Refining the DFNB7–DFNB11 deafness locus using intragenic polymorphisms in a novel gene, TMEM2

Abstract

The combined DFNB7–DFNB11 deafness locus maps to chromosome 9q13–q21 between markers D9S1806 and D9S769. We have determined the cDNA sequence and genomic structure of a novel gene, TMEM2, that maps to this interval and is expressed in the cochlea. The mouse orthologue of this gene (Tmem2) maps to the murine dn (deafness) locus on mouse chromosome 19. Screens for transmembrane helices reveal the presence of at least one putative transmembrane domain in the TMEM2 protein.

To determine whether mutations in TMEM2 cause hearing loss at the DFNB7–DFNB11 locus, we screened the coding region of this gene in DFNB7–DFNB11 affected families by direct sequencing. All DNA variants that segregated with the deafness and changed the predicted amino acid sequence of TMEM2 were common polymorphisms, as demonstrated by allele-specific amplification of pooled control DNA. Northern blot analysis showed no difference in transcript size or expression level of Tmem2 in dn/dn and control mice. The intragenic polymorphisms in TMEM2 represent a novel centromeric boundary for the DFNB7–DFNB11 interval.

Introduction

Severe congenital hearing impairment affects 0.1% of live-born infants and 1–2% of graduates of neonatal intensive care units (Marazita et al., 1993, Morton, 1991, Oudesluys-Murphy et al., 1996, Schein, 1980, Van Camp et al., 1997). Although deafness can be caused by a number of environmental and disease-related factors, in developed countries, at least 50% of cases are inherited (Reardon, 1992, Rose et al., 1977). Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common form of severe inherited childhood deafness. This genetically heterogeneous disorder can be caused by mutations in a number of different genes. To date, 28 ARNSHL loci (DFNB1–28) have been reported, and mutations in six genes — GJB2, MYO7A, MYO15, PDS, OTOF and TECTA — have been shown to cause ARNSHL (Kelsell et al., 1997, Li et al., 1998, Liu et al., 1997, Van Camp and Smith, 1999, Wang et al., 1998, Weil et al., 1997).

Five families have been described with ARNSHL linked to the DFNB7 and DFNB11 loci on chromosome 9q13–q21 (Greinwald et al., 1997, Jain et al., 1995, Scott et al., 1996). Although these loci were reported originally to map to different regions of chromosome 9, the correct placement of key markers defined a region shared by both DFNB7 and DFNB11 families (Greinwald et al., 1997), suggesting that mutations in a single gene are responsible for the DFNB7–DFNB11 hearing loss.

The DFNB7–DFNB11 interval on chromosome 9 is syntenic to that portion of mouse chromosome 19 associated with the dn mouse deafness locus (Jain et al., 1995), suggesting that these defects may be caused by mutations in orthologous genes (Jain et al., 1995, Keats et al., 1995). The dn/dn mouse mutant, which arose spontaneously from the curly-tail stock (ct), has ultrastructural abnormalities of the inner hair cells at birth, and by post-natal day 20, the organ of Corti is degenerated, and the inner and outer hair cells are lost (Bock and Steel, 1983, Dole and Kocher, 1958, Keats et al., 1995, Pujol et al., 1983). Backcross experiments and fluorescence in-situ hybridization (FISH) have demonstrated a chromosomal inversion at the dn locus (Viñas et al., 1998).

In this report, we describe the identification of a novel cochlear-expressed gene, TMEM2, which is located in the DFNB7–DFNB11 interval in humans and whose murine orthologue (Tmem2) maps to the dn region. The genomic structure and expression pattern of this gene are described, as are the results of a TMEM2 /Tmem2 mutation screen in DFNB7–DFNB11 families and dn mice.