Marjolein Kriek

Mutations in SWI/SNF chromatin remodeling complex gene ARID1B cause Coffin-Siris syndrome

We identified de novo truncating mutations in ARID1B in three individuals with Coffin-Siris syndrome (CSS) by exome sequencing. Array-based copy-number variation (CNV) analysis in 2,000 individuals with intellectual disability revealed deletions encompassing ARID1B in 3 subjects with phenotypes partially overlapping that of CSS. Taken together with published data, these results indicate that haploinsufficiency of the ARID1B gene, which encodes an epigenetic modifier of chromatin structure, is an important cause of CSS and is potentially a common cause of intellectual disability and speech impairment.

Comprehensive Detection of Genomic Duplications and Deletions in the DMD Gene, by Use of Multiplex Amplifiable Probe Hybridization

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening.

Peters Plus Syndrome Is Caused by Mutations in B3GALTL, a Putative Glycosyltransferase

Peters Plus syndrome is an autosomal recessive disorder characterized by anterior eye-chamber abnormalities, disproportionate short stature, and developmental delay. After detection of a microdeletion by array-based comparative genomic hybridization, we identified biallelic truncating mutations in the beta 1,3-galactosyltransferase-like gene (B3GALTL) in all 20 tested patients, showing that Peters Plus is a monogenic, primarily single-mutation syndrome. This finding is expected to put Peters Plus syndrome on the growing list of congenital malformation syndromes caused by glycosylation defects.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling

Mitochondrial Ca2+ uptake has key roles in cell life and death. Physiological Ca2+ signaling regulates aerobic metabolism, whereas pathological Ca2+ overload triggers cell death. Mitochondrial Ca2+ uptake is mediated by the Ca2+ uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca2+-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca2+ uptake at low cytosolic Ca2+ concentrations was increased, and cytosolic Ca2+ signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca2+ handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca2+ signaling, demonstrating the crucial role of mitochondrial Ca2+ uptake in humans.

Decreased mortality of ischaemic heart disease among carriers of haemophilia

BACKGROUND Coagulation plays an important part in ischaemic cardiovascular disease. Results of studies have shown that extremes in hypocoagulability protect against ischaemic cardiovascular disease. We have investigated overall mortality and death from cardiovascular causes in carriers of haemophilia, who in most cases have mildly decreased coagulability without clinical signs. METHODS We followed-up a cohort of 1012 mothers of all known people with haemophilia in the Netherlands from birth to death, or the end-of-study date (41984 person years of follow-up). We obtained vital status and causes of death, if deceased, and compared overall and cause-specific mortality in our cohort with that in the general Dutch female population adjusted for age and calendar period by calculating the standardised mortality ratio (SMR). FINDINGS Overall mortality was reduced by 22% (261 observed deaths, 333.74 expected; SMR 0.78 [95% CI 0.69-0.88]). Deaths from ischaemic heart disease were reduced by 36% (39 observed deaths, 60.53 expected; SMR 0.64 [0.47-0.88]). We did not note decreased mortality for cerebral stroke (ischaemic and haemorrhagic combined) (28 observed deaths, 36.82 expected; SMR 0.76 [0.53-1.10]). A separate analysis of these two types of stroke was not possible. Women in our cohort had an increased risk of death from extracranial haemorrhage (5 observed deaths, 0.18 expected; SMR 27.78 [8.49-58.18]); however, the number of deaths from this cause was much lower than that for ischaemic heart disease. CONCLUSION The results show that a mild decrease in coagulability has a protective effect against fatal ischaemic heart disease.

Copy number variation in regions flanked (or unflanked) by duplicons among patients with developmental delay and/or congenital malformations; detection of reciprocal and partial Williams-Beuren duplications.

Duplicons, that is, DNA sequences with minimum length 10 kb and a high sequence similarity, are known to cause unequal homologous recombination, leading to deletions and the reciprocal duplications. In this study, we designed a Multiplex Amplifiable Probe Hybridisation (MAPH) assay containing 63 exon-specific single-copy sequences from within a selection of the 169 regions flanked by duplicons that were identified, at a first pass, in 2001. Subsequently, we determined the frequency of chromosomal rearrangements among patients with developmental delay (DD) and/or congenital malformations (CM). In addition, we tried to identify new regions involved in DD/CM using the same assay. In 105 patients, six imbalances (5.8%) were detected and verified. Three of these were located in microdeletion-related regions, two alterations were polymorphic duplications and the effect of the last alteration is currently unknown. The same study population was tested for rearrangements in regions with no known duplicons nearby, using a set of probes derived from 58 function-selected genes. The latter screening revealed two alterations. As expected, the alteration frequency per unit of DNA is much higher in regions flanked by duplicons (fraction of the genome tested: 5.2%) compared to regions without known duplicons nearby (fraction of the genome tested: 24.5–90.2%). We were able to detect three novel rearrangements, including the previously undescribed reciprocal duplication of the Williams Beuren critical region, a subduplicon alteration within this region and a duplication on chromosome band 16p13.11. Our results support the hypothesis that regions flanked by duplicons are enriched for copy number variations.

Molecular and clinical characterization of 25 individuals with exonic deletions of NRXN1 and comprehensive review of the literature

This study aimed to elucidate the observed variable phenotypic expressivity associated with NRXN1 (Neurexin 1) haploinsufficiency by analyses of the largest cohort of patients with NRXN1 exonic deletions described to date and by comprehensively reviewing all comparable copy number variants in all disease cohorts that have been published in the peer reviewed literature (30 separate papers in all). Assessment of the clinical details in 25 previously undescribed individuals with NRXN1 exonic deletions demonstrated recurrent phenotypic features consisting of moderate to severe intellectual disability (91%), severe language delay (81%), autism spectrum disorder (65%), seizures (43%), and hypotonia (38%). These showed considerable overlap with previously reported NRXN1‐deletion associated phenotypes in terms of both spectrum and frequency. However, we did not find evidence for an association between deletions involving the β‐isoform of neurexin‐1 and increased head size, as was recently published in four cases with a deletion involving the C‐terminus of NRXN1. We identified additional rare copy number variants in 20% of cases. This study supports a pathogenic role for heterozygous exonic deletions of NRXN1 in neurodevelopmental disorders. The additional rare copy number variants identified may act as possible phenotypic modifiers as suggested in a recent digenic model of neurodevelopmental disorders.

Next-Generation Diagnostics: Gene Panel, Exome, or Whole Genome?

Although the benefits of next‐generation sequencing (NGS) for the diagnosis of heterogeneous diseases such as intellectual disability (ID) are undisputed, there is little consensus on the relative merits of targeted enrichment, whole‐exome sequencing (WES) or whole‐genome sequencing (WGS). To answer this question, WES and WGS data from the same nine samples were compared, and WES was shown not to miss any variants identified by WGS in a gene panel including ∼500 genes linked to ID (500GP). Additionally, deeply sequenced WES data were shown to adequately cover ∼99% of the 500GP; thus, little additional benefit was to be expected from a targeted enrichment approach. To reduce costs, minimal sequencing criteria were determined by investigating the relation between sequenced reads and outcome parameters such as coverage and variant yield. Our analysis indicated that 60 million reads yielded a mean coverage of ∼60×: ∼97% of the 500GP sequences were sufficiently covered to exclude variants, whereas variant yield was ∼99.5% and false‐positive and false‐negative rates were controlled. Our findings indicate that WES is currently the optimal approach to ID diagnostics. This result depends on the capture kit and sequencing strategy used. The developed framework however is amenable to other sequencing approaches.

Genomic imbalances in mental retardation

Introduction: It has been estimated that cytogenetically visible rearrangements are present in ~1% of newborns. These chromosomal changes can cause a wide range of deleterious developmental effects, including mental retardation (MR). It is assumed that many other cases exist where the cause is a submicroscopic deletion or duplication. To facilitate the detection of such cases, different techniques have been developed, which have differing efficiency as to the number of loci and patients that can be tested. Methods: We implemented multiplex amplifiable probe hybridisation (MAPH) to test areas known to be rearranged in MR patients (for example, subtelomeric/pericentromeric regions and those affected in microdeletion syndromes) and to look for new regions that might be related to MR. Results: In this study, over 30 000 screens for duplications and deletions were carried out; 162 different loci tested in each of 188 developmentally delayed patients. The analysis resulted in the detection of 19 rearrangements, of which ~65% would not have been detected by conventional cytogenetic analysis. A significant fraction (46%) of the rearrangements found were interstitial, despite the fact that only a limited number of these loci have so far been tested. Discussion: Our results strengthen the arguments for whole genome screening within this population, as it can be assumed that many more interstitial rearrangements would be detected. The strengths of MAPH for this analysis are the simplicity, the high throughput potential, and the high resolution of analysis. This combination should help in the future identification of the specific genes that are responsible for MR.

Mutations in DNMT3B Modify Epigenetic Repression of the D4Z4 Repeat and the Penetrance of Facioscapulohumeral Dystrophy

Facioscapulohumeral dystrophy (FSHD) is associated with somatic chromatin relaxation of the D4Z4 repeat array and derepression of the D4Z4-encoded DUX4 retrogene coding for a germline transcription factor. Somatic DUX4 derepression is caused either by a 1-10 unit repeat-array contraction (FSHD1) or by mutations in SMCHD1, which encodes a chromatin repressor that binds to D4Z4 (FSHD2). Here, we show that heterozygous mutations in DNA methyltransferase 3B (DNMT3B) are a likely cause of D4Z4 derepression associated with low levels of DUX4 expression from the D4Z4 repeat and increased penetrance of FSHD. Recessive mutations in DNMT3B were previously shown to cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. This study suggests that transcription of DUX4 in somatic cells is modified by variations in its epigenetic state and provides a basis for understanding the reduced penetrance of FSHD within families.