DeepPheWAS: an R package for phenotype generation and association analysis for phenome-wide association studies.

SUMMARY: DeepPheWAS is an R package for phenome-wide association studies that creates clinically curated composite phenotypes and integrates quantitative phenotypes from primary care data, longitudinal trajectories of quantitative measures, disease progression and drug response phenotypes. Tools are provided for efficient analysis of association with any genetic input, under any genetic model, with optional sex-stratified analysis, and for developing novel phenotypes. AVAILABILITY AND IMPLEMENTATION: The DeepPheWAS R package is freely available under GNU general public licence v3.0 from at https://github.com/Richard-Packer/DeepPheWAS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TMX2 Is a Crucial Regulator of Cellular Redox State, and Its Dysfunction Causes Severe Brain Developmental Abnormalities.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain.

Missense Mutations of the Pro65 Residue of PCGF2 Cause a Recognizable Syndrome Associated with Craniofacial, Neurological, Cardiovascular, and Skeletal Features.

PCGF2 encodes the polycomb group ring finger 2 protein, a transcriptional repressor involved in cell proliferation, differentiation, and embryogenesis. PCGF2 is a component of the polycomb repressive complex 1 (PRC1), a multiprotein complex which controls gene silencing through histone modification and chromatin remodelling. We report the phenotypic characterization of 13 patients (11 unrelated individuals and a pair of monozygotic twins) with missense mutations in PCGF2. All the mutations affected the same highly conserved proline in PCGF2 and were de novo, excepting maternal mosaicism in one. The patients demonstrated a recognizable facial gestalt, intellectual disability, feeding problems, impaired growth, and a range of brain, cardiovascular, and skeletal abnormalities. Computer structural modeling suggests the substitutions alter an N-terminal loop of PCGF2 critical for histone biding. Mutant PCGF2 may have dominant-negative effects, sequestering PRC1 components into complexes that lack the ability to interact efficiently with histones. These findings demonstrate the important role of PCGF2 in human development and confirm that heterozygous substitutions of the Pro65 residue of PCGF2 cause a recognizable syndrome characterized by distinctive craniofacial, neurological, cardiovascular, and skeletal features.

SCN3A-Related Neurodevelopmental Disorder: A Spectrum of Epilepsy and Brain Malformation.

OBJECTIVE: Pathogenic variants in SCN3A, encoding the voltage-gated sodium channel subunit Nav1.3, cause severe childhood onset epilepsy and malformation of cortical development. Here, we define the spectrum of clinical, genetic, and neuroimaging features of SCN3A-related neurodevelopmental disorder. METHODS: Patients were ascertained via an international collaborative network. We compared sodium channels containing wild-type versus variant Nav1.3 subunits coexpressed with ß1 and ß2 subunits using whole-cell voltage clamp electrophysiological recordings in a heterologous mammalian system (HEK-293T cells). RESULTS: Of 22 patients with pathogenic SCN3A variants, most had treatment-resistant epilepsy beginning in the first year of life (16/21, 76%; median onset, 2 weeks), with severe or profound developmental delay (15/20, 75%). Many, but not all (15/19, 79%), exhibited malformations of cortical development. Pathogenic variants clustered in transmembrane segments 4 to 6 of domains II to IV. Most pathogenic missense variants tested (10/11, 91%) displayed gain of channel function, with increased persistent current and/or a leftward shift in the voltage dependence of activation, and all variants associated with malformation of cortical development exhibited gain of channel function. One variant (p.Ile1468Arg) exhibited mixed effects, with gain and partial loss of function. Two variants demonstrated loss of channel function. INTERPRETATION: Our study defines SCN3A-related neurodevelopmental disorder along a spectrum of severity, but typically including epilepsy and severe or profound developmental delay/intellectual disability. Malformations of cortical development are a characteristic feature of this unusual channelopathy syndrome, present in >75% of affected individuals. Gain of function at the channel level in developing neurons is likely an important mechanism of disease pathogenesis. ANN NEUROL 2020;88:348-362.

Dominant Mutations in GRM1 Cause Spinocerebellar Ataxia Type 44.

The metabotropic glutamate receptor 1 (mGluR1) is abundantly expressed in the mammalian central nervous system, where it regulates intracellular calcium homeostasis in response to excitatory signaling. Here, we describe heterozygous dominant mutations in GRM1, which encodes mGluR1, that are associated with distinct disease phenotypes: gain-of-function missense mutations, linked in two different families to adult-onset cerebellar ataxia, and a de novo truncation mutation resulting in a dominant-negative effect that is associated with juvenile-onset ataxia and intellectual disability. Crucially, the gain-of-function mutations could be pharmacologically modulated in vitro using an existing FDA-approved drug, Nitazoxanide, suggesting a possible avenue for treatment, which is currently unavailable for ataxias.

De novo mutations in GRIN1 cause extensive bilateral polymicrogyria.

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

Non-Cardiac Chest Pain.

We read with interest "Non-Cardiac Chest Pain: Management in the Ambulatory Clinic setting" (Acute Med 2019;18(3)165-70). It is useful to know about the various musculoskeletal causes of chest pain, especially where specific treatment may be available.

A dominant mutation in FBXO38 causes distal spinal muscular atrophy with calf predominance.

Spinal muscular atrophies (SMAs) are a heterogeneous group of inherited disorders characterized by degeneration of anterior horn cells and progressive muscle weakness. In two unrelated families affected by a distinct form of autosomal-dominant distal SMA initially manifesting with calf weakness, we identified by genetic linkage analysis and exome sequencing a heterozygous missense mutation, c.616T>C (p.Cys206Arg), in F-box protein 38 (FBXO38). FBXO38 is a known coactivator of the transcription factor Krüppel-like factor 7 (KLF7), which regulates genes required for neuronal axon outgrowth and repair. The p.Cys206Arg substitution did not alter the subcellular localization of FBXO38 but did impair KLF7-mediated transactivation of a KLF7-responsive promoter construct and endogenous KLF7 target genes in both heterologously expressing human embryonic kidney 293T cells and fibroblasts derived from individuals with the FBXO38 missense mutation. This transcriptional dysregulation was associated with an impairment of neurite outgrowth in primary motor neurons. Together, these results suggest that a transcriptional regulatory pathway that has a well-established role in axonal development could also be critical for neuronal maintenance and highlight the importance of FBXO38 and KLF7 activity in motor neurons.

De novo point mutations in patients diagnosed with ataxic cerebral palsy.

Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies.

Common variants in WFS1 confer risk of type 2 diabetes.

We studied genes involved in pancreatic beta cell function and survival, identifying associations between SNPs in WFS1 and diabetes risk in UK populations that we replicated in an Ashkenazi population and in additional UK studies. In a pooled analysis comprising 9,533 cases and 11,389 controls, SNPs in WFS1 were strongly associated with diabetes risk. Rare mutations in WFS1 cause Wolfram syndrome; using a gene-centric approach, we show that variation in WFS1 also predisposes to common type 2 diabetes.

CGAT: computational genomics analysis toolkit.

Computational genomics seeks to draw biological inferences from genomic datasets, often by integrating and contextualizing next-generation sequencing data. CGAT provides an extensive suite of tools designed to assist in the analysis of genome scale data from a range of standard file formats. The toolkit enables filtering, comparison, conversion, summarization and annotation of genomic intervals, gene sets and sequences. The tools can both be run from the Unix command line and installed into visual workflow builders, such as Galaxy.

Mutations in the autoregulatory domain of ß-tubulin 4a cause hereditary dystonia.

Dystonia type 4 (DYT4) was first described in a large family from Heacham in Norfolk with an autosomal dominantly inherited whispering dysphonia, generalized dystonia, and a characteristic hobby horse ataxic gait. We carried out a genetic linkage analysis in the extended DYT4 family that spanned 7 generations from England and Australia, revealing a single LOD score peak of 6.33 on chromosome 19p13.12-13. Exome sequencing in 2 cousins identified a single cosegregating mutation (p.R2G) in the ß-tubulin 4a (TUBB4a) gene that was absent in a large number of controls. The mutation is highly conserved in the ß-tubulin autoregulatory MREI (methionine-arginine-glutamic acid-isoleucine) domain, highly expressed in the central nervous system, and extensive in vitro work has previously demonstrated that substitutions at residue 2, specifically R2G, disrupt the autoregulatory capability of the wild-type ß-tubulin peptide, affirming the role of the cytoskeleton in dystonia pathogenesis.

Autosomal-recessive cerebellar ataxia caused by a novel ADCK3 mutation that elongates the protein: clinical, genetic and biochemical characterisation.

BACKGROUND: The autosomal-recessive cerebellar ataxias (ARCA) are a clinically and genetically heterogeneous group of neurodegenerative disorders. The large number of ARCA genes leads to delay and difficulties obtaining an exact diagnosis in many patients and families. Ubiquinone (CoQ10) deficiency is one of the potentially treatable causes of ARCAs as some patients respond to CoQ10 supplementation. The AarF domain containing kinase 3 gene (ADCK3) is one of several genes associated with CoQ10 deficiency. ADCK3 encodes a mitochondrial protein which functions as an electron-transfer membrane protein complex in the mitochondrial respiratory chain (MRC). METHODS: We report two siblings from a consanguineous Pakistani family who presented with cerebellar ataxia and severe myoclonus from adolescence. Whole exome sequencing and biochemical assessment of fibroblasts were performed in the index patient. RESULTS: A novel homozygous frameshift mutation in ADCK3 (p.Ser616Leufs*114), was identified in both siblings. This frameshift mutation results in the loss of the stop codon, extending the coding protein by 81 amino acids. Significant CoQ10 deficiency and reduced MRC enzyme activities in the index patient's fibroblasts suggested that the mutant protein may reduce the efficiency of mitochondrial electron transfer. CoQ10 supplementation was initiated following these genetic and biochemical analyses. She gained substantial improvement in myoclonic movements, ataxic gait and dysarthric speech after treatment. CONCLUSION: This study highlights the importance of diagnosing ADCK3 mutations and the potential benefit of treatment for patients. The identification of this new mutation broadens the phenotypic spectrum associated with ADCK3 mutations and provides further understanding of their pathogenic mechanism.

Asthma exacerbations and eosinophilia in the UK Biobank: a genome-wide association study.

Background: Asthma exacerbations reflect disease severity, affect morbidity and mortality, and may lead to declining lung function. Inflammatory endotypes (e.g. T2-high (eosinophilic)) may play a key role in asthma exacerbations. We aimed to assess whether genetic susceptibility underlies asthma exacerbation risk and additionally tested for an interaction between genetic variants and eosinophilia on exacerbation risk. Methods: UK Biobank data were used to perform a genome-wide association study of individuals with asthma and at least one exacerbation compared to individuals with asthma and no history of exacerbations. Individuals with asthma were identified using self-reported data, hospitalisation data and general practitioner records. Exacerbations were identified as either asthma-related hospitalisation, general practitioner record of asthma exacerbation or an oral corticosteroid burst prescription. A logistic regression model adjusted for age, sex, smoking status and genetic ancestry via principal components was used to assess the association between genetic variants and asthma exacerbations. We sought replication for suggestive associations (p<5×10-6) in the GERA cohort. Results: In the UK Biobank, we identified 11 604 cases and 37 890 controls. While no variants reached genome-wide significance (p<5×10-8) in the primary analysis, 116 signals were suggestively significant (p<5×10-6). In GERA, two single nucleotide polymorphisms (rs34643691 and rs149721630) replicated (p<0.05), representing signals near the NTRK3 and ABCA13 genes. Conclusions: Our study has identified reproducible associations with asthma exacerbations in the UK Biobank and GERA cohorts. Confirmation of these findings in different asthma subphenotypes in diverse ancestries and functional investigation will be required to understand their mechanisms of action and potentially inform therapeutic development.

Kohlschütter-Tönz syndrome: mutations in ROGDI and evidence of genetic heterogeneity.

Kohlschütter-Tönz syndrome (KTS) is a rare autosomal recessive disorder characterized by amelogenesis imperfecta, psychomotor delay or regression and seizures starting early in childhood. KTS was established as a distinct clinical entity after the first report by Kohlschütter in 1974, and to date, only a total of 20 pedigrees have been reported. The genetic etiology of KTS remained elusive until recently when mutations in ROGDI were independently identified in three unrelated families and in five likely related Druze families. Herein, we report a clinical and genetic study of 10 KTS families. By using a combination of whole exome sequencing, linkage analysis, and Sanger sequencing, we identify novel homozygous or compound heterozygous ROGDI mutations in five families, all presenting with a typical KTS phenotype. The other families, mostly presenting with additional atypical features, were negative for ROGDI mutations, suggesting genetic heterogeneity of atypical forms of the disease.

The genetics of obesity: FTO leads the way.

In 2007, an association of single nucleotide polymorphisms (SNPs) in the fat mass and obesity-associated (FTO) gene region with body mass index (BMI) and risk of obesity was identified in multiple populations, making FTO the first locus unequivocally associated with adiposity. At the time, FTO was a gene of unknown function and it was not known whether these SNPs exerted their effect on adiposity by affecting FTO or neighboring genes. Therefore, this breakthrough association inspired a wealth of in silico, in vitro, and in vivo analyses in model organisms and humans to improve knowledge of FTO function. These studies suggested that FTO plays a role in controlling feeding behavior and energy expenditure. Here, we review the approaches taken that provide a blueprint for the study of other obesity-associated genes in the hope that this strategy will result in increased understanding of the biological mechanisms underlying body weight regulation.