The LCCL module.

Here we show that Lgl1 protein, cub-1-related proteins, coch-5b2-related proteins, coagulation factor C of horse-shoe crab and a predicted protein of Plasmodium falciparum share a homologous domain. Since this domain-type was first identified in Limulus factor C, Coch-5b2 and Lgl1 we propose the name LCCL for this domain-family. The LCCL module of coch-5b2 is of special biological interest because it has been shown recently that mutations affecting this module cause the deafness disorder DFNA9 in humans. With a view to defining the structure and function of the LCCL domain of human coch-5b2 protein, we have expressed it in Escherichia coli and subjected it to preliminary structural characterization. Structure prediction and circular dichroism studies on the recombinant protein indicate that the domain possesses both alpha helices and beta strands. It is shown that the mutations which cause hearing loss in humans affect residues that are critical for the integrity of the LCCL module of the coch-5b2 protein.     

Targeted disruption of mouse Coch provides functional evidence that DFNA9 hearing loss is not a COCH haploinsufficiency disorder

Dominant progressive hearing loss and vestibular dysfunction DFNA9 is caused by mutations of the human COCH gene. COCH encodes cochlin, a highly abundant secreted protein of unknown function in the inner ear. Cochlin has an N-terminal LCCL domain followed by two vWA domains, and all known DFNA9 mutations are either missense substitutions or an amino acid deletion in the LCCL domain. Here, we have characterized the auditory phenotype associated with a genomic deletion of mouse Coch downstream of the LCCL domain. Homozygous Coch ^−/− mice express no detectable cochlin in the inner ear. Auditory brainstem responses to click and pure-tone stimuli (8, 16, 32 kHz) were indistinguishable among wild type and homozygous Coch ^−/− mice. A Coch-LacZΔneo reporter allele detected Coch mRNA expression in nonsensory epithelial and stromal regions of the cochlea and vestibular labyrinth. These data provide functional evidence that DFNA9 is probably not caused by COCH haploinsufficiency, but via a dominant negative or gain-of-function effect, in nonsensory regions of the inner ear.Tomoko Makishima, Clara I. Rodriguez contributed equally     

Identification of a novel Cochlin isoform in the perilymph: insights to Cochlin function and the pathogenesis of DFNA9

The COCH gene mutated in DFNA9, an autosomal dominant hereditary sensorineural hearing loss and vestibular disorder, encodes Cochlin. Previously, we reported three bovine Cochlin isoforms, p63s, p44s, and p40s, which exhibit significant molecular heterogeneity in vivo. Here we have characterized Cochlin isoforms by generating four isoform-specific anti-Cochlin antibodies. The same three Cochlin isoforms, p63s, p44s, and p40s, were detected in human and cow inner ear tissue; however, p44s and p40s were not detected in perilymph. We identified a novel short 16kDa isoform in human perilymph and a 18-23kDa isoform in cow perilymph, named Cochlin-tomoprotein (CTP), corresponding to the N-terminus of full-length Cochlin (p63s) and the LCCL domain. Notably, CTP contains all of the known mutation sites associated with DFNA9. The pathogenesis of DFNA9 is not fully clarified as yet, and this novel perilymph-associated CTP isoform might provide mechanistic clues to how mutations in the COCH gene damage the inner ear function.     

Mutations in COCH that result in non-syndromic autosomal dominant deafness (DFNA9) affect matrix deposition of cochlin

The COCH gene mutated in autosomal dominant sensorineural deafness (DFNA9) encodes cochlin, a major constituent of the inner ear extracellular matrix. Sequence analysis of cochlin from DFNA9 patients identified five distinct single-amino-acid mutations within a conserved region (the LCCL domain) of cochlin. To define the molecular basis of DFNA9, we have generated myc-tagged wild-type and mutant cochlins and explored their behavior in transient transfection systems. Western blotting of cell lysates and culture media indicates that wild-type and mutant cochlins are synthesized and secreted in similar amounts. Immunofluorescent staining confirms that all are detected within the endoplasmic reticulum and the Golgi complex of transfected cells. Our findings suggest that COCH mutations are unlikely to cause abnormalities in secretion and suggest that extracellular events might cause DFNA9 pathology. In agreement, we show that wild-type cochlin accumulates in extracellular deposits that closely parallel the matrix component fibronectin, whereas mutant cochlins vary in the amount and pattern of extracellular material. Whereas some mutants exhibit an almost normal deposition pattern, some show complete lack of deposition. Our results suggest that DFNA9 results from gene products that fail to integrate correctly into the extracellular matrix. The partial or complete penetrance of integration defects suggests that DFNA9 pathology may be caused by multiple molecular mechanisms, including compromised ability of cochlin to self-assemble or to form appropriate complexes with other matrix components.     

GSDMDC家族基因功能研究进展

GSDMDC家族是近年来发现的一个全新的含有Gasdermin结构域的蛋白超家族,包括DFNA5、DFNA5L、GSDM、 GSDML 和 MLZE五个成员。研究表明GSDMDC家族可能与组织器官发育以及肿瘤,耳聋和脱发等遗传疾病相关,因而具有重要生理功能。其中,对该家族的DFNA5基因研究报道相对较多,它是常染色体显性非综合征性耳聋致病基因之一,并可能与黑色素瘤和乳腺癌相关。但对DFNA5基因在细胞和分子水平作用机制仍不清楚。对Gasdermin结构域的空间结构、特点、相互作用蛋白和生理功能也知之甚少。将来的研究将揭示此家族各成员的确切生理功能及其与疾病相关性。

     

Chromosomal Mapping of Two Members of the Human Dynein Gene Family to Chromosome Regions 7p15 and 11q13 near the Deafness Loci DFNA 5 and DFNA 11

We mapped expressed tagged sequences (ESTs) corresponding to two human dynein heavy chain genes: β heavy chain of the outer dynein arm and heavy chain isotype 1B (DYH1B), by using somatic cell hybrids and radiation hybrid panels. The EST for the β heavy chain of the outer dynein arm mapped to chromosome region 7p15, and the EST for DYH1B mapped to 11q13.5. Two loci for nonsyndromic forms of deafness, DFNA5 and DFNA11, have previously been mapped to these two chromosomal regions. Including the gene for the axonemal light chain, hp28, we have mapped three different dynein genes near loci for different forms of nonsyndromic deafness. The hypothesis that mutations in some dynein genes are associated with nonsyndromic deafness should now be tested.     

A yeast model for the study of human DFNA5, a gene mutated in nonsyndromic hearing impairment

A mutation in human DFNA5 is associated with autosomal dominant nonsyndromic hearing impairment. The function of DFNA5 protein remains unknown and no experimental model has been described so far. Here we describe fission yeast Schizosaccharomyces pombe as a model organism for studying the function of heterologously expressed DFNA5. We have expressed wild-type as well as mutant DFNA5 alleles under control of regulatable nmt1 promoter. Yeast cells tolerated expression of wild-type DFNA5, while expression of the mutant DFNA5 allele, which is responsible for nonsyndromic autosomal dominant hearing impairment, led to cell cycle arrest. We identified new rat and horse DFNA5 homologues and we describe a domain of homology shared between DFNA5 and the Mcm10 family of DNA replication proteins. Genetic interactions between heterologously expressed DFNA5 and a fission yeast cdc23 (mcm10) mutant support a possible link between DFNA5 and Mcm10 proteins.     

耳聋相关基因COCH的非同义单核苷酸多态性致聋表型预测

群体凝血因子C同源物基因(Coagulation factor C homology,COCH)是人类发现的第一个伴前庭功能障碍的耳聋基因,位于人类染色体14q12-q13上。迄今,在COCH基因上发现16个位点突变导致常染色体显性遗传非综合征型耳聋DFNA9的发生,其中包括13个非同义单核苷酸多态性(Non-synonymous single nucleotide polymorphisms,nsSNPs)位点。由于该基因其他nsSNPs的基因型与表型关系尚不清楚,因此文章采用生物信息学方法,从COCH基因全部的SNPs中分级筛选,结合已知的致病nsSNPs信息及蛋白三维结构验证,首次预测出由COCH基因编码的cochlin蛋白的vWFA (Von Willebrand factor type A domain)区的8个高风险致病性nsSNPs(I176T、R180Q、G265E、V269L、I368N、I372T、R416C和Y424D)。同时,对位于LCCL (Limulus factor C, cochlin, and late gestation lung protein Lgl1)区域的6个已知致病突变的nsSNPs ( P51S、G87W、I109N、I109T、W117R和F121S)进行了三维结构模拟,发现突变体均发生了环状结构或链状结构的改变。本研究对COCH基因的基因型与表型的相关性研究为遗传性耳聋筛查提供了相应的理论依据,也对该基因所编码的cochlin蛋白的功能研究具有一定的指导意义。     

Mutations in the WFS1 gene that cause low-frequency sensorineural hearing loss are small non-inactivating mutations

Hereditary hearing impairment is an extremely heterogeneous trait, with more than 70 identified loci. Only two of these loci are associated with an auditory phenotype that predominantly affects the low frequencies (DFNA1 and DFNA6/14). In this study, we have completed mutation screening of the WFS1 gene in eight autosomal dominant families and twelve sporadic cases in which affected persons have low-frequency sensorineural hearing impairment (LFSNHI). Mutations in this gene are known to be responsible for Wolfram syndrome or DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), which is an autosomal recessive trait. We have identified seven missense mutations and a single amino acid deletion affecting conserved amino acids in six families and one sporadic case, indicating that mutations in WFS1 are a major cause of inherited but not sporadic low-frequency hearing impairment. Among the ten WFS1 mutations reported in LFSNHI, none is expected to lead to premature protein truncation, and nine cluster in the C-terminal protein domain. In contrast, 64% of the Wolfram syndrome mutations are inactivating. Our results indicate that only non-inactivating mutations in WFS1 are responsible for non-syndromic low-frequency hearing impairment.     

Active site residues and amino acid specificity of the ubiquitin carrier protein-binding RING-H2 finger domain

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.     

Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness

Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.     

The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease.

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.     

Alpha-tectorin involvement in hearing disabilities: one gene – two phenotypes

The human alpha-tectorin (TECTA) gene has recently been cloned and proposed to be involved in autosomal dominant non-syndromic hearing impairment (NSHI) in two families linked to the DFNA12 locus. We have studied a Swedish pedigree with autosomal dominant NSHI with possible digenic inheritance of the disease, involving locus DFNA12 in chromosome 11 and locus DFNA2 in chromosome 1. Mutation analysis of the TECTA gene in this family has identified eight nucleotide substitutions indicating that TECTA is highly polymorphic. One of the changes results in a cysteine to serine (C 1057 S) mutation, in the zonadhesin domain of TECTA; this segregates with the disease haplotype on chromosome 11 and is not present in a control population. The mutation results in the replacement of a cysteine in one of the repeats of the zonadhesin/Von Willebrand domain of the protein and might cause a change in the crosslinking of the polypeptide. These findings add support to the involvement of TECTA in hearing disabilities. However, the three families carrying different TECTA mutations also show phenotypic differences: the hearing loss ranges from prelingual to progressive with late onset. The explanation for the different phenotypes and some clues regarding the functions of TECTA may lie in the localization of the mutations in the different modules of the protein. Another possibility is that the phenotype in the Swedish family is the result of two defective genes.     

KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness

Potassium channels regulate electrical signaling and the ionic composition of biological fluids. Mutations in the three known genes of the KCNQ branch of the K+ channel gene family underlie inherited cardiac arrhythmias (in some cases associated with deafness) and neonatal epilepsy. We have now cloned KCNQ4, a novel member of this branch. It maps to the DFNA2 locus for a form of nonsyndromic dominant deafness. In the cochlea, it is expressed in sensory outer hair cells. A mutation in this gene in a DFNA2 pedigree changes a residue in the KCNQ4 pore region. It abolishes the potassium currents of wild-type KCNQ4 on which it exerts a strong dominant-negative effect. Whereas mutations in KCNQ1 cause deafness by affecting endolymph secretion, the mechanism leading to KCNQ4-related hearing loss is intrinsic to outer hair cells.     

GJB2 Gene Mutations in Syndromic Skin Diseases with Sensorineural Hearing Loss

The GJB2 gene is located on chromosome 13q12 and it encodes the connexin 26, a transmembrane protein involved in cell-cell attachment of almost all tissues. GJB2 mutations cause autosomal recessive (DFNB1) and sometimes dominant (DFNA3) non-syndromic sensorineural hearing loss. Moreover, it has been demonstrated that connexins are involved in regulation of growth and differentiation of epidermis and, in fact, GJB2 mutations have also been identified in syndromic disorders with hearing loss associated with various skin disease phenotypes. GJB2 mutations associated with skin disease are, in general, transmitted with a dominant inheritance pattern. Nonsyndromic deafness is caused prevalently by a loss-of-function, while literature evidences suggest for syndromic deafness a mechanism based on gain-of-function. The spectrum of skin manifestations associated with some mutations seems to have a very high phenotypic variability. Why some mutations can lead to widely varying cutaneous manifestations is poorly understood and in particular, the reason why the skin disease-deafness phenotypes differ from each other thus remains unclear. This review provides an overview of recent findings concerning pathogenesis of syndromic deafness imputable to GJB2 mutations with an emphasis on relevant clinical genotype-phenotype correlations. After describing connexin 26 fundamental characteristics, the most relevant and recent information about its known mutations involved in the syndromic forms causing hearing loss and skin problems are summarized. The possible effects of the mutations on channel expression and function are discussed.     

Probing local environments of tryptophan residues in proteins: comparison of 19F nuclear magnetic resonance results with the intrinsic fluorescence of soluble human tissue factor

19F nuclear magnetic resonance (19F NMR) of 5-fluorotryptophan (5F-Trp) and tryptophan (Trp) fluorescence both provide information about local environment and solvent exposure of Trp residues. To compare the information provided by these spectroscopies, the four Trp residues in recombinant soluble human tissue factor (sTF) were replaced with 5F-Trp. 19F NMR assignments for the 5F-Trp residues (14, 25, 45, and 158) were based on comparison of the wild-type protein spectrum with the spectra of three single Trp-to-Phe replacement mutants. Previously we showed from fluorescence and absorption difference spectra of mutant versus wild-type sTF that the side chains of Trpl4 and Trp25 are buried, whereas those of Trp45 and Trp158 are partially exposed to bulk solvent (Hasselbacher et al., Biophys J 1995;69:20-29). 19F NMR paramagnetic broadening and solvent-induced isotope-shift experiments show that position 5 of the indole ring of 5F-Trp158 is exposed, whereas that of 5F-Trp45 is essentially inaccessible. Although 5F-Trp incorporation had no discernable effect on the procoagulant cofactor activity of either the wild-type or mutant proteins, 19F NMR chemical shifts showed that the single-Trp mutations are accompanied by subtle changes in the local environments of 5F-Trp residues residing in the same structural domain.     

The solution structure of the cytokine-binding domain of the common beta-chain of the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5

The haemopoietic cytokines, granulocyte-macrophage colony-stimulating factor, interleukin-3 and interleukin-5 bind to cell-surface receptors comprising ligand-specific alpha-chains and a shared beta-chain. The beta-chain is the critical signalling subunit of the receptor and its fourth domain not only plays a critical role in interactions with ligands, hence in receptor activation, but also contains residues whose mutation can lead to ligand-independent activation of the receptor. We have determined the NMR solution structure of the isolated human fourth domain of the beta-chain. The protein has a fibronectin type III fold with a well-defined hydrophobic core and is stabilised by an extensive network of pi-cation interactions involving Trp and Arg side-chains, including two Trp residues outside the highly conserved Trp-Ser-Xaa-Trp-Ser motif (where Xaa is any amino acid) that is found in many cytokine receptors. Most of the residues implicated in factor-independent mutants localise to the rigid core of the domain or the pi-cation stack. The loops between the B and C, and the F and G strands, that contain residues important for interactions with cytokines, lie adjacent at the membrane-distal end of the domain, consistent with their being involved cooperatively in binding cytokines. The elucidation of the structure of the cytokine-binding domain of the beta-chain provides insight into the cytokine-dependent and factor-independent activation of the receptor.     

Nonmuscle myosin heavy chain IIA mutations define a spectrum of autosomal dominant macrothrombocytopenias: May-Hegglin anomaly and Fechtner, Sebastian, Epstein, and Alport-like syndromes

May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.     

A gene for autosomal dominant nonsyndromic hearing loss (DFNA12) maps to chromosome 11q22-24.

We performed linkage analysis in a Belgian family with autosomal dominant midfrequency hearing loss, which has a prelingual onset and a nonprogressive course in most patients. We found LOD scores >6 with markers on chromosome 11q. Analysis of key recombinants maps this deafness gene (DFNA12) to a 36-cM interval on chromosome 11q22-24, between markers D11S4120 and D11S912. The critical regions for the recessive deafness locus DFNB2 and the dominant locus DFNA11, which were previously localized to the long arm of chromosome 11, do not overlap with the candidate interval of DFNA12.