Characterization of a new full length TMPRSS3 isoform and identification of mutant alleles responsible for nonsyndromic recessive deafness in Newfoundland and Pakistan

Abstract

Background: Mutant alleles of TMPRSS3 are associated with nonsyndromic recessive deafness (DFNB8/B10). TMPRSS3 encodes a predicted secreted serine protease, although the deduced amino acid sequence has no signal peptide. In this study, we searched for mutant alleles of TMPRSS3 in families from Pakistan and Newfoundland with recessive deafness co-segregating with DFNB8/B10 linked haplotypes and also more thoroughly characterized the genomic structure of TMPRSS3.

Methods: We enrolled families segregating recessive hearing loss from Pakistan and Newfoundland. Microsatellite markers flanking the TMPRSS3 locus were used for linkage analysis. DNA samples from participating individuals were sequenced for TMPRSS3. The structure of TMPRSS3 was characterized bioinformatically and experimentally by sequencing novel cDNA clones of TMPRSS3.

Results: We identified mutations in TMPRSS3 in four Pakistani families with recessive, nonsyndromic congenital deafness. We also identified two recessive mutations, one of which is novel, of TMPRSS3 segregating in a six-generation extended family from Newfoundland. The spectrum of TMPRSS3 mutations is reviewed in the context of a genotype-phenotype correlation. Our study also revealed a longer isoform of TMPRSS3 with a hitherto unidentified exon encoding a signal peptide, which is expressed in several tissues.

Conclusion: Mutations of TMPRSS3 contribute to hearing loss in many communities worldwide and account for 1.8% (8 of 449) of Pakistani families segregating congenital deafness as an autosomal recessive trait. The newly identified TMPRSS3 isoform e will be helpful in the functional characterization of the full length protein.

Figure 2

Pedigrees of Pakistani families. Four families with nonsyndromic recessive deafness from Pakistan.

Figure 1

Pedigree of Newfoundland family. There are ten hearing impaired individuals in a six generation extended family structure. Drawn below the enrolled subjects is a haplotype for STR markers around the DFNB8/B10 locus on chromosome 21. The carrier status of each person for the mutant alleles of TMPRSS3 found in this family is shown. "C" represents 207delC, while "T" stands for IVS8+8insT. Individuals V:17 and V:20 are compound heterozygotes.

Figure 3

Mutational spectrum, structure and expression of TMPRSS3 isoforms. (A) Coding and non-coding exons of all the known isoforms of TMPRSS3 are shown with black and gray rectangles, respectively. The newly identified isoform e has translation initiation codon (arrow) in exon 1, while the termination codon in exon 13 is marked with an asterisk. Shown also are predicted protein motifs encoded by the 3192 bp long mRNA of isoform e. All the known mutant alleles of TMPRSS3 causing hearing loss are shown above the protein motifs. Modified and updated from Ben-Yosef et al. 2001 [5] (B) Nucleotide sequence of the cDNA encoding the amino terminus of TMPRSS3e and its deduced amino acid sequence. The underlined nucleotide sequence represents the region predicted by SMART to encode a signal peptide. The last ATG shown is the reported translation initiation site for isoform a [4]. (C) RT-PCR specific to the TMPRSS3e transcript was performed on cDNA from seven human tissues, which include retina, lung, liver, heart, pancreas, placenta and kidney as indicated. All tissues, except heart, demonstrated expression of TMPRSS3e. G3PDH was used as a positive control.