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Item does not contain fulltext, PURPOSE: Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. METHODS: Optical genome mapping was performed to improve SV detection in short-read genome sequencing-negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. RESULTS: In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. CONCLUSION: Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.

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Item does not contain fulltext, Anophthalmia and microphthalmia (A/M) are among the most severe congenital developmental eye disorders. Despite the advancements in genome screening technologies, more than half of A/M patients do not receive a molecular diagnosis. We included seven consanguineous families affected with A/M from Pakistani cohort and an unknown molecular basis. Single gene testing of FOXE3 was performed, followed by genome sequencing for unsolved probands in order to establish a genetic diagnosis for these families. All seven families were provided with a genetic diagnosis. The identified variants were all homozygous, classified as (likely) pathogenic and present in an A/M-associated gene. Targeted FOXE3 sequencing revealed two previously reported pathogenic FOXE3 variants in four families. In the remaining families, genome sequencing revealed a known pathogenic PXDN variant, a novel 13bp deletion in VSX2, and one novel deep intronic splice variant in PXDN. An in vitro splice assay was performed for the PXDN splice variant which revealed a severe splicing defect. Our study confirmed the utility of genome sequencing as a diagnostic tool for A/M-affected individuals. Furthermore, the identification of a novel deep intronic pathogenic variant in PXDN highlights the role of non-coding variants in A/M-disorders and the value of genome sequencing for the identification of this type of variants.

Item does not contain fulltext, Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research hig

There is an increasing need for quantitative rockfall hazard and risk assessment that requires a precise definition of the terms and concepts used for this particular type of landslide. This paper suggests using terms that appear to be the most logic and explicit as possible and describes methods to derive some of the main hazards and risk descriptors. The terms and concepts presented concern the rockfall process (failure, propagation, fragmentation, modelling) and the hazard and risk descriptors, distinguishing the cases of localized and diffuse hazards. For a localized hazard, the failure probability of the considered rock compartment in a given period of time has to be assessed, and the probability for a given element at risk to be impacted with a given energy must be derived combining the failure probability, the reach probability, and the exposure of the element. For a diffuse hazard that is characterized by a failure frequency, the number of rockfalls reaching the element at risk per unit of time and with a given energy (passage frequency) can be derived. This frequency is relevant for risk assessment when the element at risk can be damaged several times. If it is not replaced, the probability that it is impacted by at least one rockfall is more relevant.

Contains fulltext : 284807.pdf (Publisher’s version ) (Open Access), Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.

Item does not contain fulltext, BACKGROUND: Inherited retinal diseases (IRDs) can be caused by variants in >270 genes. The Bardet-Biedl syndrome 1 (BBS1) gene is one of these genes and may be associated with syndromic and non-syndromic autosomal recessive retinitis pigmentosa (RP). Here, we identified a branchpoint variant in BBS1 and assessed its pathogenicity by in vitro functional analysis. METHODS: Whole genome sequencing was performed for three unrelated monoallelic BBS1 cases with non-syndromic RP. A fourth case received MGCM 105 gene panel analysis. Functional analysis using a midigene splice assay was performed for the putative pathogenic branchpoint variant in BBS1. After confirmation of its pathogenicity, patients were clinically re-evaluated, including assessment of non-ocular features of Bardet-Biedl syndrome. RESULTS: Clinical assessments of probands showed that all individuals displayed non-syndromic RP with macular involvement. Through detailed variant analysis and prioritisation, two pathogenic variants in BBS1, the most common missense variant, c.1169T>G (p.(Met390Arg)), and a branchpoint variant, c.592-21A>T, were identified. Segregation analysis confirmed that in all families, probands were compound heterozygous for c.1169T>G and c.592-21A>T. Functional analysis of the branchpoint variant revealed a complex splicing defect including exon 8 and exon 7/8 skipping, and partial in-frame deletion of exon 8. CONCLUSION: A putative severe branchpoint variant in BBS1, together with a mild missense variant, underlies non-syndromic RP in four unrelated individuals. To our knowledge, this is the first report of a pathogenic branchpoint variant in IRDs that results in a complex splice defect. In addition, this research highlights the importance of the analysis of non-coding regions in order to provide a conclusive molecular diagnosis.

Fragmentation is a common feature of rockfall that exerts a strong control on the trajectories of the generated blocks, the impact energies, and the runout. In this paper, we present a set of four real-scale rockfall tests aimed at studying the fragmentation of the rocky blocks, from the global design of the field procedure to the data analysis and the main results. A total of 124 limestone, dacite, or granite blocks ranging between 0.2 and 5 m3 were dropped from different heights (8.5 to 23.6 m) onto four slopes with different shapes (single or double bench) and slope angles (42º to 71º). The characteristics of the blocks, in particular the size, surface texture and joint condition, were measured before the drops. The trajectories of the blocks and both the initial and the impact velocities were tracked and recorded by means of three high-speed video cameras. A total of 200 block-to-ground impacts have been studied. On average, 40% of the blocks broke upon impact on the slope or on the ground, making it necessary to measure the fragments. The initial and final sizes of the blocks/fragments were measured by hand with tape, though photogrammetric techniques (UAV and terrestrial) were also used for comparison purposes. The information gathered during the field tests provides a deep insight into the fragmentation processes. On the one hand, the high-resolution slow-motion videos help to describe when and how the block breakage takes place and the spatial distribution of the pieces. On the other hand, it is possible to compute the block trajectories, the velocities, and the energy losses using videogrammetry. The results include, for instance, a block average fragmentation of 54% and 14% for the limestone and granitoids, respectively; the systematic inventory of the size fragments, which may be used for fitting the power law distributions; and after each breakage, the total angle of aperture occupied by the fragments has been measured, with values in the range

Contains fulltext : 241168.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 233581.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 237911.pdf (Publisher’s version ) (Closed access), PURPOSE: Expansions of a subset of short tandem repeats (STRs) have been implicated in approximately 30 different human genetic disorders. Despite extensive application of exome sequencing (ES) in routine diagnostic genetic testing, STRs are not routinely identified from these data. METHODS: We assessed diagnostic utility of STR analysis in exome sequencing by applying ExpansionHunter to 2,867 exomes from movement disorder patients and 35,228 other clinical exomes. RESULTS: We identified 38 movement disorder patients with a possible aberrant STR length. Validation by polymerase chain reaction (PCR) and/or repeat-primed PCR technologies confirmed the presence of aberrant expansion alleles for 13 (34%). For seven of these patients the genotype was compatible with the phenotypic description, resulting in a molecular diagnosis. We subsequently tested the remainder of our diagnostic ES cohort, including over 30 clinically and genetically heterogeneous disorders. Optimized manual curation yielded 167 samples with a likely aberrant STR length. Validations confirmed 93/167 (56%) aberrant expansion alleles, of which 48 were in the pathogenic range and 45 in the premutation range. CONCLUSION: Our work provides guidance for the implementation of STR analysis in clinical ES. Our results show that systematic STR evaluation may increase diagnostic ES yield by 0.2%, and recommend making STR evaluation a routine part of ES interpretation in genetic testing laboratories.

Contains fulltext : 227412.pdf (publisher's version ) (Open Access)

Contains fulltext : 225445.pdf (Publisher’s version ) (Open Access), Genome-wide association studies (GWAS) have identified 52 independent variants at 34 genetic loci that are associated with age-related macular degeneration (AMD), the most common cause of incurable vision loss in the elderly worldwide. However, causal genes at the majority of these loci remain unknown. In this study, we performed whole exome sequencing of 264 individuals from 63 multiplex families with AMD and analyzed the data for rare protein-altering variants in candidate target genes at AMD-associated loci. Rare coding variants were identified in the CFH, PUS7, RXFP2, PHF12 and TACC2 genes in three or more families. In addition, we detected rare coding variants in the C9, SPEF2 and BCAR1 genes, which were previously suggested as likely causative genes at respective AMD susceptibility loci. Identification of rare variants in the CFH and C9 genes in our study validated previous reports of rare variants in complement pathway genes in AMD. We then extended our exome-wide analysis and identified rare protein-altering variants in 13 genes outside the AMD-GWAS loci in three or more families. Two of these genes, SCN10A and KIR2DL4, are of interest because variants in these genes also showed association with AMD in case-control cohorts, albeit not at the level of genome-wide significance. Our study presents the first large-scale, exome-wide analysis of rare variants in AMD. Further independent replications and molecular investigation of candidate target genes, reported here, would assist in gaining novel insights into mechanisms underlying AMD pathogenesis.

Contains fulltext : 220478.pdf (publisher's version ) (Open Access), BACKGROUND: Joubert syndrome (JBTS) is a genetically heterogeneous group of neurodevelopmental syndromes caused by primary cilia dysfunction. Usually the neurological presentation starts with abnormal neonatal breathing followed by muscular hypotonia, psychomotor delay, and cerebellar ataxia. Cerebral MRI shows mid- and hindbrain anomalies including the molar tooth sign. We report a male patient with atypical presentation of Joubert syndrome type 23, thus expanding the phenotype. CASE PRESENTATION: Clinical features were consistent with JBTS already from infancy, yet the syndrome was not suspected before cerebral MRI later in childhood showed the characteristic molar tooth sign and ectopic neurohypophysis. From age 11 years seizures developed and after few years became increasingly difficult to treat, also related to inadequate compliance to therapy. He died at 23 years of sudden unexpected death in epilepsy (SUDEP). The genetic diagnosis remained elusive for many years, despite extensive genetic testing. We reached the genetic diagnosis by performing whole genome sequencing of the family trio and analyzing the data with the combination of one analysis pipeline for single nucleotide variants (SNVs)/indels and one for structural variants (SVs). This lead to the identification of the most common variant detected in patients with JBTS23 (OMIM# 616490), rs534542684, in compound heterozygosity with a 8.3 kb deletion in KIAA0586, not previously reported. CONCLUSIONS: We describe for the first time ectopic neurohypophysis and SUDEP in JBTS23, expanding the phenotype of this condition and raising the attention on the possible severity of the epilepsy in this disease. We also highlight the diagnostic power of WGS, which efficiently detects SNVs/indels and in addition allows the identification of SVs.

Contains fulltext : 223478.pdf (publisher's version ) (Open Access)

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