Esther Pomares

Autosomal Recessive Retinitis Pigmentosa with Early Macular Affectation Caused by Premature Truncation in PROM1

PURPOSE To identify the genetic basis of a large consanguineous Spanish pedigree affected with autosomal recessive retinitis pigmentosa (arRP) with premature macular atrophy and myopia. METHODS After a high-throughput cosegregation gene chip was used to exclude all known RP and Leber congenital amaurosis (LCA) candidates, genome-wide screening and linkage analysis were performed. Direct mutational screening identified the pathogenic mutation, and primers were designed to obtain the RT-PCR products for isoform characterization. RESULTS Mutational analysis detected a novel homozygous PROM1 mutation, c.869delG in exon 8 cosegregating with the disease. This variant causes a frameshift that introduces a premature stop codon, producing truncation of approximately two-thirds of the protein. Analysis of PROM1 expression in the lymphocytes of patients, carriers, and control subjects revealed an aberrant transcript that is degraded by the nonsense-mediated decay pathway, suggesting that the disease is caused by the absence of the PROM1 protein. Three (s2, s11 and s12) of the seven alternatively spliced isoforms reported in humans, accounted for 98% of the transcripts in the retina. Given that these three contained exon 8, no PROM1 isoform is expected in the affected retinas. CONCLUSIONS A remarkable clinical finding in the affected family is early macular atrophy with concentric spared areas. The authors propose that the hallmark of PROM1 truncating mutations is early and severe progressive degeneration of both rods and cones and highlight this gene as a candidate of choice to prioritize in the molecular genetic study of patients with noncanonical clinical peripheral and macular affectation.

Comprehensive SNP-chip for retinitis pigmentosa- Leber congenital amaurosis diagnosis: new mutations and detection of mutational founder effects

Fast and efficient high-throughput techniques are essential for the molecular diagnosis of highly heterogeneous hereditary diseases, such as retinitis pigmentosa (RP). We had previously approached RP genetic testing by devising a chip based on co-segregation analysis for the autosomal recessive forms. In this study, we aimed to design a diagnostic tool for all the known genes (40 up to now) responsible for the autosomal dominant and recessive RP and Leber congenital amaurosis (LCA). This new chip analyzes 240 single nucleotide polymorphisms (SNPs) (6 per gene) on a high-throughput genotyping platform (SNPlex, Applied Biosystems), and genetic diagnosis is based on the co-segregation analysis of SNP haplotypes in independent families. In a single genotyping step, the number of RP candidates to be screened for mutations is considerably reduced, and in the most informative families, all the candidates are ruled out at once. In a panel of RP Spanish pedigrees, the disease chip became a crucial tool for selecting those suitable for genome-wide RP gene search, and saved the burdensome direct mutational screening of every known RP gene. In a large adRP family, the chip allowed ruling out of all but the causative gene, and identification of an unreported null mutation (E181X) in PRPF31. Finally, on the basis of the conservation of the SNP haplotype linked to this pathogenic variant, we propose that the E181X mutation spread through a cohort of geographically isolated families by a founder effect.

Identification of an intronic single-point mutation in RP2 as the cause of semidominant X-linked retinitis pigmentosa.

PURPOSE A large family with 11 males and 2 females with X-linked retinitis pigmentosa (XLRP) was analyzed in search of pathologic mutations. METHODS Of the two major XLRP genes, RPGR was analyzed by SNP cosegregation and RP2 was directly screened for mutations. The pathogenicity of a new variant was assessed in silico, in vivo, and in vitro. RESULTS The results of cosegregation analysis with SNPs closely located to RPGR excluded this gene as the cause of the disease in this family. Sequencing of RP2 showed a putative pathogenic variant in intron 3 at the conserved polypyrimidine tract (c.1073-9T>A). This substitution cosegregated with the disease and was not found in 220 control chromosomes. In silico analyses using online resources indicated a decreased score of intron 3 acceptor splice site for the mutated sequence. Real-time RT-PCR analysis of the RP2 splicing pattern in blood samples of patients and carrier females showed skipping of exon 4, causing a frame shift that introduced a premature stop codon. Further verification of the pathogenicity of this point mutation was obtained by expression of a minigene RP2 construct in cultured cells. CONCLUSIONS A transversion (T>A) at position -9 in intron 3 of RP2 causes XLRP by altering the splicing pattern and highlights the pathogenicity of intronic variants. The single point RP2 mutation leads to a wide range of phenotypic traits in carrier females, from completely normal to severe retinal degeneration, thus supporting that RP2 is also a candidate for semidominance in XLRP.

Nonsense-Mediated Decay as the Molecular Cause for Autosomal Recessive Bestrophinopathy in Two Unrelated Families

PURPOSE To characterize the molecular basis of two novel BEST1 mutations causing autosomal recessive bestrophinopathy (ARB). Strong evidence argues in favor of the dominant negative effects of most autosomal dominantly inherited mutations, whereas there is only weak support for the molecular basis of the ARB phenotype. METHODS Patients underwent ophthalmic examination, color and autofluorescence fundus imaging, optical coherence tomography (OCT), electrooculogram, and full-field electroretinogram (ERG). BEST1 was directly screened for mutations in two ARB unrelated patients. The pathogenicity of the new BEST1 variants was assessed in silico and in vivo. RESULTS Two unrelated patients with diagnoses of ARB showed retinal pigment epithelial disturbances and abnormal ERGs. Each patient was homozygous for a novel BEST1 mutation, c.521_522del and c.1100+1G>A. A carrier sibling (WT/c.521_522del) was unaffected. Both mutations generate a frameshift and a premature stop codon that, if translated, would seriously compromise bestrophin-1 function. However, the in vivo quantitative RT-PCR assays showed that most of the mutated transcripts were eliminated before translation because the mRNA-BEST1 levels were dramatically diminished the controls. CONCLUSIONS In truncating BEST1 mutations, the null phenotype associated with ARB is attributed to a substantial decrease of BEST1 expression promoted by the nonsense-mediated decay (NMD) surveillance mechanism. Moreover, the severity of the phenotype increases with the preserved amount of altered transcript, suggesting that the clinical outcome reflects the combined null and dominant negative effects of the two mutations over the patients genetic background.

Combined Genetic and High-Throughput Strategies for Molecular Diagnosis of Inherited Retinal Dystrophies

Most diagnostic laboratories are confronted with the increasing demand for molecular diagnosis from patients and families and the ever-increasing genetic heterogeneity of visual disorders. Concerning Retinal Dystrophies (RD), almost 200 causative genes have been reported to date, and most families carry private mutations. We aimed to approach RD genetic diagnosis using all the available genetic information to prioritize candidates for mutational screening, and then restrict the number of cases to be analyzed by massive sequencing. We constructed and optimized a comprehensive cosegregation RD-chip based on SNP genotyping and haplotype analysis. The RD-chip allows to genotype 768 selected SNPs (closely linked to 100 RD causative genes) in a single cost-, time-effective step. Full diagnosis was attained in 17/36 Spanish pedigrees, yielding 12 new and 12 previously reported mutations in 9 RD genes. The most frequently mutated genes were USH2A and CRB1. Notably, RD3–up to now only associated to Leber Congenital Amaurosis– was identified as causative of Retinitis Pigmentosa. The main assets of the RD-chip are: i) the robustness of the genetic information that underscores the most probable candidates, ii) the invaluable clues in cases of shared haplotypes, which are indicative of a common founder effect, and iii) the detection of extended haplotypes over closely mapping genes, which substantiates cosegregation, although the assumptions in which the genetic analysis is based could exceptionally lead astray. The combination of the genetic approach with whole exome sequencing (WES) greatly increases the diagnosis efficiency, and revealed novel mutations in USH2A and GUCY2D. Overall, the RD-chip diagnosis efficiency ranges from 16% in dominant, to 80% in consanguineous recessive pedigrees, with an average of 47%, well within the upper range of massive sequencing approaches, highlighting the validity of this time- and cost-effective approach whilst high-throughput methodologies become amenable for routine diagnosis in medium sized labs.

Whole exome sequencing using Ion Proton system enables reliable genetic diagnosis of inherited retinal dystrophies

Inherited retinal dystrophies (IRD) comprise a wide group of clinically and genetically complex diseases that progressively affect the retina. Over recent years, the development of next-generation sequencing (NGS) methods has transformed our ability to diagnose heterogeneous diseases. In this work, we have evaluated the implementation of whole exome sequencing (WES) for the molecular diagnosis of IRD. Using Ion ProtonTM system, we simultaneously analyzed 212 genes that are responsible for more than 25 syndromic and non-syndromic IRD. This approach was used to evaluate 59 unrelated families, with the pathogenic variant(s) successfully identified in 71.18% of cases. Interestingly, the mutation detection rate varied substantially depending on the IRD subtype. Overall, we found 63 different mutations (21 novel) in 29 distinct genes, and performed in vivo functional studies to determine the deleterious impact of variants identified in MERTK, CDH23, and RPGRIP1. In addition, we provide evidences that support CDHR1 as a gene responsible for autosomal recessive retinitis pigmentosa with early macular affectation, and present data regarding the disease mechanism of this gene. Altogether, these results demonstrate that targeted WES of all IRD genes is a reliable, hypothesis-free approach, and a cost- and time-effective strategy for the routine genetic diagnosis of retinal dystrophies.

Union makes strength: a worldwide collaborative genetic and clinical study to provide a comprehensive survey of RD3 mutations and delineate the associated phenotype.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration (RD), and the most common cause of incurable blindness diagnosed in children. It is occasionally the presenting symptom of multisystemic ciliopathies which diagnosis will require a specific care of patients. Nineteen LCA genes are currently identified and three of them account for both non-syndromic and syndromic forms of the disease. RD3 (LCA12) was implicated as a LCA gene based on the identification of homozygous truncating mutations in two LCA families despite the screening of large cohorts of patients. Here we provide a comprehensive survey of RD3 mutations and of their clinical expression through the screening of a cohort of 852 patients originating worldwide affected with LCA or early-onset and severe RD. We identified three RD3 mutations in seven unrelated consanguineous LCA families - i.e., a 2 bp deletion and two nonsense mutations – predicted to cause complete loss of function. Five families originating from the Southern Shores of the Mediterranean segregated a similar mutation (c.112C>T, p.R38*) suggesting that this change may have resulted from an ancient founder effect. Considering the low frequency of RD3 carriers, the recurrence risk for LCA in non-consanguineous unions is negligible for both heterozygote and homozygote RD3 individuals. The LCA12 phenotype in our patients is highly similar to those of patients with mutant photoreceptor-specific guanylate cyclase (GUCY2D/LCA1). This observation is consistent with the report of the role of RD3 in trafficking of GUCYs and gives further support to a common mechanism of photoreceptor degeneration in LCA12 and LCA1, i.e., inability to increase cytoplasmic cGMP concentration in outer segments and thus to recover the dark-state. Similar to LCA1, LCA12 patients have no extraocular symptoms despite complete inactivation of both RD3 alleles, supporting the view that extraocular investigations in LCA infants with RD3 mutations should be avoided.

Panel‐based whole exome sequencing identifies novel mutations in microphthalmia and anophthalmia patients showing complex Mendelian inheritance patterns

Microphthalmia and anophthalmia (MA) are congenital eye abnormalities that show an extremely high clinical and genetic complexity. In this study, we evaluated the implementation of whole exome sequencing (WES) for the genetic analysis of MA patients. This approach was used to investigate three unrelated families in which previous single‐gene analyses failed to identify the molecular cause.

High-throughput approaches for the genetic diagnosis of retinal dystrophies.

The genetic diagnosis of inherited retinal dystrophies has been deeply hampered by the extreme genetic and allelic heterogeneity of this group of disorders, with most families bearing rare or private pathogenic variants. Clinicians, patients, and the affected families are becoming aware of the benefits of a reliable molecular diagnosis: it facilitates genetic counseling and prenatal screening, improves the clinical diagnosis – providing prognostic information as genotype–phenotype correlations are being drawn, opens new venues for basic research, and guides therapy as the current clinical trials for retinal disorders are mainly gene-specific. The wide range of high-throughput techniques, now increasingly available and affordable for most laboratories, poses new questions to the clinical geneticists concerning the best approach to accurate genotyping on the basis of efficiency and cost-effectiveness. The comparison between available molecular diagnosis methodologies, in this rapidly moving field, leads to the conclusion that there is not an obvious preferential approach, but a combination of prioritized strategies is at present the optimal choice.