Bart de Koning

Exome sequencing reveals a novel Moroccan founder mutation in SLC19A3 as a new cause of early-childhood fatal Leigh syndrome

Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes have been reported to cause Leigh syndrome, indicating an extreme genetic heterogeneity for this disorder, but still only explaining part of the cases. The possibility of whole exome sequencing enables not only mutation detection in known candidate genes, but also the identification of new genes associated with Leigh syndrome in small families and isolated cases. Exome sequencing was combined with homozygosity mapping to identify the genetic defect in a Moroccan family with fatal Leigh syndrome in early childhood and specific magnetic resonance imaging abnormalities in the brain. We detected a homozygous nonsense mutation (c.20C>A; p.Ser7Ter) in the thiamine transporter SLC19A3. In vivo overexpression of wild-type SLC19A3 showed an increased thiamine uptake, whereas overexpression of mutant SLC19A3 did not, confirming that the mutation results in an absent or non-functional protein. Seventeen additional patients with Leigh syndrome were screened for mutations in SLC19A3 using conventional Sanger sequencing. Two unrelated patients, both from Moroccan origin and one from consanguineous parents, were homozygous for the same p.Ser7Ter mutation. One of these patients showed the same MRI abnormalities as the patients from the first family. Strikingly, patients receiving thiamine had an improved life-expectancy. One patient in the third family deteriorated upon interruption of the thiamine treatment and recovered after reinitiating. Although unrelated, all patients came from the province Al Hoceima in Northern Morocco. Based on the recombination events the mutation was estimated to have occurred 1250-1750 years ago. Our data shows that SLC19A3 is a new candidate for mutation screening in patients with Leigh syndrome, who might benefit from high doses of thiamine and/or biotin. Especially, Moroccan patients with Leigh syndrome should be tested for the c.20C>A founder mutation in SLC19A3.

Next-generation sequencing-based genome diagnostics across clinical genetics centers: implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found ‘no causal variant’, thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of ‘greediness’ to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.

Pathogenic CWF19L1 variants as a novel cause of autosomal recessive cerebellar ataxia and atrophy.

Autosomal recessive cerebellar ataxia (ARCA) is a group of neurological disorders characterized by degeneration or abnormal development of the cerebellum and spinal cord. ARCA is clinically and genetically highly heterogeneous, with over 20 genes involved. Exome sequencing of a girl with ARCA from non-consanguineous Dutch parents revealed two pathogenic variants c.37G>C; p.D13H and c.946A>T; p.K316* in CWF19L1, a gene with an unknown function, recently reported to cause ARCA in a Turkish family. Sanger sequencing showed that the c.37G>C variant was inherited from the father and the c.946A>T variant from the mother. Pathogenicity was based on the damaging effect on protein function as the c.37G>C variant changed the highly conserved, negatively charged aspartic acid to the positively charged histidine and the c.946A>T variant introduced a premature stop codon. In addition, 27 patients with ARCA were tested for pathogenic variants in CWF19L1, however, no pathogenic variants were identified. Our data confirm CWF19L1 as a novel but rare gene causing ARCA.

A comprehensive strategy for exome-based preconception carrier screening

PURPOSE Whole-exome sequencing (WES) provides the possibility of genome-wide preconception carrier screening (PCS). Here, we propose a filter strategy to rapidly identify the majority of relevant pathogenic mutations. METHODS Our strategy was developed using WES data from eight consanguineous and five fictive nonconsanguineous couples and was subsequently applied to 20 other fictive nonconsanguineous couples. Presumably pathogenic variants based on frequency and database annotations or generic characteristics and mutation type were selected in genes shared by the couple and in the females X-chromosome. Unclassified variants were not included. RESULTS This yielded an average of 29 (19-51) variants in genes shared by the consanguineous couples and 15 (6-30) shared by the nonconsanguineous couples. For X-linked variants, the numbers per female were 3 (1-5) and 1 (0-3), respectively. Remaining variants were verified manually. The majority were able to be quickly discarded, effectively leaving true pathogenic variants. CONCLUSION We conclude that WES is applicable for PCS, both for consanguineous and nonconsanguineous couples, with the remaining number of variants being manageable in a clinical setting. The addition of gene panels for filtering was not favorable because it resulted in missing pathogenic variants. It is important to develop and continuously curate databases with pathogenic mutations to further increase the sensitivity of WES-based PCS.Genet Med advance online publication 27 October 2016Genetics in Medicine (2016); doi:10.1038/gim.2016.153.Purpose:Whole-exome sequencing (WES) provides the possibility of genome-wide preconception carrier screening (PCS). Here, we propose a filter strategy to rapidly identify the majority of relevant pathogenic mutations.Methods:Our strategy was developed using WES data from eight consanguineous and five fictive nonconsanguineous couples and was subsequently applied to 20 other fictive nonconsanguineous couples. Presumably pathogenic variants based on frequency and database annotations or generic characteristics and mutation type were selected in genes shared by the couple and in the female’s X-chromosome. Unclassified variants were not included.Results:This yielded an average of 29 (19–51) variants in genes shared by the consanguineous couples and 15 (6–30) shared by the nonconsanguineous couples. For X-linked variants, the numbers per female were 3 (1–5) and 1 (0–3), respectively. Remaining variants were verified manually. The majority were able to be quickly discarded, effectively leaving true pathogenic variants.Conclusion:We conclude that WES is applicable for PCS, both for consanguineous and nonconsanguineous couples, with the remaining number of variants being manageable in a clinical setting. The addition of gene panels for filtering was not favorable because it resulted in missing pathogenic variants. It is important to develop and continuously curate databases with pathogenic mutations to further increase the sensitivity of WES-based PCS.Genet Med advance online publication 27 October 2016

Erratum : Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects (European Journal of Human Genetics (2015) 23 (1142-1150) DOI:10.1038/ejhg.2014.279)

Terry Vrijenhoek, Ken Kraaijeveld, Martin Elferink, Joep de Ligt, Elcke Kranendonk, Gijs Santen, Isaac J Nijman, Derek Butler, Godelieve Claes, Adalberto Costessi, Wim Dorlijn, Winfried van Eyndhoven, Dicky JJ Halley, Mirjam CGN van den Hout, Steven van Hove, Lennart F Johansson, Jan DH Jongbloed, Rick Kamps, Christel EM Kockx, Bart de Koning, Marjolein Kriek, Ronald Lekanne dit Deprez, Hans Lunstroo, Marcel Mannens, Olaf R Mook, Marcel Nelen, Corrette Ploem, Marco Rijnen, Jasper J Saris, Richard Sinke, Erik Sistermans, Marjon van Slegtenhorst, Frank Sleutels, Nienke van der Stoep, Marianne van Tienhoven, Martijn Vermaat, Maartje Vogel, Quinten Waisfisz, Janneke Marjan Weiss, Arthur van den Wijngaard, Wilbert van Workum, Helger Ijntema, Bert van der Zwaag, Wilfred FJ van IJcken, Johan T den Dunnen, Joris A Veltman, Raoul Hennekam and Edwin Cuppen

Genetic defects in mtDNA-encoded protein translation cause pediatric, mitochondrial cardiomyopathy with early-onset brain disease

This study aims to identify gene defects in pediatric cardiomyopathy and early-onset brain disease with oxidative phosphorylation (OXPHOS) deficiencies. We applied whole-exome sequencing in three patients with pediatric cardiomyopathy and early-onset brain disease with OXPHOS deficiencies. The brain pathology was studied by MRI analysis. In consanguineous patient 1, we identified a homozygous intronic variant (c.850-3A > G) in the QRSL1 gene, which was predicted to cause abnormal splicing. The variant segregated with the disease and affected the protein function, which was confirmed by complementation studies, restoring OXPHOS function only with wild-type QRSL1. Patient 2 was compound heterozygous for two novel affected and disease-causing variants (c.[253G > A];[938G > A]) in the MTO1 gene. In patient 3, we detected one unknown affected and disease-causing variants (c.2872C > T) and one known disease-causing variant (c.1774C > T) in the AARS2 gene. The c.1774C > T variant was present in the paternal copy of the AARS2 gene, the c.2872C > T in the maternal copy. All genes were involved in translation of mtDNA-encoded proteins. Defects in mtDNA-encoded protein translation lead to severe pediatric cardiomyopathy and brain disease with OXPHOS abnormalities. This suggests that the heart and brain are particularly sensitive to defects in mitochondrial protein synthesis during late embryonic or early postnatal development, probably due to the massive mitochondrial biogenesis occurring at that stage. If both the heart and brain are involved, the prognosis is poor with a likely fatal outcome at young age.

Germ-line variants identified by next generation sequencing in a panel of estrogen and cancer associated genes correlate with poor clinical outcome in Lynch syndrome patients.

Background The risk to develop colorectal and endometrial cancers among subjects testing positive for a pathogenic Lynch syndrome mutation varies, making the risk prediction difficult. Genetic risk modifiers alter the risk conferred by inherited Lynch syndrome mutations, and their identification can improve genetic counseling. We aimed at identifying rare genetic modifiers of the risk of Lynch syndrome endometrial cancer. Methods A family based approach was used to assess the presence of genetic risk modifiers among 35 Lynch syndrome mutation carriers having either a poor clinical phenotype (early age of endometrial cancer diagnosis or multiple cancers) or a neutral clinical phenotype. Putative genetic risk modifiers were identified by Next Generation Sequencing among a panel of 154 genes involved in endometrial physiology and carcinogenesis. Results A simple pipeline, based on an allele frequency lower than 0.001 and on predicted non-conservative amino-acid substitutions returned 54 variants that were considered putative risk modifiers. The presence of two or more risk modifying variants in women carrying a pathogenic Lynch syndrome mutation was associated with a poor clinical phenotype. Conclusion A gene-panel is proposed that comprehends genes that can carry variants with putative modifying effects on the risk of Lynch syndrome endometrial cancer. Validation in further studies is warranted before considering the possible use of this tool in genetic counseling.

Rapid Resolution of Blended or Composite Multigenic Disease in Infants by Whole-Exome Sequencing

&NA; Whole‐exome sequencing identified multiple genetic causes in 2 infants with heterogeneous disease. Three gene defects in the first patient explained all symptoms, but manifestations were overlapping (blended phenotype). Two gene defects in the second patient explained nonoverlapping symptoms (composite phenotype). Whole‐exome sequencing rapidly and comprehensively resolves heterogeneous genetic disease.

Specific MRI abnormalities reveal severe perrault syndrome due to CLPP defects

In establishing a genetic diagnosis in heterogeneous neurological disease, clinical characterization and whole exome sequencing (WES) go hand-in-hand. Clinical data are essential, not only to guide WES variant selection and define the clinical severity of a genetic defect but also to identify other patients with defects in the same gene. In an infant patient with sensorineural hearing loss, psychomotor retardation, and epilepsy, WES resulted in identification of a novel homozygous CLPP frameshift mutation (c.21delA). Based on the gene defect and clinical symptoms, the diagnosis Perrault syndrome type 3 (PRLTS3) was established. The patient’s brain-MRI revealed specific abnormalities of the subcortical and deep cerebral white matter and the middle blade of the corpus callosum, which was used to identify similar patients in the Amsterdam brain-MRI database, containing over 3000 unclassified leukoencephalopathy cases. In three unrelated patients with similar MRI abnormalities the CLPP gene was sequenced, and in two of them novel missense mutations were identified together with a large deletion that covered part of the CLPP gene on the other allele. The severe neurological and MRI abnormalities in these young patients were due to the drastic impact of the CLPP mutations, correlating with the variation in clinical manifestations among previously reported patients. Our data show that similarity in brain-MRI patterns can be used to identify novel PRLTS3 patients, especially during early disease stages, when only part of the disease manifestations are present. This seems especially applicable to the severely affected cases in which CLPP function is drastically affected and MRI abnormalities are pronounced.

Coorigendum: Next-generation sequencing-based genome diagnostics across clinical genetics centers

Terry Vrijenhoek, Ken Kraaijeveld, Martin Elferink, Joep de Ligt, Elcke Kranendonk, Gijs Santen, Isaac J Nijman, Derek Butler, Godelieve Claes, Adalberto Costessi, Wim Dorlijn, Winfried van Eyndhoven, Dicky JJ Halley, Mirjam CGN van den Hout, Steven van Hove, Lennart F Johansson, Jan DH Jongbloed, Rick Kamps, Christel EM Kockx, Bart de Koning, Marjolein Kriek, Ronald Lekanne dit Deprez, Hans Lunstroo, Marcel Mannens, Olaf R Mook, Marcel Nelen, Corrette Ploem, Marco Rijnen, Jasper J Saris, Richard Sinke, Erik Sistermans, Marjon van Slegtenhorst, Frank Sleutels, Nienke van der Stoep, Marianne van Tienhoven, Martijn Vermaat, Maartje Vogel, Quinten Waisfisz, Janneke Marjan Weiss, Arthur van den Wijngaard, Wilbert van Workum, Helger Ijntema, Bert van der Zwaag, Wilfred FJ van IJcken, Johan T den Dunnen, Joris A Veltman, Raoul Hennekam and Edwin Cuppen