Biomedical subjects

Giuliana Silvestri

Publications and source records attributed to Giuliana Silvestri.

6 recordsLinked to original sources

PubMed2006

Development of a genotyping microarray for Usher syndrome.

BACKGROUND: Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. CONCLUSION: The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

DNA↗

PubMed2006

Fine mapping of the keratoconus with cataract locus on chromosome 15q and candidate gene analysis.

PURPOSE: To report the fine mapping of the keratoconus with cataract locus on chromosome 15q and the mutational analysis of positional candidate genes. METHODS: Genotyping of two novel microsatellite markers and a single nucleotide polymorphism (SNP) in the critical region of linkage for keratoconus with cataract on 15q was performed. Positional candidate genes (MORF4L1, KIAA1055, ETFA, AWP1, REC14, KIAA1199, RCN2, FAH, IDH3A, MTHFS, ADAMTS7, MAN2C1, PTPN9, KIAA1024, ARNT2, BCL2A1, ISL2, C15ORF22 (P24B), DNAJA4, FLJ14594, CIB2 (KIP2), C15ORF5, and PSMA4) prioritized on the basis of ocular expression and probable function were screened by PCR-based DNA sequencing methods. RESULTS: We report the refinement of the linkage region for keratoconus with cataract to an interval of approximately 5.5 Mb flanked by the MAN2C1 gene and the D15S211 marker on chromosome 15q. Mutational analysis of positional candidate genes detected many sequence variations and single nucleotide polymorphisms. None of the sequence variants were considered pathogenic as they were also found in unaffected family members and normal control DNA samples. CONCLUSIONS: Fine mapping of the keratoconus with cataract locus on 15q has reduced the linked region to 5.5 Mb, thereby excluding 28 candidate genes. A further 23 candidate genes were excluded by direct sequencing methods, although a pathogenic genomic rearrangement or exonic deletion would not have been detected.

Case-Control Studies↗

PubMed2005

A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.

Age-related macular degeneration (AMD) is the most frequent cause of irreversible blindness in the elderly in developed countries. Our previous studies implicated activation of complement in the formation of drusen, the hallmark lesion of AMD. Here, we show that factor H (HF1), the major inhibitor of the alternative complement pathway, accumulates within drusen and is synthesized by the retinal pigmented epithelium. Because previous linkage analyses identified chromosome 1q25-32, which harbors the factor H gene (HF1/CFH), as an AMD susceptibility locus, we analyzed HF1 for genetic variation in two independent cohorts comprised of approximately 900 AMD cases and 400 matched controls. We found association of eight common HF1 SNPs with AMD; two common missense variants exhibit highly significant associations (I62V, chi2 = 26.1 and P = 3.2 x 10(-7) and Y402H, chi2 = 54.4 and P = 1.6 x 10(-13)). Haplotype analysis reveals that multiple HF1 variants confer elevated or reduced risk of AMD. One common at-risk haplotype is present at a frequency of 50% in AMD cases and 29% in controls [odds ratio (OR) = 2.46, 95% confidence interval (1.95-3.11)]. Homozygotes for this haplotype account for 24% of cases and 8% of controls [OR = 3.51, 95% confidence interval (2.13-5.78)]. Several protective haplotypes are also identified (OR = 0.44-0.55), further implicating HF1 function in the pathogenetic mechanisms underlying AMD. We propose that genetic variation in a regulator of the alternative complement pathway, when combined with a triggering event, such as infection, underlie a major proportion of AMD in the human population.

Aged↗

PubMed2005

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene.

PURPOSE: Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation. METHODS: Ophthalmic examination was performed on seven family members and serially detailed in the proband over a 3-year period. Blood samples were collected and DNA extracted. All 12 coding exons and the 5' promoter region of the crumbs homologue 1 (CRB1) gene were PCR amplified and DNA sequenced. In silico homology modeling was performed on the mutated protein domain. RESULTS: Subtle symmetrical chorioretinal atrophy in the inferior quadrant was the earliest clinical sign detectable within this family. Paravenous pigmentation occurred initially in the far periphery, progressing centrally, with atrophy later becoming more widespread, involving the nasal, then the temporal, and finally the upper quadrant. A novel, dominant Val162Met mutation within the fourth EGF-like domain of CRB1 cosegregates with the PPCRA phenotype. It is thought to affect domain structure, because codon 162 is involved in hydrogen bonding between the antiparallel beta-strands of the major beta-sheet, causing sufficient perturbation of the backbone that the domain-stabilizing hydrogen bond does not form or is weakened. CONCLUSIONS: PPCRA was dominantly inherited in this family, but exhibited variable expressivity. Males are more likely to exhibit a severe phenotype, whereas females may remain virtually asymptomatic even in later years. The PPCRA phenotype is associated with a Val162Met mutation in CRB1 which is likely to affect the structure of the CRB1 protein.

Adult↗

PubMed2004

A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients.

PURPOSE: Age related macular degeneration (AMD) is a common cause of severe vision loss. Identification of genes involved in AMD will facilitate early detection and ultimately help to identify pathways for treatment for this disorder. The A16,263G mutation in the HEMICENTIN-1 gene produces a non-conservative substitution of arginine for glutamine at codon 5345 which has been implicated in familial AMD. The aim of this study is to develop a rapid diagnostic assay for the detection of this mutation and to evaluate its frequency in a sample of AMD patients. METHODS: A primer probe set was designed from exon 104 of the HEMICENTIN-1 gene to differentiate between mutant and wild type alleles. A region spanning the mutation was amplified by PCR using a LightCycler (Roche Diagnostic). The mutation was then detected by melt curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. The frequency of the mutation within the Northern Ireland population was evaluated by assaying 508 affected AMD patients, 25 possibly affected and 163 controls. RESULTS: This assay clearly discriminates between the A16,263G mutant and wild type HEMICENTIN-1 alleles. The wild type sequence has a single base mismatch with the probe which decreases the stability of the hybrid, resulting in a lower TM (TM=51.27 degrees C) than that observed for the perfectly matched mutant allele (TM=59.9 degrees C). The mutant allele was detected in only one of the 696 subjects, an affected AMD patient. CONCLUSIONS: We describe a rapid assay for the genotyping of the Gln5345Arg mutation using real-time fluorescence PCR to facilitate rapid processing of samples through combined amplification and detection steps. These characteristics are suitable for a clinical setting where high throughput diagnostic procedures are required. The frequency of this mutation within the Northern Ireland population has been estimated at 0.2%, concurring with previous findings that this mutation is a rare variant associated with AMD. A rapid diagnostic assay will facilitate a reliable and convenient evaluation of the frequency of the Gln5345Arg mutation and its association with AMD within other populations.

Aged↗

PubMed2003

Familial keratoconus with cataract: linkage to the long arm of chromosome 15 and exclusion of candidate genes.

PURPOSE: Keratoconus and cataract are common causes of visual morbidity. Both conditions show genetic predisposition. The purpose of this study was to map the disease locus in a large three-generation family affected by combined early-onset autosomal dominant anterior polar cataract and clinically severe keratoconus. Uniquely, in this family both disorders were present and fully penetrant in those affected. METHODS: Thirty members of the family were examined clinically on two occasions, at an interval of 5 years, to establish their phenotypes and determine the progression of the disease. Genomic DNA was extracted from blood samples of 16 affected and 14 unaffected individuals, and typed with more than 350 highly polymorphic microsatellite loci in a genome-wide linkage screen. Markers were amplified by PCR with fluorescently labeled primers and sized with an automated DNA analyser before calculation of lod scores. After linkage was established, several positional candidate genes were assessed by PCR-based DNA sequencing. RESULTS: The locus for keratoconus with cataract was mapped to a 6.5-Mb region of the long arm of chromosome 15, at 22.33-24.2 between CYP11A and D15S211. The positional and functional candidate genes CTSH, CRABP1, IREB2, and RASGRF1 were excluded as the cause of keratoconus with cataract in this family. CONCLUSIONS: This is the first report of a family with autosomal dominant inheritance of keratoconus in association with cataract. The causative gene maps to the long arm of chromosome 15 but has not yet been identified.

Adolescent↗