A novel locus (CORD12) for autosomal dominant cone-rod dystrophy on chromosome 2q24.2-2q33.1.

BACKGROUND: Rod-cone dystrophy, also known as retinitis pigmentosa (RP), and cone-rod dystrophy (CRD) are degenerative retinal dystrophies leading to blindness. To identify new genes responsible for these diseases, we have studied one large non consanguineous French family with autosomal dominant (ad) CRD. METHODS: Family members underwent detailed ophthalmological examination. Linkage analysis using microsatellite markers and a whole-genome SNP analysis with the use of Affymetrix 250 K SNP chips were performed. Five candidate genes within the candidate region were screened for mutations by direct sequencing. RESULTS: We first excluded the involvement of known adRP and adCRD genes in the family by genotyping and linkage analysis. Then, we undertook a whole-genome scan on 22 individuals in the family. The analysis revealed a 41.3-Mb locus on position 2q24.2-2q33.1. This locus was confirmed by linkage analysis with specific markers of this region. The maximum LOD score was 2.86 at θ = 0 for this locus. Five candidate genes, CERKL, BBS5, KLHL23, NEUROD1, and SF3B1 within this locus, were not mutated. CONCLUSION: A novel locus for adCRD, named CORD12, has been mapped to chromosome 2q24.2-2q33.1 in a non consanguineous French family.

Systematic screening of BEST1 and PRPH2 in juvenile and adult vitelliform macular dystrophies: a rationale for molecular analysis.

PURPOSE: To evaluate a genetic approach of BEST1 and PRPH2 screening according to age of onset, family history, and Arden ratio in patients with juvenile vitelliform macular dystrophy (VMD2) or adult-onset vitelliform macular dystrophy (AVMD), which are characterized by autofluorescent deposits. DESIGN: Clinical, electrophysiologic, and molecular retrospective study. PARTICIPANTS: The database of a clinic specialized in genetic sensory diseases was screened for patients with macular vitelliform dystrophy. Patients with an age of onset less than 40 years were included in the VMD2 group (25 unrelated patients), and patients with an age of onset more than 40 years were included in the AVMD group (19 unrelated patients). METHODS: Clinical, fundus photography, and electro-oculogram (EOG) findings were reviewed. Mutation screening of BEST1 and PRPH2 genes was systematically performed. MAIN OUTCOME MEASURES: Relevance of age of onset, family history, and Arden ratio were reviewed. RESULTS: Patients with VMD2 carried a BEST1 mutation in 60% of the cases. Seven novel mutations in BEST1 (p.V9L, p.F80V, p.I73V, p.R130S, pF298C, pD302A, and p.179delN) were found. Patients with VMD2 with a positive family history or a reduced Arden ratio carried a BEST1 mutation in 70.5% of cases and in 83% if both criteria were fulfilled. Patients with AVMD carried a PRPH2 mutation in 10.5% of cases and did not carry a BEST1 mutation. The probability of finding a PRPH2 mutation increased in the case of a family history (2/5 patients). Electro-oculogram was normal in 3 of 15 patients with BEST1 mutations and reduced in the 3 patients with PRPH2 mutations. CONCLUSIONS: Age of onset is a major criterion to distinguish VMD2 from AVMD. Electro-oculogram is not as relevant because decreased or normal Arden ratios have been associated with mutations in both genes and diseases. A positive family history increased the probability of finding a mutation. BEST1 screening should be recommended to patients with an age of onset less than 40 years, and PRPH2 screening should be recommended to patients with an age of onset more than 40 years. For an onset between 30 and 40 years, PRPH2 can be screened if no mutation has been detected in BEST1. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

ZZ domain of dystrophin and utrophin: topology and mapping of a beta-dystroglycan interaction site.

Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor dystroglycan. Dystrophin and its autosomal homologue utrophin interact with beta-dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and beta-dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with beta-dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and beta-dystroglycan and provide new insight into ZZ domain organization and function.

Pathological pattern of Mdx mice diaphragm correlates with gradual expression of the short utrophin isoform Up71.

Utrophin gene is transcribed in a large mRNA of 13 kb that codes for a protein of 395 kDa. It shows amino acid identity with dystrophin of up to 73% and is widely expressed in muscle and non-muscle tissues. Up71 is a short utrophin product of the utrophin gene with the same cysteine-rich and C-terminal domains as full-length utrophin (Up395). Using RT-PCR, Western blots analysis, we demonstrated that Up71 is overexpressed in the mdx diaphragm, the most pathological muscle in dystrophin-deficient mdx mice, compared to wild-type C57BL/10 or other mdx skeletal muscles. Subsequently, we demonstrated that this isoform displayed an increased expression level up to 12 months, whereas full-length utrophin (Up395) decreased. In addition, beta-dystroglycan, the transmembrane glycoprotein that anchors the cytoplasmic C-terminal domain of utrophin, showed similar increase expression in mdx diaphragm, as opposed to other components of the dystrophin-associated protein complex (DAPC) such as alpha-dystrobrevin1 and alpha-sarcoglycan. We demonstrated that Up71 and beta-dystroglycan were progressively accumulated along the extrasynaptic region of regenerating clusters in mdx diaphragm. Our data provide novel functional insights into the pathological role of the Up71 isoform in dystrophinopathies.

alpha7B integrin changes in mdx mouse muscles after L-arginine administration.

Muscle fibers attach to laminin in the basal lamina using two mechanisms, i.e., dystrophin with its associated proteins and alpha7beta1 integrin. In humans, gene-mutation defects in one member of these complexes result in muscular dystrophies. This study revealed changes after L-arginine treatment of utrophin-associated proteins and the alpha7B integrin subunit in mdx mouse, a dystrophin-deficient animal model. In the two studied muscles (cardiac muscle and diaphragm), the alpha7B integrin subunit was increased in 5-week-old treated mice. Interestingly, the diaphragm histopathological appearance was significantly improved by L-arginine administration. These results highlight a possible way to compensate for dystrophin deficiency via alpha7beta1 integrin.

Combining gene mapping and phenotype assessment for fast mutation finding in non-consanguineous autosomal recessive retinitis pigmentosa families.

Among inherited retinal dystrophies, autosomal recessive retinitis pigmentosa (arRP) is the most genetically heterogenous condition with 32 genes currently known that account for ~60 % of patients. Molecular diagnosis thus requires the tedious systematic sequencing of 506 exons. To rapidly identify the causative mutations, we devised a strategy that combines gene mapping and phenotype assessment in small non-consanguineous families. Two unrelated sibships with arRP had whole-genome scan using SNP microchips. Chromosomal regions were selected by calculating a score based on SNP coverage and genotype identity of affected patients. Candidate genes from the regions with the highest scores were then selected based on phenotype concordance of affected patients with previously described phenotype for each candidate gene. For families RP127 and RP1459, 33 and 40 chromosomal regions showed possible linkage, respectively. By comparing the scores with the phenotypes, we ended with one best candidate gene for each family, namely tubby-like protein 1 (TULP1) and C2ORF71 for RP127 and RP1459, respectively. We found that RP127 patients were compound heterozygous for two novel TULP1 mutations, p.Arg311Gln and p.Arg342Gln, and that RP1459 patients were compound heterozygous for two novel C2ORF71 mutations, p.Leu777PhefsX34 and p.Leu777AsnfsX28. Phenotype assessment showed that TULP1 patients had severe early onset arRP and that C2ORF71 patients had a cone rod dystrophy type of arRP. Only two affected individuals in each sibship were sufficient to lead to mutation identification by screening the best candidate gene selected by a combination of gene mapping and phenotype characterization.