Marta Del Pozo-Valero

Advances in whole-genome sequencing (WGS) have significantly enhanced our ability to detect genomic variants underlying inherited diseases. In this study, we performed long-read WGS on 24 patients with inherited retinal dystrophies (IRDs) to validate the utility of nanopore sequencing in detecting genomic variations. We confirmed the presence of all previously detected variants and demonstrated that this approach allows for the precise refinement of structural variants (SVs). Furthermore, we could perform genotype phasing by sequencing only the probands, confirming that the variants were inherited in trans. Moreover, nanopore sequencing enables the detection of complex variants, such as transposon insertions and structural rearrangements. This comprehensive assessment illustrates the power of long-read sequencing in capturing diverse forms of genomic variation and in improving diagnostic accuracy in IRDs.

Leber congenital amaurosis (LCA) and early‐onset retinal degeneration (EORD) are inherited retinal diseases (IRD) characterized by early‐onset vision impairment. Herein, we studied 15 Saudi families by whole exome sequencing (WES) and run‐of‐homozygosity (ROH) detection via AutoMap in 12/15 consanguineous families. This revealed (likely) pathogenic variants in 11/15 families (73%). A potential founder variant was found in RPGRIP1. Homozygous pathogenic variants were identified in known IRD genes (ATF6, CRB1, CABP4, RDH12, RIMS2, RPGRIP1, SPATA7). We established genotype‐driven clinical reclassifications for ATF6, CABP4, and RIMS2. Specifically, we observed isolated IRD in the individual with the novel RIMS2 variant, and we found a retina‐enriched RIMS2 isoform conserved but not annotated in mouse. The latter illustrates potential different phenotypic consequences of pathogenic variants depending on the particular tissue/cell‐type specific isoforms they affect. Lastly, a compound heterozygous genotype in GUCY2D in one non‐consanguineous family was demonstrated, and homozygous variants in novel candidate genes ATG2B and RUFY3 were found in the two remaining consanguineous families. Reporting these genes will allow to validate them in other IRD cohorts. Finally, the missing heritability of the two unsolved IRD cases may be attributed to variants in non‐coding regions or structural variants that remained undetected, warranting future WGS studies.

PRPH2, one of the most frequently inherited retinal dystrophy (IRD)-causing genes, implies a high phenotypic variability. This study aims to analyze the PRPH2 mutational spectrum in one of the largest cohorts worldwide, and to describe novel pathogenic variants and genotype–phenotype correlations. A study of 220 patients from 103 families recruited from a database of 5000 families. A molecular diagnosis was performed using classical molecular approaches and next-generation sequencing. Common haplotypes were ascertained by analyzing single-nucleotide polymorphisms. We identified 56 variants, including 11 novel variants. Most of them were missense variants (64%) and were located in the D2-loop protein domain (77%). The most frequently occurring variants were p.Gly167Ser, p.Gly208Asp and p.Pro221_Cys222del. Haplotype analysis revealed a shared region in families carrying p.Leu41Pro or p.Pro221_Cys222del. Patients with retinitis pigmentosa presented an earlier disease onset. We describe the largest cohort of IRD families associated with PRPH2 from a single center. Most variants were located in the D2-loop domain, highlighting its importance in interacting with other proteins. Our work suggests a likely founder effect for the variants p.Leu41Pro and p.Pro221_Cys222del in our Spanish cohort. Phenotypes with a primary rod alteration presented more severe affectation. Finally, the high phenotypic variability in PRPH2 hinders the possibility of drawing genotype–phenotype correlations.

Background: 5’ untranslated regions (5’UTRs) are essential modulators of protein translation. Predicting the impact of 5’UTR variants is challenging and typically not performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and subsequent functional assays to evaluate 5’UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). Methods: We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of these 5’UTRs by different whole exome sequencing (WES) capture kits. The selected 5’UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2,417 WGS) and an in-house database (n = 1,682 WES), respectively. Identified variants were annotated for 5’UTR-relevant features and classified into 7 distinct categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse experimental approaches. Results: Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5’UTR compared to that of their canonical isoform. Depending on the probe-design 3-20% of IRD genes have 5’UTRs fully captured by WES. After analyzing these regions in both IRD cohorts we prioritized 11 (likely) pathogenic variants in 10 genes ( ARL3 , MERTK , NDP , NMNAT1 , NPHP4 , PAX6 , PRPF31 , PRPF4 , RDH12 , RD3 ), of which 8 were novel. Functional analyses further supported the pathogenicity of 2 variants. The MERTK :c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12 :c.-123C>T variant was found in cis with the reported hypomorphic RDH12 :c.701G>A (p.Arg234His) variant in 11 patients. This 5’UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. Conclusions: This study demonstrates the importance of 5’UTR variants implicated in IRDs and provides a systematic approach for 5’UTR annotation and validation that is applicable to other inherited diseases.

Screening for pathogenic variants in the diagnosis of rare genetic diseases can now be performed on all genes thanks to the application of whole exome and genome sequencing (WES, WGS). Yet the repertoire of gene–disease associations is not complete. Several computer-based algorithms and databases integrate distinct gene–gene functional networks to accelerate the discovery of gene–disease associations. We hypothesize that the ability of every type of information to extract relevant insights is disease-dependent. We compiled 33 functional networks classified into 13 knowledge categories (KCs) and observed large variability in their ability to recover genes associated with 91 genetic diseases, as measured using efficiency and exclusivity. We developed GLOWgenes, a network-based algorithm that applies random walk with restart to evaluate KCs’ ability to recover genes from a given list associated with a phenotype and modulates the prediction of new candidates accordingly. Comparison with other integration strategies and tools shows that our disease-aware approach can boost the discovery of new gene–disease associations, especially for the less obvious ones. KC contribution also varies if obtained using recently discovered genes. Applied to 15 unsolved WES, GLOWgenes proposed three new genes to be involved in the phenotypes of patients with syndromic inherited retinal dystrophies.

The introduction of NGS in genetic diagnosis has increased the repertoire of variants and genes involved and the amount of genomic information produced. We built an allelic-frequency (AF) database for a heterogeneous cohort of genetic diseases to explore the aggregated genomic information and boost diagnosis in inherited retinal dystrophies (IRD). We retrospectively selected 5683 index-cases with clinical exome sequencing tests available, 1766 with IRD and the rest with diverse genetic diseases. We calculated a subcohort’s IRD-specific AF and compared it with suitable pseudocontrols. For non-solved IRD cases, we prioritized variants with a significant increment of frequencies, with eight variants that may help to explain the phenotype, and 10/11 of uncertain significance that were reclassified as probably pathogenic according to ACMG. Moreover, we developed a method to highlight genes with more frequent pathogenic variants in IRD cases than in pseudocontrols weighted by the increment of benign variants in the same comparison. We identified 18 genes for further studies that provided new insights in five cases. This resource can also help one to calculate the carrier frequency in IRD genes. A cohort-specific AF database assists with variants and genes prioritization and operates as an engine that provides a new hypothesis in non-solved cases, augmenting the diagnosis rate.

Background The introduction of next-generation sequencing in the diagnosis of genetic diseases has increased the known repertoire of causal variants and genes involved, as well as the amount of genomic information produced, that is not always shared or reused. Methods We built an allelic frequency database for a heterogeneous cohort of genetic diseases to explore the aggregated genomic information and boost the diagnosis in inherited retinal dystrophies (IRD). We retrospectively selected 5683 index-cases with clinical exome available, 1766 with IRD, and the rest with diverse genetic diseases. In our IRD cohort, 46% of the patients do not have conclusive diagnosis at the time of writing. We calculated the specific allele-frequencies of the solved and non-solved IRD subcohorts and compared them with suitable pseudocontrols that were used to prioritize variants. In addition, we developed a method to highlight genes with more frequent pathogenic variants in non-solved IRD cases than in pseudocontrols weighted by the increment of benign variants in the same comparison. Our resource was also used to calculate the carrier frequency of deleterious variants in IRD genes. Results Prioritized variants were significantly enriched in deleterious variants in non-solved IRD cases but not in solved. Focusing on non-solved IRD cases, we prioritized variants with a significant increment of frequencies in cases compared to pseudocontrols. Among them, eight variants may contribute to explain the phenotype of 10 cases. Applied to variants of uncertain significance (VUS) monitored in our laboratory, we detected 11 more frequent in IRD than in pseudocontrols, and 10 of them were reclassified as likely-pathogenic according to ACMG guidelines. We also identified 18 genes with an accumulated pathogenicity in non-solved IRD samples for further studies that provided new insights in five cases. Most prevalent genes carrying pathogenic mutations are ABCA4 (∼7%) and USH2A (∼3%). Conclusions A cohort-specific database of allele frequencies is able to diagnose cryptic non-solved IRD cases, reclassify VUS, propose candidate genes, and calculate CF on genes of interest. The database operates as an engine providing new hypotheses in non-solved cases as well as offering new resources for genetic counselling.

(2020). Posterior column ataxia with retinitis pigmentosa (PCARP) in an Iranian patient associated with the FLVCR1 gene. Ophthalmic Genetics. Ahead of Print.