Magdalena Harakalova

Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness.

Using exome sequencing, we identify SERAC1 mutations as the cause of MEGDEL syndrome, a recessive disorder of dystonia and deafness with Leigh-like syndrome, impaired oxidative phosphorylation and 3-methylglutaconic aciduria. We localized SERAC1 at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. A phospholipid analysis in patient fibroblasts showed elevated concentrations of phosphatidylglycerol-34:1 (where the species nomenclature denotes the number of carbon atoms in the two acyl chains:number of double bonds in the two acyl groups) and decreased concentrations of phosphatidylglycerol-36:1 species, resulting in an altered cardiolipin subspecies composition. We also detected low concentrations of bis(monoacyl-glycerol)-phosphate, leading to the accumulation of free cholesterol, as shown by abnormal filipin staining. Complementation of patient fibroblasts with wild-type human SERAC1 by lentiviral infection led to a decrease and partial normalization of the mean ratio of phosphatidylglycerol-34:1 to phosphatidylglycerol-36:1. Our data identify SERAC1 as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking.

Dominant missense mutations in ABCC9 cause Cantú syndrome

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (KATP) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the KATP channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3–5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5–6 d for array-based enrichment and 3–4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.

X-exome sequencing identifies a HDAC8 variant in a large pedigree with X-linked intellectual disability, truncal obesity, gynaecomastia, hypogonadism and unusual face

Background We present a large Dutch family with seven males affected by a novel syndrome of X-linked intellectual disability, hypogonadism, gynaecomastia, truncal obesity, short stature and recognisable craniofacial manifestations resembling but not identical to Wilson-Turner syndrome. Seven female relatives show a much milder expression of the phenotype. Methods and results We performed X chromosome exome (X-exome) sequencing in five individuals from this family and identified a novel intronic variant in the histone deacetylase 8 gene (HDAC8), c.164+5G>A, which disturbs the normal splicing of exon 2 resulting in exon skipping, and introduces a premature stop at the beginning of the histone deacetylase catalytic domain. The identified variant completely segregates in this family and was absent in 96 Dutch controls and available databases. Affected female carriers showed a notably skewed X-inactivation pattern in lymphocytes in which the mutated X-chromosome was completely inactivated. Conclusions HDAC8 is a member of the protein family of histone deacetylases that play a major role in epigenetic gene silencing during development. HDAC8 specifically controls the patterning of the skull with the mouse HDAC8 knock-out showing craniofacial deformities of the skull. The present family provides the first evidence for involvement of HDAC8 in a syndromic form of intellectual disability.

Monocarboxylate Transporter 1 Deficiency and Ketone Utilization

Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.

Expanding the spectrum of phenotypes associated with germline PIGA mutations : a child with developmental delay, accelerated linear growth, facial dysmorphisms, elevated alkaline phosphatase, and progressive CNS abnormalities

Phosphatidyl inositol glycan (PIG) enzyme subclasses are involved in distinct steps of glycosyl phosphatidyl inositol anchor protein biosynthesis. Glycolsyl phosphatidyl inositol‐anchored proteins have heterogeneous functions; they can function as enzymes, adhesion molecules, complement regulators and co‐receptors in signal transduction pathways. Germline mutations in genes encoding different members of the PIG family result in diverse conditions with (severe) developmental delay, (neonatal) seizures, hypotonia, CNS abnormalities, growth abnormalities, and congenital abnormalities as hallmark features. The variability of clinical features resembles the typical diversity of other glycosylation pathway deficiencies such as the congenital disorders of glycosylation. Here, we report the first germline missense mutation in the PIGA gene associated with accelerated linear growth, obesity, central hypotonia, severe refractory epilepsy, cardiac anomalies, mild facial dysmorphic features, mildly elevated alkaline phosphatase levels, and CNS anomalies consisting of progressive cerebral atrophy, insufficient myelinization, and cortical MRI signal abnormalities. X‐exome sequencing in the proband identified a c.278C>T (p.Pro93Leu) mutation in the PIGA gene. The mother and maternal grandmother were unaffected carriers and the mother showed 100% skewing of the X‐chromosome harboring the mutation. These results together with the clinical similarity of the patient reported here and the previously reported patients with a germline nonsense mutation in PIGA support the determination that this mutation caused the phenotype in this family.

Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy.

A systematic analysis of genetic dilated cardiomyopathy reveals numerous ubiquitously expressed and muscle-specific genes

Despite considerable progress being made in genetic diagnostics for dilated cardiomyopathy (DCM) using panels of the most prevalent genes, the cause remains unsolved in a substantial percentage of patients. We hypothesize that several previously described DCM genes with low or unknown prevalence have been neglected, which, if catalogued, could increase the yield of diagnostic DCM testing. The aim of this study is to catalogue all genetic evidence on DCM comprehensively.

Joubert syndrome: genotyping a Northern European patient cohort

Joubert syndrome (JBS) is a rare neurodevelopmental disorder belonging to the group of ciliary diseases. JBS is genetically heterogeneous, with >20 causative genes identified to date. A molecular diagnosis of JBS is essential for prediction of disease progression and genetic counseling. We developed a targeted next-generation sequencing (NGS) approach for parallel sequencing of 22 known JBS genes plus 599 additional ciliary genes. This method was used to genotype a cohort of 51 well-phenotyped Northern European JBS cases (in some of the cases, Sanger sequencing of individual JBS genes had been performed previously). Altogether, 21 of the 51 cases (41%) harbored biallelic pathogenic mutations in known JBS genes, including 14 mutations not previously described. Mutations in C5orf42 (12%), TMEM67 (10%), and AHI1 (8%) were the most prevalent. C5orf42 mutations result in a purely neurological Joubert phenotype, in one case associated with postaxial polydactyly. Our study represents a population-based cohort of JBS patients not enriched for consanguinity, providing insight into the relative importance of the different JBS genes in a Northern European population. Mutations in C5orf42 are relatively frequent (possibly due to a Dutch founder mutation) and mutations in CEP290 are underrepresented compared with international cohorts. Furthermore, we report a case with heterozygous mutations in CC2D2A and B9D1, a gene associated with the more severe Meckel–Gruber syndrome that was recently published as a potential new JBS gene, and discuss the significance of this finding.

Incomplete segregation of MYH11 variants with thoracic aortic aneurysms and dissections and patent ductus arteriosus

Thoracic aortic aneurysms and dissections (TAAD) is a serious condition with high morbidity and mortality. It is estimated that 20% of non-syndromic TAAD cases are inherited in an autosomal-dominant pattern with variable expression and reduced penetrance. Mutations in myosin heavy chain 11 (MYH11), one of several identified TAAD genes, were shown to simultaneously cause TAAD and patent ductus arteriosus (PDA). We identified two large Dutch families with TAAD/PDA and detected two different novel heterozygote MYH11 variants in the probands. These variants, a heterozygote missense variant and a heterozygote in-frame deletion, were predicted to have damaging effects on protein structure and function. However, these novel alterations did not segregate with the TAAD/PDA in 3 out of 11 cases in family TAAD01 and in 2 out of 6 cases of family TAAD02. No mutation was detected in other known TAAD genes. Thus, it is expected that within these families other genetic factors contribute to the disease either by themselves or by interacting with the MYH11 variants. Such an oligogenic model for TAAD would explain the variable onset and progression of the disorder and its reduced penetrance in general. We conclude that in familial TAAD/PDA with an MYH11 variant in the index case caution should be exercised upon counseling family members. Specialized surveillance should still be offered to the non-carriers to prevent catastrophic aortic dissections or ruptures. Furthermore, our study underscores that segregation analysis remains very important in clinical genetics. Prediction programs and mutation evaluation algorithms need to be interpreted with caution.