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Contains fulltext : 248860.pdf (Publisher’s version ) (Open Access) Reproductive counseling in facioscapulohumeral muscular dystrophy (FSHD) can be challenging due to the complexity of its underlying genetic mechanisms and due to incomplete penetrance of the disease. Full understanding of the genetic causes and potential inheritance patterns of both distinct FSHD types is essential: FSHD1 is an autosomal dominantly inherited repeat disorder, whereas FSHD2 is a digenic disorder. This has become even more relevant now that prenatal diagnosis and preimplantation genetic diagnosis options are available for FSHD1. Pregnancy and delivery outcomes in FSHD are usually favorable, but clinicians should be aware of the risks. We aim to provide clinicians with case-based strategies for reproductive counseling in FSHD, as well as recommendations for pregnancy and delivery.

PURPOSE: Few studies have systematically analyzed the structure and content of laboratory exome sequencing reports from the same patient. METHODS: We merged 8 variants from patients into "normal" exomes to create virtual patient-parent trios. We provided laboratories worldwide with the data and patient phenotype information (developmental delay, dysmorphic features, and cardiac hypertrophy). Laboratories analyzed the data and issued a diagnostic exome report. Reports were scored using a coding matrix developed from existing guidelines. RESULTS: In total, 41 laboratories representing 17 countries issued reports. Reporting of quality control statistics and technical information was poor (46.3%). Although 75.6% of the reports clearly stated the classification of all reported variants, few reports listed extensive evidence supporting variant classification. Only 53.1% of laboratories that reported unsolicited or secondary findings gave advice regarding health-related follow-up and 20.5% gave advice regarding cascade testing for relatives. Of the 147 variants reported, 105 (71.4%) were classified in agreement with classifications based on American College of Medical Genetics and Genomics/Association for Molecular Pathology and Association for Clinical Genomic Science guidelines. Concordance was higher for known pathogenic variants (86.3%) than for novel unpublished variants (56.8%). CONCLUSION: The considerable variability identified in the components that laboratories included in their reports and their classification of variants suggests that existing guidelines are not being used consistently with significant implications for patient care. ispartof: GENETICS IN MEDICINE vol:24 issue:6 pages:1306-1315 ispartof: location:United States status: published

The original version of this Article contained an error in the spelling of the author Pleuntje J. van der Sluijs, which was incorrectly given as Eline (P. J.) van der Sluijs. This has now been corrected in both the PDF and HTML versions of the Article.

Purpose: Several studies have reported diagnostic yields up to 57% for rapid exome or genome sequencing (rES/GS) as a single test in neonatal intensive care unit (NICU) patients, but the additional yield of rES/GS compared with other available diagnostic options still remains unquantified in this population. Methods: We retrospectively evaluated all genetic NICU consultations in a 2-year period. Results: In 132 retrospectively evaluated NICU consultations 27 of 32 diagnoses (84.4%) were made using standard genetic workup. Most diagnoses (65.6%) were made within 16 days. Diagnostic ES yield was 5/29 (17.2%). Genetic diagnoses had a direct effect on clinical management in 90.6% (29/32) of patients. Conclusions: Our study shows that exome sequencing has a place in NICU diagnostics, but given the associated costs and the high yield of alternative diagnostic strategies, we recommend to first perform clinical genetic consultation.

The continuous development of new genotyping technologies requires awareness of their potential advantages and limitations concerning utility for pharmacogenomics (PGx). In this review, we provide an overview of technologies that can be applied in PGx research and clinical practice. Most commonly used are single nucleotide variant (SNV) panels which contain a pre-selected panel of genetic variants. SNV panels offer a short turnaround time and straightforward interpretation, making them suitable for clinical practice. However, they are limited in their ability to assess rare and structural variants. Next-generation sequencing (NGS) and long-read sequencing are promising technologies for the field of PGx research. Both NGS and long-read sequencing often provide more data and more options with regard to deciphering structural and rare variants compared to SNV panels—in particular, in regard to the number of variants that can be identified, as well as the option for haplotype phasing. Nonetheless, while useful for research, not all sequencing data can be applied to clinical practice yet. Ultimately, selecting the right technology is not a matter of fact but a matter of choosing the right technique for the right problem.

PURPOSE: Existing research suggests that while some laboratories report variants of uncertain significance, unsolicited findings (UF), and/or secondary findings (SF) when performing exome sequencing, others do not. METHODS: To investigate reporting differences, we created virtual patient-parent trio data by merging variants from patients into "normal" exomes. We invited laboratories worldwide to analyze the data along with patient phenotype information (developmental delay, dysmorphic features, and cardiac hypertrophy). Laboratories issued a diagnostic exome report and completed questionnaires to explain their rationale for reporting (or not reporting) each of the eight variants integrated. RESULTS: Of the 39 laboratories that completed the questionnaire, 30 reported the HDAC8 variant, which was a partial cause of the patient's primary phenotype, and 26 reported the BICD2 variant, which explained another phenotypic component. Lack of reporting was often due to using a filter or a targeted gene panel that excluded the variant, or because they did not consider the variant to be responsible for the phenotype. There was considerable variation in reporting variants associated with the cardiac phenotype (MYBPC3 and PLN) and reporting UF/SF also varied widely. CONCLUSION: This high degree of variability has significant impact on whether causative variants are identified, with important implications for patient care. ispartof: GENETICS IN MEDICINE vol:23 issue:3 pages:562-570 ispartof: location:United States status: published

Existing research suggests that while some laboratories report variants of uncertain significance, unsolicited findings (UF), and/or secondary findings (SF) when performing exome sequencing, others do not.To investigate reporting differences, we created virtual patient-parent trio data by merging variants from patients into "normal" exomes. We invited laboratories worldwide to analyze the data along with patient phenotype information (developmental delay, dysmorphic features, and cardiac hypertrophy). Laboratories issued a diagnostic exome report and completed questionnaires to explain their rationale for reporting (or not reporting) each of the eight variants integrated.Of the 39 laboratories that completed the questionnaire, 30 reported the HDAC8 variant, which was a partial cause of the patient's primary phenotype, and 26 reported the BICD2 variant, which explained another phenotypic component. Lack of reporting was often due to using a filter or a targeted gene panel that excluded the variant, or because they did not consider the variant to be responsible for the phenotype. There was considerable variation in reporting variants associated with the cardiac phenotype (MYBPC3 and PLN) and reporting UF/SF also varied widely.This high degree of variability has significant impact on whether causative variants are identified, with important implications for patient care.

Contains fulltext : 229047.pdf (Publisher’s version ) (Open Access) For ~ 80 drugs, widely recognized pharmacogenetics dosing guidelines are available. However, the use of these guidelines in clinical practice remains limited as only a fraction of patients is subjected to pharmacogenetic screening. We investigated the feasibility of repurposing whole exome sequencing (WES) data for a panel of 42 variants in 11 pharmacogenes to provide a pharmacogenomic profile. Existing diagnostic WES-data from child-parent trios totaling 1,583 individuals were used. Results were successfully extracted for 39 variants. No information could be extracted for three variants, located in CYP2C19, UGT1A1, and CYP3A5, and for CYP2D6 copy number. At least one actionable phenotype was present in 86% of the individuals. Haplotype phasing proved relevant for CYP2B6 assignments as 1.5% of the phenotypes were corrected after phasing. In conclusion, repurposing WES-data can yield meaningful pharmacogenetic profiles for 7 of 11 important pharmacogenes, which can be used to guide drug treatment.

BackgroundSporadic deletions removing 100s-1000s kb of DNA, and variable numbers of poorly characterised genes, are often found in patients with a wide range of developmental abnormalities. In such cases, understanding the contribution of the deletion to an individual’s clinical phenotype is challenging.MethodsHere, as an example of this common phenomenon, we analysed 34 patients with simple deletions of ∼177 to ∼2000 kb affecting one allele of the well characterised, gene dense, distal region of chromosome 16 (16p13.3), referred to as ATR-16 syndrome. We characterised precise deletion extent and screened for genetic background effects, telomere position effect and compensatory up regulation of hemizygous genes.ResultsWe find the risk of developmental and neurological abnormalities arises from much smaller terminal chromosome 16 deletions (∼400 kb) than previously reported. Beyond this, the severity of ATR-16 syndrome increases with deletion size, but there is no evidence that critical regions determine the developmental abnormalities associated with this disorder. Surprisingly, we find no evidence of telomere position effect or compensatory upregulation of hemizygous genes, however, genetic background effects substantially modify phenotypic abnormalities.ConclusionsUsing ATR-16 as a general model of disorders caused by sporadic copy number variations, we show the degree to which individuals with contiguous gene syndromes are affected is not simply related to the number of genes deleted but also depends on their genetic background. We also show there is no critical region defining the degree of phenotypic abnormalities in ATR-16 syndrome and this has important implications for genetic counselling.

Variants in the KIF1A gene can cause autosomal recessive spastic paraplegia 30, autosomal recessive hereditary sensory neuropathy, or autosomal (de novo) dominant mental retardation type 9. More recently, variants in KIF1A have also been described in a few cases with autosomal dominant spastic paraplegia. Here, we describe 20 KIF1A variants in 24 patients from a clinical exome sequencing cohort of 347 individuals with a mostly 'pure' spastic paraplegia. In these patients, spastic paraplegia was slowly progressive and mostly pure, but with a highly variable disease onset (0-57 years). Segregation analyses showed a de novo occurrence in seven cases, and a dominant inheritance pattern in 11 families. The motor domain of KIF1A is a hotspot for disease causing variants in autosomal dominant spastic paraplegia, similar to mental retardation type 9 and recessive spastic paraplegia type 30. However, unlike these allelic disorders, dominant spastic paraplegia was also caused by loss-of-function variants outside this domain in six families. Finally, three missense variants were outside the motor domain and need further characterization. In conclusion, KIF1A variants are a frequent cause of autosomal dominant spastic paraplegia in our cohort (6-7%). The identification of KIF1A loss-of-function variants suggests haploinsufficiency as a possible mechanism in autosomal dominant spastic paraplegia.

Contains fulltext : 204157.pdf (Publisher’s version ) (Closed access) BACKGROUND: Neuromuscular disorders (NMDs) are clinically and genetically heterogeneous. Accurate molecular genetic diagnosis can improve clinical management, provides appropriate genetic counseling and testing of relatives, and allows potential therapeutic trials. OBJECTIVE: To establish the clinical utility of panel-based whole exome sequencing (WES) in NMDs in a population with children and adults with various neuromuscular symptoms. METHODS: Clinical exome sequencing, followed by diagnostic interpretation of variants in genes associated with NMDs, was performed in a cohort of 396 patients suspected of having a genetic cause with a variable age of onset, neuromuscular phenotype, and inheritance pattern. Many had previously undergone targeted gene testing without results. RESULTS: Disease-causing variants were identified in 75/396 patients (19%), with variants in the three COL6-genes (COL6A1, COL6A2 and COL6A3) as the most common cause of the identified muscle disorder, followed by variants in the RYR1 gene. Together, these four genes account for almost 25% of cases in whom a definite genetic cause was identified. Furthermore, likely pathogenic variants and/or variants of uncertain significance were identified in 95 of the patients (24%), in whom functional and/or segregation analysis should be used to confirm or reject the pathogenicity. In 18% of the cases with a disease-causing variant of which we received additional clinical information, we identified a genetic cause in genes of which the associated phenotypes did not match that of the patients. Hence, the advantage of panel-based WES is its unbiased approach. CONCLUSION: Whole exome sequencing, followed by filtering for NMD genes, offers an unbiased approach for the genetic diagnostics of NMD patients. This approach could be used as a first-tier test in neuromuscular disorders with a high suspicion of a genetic cause. With uncertain results, functional testing and segregation analysis are needed to complete the evidence.

Type 2A protein phosphatases (PP2As) are highly expressed in the brain and regulate neuronal signaling by catalyzing phospho-Ser/Thr dephosphorylations in diverse substrates. PP2A holoenzymes comprise catalytic C-, scaffolding A-, and regulatory B-type subunits, which determine substrate specificity and physiological function. Interestingly, de novo mutations in genes encoding A- and B-type subunits have recently been implicated in intellectual disability (ID) and developmental delay (DD). We now report 16 individuals with mild to profound ID and DD and a de novo mutation in PPP2CA, encoding the catalytic Cα subunit. Other frequently observed features were severe language delay (71%), hypotonia (69%), epilepsy (63%), and brain abnormalities such as ventriculomegaly and a small corpus callosum (67%). Behavioral problems, including autism spectrum disorders, were reported in 47% of individuals, and three individuals had a congenital heart defect. PPP2CA de novo mutations included a partial gene deletion, a frameshift, three nonsense mutations, a single amino acid duplication, a recurrent mutation, and eight non-recurrent missense mutations. Functional studies showed complete PP2A dysfunction in four individuals with seemingly milder ID, hinting at haploinsufficiency. Ten other individuals showed mutation-specific biochemical distortions, including poor expression, altered binding to the A subunit and specific B-type subunits, and impaired phosphatase activity and C-terminal methylation. Four were suspected to have a dominant-negative mechanism, which correlated with severe ID. Two missense variants affecting the same residue largely behaved as wild-type in our functional assays. Overall, we found that pathogenic PPP2CA variants impair PP2A-B56(δ) functionality, suggesting that PP2A-related neurodevelopmental disorders constitute functionally converging ID syndromes. ispartof: American Journal Of Human Genetics vol:104 issue:1 pages:139-156 ispartof: location:United States status: published

ObjectiveTo determine whether congenital arhinia/Bosma arhinia microphthalmia syndrome (BAMS) and facioscapulohumeral muscular dystrophy type 2 (FSHD2), 2 seemingly unrelated disorders both caused by heterozygous pathogenic missense variants in the SMCHD1 gene, might represent different ends of a broad single phenotypic spectrum associated with SMCHD1 dysfunction.MethodsWe examined and/or interviewed 14 patients with FSHD2 and 4 unaffected family members with N-terminal SMCHD1 pathogenic missense variants to identify BAMS subphenotypes.ResultsNone of the patients with FSHD2 or family members demonstrated any congenital defects or dysmorphic features commonly found in patients with BAMS. One patient became anosmic after nasal surgery and one patient was hyposmic; one man was infertile (unknown cause) but reported normal pubertal development.ConclusionThese data suggest that arhinia/BAMS and FSHD2 do not represent one phenotypic spectrum and that SMCHD1 pathogenic variants by themselves are insufficient to cause either of the 2 disorders. More likely, both arhinia/BAMS and FSHD2 are caused by complex oligogenic or multifactorial mechanisms that only partially overlap at the level of SMCHD1.

Background18p deletion syndrome is a rare disorder caused by partial or full monosomy of the short arm of chromosome 18. Clinical symptoms caused by 18p hemizygosity include cognitive impairment, mild facial dysmorphism, strabismus and ptosis. Among other genes, structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is hemizygous in most patients with 18p deletions. Digenic inheritance of a SMCHD1 mutation and a moderately sized D4Z4 repeat on a facioscapulohumeral muscular dystrophy (FSHD) permissive genetic background of chromosome 4 can cause FSHD type 2 (FSHD2).ObjectivesSince 12% of Caucasian individuals harbour moderately sized D4Z4 repeats on an FSHD permissive background, we tested if people with 18p deletions are at risk of developing FSHD.MethodsTo test our hypothesis we studied different cellular systems originating from individuals with 18p deletions not presenting FSHD2 phenotype for transcriptional and epigenetic characteristics of FSHD at D4Z4. Furthermore, individuals with an idiopathic muscle phenotype and an 18p deletion were subjected to neurological examination.ResultsPrimary fibroblasts hemizygous for SMCHD1 have a D4Z4 chromatin structure comparable with FSHD2 concomitant with DUX4 expression after transdifferentiation into myocytes. Neurological examination of 18p deletion individuals from two independent families with a moderately sized D4Z4 repeat identified muscle features compatible with FSHD.Conclusions18p deletions leading to haploinsufficiency of SMCHD1, together with a moderately sized FSHD permissive D4Z4 allele, can associate with symptoms and molecular features of FSHD. We propose that patients with 18p deletion should be characterised for their D4Z4 repeat size and haplotype and monitored for clinical features of FSHD.

Pathogenic variants in the PCDH19 gene are associated with epilepsy, intellectual disability (ID) and behavioural disturbances. Only heterozygous females and mosaic males are affected, likely due to a disease mechanism named cellular interference. Until now, only four affected mosaic male patients have been described in literature. Here, we report five additional male patients, of which four are older than the oldest patient reported so far. All reported patients were selected for genetic testing because of developmental delay and/or epilepsy. Custom-targeted next generation sequencing gene panels for epilepsy genes were used. Clinical data were collected from medical records. All patients were mosaic in blood for likely pathogenic variants in the PCDH19 gene. In most, clinical features were very similar to the female phenotype, with normal development before seizure onset, which occurred between 5 and 10 months of age, clustering of seizures and sensitivity to fever. Four out of five patients had mild to severe ID and behavioural problems. We reaffirm the similarity between male and female PCDH19-related phenotypes, now also in a later phase of the disorder (ages 10–14 years). Electronic supplementary material The online version of this article (doi:10.1007/s10048-017-0517-5) contains supplementary material, which is available to authorized users.

Item does not contain fulltext Gene-disruptive mutations contribute to the biology of neurodevelopmental disorders (NDDs), but most of the related pathogenic genes are not known. We sequenced 208 candidate genes from >11,730 cases and >2,867 controls. We identified 91 genes, including 38 new NDD genes, with an excess of de novo mutations or private disruptive mutations in 5.7% of cases. Drosophila functional assays revealed a subset with increased involvement in NDDs. We identified 25 genes showing a bias for autism versus intellectual disability and highlighted a network associated with high-functioning autism (full-scale IQ >100). Clinical follow-up for NAA15, KMT5B, and ASH1L highlighted new syndromic and nonsyndromic forms of disease.

Chudley-McCullough syndrome (CMS) is characterized by profound sensorineural hearing loss and brain anomalies. Variants in GPSM2 have recently been reported as a cause of CMS by Doherty et al. In this study we have performed exome sequencing of three CMS patients from two unrelated families from the same Dutch village. We identified one homozygous frameshift GPSM2 variants c.1473delG in all patients. We show that this variant arises from a shared, rare haplotype. Since the c.1473delG variant was found in Mennonite settlers, it likely originated in Europe. To support DNA diagnostics, we established an LOVD database for GPSM2 containing all variants thus far described.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-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 Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) 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 Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Item does not contain fulltext Spinocerebellar ataxias are phenotypically, neuropathologically, and genetically heterogeneous. The locus of autosomal recessive spinocerebellar ataxia type 7 (SCAR7) was previously linked to chromosome band 11p15. We have identified TPP1 as the causative gene for SCAR7 by exome sequencing. A missense and a splice site variant in TPP1, cosegregating with the disease, were found in a previously described SCAR7 family and also in another patient with a SCAR7 phenotype. TPP1, encoding the tripeptidyl-peptidase 1 enzyme, is known as the causative gene for late infantile neuronal ceroid lipofuscinosis disease 2 (CLN2 disease). CLN2 disease is characterized by epilepsy, loss of vision, ataxia, and a rapidly progressive course, leading to early death. SCAR7 patients showed ataxia and low activity of tripeptidyl-peptidase 1, but no ophthalmologic abnormalities or epilepsy. Also, the slowly progressive evolution of the disease until old age and absence of ultra structural curvilinear profiles is different from the known CLN2 phenotypes. Our findings now expand the phenotypes related to TPP1-variants to SCAR7. In spite of the limited sample size and measurements, a putative genotype-phenotype correlation may be drawn: we hypothesize that loss of function variants abolishing TPP1 enzyme activity lead to CLN2 disease, whereas variants that diminish TPP1 enzyme activity lead to SCAR7.

Item does not contain fulltext 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.

The detection of quantitative changes in genomic DNA, i.e. deletions and duplications or Copy Number Variants (CNVs), has recently gained considerable interest. First, detailed analysis of the human genome showed a surprising amount of CNVs, involving thousands of genes. Second, it was realised that the detection of CNVs as a cause of genetic disease was often neglected, but should be an essential part of a complete screening strategy. In both cases new efficient CNV screening methods, covering the entire range from specific loci to genome-wide, were behind these developments. This paper will briefly review the methods that are available to detect CNVs, discuss their strong and weak points, show some new developments and look ahead. Methods covered include microscopy, fluorescence in situ hybridization (including fiber-FISH), Southern blotting, PCR-based methods (including MLPA), array technology and massive parallel sequencing. In addition, we will show some new developments, including a 1400-plex CNV bead assay, fast-MLPA (from DNA to result in ∼6 h) and a simple Melting Curve Analysis assay to confirm potential CNVs. Using the 1400-plex CNV bead assay, targeting selected chromosomal regions only, we detected confirmed rearrangements in 9% of 320 mental retardation patients studied.

Trisomy of 15q26-qter is frequently associated with tall stature and mental retardation. Here we describe a patient with such trisomy, without a partial monosomy of another chromosome. The tall stature in this patient is most probably caused by duplication of the IGF1R gene. A duplication of the IGF1R gene is not a frequent finding in patients with tall stature. In 38 patients with features of Sotos syndrome without NSD1 alterations, a duplication was found only once. This patient was already known to have an unbalanced 2;15 translocation. Looking for a duplication of the 15qter region is still worth consideration in patients with tall stature and features of Sotos syndrome without an NSD1 alteration, especially when there is craniosynostosis or marked speech delay.

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.

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.European Journal of Human Genetics advance online publication, 28 January 2015; doi:10.1038/ejhg.2014.279.

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.

Background: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. Objective and methods: Array-CGH with approximately 3500 large insert clones spaced at ∼1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. Results: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. Conclusions: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.

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.

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 -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. (c) 2013 Wiley Periodicals, Inc.

De novo germline variants in several components of the SWI/SNF-like BAF complex can cause Coffin-Siris syndrome (CSS), Nicolaides-Baraitser syndrome (NCBRS), and nonsyndromic intellectual disability. We screened 63 patients with a clinical diagnosis of CSS for these genes (ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCB1, and SMARCE1) and identified pathogenic variants in 45 (71%) patients. We found a high proportion of variants in ARID1B (68%). All four pathogenic variants in ARID1A appeared to be mosaic. By using all variants from the Exome Variant Server as test data, we were able to classify variants in ARID1A, ARID1B, and SMARCB1 reliably as being pathogenic or nonpathogenic. For SMARCA2, SMARCA4, and SMARCE1 several variants in the EVS remained unclassified, underlining the importance of parental testing. We have entered all variant and clinical information in LOVD-powered databases to facilitate further genotype-phenotype correlations, as these will become increasingly important because of the uptake of targeted and untargeted next generation sequencing in diagnostics. The emerging phenotype-genotype correlation is that SMARCB1 patients have the most marked physical phenotype and severe cognitive and growth delay. The variability in phenotype seems most marked in ARID1A and ARID1B patients. Distal limbs anomalies are most marked in ARID1A patients and least in SMARCB1 patients. Numbers are small however, and larger series are needed to confirm this correlation., (undefined)

Item does not contain fulltext Congenital central hypothyroidism occurs either in isolation or in conjunction with other pituitary hormone deficits. Using exome and candidate gene sequencing, we identified 8 distinct mutations and 2 deletions in IGSF1 in males from 11 unrelated families with central hypothyroidism, testicular enlargement and variably low prolactin concentrations. IGSF1 is a membrane glycoprotein that is highly expressed in the anterior pituitary gland, and the identified mutations impair its trafficking to the cell surface in heterologous cells. Igsf1-deficient male mice show diminished pituitary and serum thyroid-stimulating hormone (TSH) concentrations, reduced pituitary thyrotropin-releasing hormone (TRH) receptor expression, decreased triiodothyronine concentrations and increased body mass. Collectively, our observations delineate a new X-linked disorder in which loss-of-function mutations in IGSF1 cause central hypothyroidism, likely secondary to an associated impairment in pituitary TRH signaling.

Heterozygous germline mutations in components of switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes were recently identified in patients with non-syndromic intellectual disability, Coffin-Siris syndrome and Nicolaides-Baraitser syndrome. The common denominator of the phenotype of these patients is severe intellectual disability and speech delay. Somatic and germline mutations in SWI/SNF components were previously implicated in tumor development. This raises the question whether patients with intellectual disability caused by SWI/SNF mutations in the germline are exposed to an increased risk of developing cancer. Here we compare the mutational spectrum of SWI/SNF components in intellectual disability syndromes and cancer, and discuss the implications of the results of this comparison for the patients.

Context: During meiosis I, the recombination frequency in the pseudoautosomal region on Xp and Yp (PAR1) in males is very high. As a result, mutated genes located within the PAR1 region can be transferred from the Y-chromosome to the X-chromosome and vice versa. Patients: Here we describe three families with SHOX abnormalities resulting in Leri-Weill dyschondrosteosis or Langer mesomelic dysplasia. Results: In about half of the segregations investigated, a transfer of the SHOX abnormality to the alternate sex chromosome was demonstrated. Conclusions: Patients with an abnormality of the SHOX gene should receive genetic counseling as to the likelihood that they may transmit the mutation or deletion to a son as well as to a daughter.

Mutations in the MICU1 gene have been shown to cause a condition with overlapping clinical and pathological features between muscular dystrophy, congenital myopathy and mitochondrial disease. The molecular defect results in mitochondrial dysfunction secondary to disruption of calcium uptake. We present the clinical/pathological features in a cohort of 18 patients. Patients presented between birth and 8 years with a mild, relatively static, proximal myopathy associated with high Creatinine Kinase (2000–10,000 iu/L), learning difficulties and frequent microcephaly. At follow up (5–28 yrs), all remained ambulant but variable extrapyramidal symptoms had developed in the majority by the end of the 1st decade. Other features suggestive of mitochondrial dysfunction included peripheral neuropathy, icthyosis, stroke like episodes, episodic weakness, ataxia and cataracts. Cardiomyopathy was not seen. Serum and CSF lactate were normal where tested. Two had low free carnitine. Other metabolic investigations were normal. One had abnormal signal change in globus pallidus on MRI at 10 yrs. All had abnormal muscle biopsies, with myopathic features; diffuse variation in fibre size, increased internal and central nuclei and clusters of regenerating fibres without pronounced fibrosis or fatty infiltration. Necrotic fibers were rare and fibre typing was preserved. Focal areas devoid of mitochondria (minicores) were observed in both type I and type II fibres. Respiratory chain enzyme activity, where tested, was normal. All had mutations in the MICU1 gene, 2 different homozygous splice site mutations (12 and 4 cases respectively) and a deletion in a kindred of 2 cases. Although the numbers are small, there appears to be some correlation between genotype and phenotype. Testing for MICU1 mutations should be considered in children with a static proximal myopathy associated with a high CK and CNS involvement when immunohistochemical studies do not identify a protein defect.

Apparently balanced chromosome abnormalities are occasionally associated with mental retardation (MR). These balanced rearrangements may disrupt genes. However, the phenotype may also be caused by small abnormalities present at the breakpoints or elsewhere in the genome. Conventional karyotyping is not instrumental for detecting small abnormalities because it only identifies genomic imbalances larger than 5-10 Mb. In contrast, high-resolution whole-genome arrays enable the detection of submicroscopic abnormalities in patients with apparently balanced rearrangements. Here, we report on the whole-genome analysis of 13 MR patients with previously detected balanced chromosomal abnormalities, five de novo, four inherited, and four of unknown inheritance, using Single Nucleotide Polymorphism (SNP) arrays. In all the cases, the patient had an abnormal phenotype. In one familial case and one unknown inheritance case, one of the parents had a phenotype which appeared identical to the patient's phenotype. Additional copy number variants (CNVs) were identified in eight patients. Three patients contained CNVs adjacent to one or either breakpoints. One of these patients showed four and two deletions near the breakpoints of a de novo pericentric inversion. In five patients we identified CNVs on chromosomes unrelated to the previously observed genomic imbalance. These data demonstrate that high-resolution array screening and conventional karyotyping is necessary to tie complex karyotypes to phenotypes of MR patients. (C) 2010 Elsevier Masson SAS. All rights reserved.

Familial mediterranean fever (FMF) is a hereditary autoinflammatory autosomal recessive disease caused by mutations in the MEFV gene. Despite the identification of many disease associated MEFV mutations, often the clinical diagnosis cannot be genetically confirmed. The currently used diagnostic sequencing techniques only allow the detection of point mutations, small deletions or duplications. The question as to whether larger genetic alterations are also involved in the pathophysiology of FMF remains to be answered. To address this question, we used multiplex ligation-dependent probe amplification (MLPA) on a total of 216 patients with FMF symptoms. This careful analysis revealed that not a single deletion/duplication could be detected in this large cohort of patients. This result suggests that single or multiexon MEFV gene copy number changes do not contribute substantially, if at all, to the MEFV mutation spectrum.

Alpha thalassemia retardation associated with chromosome16 (ATR-16 syndrome) is defined as a contiguous gene syndrome resulting from haploinsufficiency of the alpha-globin gene cluster and genes involved in mental retardation (MR). To date, only few cases have been described which result from pure monosomy for a deletion of 16p. In most of these cases the deletion was identified by densitometric analysis of Southern blot results or by Fluorescent In Situ Hybridization analysis, and these alterations have not been mapped in detail. In this study, we have fine mapped deletions causing alpha-thalassemia within 2 Mb from the telomere of 16p by multiplex ligation-dependent probe amplification (MLPA). We have developed a rapid and simple test for high resolution mapping of rearrangements involving the tip of the short arm of chromosome 16 by incorporating 62 MLPA probes spaced approximately 10-200 kb over a region of 2 Mb from the telomere. One deletion of approximately 900 kb without MR was identified in addition to three de novo deletions varying between 1.5 and 2 Mb causing ATR-16 in three patients having mild MR and alpha-thalassemia. Two were found by chance to be ATR-16 because they were included in a study to search for telomeric loss in MR and not by hematological analysis. This would plead for more alertness when a persistent microcytic hypochromic anemia at normal ferritin levels is observed as suggestive for the ATR-16 syndrome. The region on chromosome 16p for which haploinsufficiency leads to the dysmorphic features and MR typical for ATR-16, has been narrowed down to a 800 kb region localized between 0.9 and 1.7 Mb from the telomere.

The presence of highly homologous sequences, known as low copy repeats, predisposes for unequal recombination within the 22q11 region. This can lead to genomic imbalances associated with several known genetic disorders. We report here a developmentally delayed patient carrying different rearrangements on both chromosome 22 homologues, including a previously unreported rearrangement within the 22q11 region. One homologue carries a deletion of the proximal part of chromosome band 22q11. To our knowledge, a ‘pure’ deletion of this region has not been described previously. Four copies of this 22q11 region, however, are associated with Cat eye syndrome (CES). While the phenotypic impact of this deletion is unclear, familial investigation revealed five normal relatives carrying this deletion, suggesting that haplo-insufficiency of the CES region has little clinical relevance. The other chromosome 22 homologue carries a duplication of the Velocardiofacial/DiGeorge syndrome (VCFS/DGS) region. In addition, a previously undescribed deletion of 22q12.1, located in a relatively gene-poor region, was identified. As the clinical features of patients suffering from a duplication of the VCFS/DGS region have proven to be extremely variable, it is impossible to postulate as to the contribution of the 22q12.1 deletion to the phenotype of the patient. Additional patients with a deletion within this region are needed to establish the consequences of this copy number alteration. This study highlights the value of using different genomic approaches to unravel chromosomal alterations in order to study their phenotypic impact.

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.

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.

Summary 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.

Item does not contain fulltext 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.

Background Congenital central hypothyroidism occurs either as isolated thyroid-stimulating hormone (TSH) deficiency or in conjunction with other pituitary hormone deficits. Undetected central hypothyroidism is associated with developmental delay in children and adverse cardiometabolic sequelae in adults. Hitherto, mutations in the thyrotropin-releasing hormone receptor gene ( TRHR ) or the TSHb subunit gene ( TSHB ) are the only known causes of isolated TSH deficiency. Methods Using whole exome and candidate gene sequencing, we have studied 11 unrelated families with males exhibiting isolated TSH deficiency, testicular enlargement, and variably low serum prolactin levels. Findings We have identified eight distinct mutations and two whole gene deletions disrupting the X-linked immunoglobulin superfamily member 1 gene ( IGSF1 ) in affected males. IGSF1 encodes a pituitary-enriched plasma membrane glycoprotein; disease-associated mutations block trafficking of IGSF1 from the endoplasmic reticulum to the membrane, consistent with loss-of-protein function. Adult male IGSF1 null mice exhibit central hypothyroidism with decreased pituitary TSH content and circulating T3 levels; TSH secretion in response to TRH is blunted and pituitary TRHR mRNA levels are decreased, suggesting that impaired TRH signalling may provide the basis for hypothyroidism. Interpretation Our observations delineate a novel X-linked syndrome in which loss-of-function mutations in IGSF1 cause central hypothyroidism, testicular enlargement, and variable prolactin deficiency, and identify a previously unsuspected role for IGSF1 in hypothalamic-pituitary control of thyroid and testicular function. Variable biochemical penetrance in these families highlights the importance of genetic ascertainment in this syndrome, so that asymptomatic affected individuals can benefit from early initiation of thyroxine treatment. Funding Wellcome Trust and National Institute for Health Research Biomedical Research Centre.

We report a father and son with hydrocephalus, dysmorphic features and demyelinating neuropathy in whom whole exome sequencing revealed a de novo missense mutation in one of the highly conserved aminoacyl-tRNA synthetase (AARS) genes. Father was born with dysmorphic facial features and hypospadias. At 3 months he developed signs of communicating hydrocephalus treated with lumboperitoneal and ventriculoperitoneal (VP) shunting. Cognitive and motor development was delayed and running never achieved. Tendon reflexes disappeared in puberty when he complained of difficulty walking. Examination showed high arched feet and hammer toes. At 17 years, EMG revealed a demyelinating motor sensory neuropathy with nerve conduction velocities (NCV) of 20 m/s and conduction blocks. Treatment with immunoglobulins and prednisone was unsuccessful. Neurological examination, EMG and genetic studies in both parents were normal. His son was born with similar facial features and hypospadias. Prenatal ultrasound studies had been normal. Normal skull circumference at birth grew to +2.5 SD within 1 month. MRI revealed a communicating hydrocephalus treated with third ventriculostomy and VP shunting. At 17 months he underwent surgery for right coronal suture craniosynostosis. At 2.5 years, he was unable to walk without support. Hydrocephalus was marked by high CSF production (50 cc/h) and low intracranial pressure (7 cm H2O). Although EMG did not meet criteria of a demyelinating neuropathy, motor NCV of the tibial and peroneal nerve and sensory NCV of the median nerve were below the lower limit (28.1, 35.9 and 36.8 m/s). Relationships between AARS-genes and CMT disease and distal SMA have been shown. Mitochondrial AARS genes have been linked to (leuko-) encephalopathy. This apparently novel syndrome consisting of dysmorphic features, hydrocephalus, hypospadias, demyelinating neuropathy and an autosomal dominant mode of inheritance broadens the spectrum of abnormalities caused by AARS-genes.