Mutations in PRPH2, encoding peripherin‐2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have ...been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the total 252 PRPH2 variants, more than half (n = 137) were missense variants. All variants were uploaded into the Leiden Open source Variation and ClinVar databases. Our study underscores the need for experimental assays for variants of unknown significance to improve pathogenicity classification, which would allow us to better understand genotype‐phenotype correlations, and in the long‐term, hopefully also support the development of therapeutic strategies for patients with PRPH2‐associated IRD.
Mutations in Eyes shut homolog (EYS) are one of the most common causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive blinding disorder. The exact function of the EYS protein ...and the pathogenic mechanisms underlying EYS‐associated RP are still poorly understood, which hampers the interpretation of the causality of many EYS variants discovered to date. We collected all reported EYS variants present in 377 arRP index cases published before June 2017, and uploaded them in the Leiden Open Variation Database (www.LOVD.nl/EYS). We also describe 36 additional index cases, carrying 26 novel variants. Of the 297 unique EYS variants identified, almost half (n = 130) are predicted to result in premature truncation of the EYS protein. Classification of all variants using the American College of Medical Genetics and Genomics guidelines revealed that the predicted pathogenicity of these variants cover the complete spectrum ranging from likely benign to pathogenic, although especially missense variants largely fall in the category of uncertain significance. Besides the identification of likely benign alleles previously reported as being probably pathogenic, our comprehensive analysis underscores the need of functional assays to assess the causality of EYS variants, in order to improve molecular diagnostics and counseling of patients with EYS‐associated RP.
EYS‐associated RP displays a tremendous allelic heterogeneity, illustrated by the identification of almost 300 different alleles so far. As illustrated in this figure, missense variants are distributed over the entire protein coding sequence, and the pathogenicity of the majority of missense variants still remains unclear.
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.
Summary
To guide anti‐D prophylaxis, Dutch D‐ pregnant women are offered a quantitative fetal‐RHD‐genotyping assay to determine the RHD status of their fetus. This allowed us to determine the ...frequency of different maternal RHD variants in 37 782 serologically D‐ pregnant women. A variant allele is present in at least 0·96% of Dutch D‐ pregnant women The D‐ serology could be confirmed after further serological testing in only 54% of these women, which emphasizes the potential relevance of genotyping of blood donors. 43 different RHD variant alleles were detected, including 15 novel alleles (11 null‐, 2 partial D‐ and 2 DEL‐alleles). Of those novel null alleles, one allele contained a single missense mutation (RHD*443C>G) and one allele had a single amino acid deletion (RHD*424_426del). The D‐ phenotype was confirmed by transduction of human D‐ erythroblasts, consolidating that, for the first time, a single amino acid change or deletion causes the D‐ phenotype. Transduction also confirmed the phenotypes for the two new variant DEL‐alleles (RHD*721A>C and RHD*884T>C) and the novel partial RHD*492C>A allele. Notably, in three additional cases the DEL phenotype was observed but sequencing of the coding sequence, flanking introns and promoter region revealed an apparently wild‐type RHD allele without mutations.
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.
BACKGROUND
Genotyping platforms for common red blood cell (RBC) antigens have been successfully applied in Caucasian and black populations but not in Chinese populations. In this study, a genotyping ...assay based on multiplex ligation‐dependent probe amplification (MLPA) technology was applied in a Chinese population to validate the MLPA probes. Subsequently, the comprehensive distribution of 17 blood group systems also was obtained.
STUDY DESIGN AND METHODS
DNA samples from 200 Chinese donors were extracted and genotyped using the blood‐MLPA assay. To confirm the MLPA results, a second independent genotyping assay (ID Core+) was conducted in 40 donors, and serological typing of 14 blood‐group antigens was performed in 91 donors. In donors who had abnormal copy numbers of an allele (DI and GYPB) determined by MLPA, additional experiments were performed (polymerase chain reaction, sequencing, and flow cytometry analysis).
RESULTS
The genotyping results obtained using the blood‐MLPA and ID Core+ assays were consistent. Serological data were consistent with the genotyping results except for one donor who had a Lu(a−b−) phenotype. Of the 17 blood group systems, the distribution of the MNS, Duffy, Kidd, Diego, Yt, and Dombrock systems was polymorphic. The Mur and Sta antigens of the MNS system were distributed with a frequency of 9% (18 of 200) and 2% (4 of 200), respectively. One donor with chimerism and one who carried a novel DI*02(A845V) allele, which predicts the depression of Dib antigen expression, were identified.
CONCLUSIONS
The blood‐MLPA assay could easily identify the common blood‐group alleles and correctly predicted phenotype in the Chinese population. The Mur and Sta antigens were distributed with high frequency in a Southern Chinese Han population.
Abstract
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.
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
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.
To identify the genetic cause of and describe the phenotype in 4 families with autosomal recessive retinitis pigmentosa (arRP) that can be associated with pseudocoloboma.
Case series.
Seven patients ...from 4 unrelated families with arRP, among whom 3 patients had bilateral early-onset macular pseudocoloboma.
We performed homozygosity mapping and whole-exome sequencing in 5 probands and 2 unaffected family members from 4 unrelated families. Subsequently, Sanger sequencing and segregation analysis were performed in additional family members. We reviewed the medical history of individuals carrying IDH3A variants and performed additional ophthalmic examinations, including full-field electroretinography, fundus photography, fundus autofluorescence imaging, and optical coherence tomography.
IDH3A variants, age at diagnosis, visual acuity, fundus appearance, visual field, and full-field electroretinography, fundus autofluorescence, and optical coherence tomography findings.
We identified 7 different variants in IDH3A in 4 unrelated families, that is, 5 missense, 1 nonsense, and 1 frameshift variant. All participants showed symptoms early in life, ranging from night blindness to decreased visual acuity, and were diagnosed between the ages of 1 and 11 years. Four participants with biallelic IDH3A variants displayed a typical arRP phenotype and 3 participants were diagnosed with arRP and pseudocoloboma of the macula.
IDH3A variants were identified as a novel cause of typical arRP in some individuals associated with macular pseudocoloboma. We observed both phenotypes in 2 siblings carrying the same compound heterozygous variants, which could be explained by variable disease expression and warrants caution when making assertions about genotype-phenotype correlations.
Background
The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay ...to identify D variants is still lacking. We developed and evaluated a multiplex ligation–dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity.
Study design and methods
We analyzed 236 cases (73 normal and 163 selected samples) with the RH‐MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon–specific sequencing and RHD zygosity was verified by quantitative real‐time polymerase chain reaction (PCR) for 18 cases.
Results
In 99% of the cases, the RH‐MLPA assay correctly determined whether a person carried only wild‐type RHD and RHCE alleles (n = 69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n = 164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHD*DCS2, a new partial RHD allele, RHD*525T (Phe175Leu), and a new D– null allele, RHD*443G (Thr148Arg). All RHD (n = 175) and RHCE variant alleles (n = 79) indicated by the RH‐MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized.
Conclusion
The RH‐MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD– chimeras in blood donors, blood recipients, and pregnant women.
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