Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1 Δ mice) were deaf and those with a deletion of Tmc2 (Tmc2 Δ mice) were phenotypically normal, Tmc1 Δ Tmc2 Δ mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1 Δ Tmc2 Δ mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

Mitochondrial DNA (mtDNA)-depletion syndromes (MDS; OMIM 251880) are phenotypically heterogeneous, autosomal-recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected individuals with the hepatocerebral form of MDS have early progressive liver failure and neurological abnormalities, hypoglycemia and increased lactate in body fluids. Affected tissues show both decreased activity of the mtDNA-encoded respiratory chain complexes (I, III, IV, V) and mtDNA depletion. We used homozygosity mapping in three kindreds of Druze origin to map the gene causing hepatocerebral MDS to a region of 6.1 cM on chromosome 2p13, between markers D2S291 and D2S2116. This interval encompasses the gene (DGUOK) encoding the mitochondrial deoxyguanosine kinase (dGK). We identified a single-nucleotide deletion (204delA) within the coding region of DGUOK that segregates with the disease in the three kindreds studied. Western-blot analysis did not detect dGK protein in the liver of affected individuals. The main supply of deoxyribonucleotides (dNTPs) for mtDNA synthesis comes from the salvage pathway initiated by dGK and thymidine kinase-2 (TK2). The association of mtDNA depletion with mutated DGUOK suggests that the salvage-pathway enzymes are involved in the maintenance of balanced mitochondrial dNTP pools.

Thiamine-responsive megaloblastic anaemia (TRMA), also known as Rogers syndrome, is an early onset, autosomal recessive disorder defined by the occurrence of megaloblastic anaemia, diabetes mellitus and sensorineural deafness, responding in varying degrees to thiamine treatment (MIM 249270). We have previously narrowed the TRMA locus from a 16-cM to a 4-cM interval on chromosomal region 1q23.3 (refs 3,4) and this region has been further refined to a 1.4-cM interval. Previous studies have suggested that deficiency in a high-affinity thiamine transporter may cause this disorder. Here we identify the TRMA gene by positional cloning. We assembled a P1-derived artificial chromosome (PAC) contig spanning the TRMA candidate region. This clarified the order of genetic markers across the TRMA locus, provided 9 new polymorphic markers and narrowed the locus to an approximately 400-kb region. Mutations in a new gene, SLC19A2, encoding a putative transmembrane protein homologous to the reduced folate carrier proteins, were found in all affected individuals in six TRMA families, suggesting that a defective thiamine transporter protein (THTR-1) may underlie the TRMA syndrome.

Our findings demonstrate intrafamilial heterogeneity, namely the presence of GS and CBS phenotypes, in a kindred with the CLCNKB R438H mutation. We conclude that GS can be caused by a mutation in a gene other than SLC12A3. The exact role of the CLCNKB R438H mutation in the pathogenesis of the electrolyte and mineral abnormalities in GS and CBS remains to be established.

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N-and Ctermini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with the shaker-TRP superfamily of ion channels. Keywordsdeafness; endoplasmic reticulum; hearing; topology; transmembrane Mutations in transmembrane channel-like gene 1 (TMC1/Tmc1) can cause dominant or recessive hearing loss in humans and mice (1,2). Tmc1 mRNA is specifically expressed in neurosensory hair cells of the inner ear (1,2). Cochlear neurosensory hair cells of Tmc1 mutant mice fail to mature into fully functional sensory receptors (3) and exhibit concomitant structural degeneration that could be a cause or an effect of the maturational defect (2). The molecular and cellular functions of TMC1 protein remain unknown due, at least in part, to in situ expression levels that are prohibitively low for direct biochemical analysis.There are seven additional mammalian TMC paralogs whose structure and function are also unknown. There are no significant sequence similarities of any TMC protein with other proteins of known function. An initial PSORT-II analysis of human and mouse TMC proteins did not detect any N-terminal signal sequences or other trafficking signals, but it did predict that TMC proteins reside in the plasma membrane (4). The TMC proteins are all predicted to contain six to ten transmembrane domains (TMDs) and a novel, conserved * Andrew J. Griffith, MD, PhD, 5 Research Ct. Room 1A13, Rockville, MD 20850. Tel.: 301-496-1960; Fax: 301-402-7580; griffita@nidcd.nih.gov region, which we termed the TMC domain (4). TMHMM2.0 analysis of mouse and human TMC1 predicts cytoplasmically oriented N-and C-termini and six TMDs that are also predicted for the other paralogs (4). Other algorithms such as PSORTII and TopPred predict two to four additional TMDs, for a total of eight to ten TMDs, per TMC homolog (2,5). PROSITE and NetNGlyc identified several TMC sequence sites with varying probabilities of glycos...

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Atrichia with papular lesions is a rare autosomal recessive form of alopecia characterized by hair loss soon after birth and the development during childhood of a diffuse papular rash. We have previously shown that this disorder results from a deleterious mutation in the human hairless gene, a gene also involved in the pathogenesis of a related but clinically distinct form of congenital alopecia, termed alopecia universalis congenita. In this report, we describe a novel nonsense mutation in exon 4 of the human hairless gene in a consanguineous kindred affected with atrichia with papular lesions. This report provides additional evidence for phenotypic heterogeneity among inherited atrichias and for an association between the papular rash of atrichia with papular lesions and nonsense mutations in the human hairless gene.

Atrichia with papular lesions (APL) (OMIM 209500) is a hereditary form of alopecia. Hair loss occurs soon after birth and is followed years later by the development of a diffuse papular eruption. Its mode of transmission is still uncertain. A related but clinically distinct form of alopecia, known as alopecia universalis (OMIM 203655), has recently been found to be associated with a mutation in the human hairless gene. The present report describes the largest consanguineous kindred of APL reported to date and provides strong evidence for autosomal recessive inheritance of this rare disorder. On the basis of a linkage analysis of this kindred using six microsatellite markers spanning the human hairless gene region, we found that the APL locus maps to chromosome region 8p12 in a 5 cM interval between marker D8S560 and marker D8S1739. A maximum lodscore of 3.7 was obtained with marker D8S1786, at a recombination fraction of 0. Our results suggest phenotypic variability at the hairless locus although they do not rule out the existence of a gene cluster associated with hair disorders in the same region.