 5-gtcattcctcacattctgatgattg-3 5-aactgtaagggcaggatagg-3 5-gaatcttccaaaattctggc-3 5-agagccagcacacagtcaac-3 5-ttaattgcagtcttcaagcc-3 5-ctccactattgacctatgaccctg-3 5-ccctagccatgcctatgcag-3 5-atggagttagacaccgattg-3 5-gtggcagtgtattttctgcc-3 5-aatgaccattcccacctccac-3 5-gaggaagaacatcacagaggctc-3 5-ctaccctgaaagaggacctaagcag-3 5-gagtagaagatagcttaatg-3 5-gtggaatgactcgctcacagc-3 5-gcattaagcacactgaaagc-3 5-cttttcatactgaatccagcacc-3 Primers for the mass spectometry assay 5-taacacttggagggagtggata-3 5-caaaagaaacaaatgtcaggttcta-3 5-ggctggtcattgctgtc-3 5-tctcaccaccatgtaatcagt-3 Primers for extension reactions biotin-gctgtgcaaatLcctgggatc (antisense) biotin-gctgtcattttggtgaLggtgtat (sense)

TMC1ex13ms TMC1ex19ms

TMC1ex13L10 TMC1ex19G/As

L, photolinker; TMC1 GenBank accession # NT_023935.16

RESULTS

Linkage analysis yielded a peak on chromosome 9q13-q21 with a two-point LOD score of 2.06 at D9S301 (deCODE map 66.99 cM). Additional coverage with microsatellites D9S237 (67.36 cM), D9S1837, and D9S1876 (both at 68.3 cM) increased the maximum LOD score to 3.08, with of 0.05, at marker position D9S1876. D9S1876 is located within the first intron of the transmembrane channel-like gene 1 (TMC1, 9q11-q21; MIM# 606706, NCBI Locus ID 117531, GenBank accession # NM_138691.2; nt 541..2823). DNA sequencing of the TMC1 structural exons 3-22 of the twelve individuals revealed a heterozygous C>T substitution at nucleotide position 1165 of exon 13 (c.1165C>T; GenBank AY546106), resulting in the stop codon p.Arg389X, in four affected and in two normal hearing individuals. In addition, a G>A transition at the c.19+5 splice donor site (c.19+5G>A; GenBank AY546105) occurred homozygously in two deaf individuals of the pedigree without the stop codon p.Arg389X, in compound heterozygosity with TMC1 Arg389X in four deaf and, heterozygously with the normal sequence, in four hearing individuals (Fig. 1). Both variants segregated independently with the deafness phenotype linked to two different alleles of D9S1876 in small conserved haplotypes. Recombinations of both mutations with the microsatellite marker D9S1876 were not observed, although a value of 0.05 is given when calculating the LOD score. This results from the fact that in the pedigree under study two different mutations were associated with the phenotype. A priori, only a single mutation would be more likely to cause the phenotype in this pedigree, resulting in a higher LOD score at = 0.

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Fig. 1. Pedigree and genotype analysis of the DFNB7/11 Sudanese family. Informative marker alleles are shown. Marker D9S1876 is located within the first intron of TMC1. All haplotypes were inferred on the basis of minimal recombination. Haplotypes of DFNB7/B11 markers on chromosome 9q13-q21 segregate with the recessive deafness phenotype. Black boxes: haplotypes carrying the c.19+5G>A variant; hatched boxes, haplotypes carrying the c.1165C>T [p.Arg389X] variant.

Information on the frequency of the two novel TMC1 mutations among deaf and 292 normal hearing individuals was provided through the results of the MALDI-TOF MS assay. The respective MALDI-TOF spectra and the corresponding electropherograms are shown in Figure 2. The assay confirmed the occurrence of the TMC1 variants in the twelve members of the family that was subjected to linkage analysis (Fig. 1). Furthermore, c.1165C>T [p.Arg389X] in exon 13 was found homozygously in four congenitally deaf individuals apparently not related to the pedigree. The c.19+5G>A mutation was exclusively identified in members of the family as indicated. Both mutations did not occur in the normal hearing control group.
DISCUSSION

The rare occurrence of GJB2 mutations in deaf Sudanese individuals and the availability of a small consanguineous pedigree with six affected and six normal hearing members led to a linkage analysis with markers covering relevant loci associated with recessive deafness. The borderline two-point LOD score of 3.08 at a marker located within the first intron of the TMC1 gene and the previous recognition of TMC1 variants being causally involved in dominant and recessive deafness (Kurima et al., 2002) prompted DNA sequencing of the translated exons and adjacent intervening sequences. Sequence analyses eventually resulted in the identification of a novel structural and an intronic mutation in exon 13 and the intervening sequence following exon 19, respectively, of the TMC1 gene.

TMC1 Variants in Nonsydromic Deafness

Fig. 2. MALDI-TOF MS spectra with the corresponding sequencing electropherograms. MALDI-TOF MS spectra of the biplex assay designed to identify the mutations c.1165C>T (exon 13) and c.19+5G>A with the corresponding sequencing electropherograms inserted (reverse sequences given for exon 13). The intensity of peaks generated by one laser/acceleration impulse is indicated as relative intensity (r.i.), whereby the molecule yielding the highest peak is 100 %. Spectra A-C show peaks for the oligonucleotide alone used for primer extension (on, oligonucleotide) and the products that are extended by one base (wt, wild type sequence; mut, mutation). The first two peaks of each spectrum correspond to the c.19+5 variants, the third and fourth peaks relate to the exon 13 mutation. A: wild-type sequences of c.19+5G (wt) and position c.1165C of exon 13 (wt); B: c.19+5A (mut) and position c.1165C of exon 13 (wt); C: heterozygosity of c.19+5G/A (wt/mut) and position c.1165C/T of exon 13 (wt/mut). m/z, mass/charge ratio.

6 Meyer et al.

TMC genes and a variety of homologues encode families of vertebrate and invertebrate transmembrane proteins and are, most likely, channel-forming or transporter molecules or modifiers (Keresztes et al., 2003). Two members of the TMC gene family (EVER1 [MIM# 605828] and EVER2 [MIM# 605829]; syn. TMC6, TMC8) have been shown to be associated with epidermodysplasia verruciformis (Ramoz et al., 2002; Kurima et al., 2003), suggesting an analogy to the physiology of modified Cx26 molecules in deafness and dermal pathology. The first evidence that a locus on chromosome 9q13-q21 might contribute to recessive neurosensory nonsyndromic deafness came from linkage analyses in two regionally separate consanguineous Indian families (Jain et al., 1995) and two Israelian Bedouin kindreds (Scott et al., 1996). Subsequently, allelic variability of TMC1 was recognized to causally contribute to dominant progressive DFNA36 (MIM# 606705) and to recessive profound congenital DFNB7/B11 (MIM# 600974) deafness (Kurima et al., 2002). The TMC1-associated DFNA36 and DFNB7/11 phenotypes have, after first studies on the dn/dn mouse (Deol & Kocher, 1958; Webster, 1985; Keats et al., 1995) been explained by phenotyping of the Beethoven (Bth) and the deafness (dn/dn) mouse mutants, respectively (Faddis et al., 1998; Vreugde et al., 2002; Oleskevich & Walmsley, 2002). Both mutants are characterized by early cell degeneration of the organ of Corti and severe impairment of synaptic activity. The stop codon in exon 13 (c.1165G>T, [p.Arg389X]) identified in this study is compatible with the formation of a truncated protein and impaired function. The observation that c.1165G>T occurred homozygously in four individuals that were not related to the pedigree underlines the significance of TMC1 variants in deafness. The c.19+5G>A mutation affects the highly conserved donor splice site motif 5-GTATGT (> 5-GTATAT) that is characteristic of nuclear-located, spliceosomal processed pre-mRNA introns. Mutations at splice sites are commonly associated with diminished amounts or abnormal maturation of mRNA, resulting in either exon skipping or cryptic splice site activation (Krawczak et al., 1992). The substitution of a G at the donor splice site position c.+5 substantially impairs the binding affinity of the splice site with the corresponding U1 snRNA (Lear et al., 1990). Applying the statistical Shapiro-Senapathy splicing algorithm (Senapathy et al., 1990), splice scores of < 0.1 and 0.88 result for the mutant and the wildtype variant, respectively, compatible with skipping of exon 19 and the generation of a truncated protein. While mutations of the first two invariant nucleotides (GT) of the donor splice site are frequently associated with genetic disorders, the c.+ 5-GTATAT variant has, to our knowledge, so far been implicated only in a type II/III protein S-deficient phenotype in two related individuals with recurrent deep venous thromboses (Leroy-Matheron et al., 1998). The identification of novel TMC1 structural and splice site variants segregating with deafness in a Sudanese pedigree and the additional identification of the TMC1-c.1165C>T [p.Arg389X] mutation homozygously in four unrelated deaf individuals confirms the role of TMC1 in non-syndromic DFNB7/B11 deafness. It furthermore demonstrates that TMC1 associated DFNB7/B11 is not restricted to those ethnic groups where it has first been observed, but occurs in other parts of the world as well.
ACKNOWLEDGMENTS

The comments of B. Mueller-Myhsok and the clinical advice of O.M. El Mustafa are gratefully acknowleged.
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