Rapid exome sequencing as a first-tier test in neonates with suspected genetic disorder: results of a prospective multicenter clinical utility study in the Netherlands.

The introduction of rapid exome sequencing (rES) for critically ill neonates admitted to the neonatal intensive care unit has made it possible to impact clinical decision-making. Unbiased prospective studies to quantify the impact of rES over routine genetic testing are, however, scarce. We performed a clinical utility study to compare rES to conventional genetic diagnostic workup for critically ill neonates with suspected genetic disorders. In a multicenter prospective parallel cohort study involving five Dutch NICUs, we performed rES in parallel to routine genetic testing for 60 neonates with a suspected genetic disorder and monitored diagnostic yield and the time to diagnosis. To assess the economic impact of rES, healthcare resource use was collected for all neonates. rES detected more conclusive genetic diagnoses than routine genetic testing (20% vs. 10%, respectively), in a significantly shorter time to diagnosis (15 days (95% CI 10-20) vs. 59 days (95% CI 23-98, p < 0.001)). Moreover, rES reduced genetic diagnostic costs by 1.5% (€85 per neonate). CONCLUSION:  Our findings demonstrate the clinical utility of rES for critically ill neonates based on increased diagnostic yield, shorter time to diagnosis, and net healthcare savings. Our observations warrant the widespread implementation of rES as first-tier genetic test in critically ill neonates with disorders of suspected genetic origin. WHAT IS KNOWN: • Rapid exome sequencing (rES) enables diagnosing rare genetic disorders in a fast and reliable manner, but retrospective studies with neonates admitted to the neonatal intensive care unit (NICU) indicated that genetic disorders are likely underdiagnosed as rES is not routinely used. • Scenario modeling for implementation of rES for neonates with presumed genetic disorders indicated an expected increase in costs associated with genetic testing. WHAT IS NEW: • This unique prospective national clinical utility study of rES in a NICU setting shows that rES obtained more and faster diagnoses than conventional genetic tests. • Implementation of rES as replacement for all other genetic tests does not increase healthcare costs but in fact leads to a reduction in healthcare costs.

Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.

Detection of clinically relevant copy-number variants by exome sequencing in a large cohort of genetic disorders.

PURPOSE: Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic test for patients with neurodevelopmental disorders, with a diagnostic yield of 10-20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test. METHODS: We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting. RESULTS: In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to -5.8% per disorder). CONCLUSIONS: This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016.

Mutations in BICD2, which encodes a golgin and important motor adaptor, cause congenital autosomal-dominant spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.

Identification of Patients With Variants in TPMT and Dose Reduction Reduces Hematologic Events During Thiopurine Treatment of Inflammatory Bowel Disease.

BACKGROUND & AIMS: More than 20% of patients with inflammatory bowel disease (IBD) discontinue thiopurine therapy because of severe adverse drug reactions (ADRs); leukopenia is one of the most serious ADRs. Variants in the gene encoding thiopurine S-methyltransferase (TPMT) alter its enzymatic activity, resulting in higher levels of thiopurine metabolites, which can cause leukopenia. We performed a prospective study to determine whether genotype analysis of TPMT before thiopurine treatment, and dose selection based on the results, affects the outcomes of patients with IBD. METHODS: In a study performed at 30 Dutch hospitals, patients were assigned randomly to groups that received standard treatment (control) or pretreatment screening (intervention) for 3 common variants of TPMT (TPMT*2, TPMT*3A, and TPMT*3C). Patients in the intervention group found to be heterozygous carriers of a variant received 50% of the standard dose of thiopurine (azathioprine or 6-mercaptopurine), and patients homozygous for a variant received 0%-10% of the standard dose. We compared, in an intention-to-treat analysis, outcomes of the intervention (n = 405) and control groups (n = 378) after 20 weeks of treatment. Primary outcomes were the occurrence of hematologic ADRs (leukocyte count < 3.0*10(9)/L or reduced platelet count < 100*10(9)/L) and disease activity (based on the Harvey-Bradshaw Index for Crohn's disease [n = 356] or the partial Mayo score for ulcerative colitis [n = 253]). RESULTS: Similar proportions of patients in the intervention and control groups developed a hematologic ADR (7.4% vs 7.9%; relative risk, 0.93; 95% confidence interval, 0.57-1.52) in the 20 weeks of follow-up evaluation; the groups also had similar mean levels of disease activity (P = .18 for Crohn's disease and P = .14 for ulcerative colitis). However, a significantly smaller proportion of carriers of the TPMT variants in the intervention group (2.6%) developed hematologic ADRs compared with patients in the control group (22.9%) (relative risk, 0.11; 95% confidence interval, 0.01-0.85). CONCLUSIONS: Screening for variants in TPMT did not reduce the proportions of patients with hematologic ADRs during thiopurine treatment for IBD. However, there was a 10-fold reduction in hematologic ADRs among variant carriers who were identified and received a dose reduction, compared with variant carriers who did not, without differences in treatment efficacy. ClinicalTrials.gov number: NCT00521950.

Diagnostic exome sequencing in persons with severe intellectual disability.

BACKGROUND: The causes of intellectual disability remain largely unknown because of extensive clinical and genetic heterogeneity. METHODS: We evaluated patients with intellectual disability to exclude known causes of the disorder. We then sequenced the coding regions of more than 21,000 genes obtained from 100 patients with an IQ below 50 and their unaffected parents. A data-analysis procedure was developed to identify and classify de novo, autosomal recessive, and X-linked mutations. In addition, we used high-throughput resequencing to confirm new candidate genes in 765 persons with intellectual disability (a confirmation series). All mutations were evaluated by molecular geneticists and clinicians in the context of the patients' clinical presentation. RESULTS: We identified 79 de novo mutations in 53 of 100 patients. A total of 10 de novo mutations and 3 X-linked (maternally inherited) mutations that had been previously predicted to compromise the function of known intellectual-disability genes were found in 13 patients. Potentially causative de novo mutations in novel candidate genes were detected in 22 patients. Additional de novo mutations in 3 of these candidate genes were identified in patients with similar phenotypes in the confirmation series, providing support for mutations in these genes as the cause of intellectual disability. We detected no causative autosomal recessive inherited mutations in the discovery series. Thus, the total diagnostic yield was 16%, mostly involving de novo mutations. CONCLUSIONS: De novo mutations represent an important cause of intellectual disability; exome sequencing was used as an effective diagnostic strategy for their detection. (Funded by the European Union and others.).

Translating sanger-based routine DNA diagnostics into generic massive parallel ion semiconductor sequencing.

BACKGROUND: Dideoxy-based chain termination sequencing developed by Sanger is the gold standard sequencing approach and allows clinical diagnostics of disorders with relatively low genetic heterogeneity. Recently, new next generation sequencing (NGS) technologies have found their way into diagnostic laboratories, enabling the sequencing of large targeted gene panels or exomes. The development of benchtop NGS instruments now allows the analysis of single genes or small gene panels, making these platforms increasingly competitive with Sanger sequencing. METHODS: We developed a generic automated ion semiconductor sequencing work flow that can be used in a clinical setting and can serve as a substitute for Sanger sequencing. Standard amplicon-based enrichment remained identical to PCR for Sanger sequencing. A novel postenrichment pooling strategy was developed, limiting the number of library preparations and reducing sequencing costs up to 70% compared to Sanger sequencing. RESULTS: A total of 1224 known pathogenic variants were analyzed, yielding an analytical sensitivity of 99.92% and specificity of 99.99%. In a second experiment, a total of 100 patient-derived DNA samples were analyzed using a blind analysis. The results showed an analytical sensitivity of 99.60% and specificity of 99.98%, comparable to Sanger sequencing. CONCLUSIONS: Ion semiconductor sequencing can be a first choice mutation scanning technique, independent of the genes analyzed.

A novel SLC2A1 mutation linking hemiplegic migraine with alternating hemiplegia of childhood.

BACKGROUND: Hemiplegic migraine (HM) and alternating hemiplegia of childhood (AHC) are rare episodic neurological brain disorders with partial clinical and genetic overlap. Recently, ATP1A3 mutations were shown to account for the majority of AHC patients. In addition, a mutation in the SLC2A1 gene was reported in a patient with atypical AHC. We therefore investigated whether mutations in these genes may also be involved in HM. Furthermore, we studied the role of SLC2A1 mutations in a small set of AHC patients without ATP1A3 mutations. METHODS: We screened 42 HM patients (21 familial and 21 sporadic patients) for ATP1A3 and SLC2A1 mutations. In addition, four typical AHC patients and one atypical patient with overlapping symptoms of both disorders were screened for SLC2A1 mutations. RESULTS: A pathogenic de novo SLC2A1 mutation (p.Gly18Arg) was found in the atypical patient with overlapping symptoms of AHC and hemiplegic migraine. No mutations were found in the HM and the other AHC patients. CONCLUSION: Screening for a mutation in the SLC2A1 gene should be considered in patients with a complex phenotype with overlapping symptoms of hemiplegic migraine and AHC.

A post-hoc comparison of the utility of sanger sequencing and exome sequencing for the diagnosis of heterogeneous diseases.

The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene-specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger-based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite-stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost.

Next-generation sequencing of a 40 Mb linkage interval reveals TSPAN12 mutations in patients with familial exudative vitreoretinopathy.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of approximately 40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia.

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.

Genome-wide association analysis of anti-TNF drug response in patients with rheumatoid arthritis.

BACKGROUND: Treatment strategies blocking tumour necrosis factor (anti-TNF) have proven very successful in patients with rheumatoid arthritis (RA). However, a significant subset of patients does not respond for unknown reasons. Currently, there are no means of identifying these patients before treatment. This study was aimed at identifying genetic factors predicting anti-TNF treatment outcome in patients with RA using a genome-wide association approach. METHODS: We conducted a multistage, genome-wide association study with a primary analysis of 2 557 253 single-nucleotide polymorphisms (SNPs) in 882 patients with RA receiving anti-TNF therapy included through the Dutch Rheumatoid Arthritis Monitoring (DREAM) registry and the database of Apotheekzorg. Linear regression analysis of changes in the Disease Activity Score in 28 joints after 14 weeks of treatment was performed using an additive model. Markers with p<10(-3) were selected for replication in 1821 patients from three independent cohorts. Pathway analysis including all SNPs with p<10(-3) was performed using Ingenuity. RESULTS: 772 markers showed evidence of association with treatment outcome in the initial stage. Eight genetic loci showed improved p value in the overall meta-analysis compared with the first stage, three of which (rs1568885, rs1813443 and rs4411591) showed directional consistency over all four cohorts studied. We were unable to replicate markers previously reported to be associated with anti-TNF outcome. Network analysis indicated strong involvement of biological processes underlying inflammatory response and cell morphology. CONCLUSIONS: Using a multistage strategy, we have identified eight genetic loci associated with response to anti-TNF treatment. Further studies are required to validate these findings in additional patient collections.

Genetic variation in ataxia gene ATXN7 influences cerebellar grey matter volume in healthy adults.

An increasing number of candidate genes for common and rare brain disorders are discovered, but the mechanism of action through which these genes cause disease is often still unclear. Some of the genetic factors known to increase the risk for common brain disorders affect brain structure, even in healthy individuals, and therefore possibly have a role in the normal development of specific brain regions. In this study, we explored this principle for a group of rare brain disorders, the spinocerebellar ataxias (SCAs). As a proof of concept, we investigated whether genetic variation in a gene known to cause a polyglutamine-expansion SCA is associated with cerebellar volume in healthy adults. The functional single nucleotide polymorphism (SNP) rs3774729 located in ATXN7 was selected as the variant of interest. Cerebellar grey matter volume was determined using volumetry on magnetic resonance imaging data in a discovery sample scanned at 1.5 T (n = 680) and a replication sample scanned at 3 T (n = 683), both consisting of healthy adults aged 18 to 35 years. The volumes were compared as a function of the presence of the minor allele of SNP rs3774729, which was associated with significantly smaller cerebellar grey matter volume in both the discovery and replication sample (p = 0.033 and p = 0.024, respectively). Our results demonstrate that a common genetic variant in the ataxia-causing gene ATXN7 influences cerebellar grey matter volume in healthy young adults. This finding may also imply that genes associated with cerebellar volume in healthy subjects are valid candidates for causing or modifying ataxia.

Genotype-phenotype correlations in spastic paraplegia type 7: a study in a large Dutch cohort.

Spastic paraplegia type 7 is an autosomal recessive neurodegenerative disorder mainly characterized by progressive bilateral lower limb spasticity and referred to as a form of hereditary spastic paraplegia. Additional disease features may also be observed as part of a more complex phenotype. Many different mutations have already been identified, but no genotype-phenotype correlations have been found so far. From a total of almost 800 patients referred for testing, we identified 60 patients with mutations in the SPG7 gene. We identified 14 previously unreported mutations and detected a high recurrence rate of several earlier reported mutations. We were able to collect detailed clinical data for 49 patients, who were ranked based on a pure versus complex phenotype, ataxia versus no ataxia and missense versus null mutations. A generally complex phenotype occurred in 69% of all patients and was associated with a younger age at onset (trend with P = 0.07). Ataxia was observed in 57% of all patients. We found that null mutations were associated with the co-occurrence of cerebellar ataxia (trend with P = 0.06). The c.1409 G > A (p.Arg470Gln) mutation, which was found homozygously in two sibs, was associated with a specific complex phenotype that included predominant visual loss due to optical nerve atrophy. Neuropathology in one of these cases showed severe degeneration of the optic system, with less severe degeneration of the ascending tracts of the spinal cord and cerebellum. Other disease features encountered in this cohort included cervical dystonia, vertical gaze palsy, ptosis and severe intellectual disability. In this large Dutch cohort, we seem to have identified the first genotype-phenotype correlation in spastic paraplegia type 7 by observing an association between the cerebellar phenotype of spastic paraplegia type 7 and SPG7 null alleles. An overlapping phenotypic presentation with its biological counterpart AFG3L2, which when mutated causes spinocerebellar ataxia type 28, is apparent and possibly suggests that abnormal levels of the SPG7 protein impact the function of the mitochondrial ATPases associated with diverse cellular activities-protease complex (formed by SPG7 and AFG3L2) in the cerebellum. In addition, a missense mutation in exon 10 resulted in predominant optical nerve atrophy, which might suggest deleterious interactions of this SPG7 variant with its substrate OPA1, the mutated gene product in optic atrophy type 1. Functional studies are required to further investigate these interactions.

Next-generation genetic testing for retinitis pigmentosa.

Molecular diagnostics for patients with retinitis pigmentosa (RP) has been hampered by extreme genetic and clinical heterogeneity, with 52 causative genes known to date. Here, we developed a comprehensive next-generation sequencing (NGS) approach for the clinical molecular diagnostics of RP. All known inherited retinal disease genes (n = 111) were captured and simultaneously analyzed using NGS in 100 RP patients without a molecular diagnosis. A systematic data analysis pipeline was developed and validated to prioritize and predict the pathogenicity of all genetic variants identified in each patient, which enabled us to reduce the number of potential pathogenic variants from approximately 1,200 to zero to nine per patient. Subsequent segregation analysis and in silico predictions of pathogenicity resulted in a molecular diagnosis in 36 RP patients, comprising 27 recessive, six dominant, and three X-linked cases. Intriguingly, De novo mutations were present in at least three out of 28 isolated cases with causative mutations. This study demonstrates the enormous potential and clinical utility of NGS in molecular diagnosis of genetically heterogeneous diseases such as RP. De novo dominant mutations appear to play a significant role in patients with isolated RP, having major implications for genetic counselling.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis.

OBJECTIVE: Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD. METHODS: We reviewed all previous studies on ANG in ALS and performed sequence experiments on additional samples, which allowed us to analyze data from 6,471 ALS patients and 7,668 controls from 15 centers (13 from Europe and 2 from the USA). We sequenced DNA samples from 3,146 PD patients from 6 centers (5 from Europe and 1 from the USA). Statistical analysis was performed using the variable threshold test, and the Mantel-Haenszel procedure was used to estimate odds ratios. RESULTS: Analysis of sequence data from 17,258 individuals demonstrated a significantly higher frequency of ANG variants in both ALS and PD patients compared to control subjects (p = 9.3 × 10(-6) for ALS and p = 4.3 × 10(-5) for PD). The odds ratio for any ANG variant in patients versus controls was 9.2 for ALS and 6.7 for PD. INTERPRETATION: The data from this multicenter study demonstrate that there is a strong association between PD, ALS, and ANG variants. ANG is a genetic link between ALS and PD.

The inversa type of recessive dystrophic epidermolysis bullosa is caused by specific arginine and glycine substitutions in type VII collagen.

BACKGROUND: The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I) is a rare variant of dystrophic epidermolysis bullosa, characterised by blistering in the body flexures, trunk, and mucosa. The cause of this specific distribution is unknown. So far, 20 COL7A1 genotypes have been described in RDEB-I and genotype-phenotype correlations have not been studied extensively. The aim of the study was to gain more insight into the pathophysiology of this intriguing RDEB-I phenotype. METHODS: Twenty Dutch and British RDEB-I patients, and full genotypes in 18 of them, were identified. The literature on RDEB-I genotypes was reviewed and an extensive genotype-phenotype correlation study for RDEB-I was conducted. RESULTS: All 20 patients had generalised blistering at birth and during early infancy. In most patients, the age of transition from generalised to inversa distribution was before the age of 4 years. A spectrum of disease severity, ranging from the mildest 'mucosal only' phenotype to the severest phenotype with limited acral involvement, was noted. The 29 genotypes of these RDEB-I patients and those reported in the literature revealed that RDEB-I is associated with specific recessive arginine and glycine substitutions in the triple helix domain of type VII collagen. DISCUSSION AND CONCLUSION: Why these specific arginine and glycine substitutions cause the inversa distribution remains unknown. It was not possible to identify clear differences in location and nature of substituting amino acids between these mutations and missense mutations causing other RDEB phenotypes. It is hypothesised that the higher skin temperature in the affected areas plays an important role in the pathophysiology of RDEB-I.

Stepwise ABC system for classification of any type of genetic variant.

The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) system for variant classification is score based with five classes: benign, likely benign, variant of unknown significance (VUS), likely pathogenic, and pathogenic. Here, we present a variant classification model that can be an add-on or alternative to ACMG classification: A stepwise system that can classify any type of genetic variant (e.g., hypomorphic alleles, imprinted alleles, copy number variants, runs of homozygosity, enhancer variants, and variants related to traits). We call it the ABC system because classification is first functional (A), then clinical (B), and optionally a standard comment that fits the clinical question is selected (C). Both steps A and B have 1-5 grading when knowledge is sufficient, if not, class "zero" is assigned. Functional grading (A) only concerns biological consequences with the stages normal function (1), likely normal function (2), hypothetical functional effect (3), likely functional effect (4), and proven functional effect (5). Clinical grading (B) is genotype-phenotype focused with the stages "right type of gene" (1), risk factor (2), and pathogenic (3-5, depending on penetrance). Both grades are listed for each variant and combined to generate a joint class ranging from A to F. Importantly, the A-F classes are linked to standard comments, reflecting laboratory or national policy. In step A, the VUS class is split into class 0 (true unknown) and class 3 (hypothetical functional effect based on molecular predictions or de novo occurrence), providing a rationale for variant-of-interest reporting when the clinical picture could fit the finding. The system gives clinicians a better guide to variant significance.

Recommendations for whole genome sequencing in diagnostics for rare diseases.

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.

Milder phenotypes of glucose transporter type 1 deficiency syndrome.

Glucose transporter type 1 deficiency syndrome (GLUT1DS) is a treatable condition resulting from impaired glucose transport into the brain. The classical presentation is with infantile-onset epilepsy and severe developmental delay. Non-classical phenotypes with movement disorders and early-onset absence epilepsy are increasingly recognized and the clinical spectrum is expanding. The hallmark is hypoglycorrhachia (cerebrospinal fluid [CSF] glucose<2.2 mmol/l) in the presence of normoglycaemia with a CSF/blood glucose ratio of less than 0.4. GLUT1DS is due to a mutation in the solute carrier family 2, member 1 gene (SLC2A1). We present five individuals (four males, one female), all of whom had a mild phenotype, highlighting the importance of considering this diagnosis in unexplained neurological disorders associated with mild learning difficulties, subtle motor delay, early-onset absence epilepsy, fluctuating gait disorders, and/or dystonia. The mean age at diagnosis was 8 years 8 months. This paper also shows phenotypical parallels between GLUT1DS and paroxysmal exertion-induced dyskinesia.