Many proteins necessary for sound transduction have been discovered through positional cloning of genes that cause deafness1–3. In this study, we report that mutations of LRTOMT are associated with profound non-syndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, that are detected by Western blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, novel transcripts can arise through partial or complete coalescence of genes4. We provide evidence that in the primate lineage LRTOMT evolved from the fusion of two neighboring ancestral genes, which exist as separate genes (Lrrc51and Tomt) in rodents.

Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor β2 (RARβ2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and 9 normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RARβ2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RARβ2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RARβ2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RARβ2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis.To assess the functional significance of the epigenetic silencing of RARβ2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RARβ2 through aberrant methylation (p = 0.003) in NPC biopsies.We concluded that the inactivation of RASSF1A, RARβ2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.

SummaryOtosclerosis is a condition characterized by an abnormal bone metabolism in the otic capsule, resulting in conductive and/or sensorineural hearing loss. Otosclerosis is a common disorder in which genes play an important role. Case-control association studies have implicated several genes in the abnormal bone metabolism associated with otosclerosis: COL1A1, TGFB1, BMP2, and BMP4. To investigate the association of these genes with otosclerosis in the Tunisian population, we examined nine single nucleotide polymorphisms (SNPs) in 159 unrelated otosclerosis patients and 155 unrelated controls. We found an association of rs11327935 in COL1A1 with otosclerosis that was shown to be sex specific. The coding polymorphism T263I in TGFB1 was also associated with otosclerosis in the Tunisian population. The effect sizes of both the associations were consistent with previous studies, as the same effect was found in all cases. The association of BMP2 and BMP4 was not significant. However, a trend towards association was found for the BMP4 gene that was consistent with earlier reports. In conclusion, this study replicates and strengthens the evidence for association between polymorphisms of COL1A1 and TGFB1 in the genetic aetiology of otosclerosis.

SummaryOtosclerosis is a common form of conductive hearing loss, caused by an abnormal bone remodelling in the otic capsule. Both environmental and genetic factors have been implicated in the etiology of this disease. A recent genome wide association study identified two regions associated with otosclerosis, one on chr7q22.1, located in the RELN gene, and one on chr11q13.1. A second study in four European populations has replicated the association of the RELN gene with otosclerosis. To investigate the association of these loci with otosclerosis in a non-European population, we tested 11 SNPs from the two regions in 149 unrelated Tunisian patients and 152 controls. Four SNPs were significantly associated with otosclerosis. Three SNPs are located in the RELN region and the last one is located in the region on chromosome 11. We also observed a significant interaction with gender for rs3914132. This suggests an influence of sex on the association of RELN with otosclerosis. A meta-analysis showed that the disease-associated alleles in the Tunisian sample are the same as in all previously reported associations. Our study provides additional evidence implicating RELN in the development of otosclerosis. Additional functional studies should determine the role of RELN in the physiopathology of this disease.

Hereditary nonsyndromic hearing impairment (HI) is extremely heterogeneous. Mutations of the transmembrane channel-like gene 1 (TMC1) have been shown to cause autosomal dominant and recessive forms of nonsyndromic HI linked to the loci DFNA36 and DFNB7/B11, respectively. TMC1 is 1 member of a family of 8 genes encoding transmembrane proteins. In the mouse, MmTmc1 and MmTmc2 are both members of Tmc subfamily A and are highly and almost exclusively expressed in the cochlea. The restricted expression of Tmc2 in the cochlea and its close phylogenetic relationship to Tmc1 makes it a candidate gene for nonsyndromic HI. We analyzed 3 microsatellite markers linked to the TMC1 and TMC2 genes in 85 Tunisian families with autosomal recessive nonsyndromic HI and without mutations in the protein-coding region of the GJB2 gene. Autozygosity by descent analysis of 2 markers bordering the TMC2 gene allowed us to rule out its association with deafness within these families. However, 5 families were found to segregate deafness with 3 different alleles of marker D9S1837, located within the first intron of the TMC1 gene. By DNA sequencing of coding exons of TMC1 in affected individuals, we identified 3 homozygous mutations, c.100C→T (p.R34X), c.1165C→T (p.R389X) and the novel mutation c.1764G→A (p.W588X). We additionally tested 60 unrelated deaf Tunisian individuals for the c.100C→T mutation. We detected this mutation in a homozygous state in 2 cases. This study confirms that mutations in the TMC1 gene may be a common cause for autosomal recessive nonsyndromic HI.