Molecular screening of deafness in Algeria: High genetic heterogeneity involving DFNB1 and the Usher loci,DFNB2/USH1B,DFNB12/USH1D and DFNB23/USH1F

A systematic approach, involving haplotyping and genotyping, to the molecular diagnosis of non-syndromic deafness within 50 families and 9 sporadic cases from Algeria is described.Mutations at the DFNB1 locus (encompassing the GJB2 and GJB6 genes) are responsible for more than half of autosomal recessive prelingual non-syndromic deafness in various populations. A c.35delG mutation can account for up to 85% of GJB2 mutations and two large deletions del(GJB6-D13S1830) and del(GJB6-D13S1854) have also been reported in several population groups.In view of the genetic heterogeneity a strategy was developed which involved direct analysis of DFNB1. In negative familial cases, haplotype analysis was carried out, where possible, to exclude DFNB1 mutations. Following this, haplotype analysis of five Usher syndrome loci, sometimes involved in autosomal non-syndromic hearing loss, was carried out to identify cases in which Usher gene sequencing was indicated. When homozygosity was observed at a locus in a consanguineous family, the corresponding gene was exhaustively sequenced.Pathogenic DFNB1 genotypes were identified in 40% of the cases. Of the 21 cases identified with 2 pathogenic mutations, c.35delG represented 76% of the mutated alleles. The additional mutations were one nonsense, two missense and one splicing mutation. Four additional patients were identified with a single DFNB1 mutation. None carried the large deletions.Three families with non-syndromic deafness carried novel unclassified variants (UVs) in MYO7A (1 family) and CDH23 (2 families) of unknown pathogenic effect.Additionally, molecular diagnosis was carried out on two Usher type I families and pathogenic mutations in MYO7A and PCDH15 were found.     

MPP1 links the Usher protein network and the Crumbs protein complex in the retina

The highly ordered distribution of neurons is an essential feature of a functional mammalian retina. Disruptions in the apico-basal polarity complexes at the outer limiting membrane (OLM) of the retina are associated with retinal patterning defects in vertebrates. We have analyzed the binding repertoire of MPP5/Pals1, a key member of the apico-basal Crumbs polarity complex, that has functionally conserved counterparts in zebrafish (nagie oko) and Drosophila (Stardust). We show that MPP5 interacts with its MAGUK family member MPP1/p55 at the OLM. Mechanistically, this interaction involves heterodimerization of both MAGUK modules in a directional fashion. MPP1 expression in the retina throughout development resembles the expression of whirlin, a multi-PDZ scaffold protein and an important organizer in the Usher protein network. We demonstrate that both proteins interact strongly by both a classical PDZ domain-to-PDZ binding motif (PBM) mechanism, and a mechanism involving internal epitopes. MPP1 and whirlin colocalize in the retina at the OLM, at the outer synaptic layer and at the basal bodies and the ciliary axoneme. In view of the known roles of the Crumbs and Usher protein networks, our findings suggest a novel link of the core developmental processes of actin polymerization and establishment/maintenance of apico-basal cell polarity through MPP1. These processes, essential in neural development and patterning of the retina, may be disrupted in eye disorders that are associated with defects in these protein networks.     

Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS,a protein that associates with the USH1C protein,harmonin

Usher syndrome type I (USH1) is the most frequent cause of hereditary deaf-blindness in humans. Seven genetic loci (USH1A-G) have been implicated in this disease to date, and four of the corresponding genes have been identified: USH1B, C, D and F. We carried out fine mapping of USH1G (chromosome 17q24-25), restricting the location of this gene to an interval of 2.6 Mb and then screened genes present within this interval for mutations. The genes screened included the orthologue of the Sans gene, which is defective in the Jackson shaker deaf mutant and maps to the syntenic region in mice. In two consanguineous USH1G-affected families, we detected two different frameshift mutations in the SANS gene. Two brothers from a German family affected with USH1G were found to be compound heterozygotes for a frameshift and a missense mutation. These results demonstrate that SANS underlies USH1G. The SANS protein contains three ankyrin domains and a sterile alpha motif, and its C-terminal tripeptide presents a class I PDZ-binding motif. We showed, by means of co-transfection experiments, that SANS associates with harmonin, a PDZ domain-containing protein responsible for USH1C. In Jackson shaker mice the hair bundles, the mechanoreceptive structures of inner ear sensory cells, are disorganized. Based on the known interaction between USH1B (myosin VIIa), USH1C (harmonin) and USH1D (cadherin 23) proteins and the results obtained in this study, we suggest that a functional network formed by the USH1B, C, D and G proteins is responsible for the correct cohesion of the hair bundle.     

Three novel mutations and twelve polymorphisms identified in the USH2A gene in Israeli USH2 families

The Usher syndromes are autosomal recessive hereditary disorders characterized by hearing impairment and progressive visual loss due to Retinitis Pigmentosa (RP). Moderate to severe sensorineural hearing loss and progressive RP characterizes Usher syndrome type IIa (USH2A), which maps to the long arm of chromosome 1q41. Recently, three deletions carried by USH2 patients, which were found in a novel gene isolated from the critical 1q41 region, defined this gene as responsible for USH2A. The USH2A gene is predicted to encode a 1546 amino acid protein which possesses domains that are observed in basal lamina and extracellular matrix proteins and in cell adhesion molecules. Affected individuals and additional members from eleven USH2 Israeli families of diverse ethnic origin were screened for the presence of changes in all 20 coding exons of the USH2A gene. Three novel mutations (239-242insCGTA, R334W, T1515M) were identified in three families of Jewish Moroccan and Jewish Iranian origins. Twelve polymorphisms were found in the families, four of which are novel. None of the known USH2 mutations were identified in the families studied in this work. Hum Mutat 15:388, 2000.     

Mutation screening of USH3 gene (clarin-1) in Spanish patients with Usher syndrome: low prevalence and phenotypic variability

Usher syndrome type III is an autosomal recessive disorder clinically characterized by the association of retinitis pigmentosa (RP), variable presence of vestibular dysfunction and progressive hearing loss, being the progression of the hearing impairment the critical parameter classically used to distinguish this form from Usher syndrome type I and Usher syndrome type II. Usher syndrome type III clinical subtype is the rarest form of Usher syndrome in Spain, accounting only for 6% of all Usher syndrome Spanish cases. The gene responsible for Usher syndrome type III is named clarin-1 and it is thought to be involved in hair cell and photoreceptor cell synapses. Here, we report a screening for mutations in clarin-1 gene among our series of Usher syndrome Spanish patients. Clarin-1 has been found to be responsible for the disease in only two families: the first one is a previously reported family homozygous for Y63X mutation and the second one, described here, is homozygous for C40G. This accounts for 1.7% of Usher syndrome Spanish families. It is noticeable that, whereas C40G family is clinically compatible with Usher syndrome type III due to the progression of the hearing loss, Y63X family could be diagnosed as Usher syndrome type I because the hearing impairment is profound and stable. Thus, we consider that the progression of hearing loss is not the definitive key parameter to distinguish Usher syndrome type III from Usher syndrome type I and Usher syndrome type II.     

Identification of 14 novel mutations in the long isoform of USH2A in Spanish patients with Usher syndrome type II

Mutations in USH2A gene have been shown to be responsible for Usher syndrome type II, an autosomal recessive disorder characterised by hearing loss and retinitis pigmentosa. USH2A was firstly described as consisting of 21 exons, but 52 novel exons at the 3' end of the gene were recently identified. In this report, a mutation analysis of the new 52 exons of USH2A gene was carried out in 32 unrelated patients in which both disease-causing mutations could not be found after the screening of the first 21 exons of the USH2A gene. On analysing the new 52 exons, fourteen novel mutations were identified in 14 out of the 32 cases studied, including 7 missense, 5 frameshift, 1 duplication and a putative splice-site mutation.     

Novel USH2A mutations in Japanese Usher syndrome type 2 patients: marked differences in the mutation spectrum between the Japanese and other populations

Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A. In a recent mutation screening of USH2A in Japanese USH2 patients, we identified 11 novel mutations in 10 patients and found the possible frequent mutation c.8559-2A>G in 4 of 10 patients. To obtain a more precise mutation spectrum, we analyzed further nine Japanese patients in this study. We identified nine mutations, of which eight were novel. This result indicates that the mutation spectrum for USH2A among Japanese patients largely differs from Caucasian, Jewish and Palestinian patients. Meanwhile, we did not find the c.8559-2A>G in this study. Haplotype analysis of the c.8559-2G (mutated) alleles using 23 single nucleotide polymorphisms surrounding the mutation revealed an identical haplotype pattern of at least 635?kb in length, strongly suggesting that the mutation originated from a common ancestor. The fact that all patients carrying c.8559-2A>G came from western Japan suggests that the mutation is mainly distributed in that area; indeed, most of the patients involved in this study came from eastern Japan, which contributed to the absence of c.8559-2A>G.     

Junctional epidermolysis bullosa inversa (locus EBR2A) assigned to 1q31 by linkage and association to LAMC1

Junctional epidermolysis bullosa Inversa is an autosomal recessiveblistering skin disease with an ultrastructural hemldesmosomedefect similar to that of the Heriltz disease, yet with a non-lethaland different course of the disease. Its delineation is basedon five geographically associated Norwegian families where allparents are likely to carry a mutant EBR2A allele Identicalin descent. Three informative families show a lod score of +1.65at zero recombination to a trinucleotide repeat marker In Intron20 of the laminin gamma 1 (LAMC1, previously LAMB2) locus on1q31. The four patients of these families are all homozygousfor the 146 bp LAMC1 allele present only on 5% of random Norwegianchromosomes. The daughter of a deceased patient in a fourthfamily carries the same 146 bp allele. This extreme associationconfirms that the disease locus, EBR2A, Is at or closely linkedto LAMC1. Localized and generalized Mitis types as well as themajority of tested families with the Herlitz type of junctionalepidermolysis bullosa appeared not to be similarly linked orassociated to LAMC1. The Msp\ and Alu\ RFLPs of LAMC1 showedabsolute allelic association. Each of the two RFLP haplotypesshowed association to either ‘long’ or ‘short’intron 20 STR alleles.     

Genetic association to the amyloid plaque associated protein gene COL25A1 in Alzheimer's disease

The COL25A1 gene, located in 4q25, encodes the CLAC protein, which has been implicated in Alzheimer's disease (AD) pathogenesis. CLAC was originally identified in amyloid preparations from AD brain and has been shown to be associated with amyloid plaques, inhibition of Abeta-fibril elongation and increased protease resistance of Abeta-fibrils through direct binding to Abeta. These biochemical data as well as the genomic location of the COL25A1 gene in chromosome 4q25 where we previously have reported a weak linkage-signal in Swedish AD families encouraged us to perform a case-control association study of two LD blocks in COL25A1 using 817 AD cases and 364 controls. The LD blocks cover a putative Abeta-binding motif and the variable 3' end of the gene. The analyses indicated association to three of eight analysed SNPs. We found further support for the association by replication in a Swedish population-based longitudinal sample set (n=926). Thus, in addition to the biochemical data, there is now genetic evidence of association between COL25A1 and risk for Alzheimer's disease.     

The equine herpesvirus 1 UL11 gene product localizes to the trans-golgi network and is involved in cell-to-cell spread

Experiments were conducted to identify and characterize the equine herpesvirus type 1 (EHV-1) UL11 homologous protein. At early-late times after EHV-1 infection of Rk13 cells several proteins at an M(r) of 8000 to 12,000 were detected using a UL11 protein-specific antiserum. Particularly, an M(r) of 11,000 protein was found abundantly in purified virions and could be assigned to the tegument fraction. As demonstrated by confocal laser scanning microscopy, UL11 reactivity localized predominantly to the trans-Golgi network of infected cells, but was also noted at the plasma membrane, specifically of transfected cells. Deletion of UL11 sequences in EHV-1 vaccine strain RacH (Hdelta11) and in the virulent isolate RacL22 (Ldelta11) resulted in viruses that were able to replicate on noncomplementing cells. It was shown in one-step growth kinetics on Rk13 cells that the reduction of intracellular and of extracellular virus titers caused by the absence of UL11 expression in either virus was somewhat variable, but approximately 10- to 20-fold. In contrast, a marked influence on the plaque phenotype was noted, as mean maximal diameters of plaques were reduced to 23.2% (RacL22) or 34.7% (RacH) of parental virus plaques and as an effect on the ability of RacH to cause syncytia upon infection was noted. It was therefore concluded that the EHV-1 UL11 product is not essential for virus replication in Rk13 cells but is involved in cell-to-cell spread.     

Mutations in myosin VIIA (MYO7A) and usherin (USH2A) in Spanish patients with Usher syndrome types I and II,respectively

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment and retinitis pigmentosa. Three clinical types are known (USH1, USH2 and USH3), and there is an extensive genetic heterogeneity, with at least ten genes implicated. The most frequently mutated genes are MYO7A, which causes USH1B, and usherin, which causes USH2A. We carried out a mutation analysis of these two genes in the Spanish population. Analysis of the MYO7A gene in patients from 30 USH1 families and sporadic cases identified 32% of disease alleles, with mutation Q821X being the most frequent. Most of the remaining variants are private mutations. With regard to USH2, mutation 2299delG was detected in 25% of the Spanish patients. Altogether the mutations detected in USH2A families account for 23% of the disease alleles.     

An USH2A founder mutation is the major cause of Usher syndrome type 2 in Canadians of French origin and confirms common roots of Quebecois and Acadians

Congenital hearing loss affects approximately one child in 1000. About 10% of the deaf population have Usher syndrome (USH). In USH, hearing loss is complicated by retinal degeneration with onset in the first (USH1) or second (USH2) decade. In most populations, diagnostic testing is hampered by a multitude of mutations in nine genes. We have recently shown that in French Canadians from Quebec, USH1 largely results from a single USH1C founder mutation, c.216G>A ('Acadian allele'). The genetic basis of USH2 in Canadians of French descent, however, has remained elusive. Here, we have investigated nine USH2 families from Quebec and New Brunswick (the former Acadia) by haplotype analyses of the USH2A locus and sequencing of the three known USH2 genes. Seven USH2A mutations were identified in eight patients. One of them, c.4338_4339delCT, accounts for 10 out of 18 disease alleles (55.6%). This mutation has previously been reported in an Acadian USH2 family, and it was found in homozygous state in the three Acadians of our sample. As in the case of c.216G>A (USH1C), a common haplotype is associated with c.4338_4339delCT. With a limited number of molecular tests, it will now be possible in these populations to estimate whether children with congenital hearing impairment of different degrees will develop retinal disease - with important clinical and therapeutic implications. USH2 is the second example that reveals a significant genetic overlap between Quebecois and Acadians: in contrast to current understanding, other genetic disorders present in both populations are likely based on common founder mutations as well.     

The neutralizing antibody response to the vaccinia virus A28 protein is specifically enhanced by its association with the H2 protein

The vaccinia virus (VACV) entry-fusion complex (EFC) is composed of at least nine membrane proteins. Immunization of mice with individual EFC genes induced corresponding protein-binding antibody but failed to protect against VACV intranasal challenge and only DNA encoding A28 elicited low neutralizing antibody. Because the A28 and H2 proteins interact, we determined the effect of immunizing with both genes simultaneously. This procedure greatly enhanced the amount of antibody that bound intact virions, neutralized infectivity, and provided partial protection against respiratory challenge. Neither injection of A28 and H2 plasmids at different sites or mixing A28 and H2 sera enhanced neutralizing antibody. The neutralizing antibody could be completely removed by binding to the A28 protein alone and the epitope was located in the C-terminal segment. These data suggest that the interaction of H2 with A28 stabilizes the immunogenic form of A28, mimicking an exposed region of the entry-fusion complex on infectious virions.     

Varicella-zoster virus ORF1 gene product is a tail-anchored membrane protein localized to plasma membrane and trans-Golgi network in infected cells

Varicella-zoster virus (VZV) encodes five genes that do not have herpes simplex virus homologs. One of these genes, VZV open reading frame 1 (ORF1), encodes a membrane protein with a hydrophobic domain at its C-terminus that is predicted to be the transmembrane domain. However, the detailed characterization of ORF1 protein in infected cells has not been reported. Here, we produced mono-specific antibodies against ORF1 protein and characterized the gene products in infected cells. Western blot analyses showed the ORF1 polypeptides had apparent molecular masses of approximately 14-17 kDa. Furthermore, ORF1 was found to be a phosphoprotein by immunoprecipitation assay. In immunofluorescence assays, the VZV ORF1 protein was detected at both the plasma membrane and trans-Golgi network in both VZV-infected and ORF1-transfected cells. Moreover, ORF1 proteins associated with each other to form homodimer, and were incorporated into viral particles. The C-terminal hydrophobic domain was required for the association of ORF1 with the membrane structures, indicating that ORF1 protein is anchored to the membrane thorough its C-terminus, which is a transmembrane domain. Because ORF1 possesses a C-terminal transmembrane domain without an N-terminal signal sequence for its translocation to the ER lumen, ORF1 can be classified as a tail-anchored membrane protein. These results show that the N terminus of ORF1 protein faces the cytoplasm in infected cells and the tegument region in mature virions.     

Mutations in CIB2 calcium and integrin‐binding protein disrupt auditory hair cell calcium homeostasis in Usher syndrome type 1J and non‐syndromic deafness DFNB48

Alterations of the CIB2 calcium‐ and integrin‐binding protein cause Usher syndrome type 1J and non‐syndromic deafness DFNB48 Riazuddin et al. (2012) Nature Genetics 44(11):1265–1271     

The Leber congenital amaurosis gene product AIPL1 is localized exclusively in rod photoreceptors of the adult human retina

Leber congenital amaurosis (LCA) is the most severe inherited retinal dystrophy resulting in markedly impaired vision or blindness at birth. LCA is characterized by an extinguished electroretinogram in infancy, which is thought to be indicative of an early and severe impairment of both the rod and cone photoreceptors in the human retina. Recently, the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene was identified as the fourth causative gene of LCA. AIPL1 encodes a 384 amino acid protein of unknown function. We have generated a polyclonal antibody against a peptide from a unique region within the primate AIPL1 protein, which detects a protein of approximately 43 kDa in human retinal extracts. A screen of human tissues and immortalized cell lines with this antibody reveals AIPL1 to be specific to human retina and cell lines of retinal origin (Y79 retinoblastoma cells). Within the retina, AIPL1 was detected only in the rod photoreceptor cells of the peripheral and central human retina. The AIPL1 staining pattern extended within the rod photoreceptor cells from the inner segments, through the rod nuclei to the rod photoreceptor synaptic spherules in the outer plexiform layer. AIPL1 was not detected in the cone photoreceptors of peripheral or central human retina. This study is the first to suggest that AIPL1 performs a function essential to the maintenance of rod photoreceptor function.     

Protein gene product 9.5 in the developing cochlea of the rat: cellular distribution and relation to the cochlear cytoskeleton

Summary Protein gene product 9.5 was immunolocalized in the adult and early postnatal (P2-P15) rat cochlea, and its distribution compared with a 200 kDa highly phosphorylated neurofilament subunit (neurofilament 200) and -tubulin. In the adult, Protein gene product 9.5 was expressed exclusively in cochlear nerve fibres and ganglion cells, a small percentage of these (Type II ganglion cells and olivocochlear bundle fibres) being intensely positive for both protein gene product and neurofilament 200. In postnatal development, pillar and Deiters' cells were at first (P2-P15) strongly positive for protein gene product 9.5, and hair cells moderately so. At P2, all nerve fibres and ganglion cells showed co-expression of protein gene product 9.5 and neurofilament 200, but at later stages, the subset of intensely co-labelled neurons appeared, nerve fibres at P7 onwards and ganglion cells from P12. There was no overt correlation between the onset of protein gene product 9.5 and a-tubulin expression in any cochlear component. Protein gene product 9.5 expression in ganglion cells was at first (P2 and P7) mainly nuclear, and later also cytoplasmic. It is concluded that there is a clear correlation of high levels of protein gene product 9.5 and neurofilament protein expression, and that protein gene product 9.5 is expressed in some non-neuronal cells of the cochlea during its early development, persisting until after hearing has commenced.     

白细胞介素1基因多态性与维吾尔族群体慢性牙周炎易感性的关系

目的：探讨白细胞介素1（interleukin-1，IL-1）基因多态性与新疆地区维吾尔族居民慢性牙周炎（chronic periodontitis，CP）易感性的关系。方法：收集维吾尔族41例重度CP患者、43例中度CP患者、49例轻度CP患者和92名健康对照者的颊粘膜拭子，提取DNA。采用序列特异引物聚合酶链反应（sequence specific primers-polymerase chain reaction，SSP-PCR）和聚合酶链反应-限制性片段长度多态性（polymerase chain reaction-retriction fragment length pohymorphism，PCR-RFLP）方法对其进行IL-1A-889/NcoI和IL-1B 3954/TaqI位点的基因型测定，分析各基因型的分布。结果：IL-1A-889/NcoI基因型在重度CP、中度CP、轻度CP和对照组之间的分布差异无显著性；IL-1B 3954/TaqI等位基因2在重度CP中的检出率显著高于对照组，而在中度CP、轻度CP与对照组之间的分布差异无显著性。结论：IL-1B 3954/TaqI等位基因2可能与新疆维吾尔族重度CP遗传易感性相关。     

The contribution of USH1C mutations to syndromic and non-syndromic deafness in the UK

Denaturing high-performance liquid chromatography (DHPLC) was used to screen 14 UK patients with Usher syndrome type 1, in order to assess the contribution of mutations in USH1C to type 1 Usher. In addition, 16 Caucasian sib pairs and two small consanguineous families with non-syndromic deafness, who were concordant for haplotypes around DFNB18, were also screened for mutations in the USH1C gene. Two Usher type 1 patients were found to have the 238-239insC mutation reported previously; one of Greek Cypriot origin was homozygous for the mutation and another Caucasian was heterozygous. This indicates that mutations in the USH1C gene make a greater contribution to Usher syndrome type 1 than originally thought, which has implications for the genetic testing of families with Usher syndrome in the UK. Analysis using intragenic single nucleotide polymorphisms (SNPs) revealed that the haplotypic background bearing this common mutation was not consistent across the gene in two families, and that there are either two haplotypes on which the mutation has arisen or that there has been a recombination on a single haplotype. We found no evidence of mutations in USH1C in the patients with non-syndromic deafness, suggesting that the gene is not a major contributor to autosomal-recessive non-syndromic deafness in the UK.