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01 januari 2023, Item does not contain fulltext, Genome sequencing (GS) can identify novel diagnoses for patients who remain undiagnosed after routine diagnostic procedures. We tested whether GS is a better first-tier genetic diagnostic test than current standard of care (SOC) by assessing the technical and clinical validity of GS for patients with neurodevelopmental disorders (NDD). We performed both GS and exome sequencing in 150 consecutive NDD patient-parent trios. The primary outcome was diagnostic yield, calculated from disease-causing variants affecting exonic sequence of known NDD genes. GS (30%, n = 45) and SOC (28.7%, n = 43) had similar diagnostic yield. All 43 conclusive diagnoses obtained with SOC testing were also identified by GS. SOC, however, required integration of multiple test results to obtain these diagnoses. GS yielded two more conclusive diagnoses, and four more possible diagnoses than ES-based SOC (35 vs. 31). Interestingly, these six variants detected only by GS were copy number variants (CNVs). Our data demonstrate the technical and clinical validity of GS to serve as routine first-tier genetic test for patients with NDD. Although the additional diagnostic yield from GS is limited, GS comprehensively identified all variants in a single experiment, suggesting that GS constitutes a more efficient genetic diagnostic workflow.

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Contains fulltext : 292751.pdf (Publisher’s version ) (Open Access), BACKGROUND: Long-read sequencing (LRS) techniques have been very successful in identifying structural variants (SVs). However, the high error rate of LRS made the detection of small variants (substitutions and short indels < 20 bp) more challenging. The introduction of PacBio HiFi sequencing makes LRS also suited for detecting small variation. Here we evaluate the ability of HiFi reads to detect de novo mutations (DNMs) of all types, which are technically challenging variant types and a major cause of sporadic, severe, early-onset disease. METHODS: We sequenced the genomes of eight parent-child trios using high coverage PacBio HiFi LRS (~ 30-fold coverage) and Illumina short-read sequencing (SRS) (~ 50-fold coverage). De novo substitutions, small indels, short tandem repeats (STRs) and SVs were called in both datasets and compared to each other to assess the accuracy of HiFi LRS. In addition, we determined the parent-of-origin of the small DNMs using phasing. RESULTS: We identified a total of 672 and 859 de novo substitutions/indels, 28 and 126 de novo STRs, and 24 and 1 de novo SVs in LRS and SRS respectively. For the small variants, there was a 92 and 85% concordance between the platforms. For the STRs and SVs, the concordance was 3.6 and 0.8%, and 4 and 100% respectively. We successfully validated 27/54 LRS-unique small variants, of which 11 (41%) were confirmed as true de novo events. For the SRS-unique small variants, we validated 42/133 DNMs and 8 (19%) were confirmed as true de novo event. Validation of 18 LRS-unique de novo STR calls confirmed none of the repeat expansions as true DNM. Confirmation of the 23 LRS-unique SVs was possible for 19 candidate SVs of which 10 (52.6%) were true de novo events. Furthermore, we were able to assign 96% of DNMs to their parental allele with LRS data, as opposed to just 20% with SRS data. CONCLUSIONS: HiFi LRS can now produce the most comprehensive variant dataset obtainable by a single technology in a single laboratory

01 december 2023, Contains fulltext : 293290.pdf (Publisher’s version ) (Open Access), PURPOSE: Myopia (nearsightedness) is a condition in which a refractive error (RE) affects vision. Although common variants explain part of the genetic predisposition (18%), most of the estimated 70% heritability is missing. Here, we investigate the contribution of rare genetic variation because this might explain more of the missing heritability in the more severe forms of myopia. In particular, high myopia can lead to blindness and has a tremendous impact on a patient and at the societal level. The exact molecular mechanisms behind this condition are not yet completely unraveled, but whole genome sequencing (WGS) studies have the potential to identify novel (rare) disease genes, explaining the high heritability. DESIGN: Cross-sectional study performed in the Netherlands. PARTICIPANTS: We investigated 159 European patients with high myopia (RE > -10 diopters). METHODS: We performed WGS using a stepwise filtering approach and burden analysis. The contribution of common variants was calculated as a genetic risk score (GRS). MAIN OUTCOME MEASURES: Rare variant burden, GRS. RESULTS: In 25% (n = 40) of these patients, there was a high (> 75th percentile) contribution of common predisposing variants; that is, these participants had higher GRSs. In 7 of the remaining 119 patients (6%), deleterious variants in genes associated with known (ocular) disorders, such as retinal dystrophy disease (prominin 1 [PROM1]) or ocular development (ATP binding cassette subfamily B member 6 [ABCB6], TGFB induced factor homeobox 1 [TGIF1]), were identified. Furthermore, without using a gene panel, we identified a high burden of rare variants in 8 novel genes associated with myopia. The genes heparan sulfate 6-O-sulfotransferase 1 (HS6ST1) (proportion in study population vs. the Genome Aggregation Database (GnomAD) 0.14 vs. 0.03, P = 4.22E-17), RNA binding motif protein 20 (RBM20) (0.15 vs. 0.06, P = 4.98E-05), and MAP7 domain containing 1 (MAP7D1) (0.19 vs. 0.06, P = 1.16E-10) were involved

01 juni 2023, Contains fulltext : 293778.pdf (Publisher’s version ) (Open Access), Various groups of neurological disorders, including movement disorders and neuromuscular diseases, are clinically and genetically heterogeneous. Diagnostic panel-based exome sequencing is a routine test for these disorders. Despite the success rates of exome sequencing, it results in the detection of causative sequence variants in 'only' 25-30% of cases. Copy number variants (CNVs), i.e. deletion or duplications, explain 10-20% of individuals with multisystemic phenotypes, such as co-existing intellectual disability, but may also have a role in disorders affecting a single system (organ), like neurological disorders with normal intelligence. In this study, CNVs were extracted from clinical exome sequencing reports of 4800 probands primarily with a movement disorder, myopathy or neuropathy. In 88 (~2%) probands, phenotype-matching CNVs were detected, representing ~7% of genetically confirmed cases. CNVs varied from involvement of over 100 genes to single exons and explained X-linked, autosomal dominant, or - recessive disorders, the latter due to either a homozygous CNV or a compound heterozygous CNV with a sequence variant on the other allele. CNVs were detected affecting genes where deletions or duplications are established as a common mechanism, like PRKN (in Parkinson's disease), DMD (in Duchenne muscular dystrophy) and PMP22 (in neuropathies), but also genes in which no intragenic CNVs have been reported to date. Analysis of CNVs as part of panel-based exome sequencing for genetically heterogeneous neurological diseases provides an additional diagnostic yield of ~2% without extra laboratory costs. Therefore it is recommended to perform CNV analysis for movement disorders, muscle disease, neuropathies, or any other single-system disorder.

01 juli 2023, Item does not contain fulltext, INTRODUCTION: Patients with early-stage and locally advanced rectal cancer are often treated with neoadjuvant therapy followed by surgery or watch and wait. This study evaluated the role of circulating tumor DNA (ctDNA) to measure disease after neoadjuvant treatment and surgery to optimize treatment choices. MATERIALS AND METHODS: Patients with rectal cancer treated with both chemotherapy and radiotherapy were included and diagnostic biopsies were analyzed for tumor-specific mutations. Presence of ctDNA was measured in plasma by tracing the tumor-informed mutations using a next-generation sequencing panel. The association between ctDNA detection and clinicopathological characteristics and progression-free survival was measured. RESULTS: Before treatment ctDNA was detected in 69% (35/51) of patients. After neoadjuvant therapy ctDNA was detected in only 15% (5/34) of patients. In none of the patients with a complete clinical response who were selected for a watch and wait strategy (0/10) or patients with ypN0 disease (0/8) ctDNA was detected, whereas it was detected in 31% (5/16) of patients with ypN + disease. After surgery ctDNA was detected in 16% (3/19) of patients, of which all (3/3) developed recurrent disease compared to only 13% (2/16) in patients with undetected ctDNA after surgery. In an exploratory survival analysis, both ctDNA detection after neoadjuvant therapy and after surgery was associated with worse progression-free survival (p = 0.01 and p = 0.007, respectively, Cox-regression). CONCLUSION: These data show that in patients with early-stage and locally advanced rectal cancer tumor-informed ctDNA detection in plasma using ultradeep sequencing may have clinical value to complement response prediction after neoadjuvant therapy and surgery.

01 juni 2023, Contains fulltext : 293878.pdf (Publisher’s version ) (Open Access), BACKGROUND: Haematopoietic clones caused by somatic mutations with ≥2% variant allele frequency (VAF) increase with age and are linked to risk of haematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones (VAF<2%) are also associated with adverse outcomes. Our aims were to determine the prevalence of clonal haematopoiesis driven by clones of variable sizes in individuals with obesity treated by usual care or bariatric surgery (a treatment that improves metabolic status), and to examine the expansion of clones in relation to age and metabolic dysregulation over up to 20 years. METHODS: Clonal haematopoiesis-driver mutations (CHDMs) were identified in blood samples from participants of the Swedish Obese Subjects intervention study. Using an ultrasensitive assay, we analysed single-timepoint samples from 1050 individuals treated by usual care and 841 individuals who had undergone bariatric surgery, and multiple-timepoint samples taken over 20 years from a subset (n = 40) of the individuals treated by usual care. FINDINGS: In this explorative study, prevalence of CHDMs was similar in the single-timepoint usual care and bariatric surgery groups (20.6% and 22.5%, respectively, P = 0.330), with VAF ranging from 0.01% to 31.15%. Clone sizes increased with age in individuals with obesity, but not in those who underwent bariatric surgery. In the multiple-timepoint analysis, VAF increased by on average 7% (range -4% to 24%) per year and rate of clone growth was negatively associated with HDL-cholesterol (R = -0.68, 1.74 E(-04)). INTERPRETATION: Low HDL-C was associated with growth of haematopoietic clones in individuals with obesity treated by usual care. FUNDING: The Swedish Research Council, The Swedish state under an agreement between the Swedish government and the county councils, the ALF (Avtal om Läkarutbildning och Forskning) agreement, The Swedish Heart-Lung Foundation, The Novo Nordisk Foundation, The European Research Coun

01 april 2023, Item does not contain fulltext, PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.

01 augustus 2023, Contains fulltext : 295912.pdf (Publisher’s version ) (Open Access), Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10-7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31-8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62-31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searching for gen

Contains fulltext : 294552.pdf (Publisher’s version ) (Open Access), PURPOSE: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. METHODS: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. RESULTS: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. CONCLUSIONS: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.

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

Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to be causal or underlie predisposition to various diseases. However, the role of CNVs in inherited breast cancer susceptibility has not been thoroughly investigated. To address this, we performed whole-exome sequencing based analysis of rare CNVs in 98 high-risk Northern Finnish breast cancer cases. After filtering, selected candidate alleles were validated and characterized with a combination of orthogonal methods, including PCR-based approaches, optical genome mapping and long-read sequencing. This revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. Notably, all these genes encode proteins involved in pathways previously identified as essential for breast cancer development. Variants were genotyped in geographically matched cases and controls (altogether 278 hereditary and 1983 unselected breast cancer cases, and 1229 controls). The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (2.5%, P = 0.034, OR = 2.86, 95% CI = 1.10–7.45) and HSD17B14 deletion in 8/278 cases (2.9%, P = 0.014, OR = 3.28, 95% CI = 1.31–8.23), the frequency of both variants in the controls being 11/1229 (0.9%). This suggests a role for RAD52 and HSD17B14 in hereditary breast cancer susceptibility. The RAD51C duplication was very rare, identified only in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45, 95% CI = 0.62–31.70). The identification of recurrent CNVs in these genes, and especially the relatively high frequency of RAD52 and HSD17B14 alterations in the Finnish population, highlights the importance of studying CNVs alongside single nucleotide variants when searchin

Item does not contain fulltext, De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10(-5)) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10(-4)) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Contains fulltext : 251115.pdf (Publisher’s version ) (Closed access)

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Contains fulltext : 251982.pdf (Publisher’s version ) (Open Access), STUDY QUESTION: Are there more de novo mutations (DNMs) present in the genomes of children born through medical assisted reproduction (MAR) compared to spontaneously conceived children? SUMMARY ANSWER: In this pilot study, no statistically significant difference was observed in the number of DNMs observed in the genomes of MAR children versus spontaneously conceived children. WHAT IS KNOWN ALREADY: DNMs are known to play a major role in sporadic disorders with reduced fitness such as severe developmental disorders, including intellectual disability and epilepsy. Advanced paternal age is known to place offspring at increased disease risk, amongst others by increasing the number of DNMs in their genome. There are very few studies reporting on the effect of MAR on the number of DNMs in the offspring, especially when male infertility is known to be affecting the potential fathers. With delayed parenthood an ongoing epidemiological trend in the 21st century, there are more children born from fathers of advanced age and more children born through MAR every day. STUDY DESIGN, SIZE, DURATION: This observational pilot study was conducted from January 2015 to March 2019 in the tertiary care centre at Radboud University Medical Center. We included a total of 53 children and their respective parents, forming 49 trios (mother, father and child) and two quartets (mother, father and two siblings). One group of children was born after spontaneous conception (n = 18); a second group of children born after IVF (n = 17) and a third group of children born after ICSI combined with testicular sperm extraction (ICSI-TESE) (n = 18). In this pilot study, we also subdivided each group by paternal age, resulting in a subgroup of children born to younger fathers (<35 years of age at conception) and older fathers (>45 years of age at conception). PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole-genome sequencing (WGS) was performed on all parent-offspring trios to identify DNMs. For 34 of 53 tri

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Item does not contain fulltext, Macular degenerations (MDs) are a subgroup of retinal disorders characterized by central vision loss. Knowledge is still lacking on the extent of genetic and nongenetic factors influencing inherited MD (iMD) and age-related MD (AMD) expression. Single molecule Molecular Inversion Probes (smMIPs) have proven effective in sequencing the ABCA4 gene in patients with Stargardt disease to identify associated coding and noncoding variation, however many MD patients still remain genetically unexplained. We hypothesized that the missing heritability of MDs may be revealed by smMIPs-based sequencing of all MD-associated genes and risk factors. Using 17,394 smMIPs, we sequenced the coding regions of 105 iMD and AMD-associated genes and noncoding or regulatory loci, known pseudo-exons, and the mitochondrial genome in two test cohorts that were previously screened for variants in ABCA4. Following detailed sequencing analysis of 110 probands, a diagnostic yield of 38% was observed. This established an ''MD-smMIPs panel," enabling a genotype-first approach in a high-throughput and cost-effective manner, whilst achieving uniform and high coverage across targets. Further analysis will identify known and novel variants in MD-associated genes to offer an accurate clinical diagnosis to patients. Furthermore, this will reveal new genetic associations for MD and potential genetic overlaps between iMD and AMD.

Contains fulltext : 282840.pdf (Publisher’s version ) (Open Access), Untargeted metabolomics (UM) allows for the simultaneous measurement of hundreds of metabolites in a single analytical run. The sheer amount of data generated in UM hampers its use in patient diagnostics because manual interpretation of all features is not feasible. Here, we describe the application of a pathway-based metabolite set enrichment analysis method to prioritise relevant biological pathways in UM data. We validate our method on a set of 55 patients with a diagnosed inherited metabolic disorder (IMD) and show that it complements feature-based prioritisation of biomarkers by placing the features in a biological context. In addition, we find that by taking enriched pathways shared across different IMDs, we can identify common drugs and compounds that could otherwise obscure genuine disease biomarkers in an enrichment method. Finally, we demonstrate the potential of this method to identify novel candidate biomarkers for known IMDs. Our results show the added value of pathway-based interpretation of UM data in IMD diagnostics context.

Contains fulltext : 282710.pdf (Publisher’s version ) (Open Access), In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

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.

Contains fulltext : 243955.pdf (Publisher’s version ) (Open Access), PURPOSE: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. METHODS: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. RESULTS: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. CONCLUSION: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

Contains fulltext : 244033.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 237830.pdf (Publisher’s version ) (Open Access), Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Contains fulltext : 234188.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 237810.pdf (Publisher’s version ) (Open Access), The genetic etiology of intellectual disability remains elusive in almost half of all affected individuals. Within the Solve-RD consortium, systematic re-analysis of whole exome sequencing (WES) data from unresolved cases with (syndromic) intellectual disability (n = 1,472 probands) was performed. This re-analysis included variant calling of mitochondrial DNA (mtDNA) variants, although mtDNA is not specifically targeted in WES. We identified a functionally relevant mtDNA variant in MT-TL1 (NC_012920.1:m.3291T > C; NC_012920.1:n.62T > C), at a heteroplasmy level of 22% in whole blood, in a 23-year-old male with severe intellectual disability, epilepsy, episodic headaches with emesis, spastic tetraparesis, brain abnormalities, and feeding difficulties. Targeted validation in blood and urine supported pathogenicity, with heteroplasmy levels of 23% and 58% in index, and 4% and 17% in mother, respectively. Interestingly, not all phenotypic features observed in the index have been previously linked to this MT-TL1 variant, suggesting either broadening of the m.3291T > C-associated phenotype, or presence of a co-occurring disorder. Hence, our case highlights the importance of underappreciated mtDNA variants identifiable from WES data, especially for cases with atypical mitochondrial phenotypes and their relatives in the maternal line.

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 : 237907.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 217770.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 229283.pdf (Publisher’s version ) (Closed access), Upon the discovery of numerous genes involved in the pathogenesis of neurodevelopmental disorders, several studies showed that a significant proportion of these genes converge on common pathways and protein networks. Here, we used a reversed approach, by screening the AnkyrinG protein-protein interaction network for genetic variation in a large cohort of 1009 cases with neurodevelopmental disorders. We identified a significant enrichment of de novo potentially disease-causing variants in this network, confirming that this protein network plays an important role in the emergence of several neurodevelopmental disorders.