Bi-allelic Mutations in NDUFA6 Establish Its Role in Early-Onset Isolated Mitochondrial Complex I Deficiency.

Isolated complex I deficiency is a common biochemical phenotype observed in pediatric mitochondrial disease and often arises as a consequence of pathogenic variants affecting one of the ∼65 genes encoding the complex I structural subunits or assembly factors. Such genetic heterogeneity means that application of next-generation sequencing technologies to undiagnosed cohorts has been a catalyst for genetic diagnosis and gene-disease associations. We describe the clinical and molecular genetic investigations of four unrelated children who presented with neuroradiological findings and/or elevated lactate levels, highly suggestive of an underlying mitochondrial diagnosis. Next-generation sequencing identified bi-allelic variants in NDUFA6, encoding a 15 kDa LYR-motif-containing complex I subunit that forms part of the Q-module. Functional investigations using subjects' fibroblast cell lines demonstrated complex I assembly defects, which were characterized in detail by mass-spectrometry-based complexome profiling. This confirmed a marked reduction in incorporated NDUFA6 and a concomitant reduction in other Q-module subunits, including NDUFAB1, NDUFA7, and NDUFA12. Lentiviral transduction of subjects' fibroblasts showed normalization of complex I. These data also support supercomplex formation, whereby the ∼830 kDa complex I intermediate (consisting of the P- and Q-modules) is in complex with assembled complex III and IV holoenzymes despite lacking the N-module. Interestingly, RNA-sequencing data provided evidence that the consensus RefSeq accession number does not correspond to the predominant transcript in clinically relevant tissues, prompting revision of the NDUFA6 RefSeq transcript and highlighting not only the importance of thorough variant interpretation but also the assessment of appropriate transcripts for analysis.

Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data.

PURPOSE: Spinal muscular atrophy (SMA), caused by loss of the SMN1 gene, is a leading cause of early childhood death. Due to the near identical sequences of SMN1 and SMN2, analysis of this region is challenging. Population-wide SMA screening to quantify the SMN1 copy number (CN) is recommended by the American College of Medical Genetics and Genomics. METHODS: We developed a method that accurately identifies the CN of SMN1 and SMN2 using genome sequencing (GS) data by analyzing read depth and eight informative reference genome differences between SMN1/2. RESULTS: We characterized SMN1/2 in 12,747 genomes, identified 1568 samples with SMN1 gains or losses and 6615 samples with SMN2 gains or losses, and calculated a pan-ethnic carrier frequency of 2%, consistent with previous studies. Additionally, 99.8% of our SMN1 and 99.7% of SMN2 CN calls agreed with orthogonal methods, with a recall of 100% for SMA and 97.8% for carriers, and a precision of 100% for both SMA and carriers. CONCLUSION: This SMN copy-number caller can be used to identify both carrier and affected status of SMA, enabling SMA testing to be offered as a comprehensive test in neonatal care and an accurate carrier screening tool in GS sequencing projects.

Male child with somatic mosaic Osteopathia Striata with Cranial Sclerosis caused by a novel pathogenic AMER1 frameshift mutation.

Osteopathia striata with cranial sclerosis (OSCS; OMIM #300373) is a rare X-linked dominant condition caused by mutations in the AMER1 gene (also known as WTX or FAM123B). It is a condition which usually affects females in whom the clinical phenotype can be extremely variable. Conversely affected males typically die in utero or during the neonatal period [Perdu et al. (); Clinical Genetics 80: 383-388; Vasiljevic et al. (); Prenatal Diagnosis 35: 302-304]. There have been a small number of reported cases of surviving males, including three patients who are somatic mosaic for the condition [Chénier, Noor, Dupuis, Stavropoulos, & Mendoza-Londono, (); American Journal of Medical Genetics Part A 158A: 2946-2952; Holman et al. (); American Journal of Medical Genetics Part A 155A: 2397-2408; Joseph, Shoji, & Econs, (); The Journal of Clinical Endocrinology and Metabolism 95: 1506-1507]. We report a case of a male child who has proven somatic mosaicism for OSCS associated with a novel pathogenic frameshift mutation, c.607_611delAGGCC (p.Arg203 fs) in AMER1. We describe the multisystemic clinical features which include macrocephaly with ventriculomegaly and requirement for ventriculoperitoneal shunt, cleft palate, and respiratory difficulties after birth requiring tracheostomy insertion, persistent patent ductus arteriosus, failure to thrive and gastrostomy insertion, growth retardation, ophthalmoplegia, kidney malformation, cryptorchidism, and developmental delay. The use of new technologies with next generation sequencing (NGS) may improve the detection rate of mosaicism in rare conditions.

Molecularly proven mosaicism in phenotypically normal parent of a girl with Freeman-Sheldon Syndrome caused by a pathogenic MYH3 mutation.

We report a case of a female child who has classical Freeman-Sheldon syndrome (FSS) associated with a previously reported recurrent pathogenic heterozygous missense mutation, c.2015G > A, p. (Arg672His), in MYH3 where the phenotypically normal mother is a molecularly confirmed mosaic. To the best of our knowledge, this is the first report in the medical literature of molecularly confirmed parental mosaicism for a MYH3 mutation causing FSS. Since proven somatic mosaicism after having an affected child is consistent with gonadal mosaicism, a significantly increased recurrence risk is advised. Parental testing is thus essential for accurate risk assessment for future pregnancies and the use of new technologies with next generation sequencing (NGS) may improve the detection rate of mosaicism. © 2016 Wiley Periodicals, Inc.

Closing the tau loop: the missing tau mutation.

Frontotemporal lobar degeneration comprises a group of disorders characterized by behavioural, executive, language impairment and sometimes features of parkinsonism and motor neuron disease. In 1994 we described an Irish-American family with frontotemporal dementia linked to chromosome 17 associated with extensive tau pathology. We named this disinhibition-dementia-parkinsonism-amyotrophy complex. We subsequently identified mutations in the MAPT gene. Eleven MAPT gene splice site stem loop mutations were identified over time except for 5' splice site of exon 10. We recently identified another Irish family with autosomal dominant early amnesia and behavioural change or parkinsonism associated with the 'missing' +15 mutation at the intronic boundary of exon 10. We performed a clinical, neuropsychological and neuroimaging study on the proband and four siblings, including two affected siblings. We sequenced MAPT and performed segregation analysis. We looked for a biological effect of the tau variant by performing real-time polymerase chain reaction analysis of RNA extracted from human embryonic kidney cells transfected with exon trapping constructs. We found a c.915+15A>C exon 10/intron 10 stem loop mutation in all affected subjects but not in the unaffected. The c.915+15A>C variant caused a shift in tau splicing pattern to a predominantly exon 10+ pattern presumably resulting in predominant 4 repeat tau and little 3 repeat tau. This strongly suggests that the c.915+15A>C variant is a mutation and that it causes frontotemporal dementia linked to chromosome 17 in this pedigree by shifting tau transcription and translation to +4 repeat tau. Tau (MAPT) screening should be considered in families where amnesia or atypical parkinsonism coexists with behavioural disturbance early in the disease process. We describe the final missing stem loop tau mutation predicted 15 years ago. Mutations have now been identified at all predicted sites within the 'stem' when the stem-loop model was first proposed and no mutations have been found within the 'loop' region as expected. Therefore we 'close the tau loop' having 'opened the loop' 21 years ago.

Integrins and the actin cytoskeleton.

The ability to connect to the actin cytoskeleton is a key part of the adhesive function of integrins. This linkage between integrins and the cytoskeleton involves a large complex of integrin-associated proteins that function in both the assembly and disassembly of the link. Genetic evidence has helped to clarify the relative contributions of different components of this link. In different contexts integrins can either stimulate or suppress actin based structures, indicating the variety of pathways leading from integrins to the cytoskeleton. The cytoskeleton also contributes to the extent of the integrin junction, allowing an adhesive contact to attain sufficient strength to resist contractile forces involved in cellular movement and function.

Morphological evolution through multiple cis-regulatory mutations at a single gene.

One central, and yet unsolved, question in evolutionary biology is the relationship between the genetic variants segregating within species and the causes of morphological differences between species. The classic neo-darwinian view postulates that species differences result from the accumulation of small-effect changes at multiple loci. However, many examples support the possible role of larger abrupt changes in the expression of developmental genes in morphological evolution. Although this evidence might be considered a challenge to a neo-darwinian micromutationist view of evolution, there are currently few examples of the actual genes causing morphological differences between species. Here we examine the genetic basis of a trichome pattern difference between Drosophila species, previously shown to result from the evolution of a single gene, shavenbaby (svb), probably through cis-regulatory changes. We first identified three distinct svb enhancers from D. melanogaster driving reporter gene expression in partly overlapping patterns that together recapitulate endogenous svb expression. All three homologous enhancers from D. sechellia drive expression in modified patterns, in a direction consistent with the evolved svb expression pattern. To test the influence of these enhancers on the actual phenotypic difference, we conducted interspecific genetic mapping at a resolution sufficient to recover multiple intragenic recombinants. This functional analysis revealed that independent genetic regions upstream of svb that overlap the three identified enhancers are collectively required to generate the D. sechellia trichome pattern. Our results demonstrate that the accumulation of multiple small-effect changes at a single locus underlies the evolution of a morphological difference between species. These data support the view that alleles of large effect that distinguish species may sometimes reflect the accumulation of multiple mutations of small effect at select genes.

Refinements and considerations for trio whole-genome sequence analysis when investigating Mendelian diseases presenting in early childhood.

To facilitate early deployment of whole-genome sequencing (WGS) for severely ill children, a standardized pipeline for WGS analysis with timely turnaround and primary care pediatric uptake is needed. We developed a bioinformatics pipeline for comprehensive gene-agnostic trio WGS analysis of children suspected of having an undiagnosed monogenic disease that included detection and interpretation of primary genetic mechanisms of disease, including SNVs/indels, CNVs/SVs, uniparental disomy (UPD), imprinted genes, short tandem repeat expansions, mobile element insertions, SMN1/2 copy number calling, and mitochondrial genome variants. We assessed primary care practitioner experience and competence in a large cohort of 521 families (comprising 90% WGS trios). Children were identified by primary practitioners for recruitment, and we used the UK index of multiple deprivation to confirm lack of patient socio-economic status ascertainment bias. Of the 521 children sequenced, 176 (34%) received molecular diagnoses, with rates as high as 45% for neurology clinics. Twenty-three of the diagnosed cases (13%) required bespoke methods beyond routine SNV/CNV analysis. In our multidisciplinary clinician user experience assessment, both pediatricians and clinical geneticists expressed strong support for rapid WGS early in the care pathway, but requested further training in determining patient selection, consenting, and variant interpretation. Rapid trio WGS provides an efficacious single-pass screening test for children when deployed by primary practitioners in clinical settings that carry high a priori risk for rare pediatric disease presentations.

Cell-matrix adhesion: the Wech connection.

Integrins link the extracellular matrix to the cytoskeleton via a complex of proteins: the integrin-cytoskeleton link. A recent study in Drosophila has uncovered a new component of the link, Wech, and shown that it is essential for integrin-mediated adhesion.

The integrin adhesion complex changes its composition and function during morphogenesis of an epithelium.

Cell adhesion to the extracellular matrix (ECM) is mediated by the integrin family of transmembrane receptors. Integrins link ECM ligands to the cytoskeleton, providing strong attachment to enable cell-shape change and tissue integrity. This connection is made possible by an intracellular complex of proteins, which links to actin filaments and controls signalling cascades that regulate cytoskeletal rearrangements. We have identified stress-fibre-associated focal adhesions that change their composition during tissue morphogenesis. Early expression of alphaPS1betaPS integrin decreases the levels of the actin-nucleating factors Enabled, Diaphanous and profilin, as well as downregulating the amount of F-actin incorporated into the stress fibres. As follicle cells mature in their developmental pathway and become squamous, the integrin in the focal adhesions changes from alphaPS1betaPS to alphaPS2betaPS. During the switch, stress fibres increase their length and change orientation, first changing by 90 degrees and then reorienting back. The normal rapid reorientation requires new expression of alphaPS2betaPS, which also permits recruitment of the adaptor protein tensin. Unexpectedly, it is the extracellular portion of the alphaPS2 subunit that provides the specificity for intracellular recruitment of tensin. Molecular variation of the integrin complex is thus a key component of developmentally programmed morphogenesis.

Dmoesin controls actin-based cell shape and polarity during Drosophila melanogaster oogenesis.

Ezrin, Radixin and Moesin (ERM) proteins are thought to constitute a bridge between the actin cytoskeleton and the plasma membrane (PM). Here we report a genetic analysis of Dmoesin, the sole member of the ERM family in Drosophila. We show that Dmoesin is required during oogenesis for anchoring microfilaments to the oocyte cortex. Alteration of the actin cytoskeleton resulting from Dmoesin mutations impairs the localization of maternal determinants, thus disrupting antero-posterior polarity. This study also demonstrates the requirement of Dmoesin for the specific organization of cortical microfilaments in nurse cells and, consequently, mutations in Dmoesin produce severe defects in cell shape.

A clinical, molecular genetics and pathological study of a FTDP-17 family with a heterozygous splicing variant c.823-10G>T at the intron 9/exon 10 of the MAPT gene.

We report the first clinical-radiological-genetic-molecular-pathological study of a kindred with c.823-10G>T MAPT intronic variant (rs63749974) associated with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We describe the clinical spectrum within this family and emphasize the association between MAPT gene variants and motor neuron disease. This report of a second family with FTDP-17 associated with c.823-10G>T MAPT variant strongly supports pathogenicity of the variant and confirms it is a 4-repeat (4R) tauopathy. This intronic point mutation, probably strengthens the polypyrimidine tract and alters the splicing of exon 10 (10 nucleotides into intron 9) close to the 3' splice site.

Regulatory evolution of shavenbaby/ovo underlies multiple cases of morphological parallelism.

Cases of convergent evolution that involve changes in the same developmental pathway, called parallelism, provide evidence that a limited number of developmental changes are available to evolve a particular phenotype. To our knowledge, in no case are the genetic changes underlying morphological convergence understood. However, morphological convergence is not generally assumed to imply developmental parallelism. Here we investigate a case of convergence of larval morphology in insects and show that the loss of particular trichomes, observed in one species of the Drosophila melanogaster species group, has independently evolved multiple times in the distantly related D. virilis species group. We present genetic and gene expression data showing that regulatory changes of the shavenbaby/ovo (svb/ovo) gene underlie all independent cases of this morphological convergence. Our results indicate that some developmental regulators might preferentially accumulate evolutionary changes and that morphological parallelism might therefore be more common than previously appreciated.

Whole genome sequencing reveals that genetic conditions are frequent in intensively ill children.

PURPOSE: With growing evidence that rare single gene disorders present in the neonatal period, there is a need for rapid, systematic, and comprehensive genomic diagnoses in ICUs to assist acute and long-term clinical decisions. This study aimed to identify genetic conditions in neonatal (NICU) and paediatric (PICU) intensive care populations. METHODS: We performed trio whole genome sequence (WGS) analysis on a prospective cohort of families recruited in NICU and PICU at a single site in the UK. We developed a research pipeline in collaboration with the National Health Service to deliver validated pertinent pathogenic findings within 2-3 weeks of recruitment. RESULTS: A total of 195 families had whole genome analysis performed (567 samples) and 21% received a molecular diagnosis for the underlying genetic condition in the child. The phenotypic description of the child was a poor predictor of the gene identified in 90% of cases, arguing for gene agnostic testing in NICU/PICU. The diagnosis affected clinical management in more than 65% of cases (83% in neonates) including modification of treatments and care pathways and/or informing palliative care decisions. A 2-3 week turnaround was sufficient to impact most clinical decision-making. CONCLUSIONS: The use of WGS in intensively ill children is acceptable and trio analysis facilitates diagnoses. A gene agnostic approach was effective in identifying an underlying genetic condition, with phenotypes and symptomatology being primarily used for data interpretation rather than gene selection. WGS analysis has the potential to be a first-line diagnostic tool for a subset of intensively ill children.

Complex structural variants in Mendelian disorders: identification and breakpoint resolution using short- and long-read genome sequencing.

BACKGROUND: Studies have shown that complex structural variants (cxSVs) contribute to human genomic variation and can cause Mendelian disease. We aimed to identify cxSVs relevant to Mendelian disease using short-read whole-genome sequencing (WGS), resolve the precise variant configuration and investigate possible mechanisms of cxSV formation. METHODS: We performed short-read WGS and analysis of breakpoint junctions to identify cxSVs in a cohort of 1324 undiagnosed rare disease patients. Long-read WGS and gene expression analysis were used to resolve one case. RESULTS: We identified three pathogenic cxSVs: a de novo duplication-inversion-inversion-deletion affecting ARID1B, a de novo deletion-inversion-duplication affecting HNRNPU and a homozygous deletion-inversion-deletion affecting CEP78. Additionally, a de novo duplication-inversion-duplication overlapping CDKL5 was resolved by long-read WGS demonstrating the presence of both a disrupted and an intact copy of CDKL5 on the same allele, and gene expression analysis showed both parental alleles of CDKL5 were expressed. Breakpoint analysis in all the cxSVs revealed both microhomology and longer repetitive elements. CONCLUSIONS: Our results corroborate that cxSVs cause Mendelian disease, and we recommend their consideration during clinical investigations. We show that resolution of breakpoints can be critical to interpret pathogenicity and present evidence of replication-based mechanisms in cxSV formation.

The Ovo/Shavenbaby transcription factor specifies actin remodelling during epidermal differentiation in Drosophila.

In Drosophila, differentiation of the epidermis results in a stereotyped array of cells with F-actin-based extensions at their apical face. We identified Ovo/Shavenbaby (Svb) as a transcription factor that governs changes in epidermal cell shape. Svb is required for the formation of apical extensions and cells deficient in svb differentiate a smooth surface. In both the embryo and the adult, we show that Svb is necessary and sufficient for the cells to grow extensions and that the tight regulation of ovo/svb activity is critical for morphogenesis to occur correctly. We establish that Svb triggers early F-actin redistribution and is able to initiate the entire process of cytoskeletal remodelling, thereby defining it as a major regulator of epidermal differentiation.

Malignant peripheral nerve sheath tumor in cowden syndrome: a first report.

Malignant peripheral nerve sheath tumor is a rare malignancy, accounting for 3% to 10% of all soft-tissue sarcomas. We describe a previously healthy 48-year-old man who was diagnosed as having a high-grade malignant neoplasm involving the facial nerve in the right petrous canal after a 4-year history of deafness. The tumor was resected; histologic appearance and immunophenotype, including patchy but strong positivity for S100 protein, indicated a diagnosis of malignant peripheral nerve sheath tumor. A PTEN mutation, c.1003C>T p.(Arg335Ter), was subsequently identified as the cause of Cowden syndrome in another family member (a nephew) with dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos disease), and genetic testing in the proband's daughter indicated that he was an obligate carrier of the mutation. Sequencing of the tumor showed homozygosity for c.1003C>T, confirming the presence of a germline mutation and implying loss of the second allele. With the exception of Lhermitte-Duclos disease, tumors of the nervous system are not a prominent feature of Cowden syndrome, and this is the first report of malignant peripheral nerve sheath tumor in Cowden syndrome. Sequencing results in the tumor lend evidence to PTEN gene inactivation being implicated in tumorigenesis in this case, suggesting causality rather than chance association.

Downstream of identity genes: muscle-type-specific regulation of the fusion process.

In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. Here, to understand how cell fate information residing within founders is translated during differentiation, we focus on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Mp20, Pax, and mspo, three genes that regulate actin dynamics and cell adhesion and, as we show here, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation.