C. Ayuso

Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

Overview of the Mutation Spectrum in Familial Exudative Vitreoretinopathy and Norrie Disease with Identification of 21 Novel Variants in FZD4, LRP5, and NDP

Wnt signaling is a crucial component of the cell machinery orchestrating a series of physiological processes such as cell survival, proliferation, and migration. Among the plethora of roles that Wnt signaling plays, its canonical branch regulates eye organogenesis and angiogenesis. Mutations in the genes encoding the low density lipoprotein receptor protein 5 (LRP5) and frizzled 4 (FZD4), acting as coreceptors for Wnt ligands, cause familial exudative vitreoretinopathy (FEVR). Moreover, mutations in the gene encoding NDP, a ligand for these Wnt receptors, cause Norrie disease and FEVR. Both FEVR and Norrie disease share similar phenotypic characteristics, including abnormal vascularization of the peripheral retina and formation of fibrovascular masses in the eye that can lead to blindness. In this mutation update, we report 21 novel variants for FZD4, LRP5, and NDP, and discuss the putative functional consequences of missense mutations. In addition, we provide a comprehensive overview of all previously published variants in the aforementioned genes and summarize the phenotypic characteristics in mouse models carrying mutations in the orthologous genes. The increasing molecular understanding of Wnt signaling, related to ocular development and blood supply, offers more tools for accurate disease diagnosis that may be important in the development of therapeutic interventions. Hum Mutat 31:656–666, 2010.

Foetal sex determination in maternal blood from the seventh week of gestation and its role in diagnosing haemophilia in the foetuses of female carriers

Summary.u2002 The existence of foetal DNA in maternal blood, discovered in 1997, opened new possibilities for noninvasive prenatal diagnosis. This includes foetal sex assessment by the detection of specific Y chromosome sequences in maternal blood, particularly important when a foetus may be affected by an X‐linked disorder such as haemophilia. This study aims to validate this sex assessment method and to test its clinical utility in the diagnosis of 15 potentially affected pregnancies in female carriers of haemophilia. In the validation study, 316 maternal blood samples from 196 pregnant women at gestations ranging from 5u2003weeks to 12u2003weeks were analysed. In the clinical study, 15 pregnancies at risk of having a haemophilic foetus were tested. All pregnancies in the validation study were correctly diagnosed. The accuracy and specificity of the methodology from the seventh week of gestation was 100%. The sex of all 15 pregnancies identified as being at risk of bearing a haemophilic foetus was correctly diagnosed. Foetal sex assessment by detecting specific Y chromosome sequences in maternal blood is now routinely used in our hospital because of its high accuracy from the seventh week of gestation. Reliable foetal gender determination from maternal blood of pregnant women carriers of haemophilia in the first trimester of gestation can avoid more conventional, invasive methods of prenatal diagnosis.

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.

Microarray-based mutation analysis of 183 Spanish families with Usher syndrome.

PURPOSEnThe purpose of this study was to test the ability of the genotyping microarray for Usher syndrome (USH) to identify the mutations responsible for the disease in a cohort of 183 patients with USH.nnnMETHODSnDNA from 183 patients with Usher syndrome from the Spanish population was analyzed using a genotyping microarray containing 429 previously identified disease-associated variants in eight USH genes. Mutations detected by the array were confirmed by direct sequencing. Haplotype analysis was also performed in families carrying common Spanish mutations.nnnRESULTSnThe genotyping microarray identified 43 different variants, divided into 32 disease causative and 11 probably nonpathologic. Mutations were detected in 62 patients with USH (33.9%). According to the clinical classification of patients, pathologic variants were detected in 31.4% patients with USH1, 39.4% of with USH2, 22.2% with USH3 and 15.8% with unclassified Usher syndrome. Ninety-seven pathologic alleles were detected, corresponding to 26.5% of expected alleles. The USH2A mutations p.C3267R and p.T3571M were revealed as common in the Spanish population, and two major haplotypes linked to these mutations were observed.nnnCONCLUSIONSnThe genotyping microarray is a robust, low-cost, rapid technique that is effective for the genetic study of patients with USH. However, it also indicates variants of unclear pathologic nature and detection failures have also been observed. Results must be confirmed by direct sequencing to avoid misdiagnosis, and continuous updates of the microarray should be performed to increase the efficiency and rate of detection of mutations.

Study of the involvement of the RGR, CRPB1, and CRB1 genes in the pathogenesis of autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP), which occurs in about 1 in 3000-7000 people in Spain, is inherited in an autosomal dominant manner in 12% of cases, in an autosomal recessive way in 39%, and in an X linked manner in 4% of cases.1 This leaves 41% of RP cases with a simplex form and 4% in which the transmission pattern is unclear.nnThe different genes that have been implicated in retinal degeneration are known or assumed to be expressed in the photoreceptor cells of the retina or in the retinal pigment epithelium (RPE). The large number of RP genes identified can be grouped into a number of functional classes: (1) proteins of the visual cascade, (2) proteins of the visual cycle, (3) photoreceptor cell transcription factors, (4) proteins related to catabolic processes, and (5) genes of unknown function.nnPrevious studies performed in autosomal recessive retinitis pigmentosa (ARRP) Spanish families have shown that genes coding for recoverin,2 rhodopsin, rod outer segment membrane protein and peripherin/RDS,3 S antigen and the gamma subunit of rod cGMP-phosphodiesterase,4 interstitial retinol binding protein,5 the alpha subunit of rod cGMP-phosphodiesterase and NRL,6 and the retinaldehyde binding protein7 do not play a role in this disorder. However, mutations in the beta subunit of the rod cGMP-phosphodiesterase gene,8–11 in the ATP binding cassette receptor gene,12 in the TULP1 gene,13 in the alpha subunit of the rod cGMP gated channel,14 and in the USH2A gene15 have been detected in a small percentage of Spanish ARRP families. These data indicate that other genes play a part in the degeneration process of the retina in the remaining families.nnWe analysed the involvement of three additional genes, the RPE retinal G protein coupled receptor ( RGR ), the cellular retinol binding protein ( CRBP1 ), and …

Prenatal diagnosis of Huntington disease in maternal plasma: direct and indirect study

Background and purpose:u2002 The presence of cell‐free fetal DNA in maternal plasma could allow performing a non‐invasive prenatal diagnosis of Huntington disease (HD). The great advantage of this diagnosis is the absence of risk of fetal loss that it entails.

Clinical and genetic studies in Spanish patients with Usher syndrome type II: description of new mutations and evidence for a lack of genotype--phenotype correlation.

Patients with Usher syndrome type II (USH2) show moderate‐to‐severe hearing loss (HL), retinitis pigmentosa and normal vestibular function. The progression of HL remains controversial. To evaluate whether a phenotype–genotype correlation exists regarding the issue of progression of HL, only USH2 patients with a defined genotype were selected. Ophthalmologic, vestibular and audiometric examination along with a mutation analysis of the USH2A gene (exons 1–21) was performed in twenty‐eight Spanish USH2 patients. Ten different pathogenic mutations and 17 sequence variants not associated with the disease were found. Six of the 10 mutations are novel. Disease alleles were identified in 13 of the 28 families tested. Eight of these 13 families had a mutation found in both alleles. In the other five families, only one mutation was identified. The phenotypic data provide evidence for the existence of phenotypic differences between patients with the same genotype. These differences were observed at both the interfamilial and intrafamilial levels.

Analysis of the ABCA4 genomic locus in Stargardt disease

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses. The entire 140 kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation revealing, on average, 200 intronic variants per sample. Filtering of these data resulted in 141 candidates for new mutations. Two variants were detected in four samples, two in three samples, and 20 variants in two samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 12 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. No copy number variation (large deletions and insertions) was detected in any patient suggesting that it is a very rare event in the ABCA4 locus. Many variants were excluded since they were not conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants. The sequence variability in the ABCA4 locus is extensive and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Defining disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and extensive analyses by a combination of various approaches.

Targeted next generation sequencing for molecular diagnosis of Usher syndrome

BackgroundUsher syndrome is an autosomal recessive disease that associates sensorineural hearing loss, retinitis pigmentosa and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous. To date, 10 genes have been associated with the disease, making its molecular diagnosis based on Sanger sequencing, expensive and time-consuming. Consequently, the aim of the present study was to develop a molecular diagnostics method for Usher syndrome, based on targeted next generation sequencing.MethodsA custom HaloPlex panel for Illumina platforms was designed to capture all exons of the 10 known causative Usher syndrome genes (MYO7A, USH1C, CDH23, PCDH15, USH1G, CIB2, USH2A, GPR98, DFNB31 and CLRN1), the two Usher syndrome-related genes (HARS and PDZD7) and the two candidate genes VEZT and MYO15A. A cohort of 44 patients suffering from Usher syndrome was selected for this study. This cohort was divided into two groups: a test group of 11 patients with known mutations and another group of 33 patients with unknown mutations.ResultsForty USH patients were successfully sequenced, 8 USH patients from the test group and 32 patients from the group composed of USH patients without genetic diagnosis. We were able to detect biallelic mutations in one USH gene in 22 out of 32 USH patients (68.75%) and to identify 79.7% of the expected mutated alleles. Fifty-three different mutations were detected. These mutations included 21 missense, 8 nonsense, 9 frameshifts, 9 intronic mutations and 6 large rearrangements.ConclusionsTargeted next generation sequencing allowed us to detect both point mutations and large rearrangements in a single experiment, minimizing the economic cost of the study, increasing the detection ratio of the genetic cause of the disease and improving the genetic diagnosis of Usher syndrome patients.