Identification of candidate regions for a novel Usher syndrome type II locus

PURPOSE: Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin. METHODS: Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes. RESULTS: Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q. CONCLUSIONS: Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q

Endoplasmic Reticulum Quality Control Is Involved in the Mechanism of Endoglin-Mediated Hereditary Haemorrhagic Telangiectasia

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominant genetic condition affecting the vascular system and is characterised by epistaxis, arteriovenous malformations and mucocutaneous and gastrointestinal telangiectases. This disorder affects approximately 1 in 8,000 people worldwide. Significant morbidity is associated with this condition in affected individuals, and anaemia can be a consequence of repeated haemorrhages from telangiectasia in the gut and nose. In the majority of the cases reported, the condition is caused by mutations in either ACVRL1 or endoglin genes, which encode components of the TGF-beta signalling pathway. Numerous missense mutations in endoglin have been reported as causative defects for HHT but the exact underlying cellular mechanisms caused by these mutations have not been fully established despite data supporting a role for the endoplasmic reticulum (ER) quality control machinery. For this reason, we examined the subcellular trafficking of twenty-five endoglin disease-causing missense mutations. The mutant proteins were expressed in HeLa and HEK293 cell lines, and their subcellular localizations were established by confocal fluorescence microscopy alongside the analysis of their N-glycosylation profiles. ER quality control was found to be responsible in eight (L32R, V49F, C53R, V125D, A160D, P165L, I271N and A308D) out of eleven mutants located on the orphan extracellular domain in addition to two (C363Y and C382W) out of thirteen mutants in the Zona Pellucida (ZP) domain. In addition, a single intracellular domain missense mutant was examined and found to traffic predominantly to the plasma membrane. These findings support the notion of the involvement of the ER's quality control in the mechanism of a significant number, but not all, missense endoglin mutants found in HHT type 1 patients. Other mechanisms including loss of interactions with signalling partners as well as adverse effects on functional residues are likely to be the cause of the mutant proteins' loss of function

Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis

International audiencePurpose Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, cadherin-23, protocadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively. Methods In the present study, we recruited four Tunisian families with a diagnosis of USH1, together with healthy unrelated controls. Affected members underwent detailed audiologic and ocular examinations. We used the North African Deafness (NADf) chip to search for known North African mutations associated with USH. Then, we selected microsatellite markers covering USH1 known loci to genotype the DNA samples. Finally, we performed DNA sequencing of three known USH1 genes: MYO7A, PCDH15, and USH1C. Results Four biallelic mutations, all single base changes, were found in the MYO7A, USH1C, and PCDH15 genes. These mutations consist of a previously reported splicing defect c.470+1G>A in MYO7A, three novel variants, including two nonsense (p.Arg3X and p.Arg134X) in USH1C and PCDH15, respectively, and one frameshift (p.Lys615Asnfs*6) in MYO7A. Conclusions We found a remarkable genetic heterogeneity in the studied families with USH1 with a variety of mutations, among which three were novel. These novel mutations will be included in the NADf mutation screening chip that will allow a higher diagnosis efficiency of this extremely genetically heterogeneous disease. Ultimately, efficient molecular diagnosis of USH in a patient’s early childhood is of utmost importance, allowing better educational and therapeutic management

Comparison of the subcellular localization of wild type Endoglin with two (L32R and C53R) orphan domain HHT1-causing mutants.

<p>HeLa cells were transiently transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids (panels A-C, G-I and M-O) or co-transfected with the same plasmids and the EGFP-tagged H-Ras plasmid (Panels D–F, J–L and P–R) and processed for fluorescence confocal microscopy as described in the methods. The HA tagged proteins were detected with Anti-HA monoclonal antibodies (red panels A, D, G, J. M and P) and the ER marker calnexin was detected with anti-calnexin polyclonal antibodies (panels B, H and N). GFP-H-Ras staining is shown in panels E, K and Q. The wild type predominantly showed plasma membrane localization as evidenced by its co-localization with Ras (D–F). On the other hand the two mutants (L32R and C53R) showed ER localization (G–R).</p

The majority of the missense mutants in the ZP and intraceullar domains traffic normally to the plasma membrane.

<p>HeLa cells were transiently co-transfected with the C-terminally HA-tagged Endoglin pCMV5 plasmids and the EGFP-tagged H-Ras plasmid and 24 hours later the cells were processed for fluorescence confocal microscopy as described in the methods. The following missense mutanst were shown to traffic predominantly to the plama membrane as eviednced by their co-localization with GFP-H-Ras: C382G ( data not shoiwn due to space limitations), F403S (panles A–C), S407N (not shown), C412S (not shown), G413V (not shown), N423S (panels D–F), R437W (not shown), A452G (not shown), Q476H (not shown), V504M (panels J–L), R571H (panels M–O ) and P615L (panels P–R).</p