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Inherited retinal diseases (IRDs) are a major cause of visual impairment. These clinically heterogeneous disorders are caused by pathogenic variants in more than 270 genes. As 30–40% of cases remain genetically unexplained following conventional genetic testing, we aimed to obtain a genetic diagnosis in an IRD cohort in which the genetic cause was not found using whole-exome sequencing or targeted capture sequencing. We performed whole-genome sequencing (WGS) to identify causative variants in 100 unresolved cases. After initial prioritization, we performed an in-depth interrogation of all noncoding and structural variants in genes when one candidate variant was detected. In addition, functional analysis of putative splice-altering variants was performed using in vitro splice assays. We identified the genetic cause of the disease in 24 patients. Causative coding variants were observed in genes such as ATXN7, CEP78, EYS, FAM161A, and HGSNAT. Gene disrupting structural variants were also detected in ATXN7, PRPF31, and RPGRIP1. In 14 monoallelic cases, we prioritized candidate noncanonical splice sites or deep-intronic variants that were predicted to disrupt the splicing process based on in silico analyses. Of these, seven cases were resolved as they carried pathogenic splice defects. WGS is a powerful tool to identify causative variants residing outside coding regions or heterozygous structural variants. This approach was most efficient in cases with a distinct clinical diagnosis. In addition, in vitro splice assays provide important evidence of the pathogenicity of rare variants.

The short lengths of short-read sequencing reads challenge the analysis of paralogous genomic regions in exome and genome sequencing data. Most genetic variants within these homologous regions therefore remain unidentified in standard analyses. Here, we present a method (Chameleolyser) that accurately identifies single nucleotide variants and small insertions/deletions (SNVs/Indels), copy number variants and ectopic gene conversion events in duplicated genomic regions using whole-exome sequencing data. Application to a cohort of 41,755 exome samples yields 20,432 rare homozygous deletions and 2,529,791 rare SNVs/Indels, of which we show that 338,084 are due to gene conversion events. None of the SNVs/Indels are detectable using regular analysis techniques. Validation by high-fidelity long-read sequencing in 20 samples confirms >88% of called variants. Focusing on variation in known disease genes leads to a direct molecular diagnosis in 25 previously undiagnosed patients. Our method can readily be applied to existing exome data. Chameleolyser enables the accurate identification of genetic variants hidden within complex regions of the genome. Its application uncovers the disease-explanatory variant in 25 previously undiagnosed patients.

Unsolicited findings (UFs) are uncovered unintentionally and predispose to a disease unrelated to the clinical question. The frequency and nature of UFs uncovered in clinical practice remain largely unexplored. We here evaluated UFs identified during a 5-year period in which 16,482 index patients received clinical whole-exome sequencing (WES). UFs were identified in 0.58% (95/16,482) of index patients, indicating that the overall frequency of UFs in clinical WES is low. Fewer UFs were identified using restricted disease-gene panels (0.03%) than when using whole-exome/Mendeliome analysis (1.03%). The UF was disclosed to 86 of 95 individuals, for reasons of medical actionability. Only 61% of these UFs reside in a gene that is listed on the “ACMG59”-list, representing a list of 59 genes for which the American College of Medical Genetics recommends UF disclosure. The remaining 39% were grouped into four categories: disorders similar to “ACMG59”-listed disorders (25%); disorders for which disease manifestation could be influenced (7%); UFs providing reproductive options (2%); and UFs with pharmacogenetic implications (5%). Hence, our experience shows that UFs predisposing to medically actionable disorders affect a broader range of genes than listed on the “ACMG59”, advocating that a pre-defined gene list is too restrictive, and that UFs may require ad hoc evaluation of medical actionability. While both the identification and disclosure of UFs depend on local policy, our lessons learned provide general essential insight into the nature and odds of UFs in clinical exome sequencing.

Background Rare Eye Diseases (RED) are the leading cause of visual impairment and blindness for children and young adults in Europe. This heterogeneous group of conditions includes over 900 disorders ranging from relatively prevalent disorders such as retinitis pigmentosa to very rare entities such as developmental eye anomalies. A significant number of patients with RED have an underlying genetic etiology. One of the aims of the European Reference Network for Rare Eye Diseases (ERN–EYE) is to facilitate improvement in diagnosis of RED in European member states. Main body Technological advances have allowed genetic and genomic testing for RED. The outcome of genetic testing allows better understanding of the condition and allows reproductive and therapeutic options. The increase of the number of clinical trials for RED has provided urgency for genetic testing in RED. A survey of countries participating in ERN-EYE demonstrated that the majority are able to access some forms of genomic testing. However, there is significant variability, particularly regarding testing as part of clinical service. Some countries have a well-delineated rare disease pathway and have a national plan for rare diseases combined or not with a national plan for genomics in medicine. In other countries, there is a well-established organization of genetic centres that offer reimbursed genomic testing of RED and other rare diseases. Clinicians often rely upon research-funded laboratories or private companies. Notably, some member states rely on cross-border testing by way of an academic research project. Consequently, many clinicians are either unable to access testing or are confronted with long turnaround times. Overall, while the cost of sequencing has dropped, the cumulative cost of a genomic testing service for populations remains considerable. Importantly, the majority of countries reported healthcare budgets that limit testing. Short conclusion Despite technological advances, critical gaps in genomic testing remain in Europe, especially in smaller countries where no formal genomic testing pathways exist. Even within larger countries, the existing arrangements are insufficient to meet the demand and to ensure access. ERN-EYE promotes access to genetic testing in RED and emphasizes the clinical need and relevance of genetic testing in RED.

Myotonic dystrophy type 1 is a multisystem disorder caused by the expansion of a trinucleotide repeat in the DMPK gene. In this study we evaluated the performance of the FastDM1 TM DMPK sizing kit in myotonic dystrophy type 1 testing. This commercially available triplet repeat-primed PCR based kit was validated using reference and clinical samples. Based on testing with 19 reference samples, the assay yielded repeat sizes within three repeats from the consensus reference length, demonstrating an accuracy of 100%. Additionally, the assay generated consistent repeat size information with a concentration range of template-DNA, and upon repetition and reproduction (CV 0.36% to 0.41%). Clinical performance was established with 235 archived prenatal and postnatal clinical samples, yielding results of 100% sensitivity (95% CI, 97.29% to 100%) and 100% specificity (95% CI, 96.19% to 100%) in classifying the samples into the respective genotype groups of 5–35 (normal), 36–50 (non-pathogenic pre-expansion), 51–150 (unstable intermediate-sized pathogenic) or >150 (unstable pathogenic) CTG repeats, respectively. Furthermore, the assay identified interrupted repeat expansions in all samples known to have interruptions, and also identified interruptions in a subset of the clinical samples.

Senior-Løken syndrome is a rare ciliopathy characterized by retinal dystrophy and nephronophthisis. This autosomal recessive inherited disease is caused by pathogenic variants in several genes, including IQCB1 . We present a Senior-Løken case that remained genetically unexplained after routine genetic testing, including exome and genome sequencing. To identify the genetic cause for this individual, a combination of innovative long-read technologies was employed. Using optical genome mapping, an intronic 6.2-kb insertion in IQCB1 was revealed. Validation by long-read genome sequencing determined that this insertion consisted of a LINE-1/ERV1-mobile element. The variant was found in trans with a pathogenic IQCB1 2-bp deletion previously identified by exome sequencing. To investigate the consequences of the insertion, targeted long-read RNA-sequencing was performed, revealing a complex splice defect causing the introduction of a premature stop codon. This finding suggests that mobile element insertions represent a yet underestimated variant type that is difficult to detect using short-read sequencing.

Pathogenic variants in the OPN1LW/OPN1MW gene cluster are causal for a range of mild to severe visual impairments with color deficiencies. The widely utilized short-read next-generation sequencing (NGS) is inappropriate for the analysis of the OPN1LW/OPN1MW gene cluster and many patients with pathogenic variants stay underdiagnosed. A diagnostic genetic assay was developed for the OPN1LW/OPN1MW gene cluster, consisting of copy number analysis via multiplex ligation-dependent probe amplification and sequence analysis via long-read circular consensus sequencing. Performance was determined on 50 clinical samples referred for genetic confirmation of the clinical diagnosis (n = 43) or carrier status analysis (n = 7). A broad range of pathogenic haplotypes were detected, including deletions, hybrid genes, single variants and combinations of variants. The developed genetic assay for the OPN1LW/OPN1MW gene cluster is a diagnostic test that can detect both structural and nucleotide variants with a straightforward analysis, improving diagnostic care of patients with visual impairment.

Homozygosity mapping has facilitated the identification of the genetic causes underlying inherited diseases, particularly in consanguineous families with multiple affected individuals. This knowledge has also resulted in a mutation dataset that can be used in a cost and time effective manner to screen frequent population-specific genetic variations associated with diseases such as inherited retinal disease (IRD). We genetically screened 13 families from a cohort of 81 Pakistani IRD families diagnosed with Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), congenital stationary night blindness (CSNB), or cone dystrophy (CD). We employed genome-wide single nucleotide polymorphism (SNP) array analysis to identify homozygous regions shared by affected individuals and performed Sanger sequencing of IRD-associated genes located in the sizeable homozygous regions. In addition, based on population specific mutation data we performed targeted Sanger sequencing (TSS) of frequent variants in AIPL1, CEP290, CRB1, GUCY2D, LCA5, RPGRIP1 and TULP1, in probands from 28 LCA families. Homozygosity mapping and Sanger sequencing of IRD-associated genes revealed the underlying mutations in 10 families. TSS revealed causative variants in three families. In these 13 families four novel mutations were identified in CNGA1, CNGB1, GUCY2D, and RPGRIP1. Homozygosity mapping and TSS revealed the underlying genetic cause in 13 IRD families, which is useful for genetic counseling as well as therapeutic interventions that are likely to become available in the near future.

Biallelic variants in ABCA4 cause Stargardt disease (STGD1), the most frequent heritable macular disease. Determination of the pathogenicity of variants in ABCA4 proves to be difficult due to (1) the high number of benign and pathogenic variants in the gene; (2) the presence of many rare ABCA4 variants; (3) the presence of complex alleles for which phasing data are absent; (4) the extensive variable expressivity of this disease and (5) reduced penetrance of hypomorphic variants. Therefore, the classification of many variants in ABCA4 is currently of uncertain significance. Here, we complemented the ABCA4 Leiden Open Variation Database (LOVD) with data from ~11,000 probands with ABCA4-associated inherited retinal diseases from literature up to the end of 2020. We carefully adapted the ACMG/AMP classifications to ABCA4 incorporating ClinGen recommendations and assigned these classifications to all 2,246 unique variants from the ABCA4 LOVD to increase the knowledge of pathogenicity. In total, 1,248 variants were categorized with a likely pathogenic or pathogenic classification, whereas 194 variants were categorized with a likely benign or benign classification. This uniform and improved structured reclassification, incorporating the largest dataset of ABCA4-associated retinopathy cases so far, will improve both the diagnosis as well as genetic counselling for individuals with ABCA4-associated retinopathy.