Synergism between remdesivir and ribavirin leads to SARS-CoV-2 extinction in cell culture.

BACKGROUND AND PURPOSE: There is a need for effective anti-COVID-19 treatments, mainly for individuals at risk of severe disease such as the elderly and the immunosuppressed. Drug repositioning has proved effective in identifying drugs that can find a new application for the control of coronavirus disease, in particular COVID-19. The purpose of the present study was to find synergistic antiviral combinations for COVID-19 based on lethal mutagenesis. EXPERIMENTAL APPROACH: The effect of combinations of remdesivir and ribavirin on the infectivity of SARS-CoV-2 in cell culture has been tested. Viral populations were monitored by ultra-deep sequencing, and the decrease of infectivity as a result of the treatment was measured. KEY RESULTS: Remdesivir and ribavirin exerted a synergistic inhibitory activity against SARS-CoV-2, quantified both by CompuSyn (Chou-Talalay method) and Synergy Finder (ZIP-score model). In serial passage experiments, virus extinction was readily achieved with remdesivir-ribavirin combinations at concentrations well below their cytotoxic 50 value, but not with the drugs used individually. Deep sequencing of treated viral populations showed that remdesivir, ribavirin, and their combinations evoked significant increases of the number of viral mutations and haplotypes, as well as modification of diversity indices that characterize viral quasi-species. CONCLUSION AND IMPLICATIONS: SARS-CoV-2 extinction can be achieved by synergistic combination treatments based on lethal mutagenesis. In addition, the results offer prospects of triple drug treatments for effective SARS-CoV-2 suppression.

SARS-CoV-2 mutant spectra as variant of concern nurseries: endless variation?

Introduction: SARS-CoV-2 isolates of a given clade may contain low frequency genomes that encode amino acids or deletions which are typical of a different clade. Methods: Here we use high resolution ultra-deep sequencing to analyze SARS-CoV-2 mutant spectra. Results: In 6 out of 11 SARS-CoV-2 isolates from COVID-19 patients, the mutant spectrum of the spike (S)-coding region included two or more amino acids or deletions, that correspond to discordant viral clades. A similar observation is reported for laboratory populations of SARS-CoV-2 USA-WA1/2020, following a cell culture infection in the presence of remdesivir, ribavirin or their combinations. Moreover, some of the clade-discordant genome residues are found in the same haplotype within an amplicon. Discussion: We evaluate possible interpretations of these findings, and reviewed precedents for rapid selection of genomes with multiple mutations in RNA viruses. These considerations suggest that intra-host evolution may be sufficient to generate minority sequences which are closely related to sequences typical of other clades. The results provide a model for the origin of variants of concern during epidemic spread─in particular Omicron lineages─that does not require prolonged infection, involvement of immunocompromised individuals, or participation of intermediate, non-human hosts.

Detection of elusive DNA copy-number variations in hereditary disease and cancer through the use of noncoding and off-target sequencing reads.

Copy-number variants (CNVs) play a substantial role in the molecular pathogenesis of hereditary disease and cancer, as well as in normal human interindividual variation. However, they are still rather difficult to identify in mainstream sequencing projects, especially involving exome sequencing, because they often occur in DNA regions that are not targeted for analysis. To overcome this problem, we developed OFF-PEAK, a user-friendly CNV detection tool that builds on a denoising approach and the use of "off-target" DNA reads, which are usually discarded by sequencing pipelines. We benchmarked OFF-PEAK on data from targeted sequencing of 96 cancer samples, as well as 130 exomes of individuals with inherited retinal disease from three different populations. For both sets of data, OFF-PEAK demonstrated excellent performance (>95% sensitivity and >80% specificity vs. experimental validation) in detecting CNVs from in silico data alone, indicating its immediate applicability to molecular diagnosis and genetic research.

Clinical and Genetic Analysis of Patients With TK2 Deficiency.

Objectives: Thymidine kinase 2 deficiency (TK2d) is a rare autosomal recessive disorder that stems from a perturbation of the mitochondrial DNA maintenance. Nucleoside treatment has recently shown promise as a disease-modifying therapy. TK2d was initially associated with rapidly progressive fatal myopathy in children featuring mitochondrial DNA depletion. Subsequently, less severe variants of the disease were described, with onset of symptoms during adolescence or adulthood and associated with the presence of multiple mtDNA deletions. These less severe phenotypes have been reported in only 15% of the approximately 120 patients described worldwide. However, some reports suggest that these juvenile and adult-onset presentations may be more common. The objective of this study was to describe the clinical phenotype in a sample of patients from Spain. Methods: This study includes 53 patients harboring biallelic TK2 pathogenic variants, compiling data retrospectively from 7 Spanish centers. We analyzed allele frequency, investigated the most recent common ancestor of core haplotypes, and used the Runs of Homozygosity approach to investigate variant coalescence. Results: Symptom onset distribution revealed that 32 patients (60%) experienced symptoms beyond 12 years of age. Approximately 30% of patients died of respiratory insufficiency, while 56% of surviving patients needed mechanical ventilation. Genetic analysis identified 16 distinct variants in TK2. Two variants, p.Lys202del and p.Thr108Met, exhibited significantly higher prevalence in the Spanish population than that reported in gnomAD database (86-fold and 13-fold, respectively). These variants are estimated to have originated approximately 16.8 generations ago for p.Thr108Met and 95.2 generations ago for p.Lys202del within the Spanish population, with the increase in frequency attributed to various forms of inbreeding. In late-onset cases, 46.9% carried the p.Lys202del variant. Discussion: The higher frequency of TK2d in Spain can be partially attributed to the increased prevalence of 2 variants and consanguinity. Notably, in 60% of the cohort, the disease was late-onset, emphasizing the potential underdiagnosis of this subgroup of patients in other regions. Raising awareness of this potentially treatable disorder is of utmost importance because early interventions can significantly affect the quality of life and survival of affected individuals.

PRPH2-Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort.

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.

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing.

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples.

Long-read genome sequencing identifies cryptic structural variants in congenital aniridia cases.

BACKGROUND: Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches. RESULTS: Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1. CONCLUSIONS: In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.

Comprehensive Genotyping and Phenotyping Analysis of GUCY2D-Associated Rod- and Cone-Dominated Dystrophies.

PURPOSE: To describe the genetic and clinical spectrum of GUCY2D-associated retinopathies and to accurately establish their prevalence in a large cohort of patients. DESIGN: Retrospective case series. METHODS: Institutional study of 47 patients from 27 unrelated families with retinal dystrophies carrying disease-causing GUCY2D variants from the Fundación Jiménez Díaz hospital dataset of 8000 patients. Patients underwent ophthalmological examination and molecular testing by Sanger or exome sequencing approaches. Statistical and principal component analyses were performed to determine genotype-phenotype correlations. RESULTS: Four clinically different associated phenotypes were identified: 66.7% of families with cone/cone-rod dystrophy, 22.2% with Leber congenital amaurosis, 7.4% with early-onset retinitis pigmentosa, and 3.7% with congenital night blindness. Twenty-three disease-causing GUCY2D variants were identified, including 6 novel variants. Biallelic variants accounted for 28% of patients, whereas most carried dominant alleles associated with cone/cone-rod dystrophy. The disease onset had statistically significant differences according to the functional variant effect. Patients carrying GUCY2D variants were projected into 3 subgroups by allelic combination, disease onset, and presence of nystagmus or night blindness. In contrast to patients with the most severe phenotype of Leber congenital amaurosis, 7 patients with biallelic GUCY2D had a later and milder rod form with night blindness in infancy as the first symptom. CONCLUSIONS: This study represents the largest GUCY2D cohort in which 4 distinctly different phenotypes were identified, including rare intermediate presentations of rod-dominated retinopathies. We established that GUCY2D is linked to about 1% of approximately 3000 molecularly characterized families of our cohort. All of these findings are critical for defining cohorts for inclusion in future clinical trials.

VIsoQLR: an interactive tool for the detection, quantification and fine-tuning of isoforms in selected genes using long-read sequencing.

DNA variants altering the pre-mRNA splicing process represent an underestimated cause of human genetic diseases. Their association with disease traits should be confirmed using functional assays from patient cell lines or alternative models to detect aberrant mRNAs. Long-read sequencing is a suitable technique to identify and quantify mRNA isoforms. Available isoform detection and/or quantification tools are generally designed for the whole transcriptome analysis. However experiments focusing on genes of interest need more precise data fine-tuning and visualization tools.Here we describe VIsoQLR, an interactive analyzer, viewer and editor for the semi-automated identification and quantification of known and novel isoforms using long-read sequencing data. VIsoQLR is tailored to thoroughly analyze mRNA expression in splicing assays of selected genes. Our tool takes sequences aligned to a reference, and for each gene, it defines consensus splice sites and quantifies isoforms. VIsoQLR introduces features to edit the splice sites through dynamic and interactive graphics and tables, allowing accurate manual curation. Known isoforms detected by other methods can also be imported as references for comparison. A benchmark against two other popular transcriptome-based tools shows VIsoQLR accurate performance on both detection and quantification of isoforms. Here, we present VIsoQLR principles and features and its applicability in a case study example using nanopore-based long-read sequencing. VIsoQLR is available at https://github.com/TBLabFJD/VIsoQLR .

Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.

Analysis of Differentially Expressed MicroRNAs in Serum and Lung Tissues from Individuals with Severe Asthma Treated with Oral Glucocorticoids.

Nowadays, microRNAs (miRNAs) are increasingly used as biomarkers due to their potential contribution to the diagnosis and targeted treatment of a range of diseases. The aim of the study was to analyze the miRNA expression profiles in serum and lung tissue from patients with severe asthma treated with oral corticosteroids (OCS) and those without OCS treatment. For this purpose, serum and lung tissue miRNAs of OCS and non-OCS asthmatic individuals were evaluated by miRNAs-Seq, and subsequently miRNA validation was performed using RT-qPCR. Additionally, pathway enrichment analysis of deregulated miRNAs was conducted. We observed altered expression by the next-generation sequencing (NGS) of 11 miRNAs in serum, of which five (hsa-miR-148b-3p, hsa-miR-221-5p, hsa-miR-618, hsa-miR-941, and hsa-miR-769-5p) were validated by RT-qPCR, and three miRNAs in lung tissue (hsa-miR-144-3p, hsa-miR-144-5p, and hsa-miR-451a). The best multivariate logistic regression model to differentiate individuals with severe asthma, treated and untreated with OCS, was to combine the serum miRNAs hsa-miR-221-5p and hsa-miR-769-5p. Expression of hsa-miR-148b-3p and hsa-miR-221-5p correlated with FEV1/FVC (%) and these altered miRNAs act in key signaling pathways for asthma disease and the regulated expression of some genes (FOXO3, PTEN, and MAPK3) involved in these pathways. In conclusion, there are miRNA profiles differentially expressed in OCS-treated individuals with asthma and could be used as biomarkers of OCS treatment.

Prioritization of New Candidate Genes for Rare Genetic Diseases by a Disease-Aware Evaluation of Heterogeneous Molecular Networks.

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.

Atypical Mutational Spectrum of SARS-CoV-2 Replicating in the Presence of Ribavirin.

We report that ribavirin exerts an inhibitory and mutagenic activity on SARS-CoV-2-infecting Vero cells, with a therapeutic index higher than 10. Deep sequencing analysis of the mutant spectrum of SARS-CoV-2 replicating in the absence or presence of ribavirin indicated an increase in the number of mutations, but not in deletions, and modification of diversity indices, expected from a mutagenic activity. Notably, the major mutation types enhanced by replication in the presence of ribavirin were A→G and U→C transitions, a pattern which is opposite to the dominance of G→A and C→U transitions previously described for most RNA viruses. Implications of the inhibitory activity of ribavirin, and the atypical mutational bias produced on SARS-CoV-2, for the search for synergistic anti-COVID-19 lethal mutagen combinations are discussed.

Biallelic intragenic tandem duplication of CPLANE1 in Joubert syndrome: A case report.

Joubert syndrome (JS) is a clinically and genetically heterogeneous genetic disorder. To date, 40 JS-causing genes have been reported and CPLANE1 is one of the most frequently mutated, with biallelic pathogenic missense and truncating variants explaining up to 14% of JS cases. We present a case of JS diagnosed after the identification of a novel biallelic intragenic duplication of exons 20-46 of CPLANE1. The quadruplication was identified by short-read sequencing and copy number variant analysis and confirmed in tandem by long PCR with the breakpoints defined by a nanopore-based long-read sequencing approach. Based on the genetic findings and the clinical presentation of the patient, a brain MRI was ordered, evidencing the molar tooth sign, which confirmed the diagnosis of JS in the patient. This is, to the best of our knowledge, the first report of an intragenic duplication in this gene as the potential molecular mechanism of JS.

Androgen receptor polyQ alleles and COVID-19 severity in men: A replication study.

BACKGROUND: Ample evidence indicates a sex-related difference in severity of COVID-19, with less favorable outcomes observed in men. Genetic factors have been proposed as candidates to explain this difference. The polyglutamine (polyQ) polymorphism in the androgen receptor gene has been recently described as a genetic biomarker of COVID-19 severity. OBJECTIVE: To test the association between the androgen receptor polyQ polymorphism and COVID-19 severity in a large cohort of COVID-19 male patients. MATERIALS AND METHODS: This study included 1136 male patients infected with SARS-CoV-2 as confirmed by positive PCR. Patients were retrospectively and prospectively enrolled from March to November 2020. Patients were classified according to their severity into three categories: oligosymptomatic, hospitalized and severe patients requiring ventilatory support. The number of CAG repeats (polyQ polymorphism) at the androgen receptor was obtained by PCR and patients were classified as either short (<23 repeats) or long (≥23 repeats) allele carriers. The association between polyQ alleles (short or long) and COVID-19 severity was assessed by Chi-squared (Chi2 ) and logistic regression analysis. RESULTS: The mean number of polyQ CAG repeats was 22 (±3). Patients were classified as oligosymptomatic (15.5%), hospitalized (63.2%), and severe patients (21.3%) requiring substantial respiratory support. PolyQ alleles distribution did not show significant differences between severity classes in our cohort (Chi2 test p > 0.05). Similar results were observed after adjusting by known risk factors such as age, comorbidities, and ethnicity (multivariate logistic regression analysis). DISCUSSION: Androgen sensitivity may be a critical factor in COVID-19 disease severity. However, we did not find an association between the polyQ polymorphism and the COVID-19 severity. Additional studies are needed to clarify the mechanism underlying the association between androgens and COVID-19 outcome. CONCLUSIONS: The results obtained in our study do not support the role of this polymorphism as biomarker of COVID-19 severity.

Age-dependent association of clonal hematopoiesis with COVID-19 mortality in patients over 60 years.

Clonal hematopoiesis, especially that of indeterminate potential (CHIP), has been associated with age-related diseases, such as those contributing to a more severe COVID-19. Four studies have attempted to associate CHIP with COVID-19 severity without conclusive findings. In the present work, we explore the association between CHIP and COVID-19 mortality. Genomic DNA extracted from peripheral blood of COVID-19 patients (n = 241 deceased, n = 239 survivors) was sequenced with the Myeloid Solutions™ panel of SOPHiA Genetics. The association between clonality and age and clonality and mortality was studied using logistic regression models adjusted for sex, ethnicity, and comorbidities. The association with mortality was performed with patients stratified into four groups of age according to the quartiles of the distribution: 60-74 years, 75-84 years, 85-91 years, and 92-101 years. Clonality was found in 38% of the cohort. The presence of CHIP variants, but not the number, significantly increased with age in the entire cohort of COVID-19 patients, as well as in the group of survivors (p < 0.001). When patients were stratified by age and the analysis adjusted, CHIP classified as pathogenic/likely pathogenic was significantly more represented in deceased patients compared with survivors in the group of 75-84 years (34.6% vs 13.7%, p = 0.020). We confirmed the well-established linear relationship between age and clonality in the cohort of COVID-19 patients and found a significant association between pathogenic/likely pathogenic CHIP and mortality in patients from 75 to 84 years that needs to be further validated.

A Novel PiRNA Enhances CA19-9 Sensitivity for Pancreatic Cancer Identification by Liquid Biopsy.

Pancreatic cancer is one of the deadliest tumours worldwide, and its poor prognosis is due to an inability to detect the disease at the early stages, thereby creating an urgent need to develop non-invasive biomarkers. P-element-induced wimpy testis (PIWI) proteins work together with piwi-interacting RNAs (piRNAs) to perform epigenetic regulation and as such hold great potential as biomarkers for pancreatic cancer. PIWIL2 and PIWIL4 are associated with better prognosis, while PIWIL1 and PIWIL3 involvement appears to be associated with carcinogenesis. We aimed to discover PIWIL3- and PIWIL4-modulated piRNAs and determine their potential mechanisms in pancreatic cancer and the clinical implications. PIWIL3 or PIWIL4 was downregulated in pancreatic cancer-derived cell lines or in a non-tumour cell line. Differentially expressed piRNAs were analysed by next generation sequencing of small RNA. Nine fresh-frozen samples from solid human pancreases (three healthy pancreases, three intraductal papillary mucinous neoplasms, and three early-stage pancreatic cancers) were included in the sequencing analysis. Two piRNAs associated with PIWIL3 (piR-168112 and piR-162725) were identified in the neoplastic cells; in untransformed samples, we identified one piRNA associated with PIWIL4 (pir-366845). After validation in pancreatic cancer-derived cell lines and one untransformed pancreatic cell line, these piRNAs were evaluated in plasma samples from healthy donors (n = 27) or patients with pancreatic cancer (n = 45). Interestingly, piR-162725 expression identified pancreatic cancer patients versus healthy donors in liquid biopsies. Moreover, the potential of the serum carbohydrate antigen 19-9 (CA19-9) biomarker to identify pancreatic cancer patients was greatly enhanced when combined with piR-162725 detection. The enhanced diagnostic potential for the early detection of pancreatic cancer in liquid biopsies of these new small non-coding RNAs will likely improve the prognosis and management of this deadly cancer.

Aggregated Genomic Data as Cohort-Specific Allelic Frequencies can Boost Variants and Genes Prioritization in Non-Solved Cases of 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.

Allelic overload and its clinical modifier effect in Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy characterized by extensive inter- and intra-familial variability, in which oligogenic interactions have been also reported. Our main goal is to elucidate the role of mutational load in the clinical variability of BBS. A cohort of 99 patients from 77 different families with biallelic pathogenic variants in a BBS-associated gene was retrospectively recruited. Human Phenotype Ontology terms were used in the annotation of clinical symptoms. The mutational load in 39 BBS-related genes was studied in index cases using different molecular and next-generation sequencing (NGS) approaches. Candidate allele combinations were analysed using the in silico tools ORVAL and DiGePred. After clinical annotation, 76 out of the 99 cases a priori fulfilled established criteria for diagnosis of BBS or BBS-like. BBS1 alleles, found in 42% of families, were the most represented in our cohort. An increased mutational load was excluded in 41% of the index cases (22/54). Oligogenic inheritance was suspected in 52% of the screened families (23/45), being 40 tested by means of NGS data and 5 only by traditional methods. Together, ORVAL and DiGePred platforms predicted an oligogenic effect in 44% of the triallelic families (10/23). Intrafamilial variable severity could be clinically confirmed in six of the families. Our findings show that the presence of more than two alleles in BBS-associated genes correlated in six families with a more severe phenotype and associated with specific findings, highlighting the role of the mutational load in the management of BBS cases.