Publications by authors named "Daniel MacArthur"

176 Publications

Novel variants in TUBA1A cause congenital fibrosis of the extraocular muscles with or without malformations of cortical brain development.

Eur J Hum Genet 2021 Mar 1. Epub 2021 Mar 1.

F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA.

Variants in multiple tubulin genes have been implicated in neurodevelopmental disorders, including malformations of cortical development (MCD) and congenital fibrosis of the extraocular muscles (CFEOM). Distinct missense variants in the beta-tubulin encoding genes TUBB3 and TUBB2B cause MCD, CFEOM, or both, suggesting substitution-specific mechanisms. Variants in the alpha tubulin-encoding gene TUBA1A have been associated with MCD, but not with CFEOM. Using exome sequencing (ES) and genome sequencing (GS), we identified 3 unrelated probands with CFEOM who harbored novel heterozygous TUBA1A missense variants c.1216C>G, p.(His406Asp); c.467G>A, p.(Arg156His); and c.1193T>G, p.(Met398Arg). MRI revealed small oculomotor-innervated muscles and asymmetrical caudate heads and lateral ventricles with or without corpus callosal thinning. Two of the three probands had MCD. Mutated amino acid residues localize either to the longitudinal interface at which α and β tubulins heterodimerize (Met398, His406) or to the lateral interface at which tubulin protofilaments interact (Arg156), and His406 interacts with the motor domain of kinesin-1. This series of individuals supports TUBA1A variants as a cause of CFEOM and expands our knowledge of tubulinopathies.
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http://dx.doi.org/10.1038/s41431-020-00804-7DOI Listing
March 2021

Author Correction: Evaluating drug targets through human loss-of-function genetic variation.

Nature 2021 Feb;590(7846):E56

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41586-020-03177-5DOI Listing
February 2021

Author Correction: Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes.

Nat Commun 2021 Feb 2;12(1):827. Epub 2021 Feb 2.

Program in Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

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http://dx.doi.org/10.1038/s41467-021-21077-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854597PMC
February 2021

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay, seizures, and hypotonia.

Genet Med 2021 Jan 20. Epub 2021 Jan 20.

Department of Pediatrics, Division of Genetic Medicine, University of Washington, Seattle, WA, USA.

Purpose: Neurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene.

Methods: Through large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual.

Results: We identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and 1 was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy.

Conclusion: This collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.
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http://dx.doi.org/10.1038/s41436-020-01076-8DOI Listing
January 2021

Autosomal recessive variants in alter the γ-tubulin ring complex leading to neurodevelopmental disease.

iScience 2021 Jan 30;24(1):101948. Epub 2020 Dec 30.

John Walton Muscular Dystrophy Research Centre, Institute of Translational and Clinical Research, Newcastle University, Newcastle upon Tyne, UK.

Microtubules help building the cytoskeleton of neurons and other cells. Several components of the gamma-tubulin (γ-tubulin) complex have been previously reported in human neurodevelopmental diseases. We describe two siblings from a consanguineous Turkish family with dysmorphic features, developmental delay, brain malformation, and epilepsy carrying a homozygous mutation (p.Glu311Lys) in encoding the γ-tubulin complex 2 (GCP2) protein. This variant is predicted to disrupt the electrostatic interaction of GCP2 with GCP3. In primary fibroblasts carrying the variant, we observed a faint delocalization of γ-tubulin during the cell cycle but normal GCP2 protein levels. Through mass spectrometry, we observed dysregulation of multiple proteins involved in the assembly and organization of the cytoskeleton and the extracellular matrix, controlling cellular adhesion and of proteins crucial for neuronal homeostasis including axon guidance. In summary, our functional and proteomic studies link TUBGCP2 and the γ-tubulin complex to the development of the central nervous system in humans.
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http://dx.doi.org/10.1016/j.isci.2020.101948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7797523PMC
January 2021

Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations.

Authors:
Dervla M Connaughton Rufeng Dai Danielle J Owen Jonathan Marquez Nina Mann Adda L Graham-Paquin Makiko Nakayama Etienne Coyaud Estelle M N Laurent Jonathan R St-Germain Lot Snijders Blok Arianna Vino Verena Klämbt Konstantin Deutsch Chen-Han Wilfred Wu Caroline M Kolvenbach Franziska Kause Isabel Ottlewski Ronen Schneider Thomas M Kitzler Amar J Majmundar Florian Buerger Ana C Onuchic-Whitford Mao Youying Amy Kolb Daanya Salmanullah Evan Chen Amelie T van der Ven Jia Rao Hadas Ityel Steve Seltzsam Johanna M Rieke Jing Chen Asaf Vivante Daw-Yang Hwang Stefan Kohl Gabriel C Dworschak Tobias Hermle Mariëlle Alders Tobias Bartolomaeus Stuart B Bauer Michelle A Baum Eva H Brilstra Thomas D Challman Jacob Zyskind Carrie E Costin Katrina M Dipple Floor A Duijkers Marcia Ferguson David R Fitzpatrick Roger Fick Ian A Glass Peter J Hulick Antonie D Kline Ilona Krey Selvin Kumar Weining Lu Elysa J Marco Ingrid M Wentzensen Heather C Mefford Konrad Platzer Inna S Povolotskaya Juliann M Savatt Natalia V Shcherbakova Prabha Senguttuvan Audrey E Squire Deborah R Stein Isabelle Thiffault Victoria Y Voinova Michael J G Somers Michael A Ferguson Avram Z Traum Ghaleb H Daouk Ankana Daga Nancy M Rodig Paulien A Terhal Ellen van Binsbergen Loai A Eid Velibor Tasic Hila Milo Rasouly Tze Y Lim Dina F Ahram Ali G Gharavi Heiko M Reutter Heidi L Rehm Daniel G MacArthur Monkol Lek Kristen M Laricchia Richard P Lifton Hong Xu Shrikant M Mane Simone Sanna-Cherchi Andrew D Sharrocks Brian Raught Simon E Fisher Maxime Bouchard Mustafa K Khokha Shirlee Shril Friedhelm Hildebrandt

Am J Hum Genet 2020 10 4;107(4):727-742. Epub 2020 Sep 4.

Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.
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http://dx.doi.org/10.1016/j.ajhg.2020.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536580PMC
October 2020

Pathogenic deep intronic MTM1 variant activates a pseudo-exon encoding a nonsense codon resulting in severe X-linked myotubular myopathy.

Eur J Hum Genet 2021 Jan 29;29(1):61-66. Epub 2020 Aug 29.

Kids Neuroscience Centre, Kids Research Institute, The Children's Hospital at Westmead, Westmead, NSW, Australia.

X-linked myotubular myopathy (XLMTM) is a severe congenital myopathy characterised by generalised weakness and respiratory insufficiency. XLMTM is associated with pathogenic variants in MTM1; a gene encoding the lipid phosphatase myotubularin. Whole genome sequencing (WGS) of an exome-negative male proband with severe hypotonia, respiratory insufficiency and centralised nuclei on muscle biopsy identified a deep intronic MTM1 variant NG_008199.1(NM_000252.2):c.1468-577A>G, which strengthened a cryptic 5' splice site (A>G substitution at the +5 position). Muscle RNA sequencing was non-diagnostic due to low read depth. Reverse transcription PCR (RT-PCR) of muscle RNA confirmed the c.1468-577A>G variant activates inclusion of a pseudo-exon encoding a premature stop codon into all detected MTM1 transcripts. Western blot analysis establishes deficiency of myotubularin protein, consistent with the severe XLMTM phenotype. We expand the genotypic spectrum of XLMTM and highlight benefits of screening non-coding regions of MTM1 in male probands with phenotypically concordant XLMTM who remain undiagnosed following exome sequencing.
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http://dx.doi.org/10.1038/s41431-020-00715-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852879PMC
January 2021

Sequential targeted exome sequencing of 1001 patients affected by unexplained limb-girdle weakness.

Genet Med 2020 Sep 11;22(9):1478-1488. Epub 2020 Jun 11.

John Walton Muscular Dystrophy Research Centre, Translational and Clinical Research Institute, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.

Purpose: Several hundred genetic muscle diseases have been described, all of which are rare. Their clinical and genetic heterogeneity means that a genetic diagnosis is challenging. We established an international consortium, MYO-SEQ, to aid the work-ups of muscle disease patients and to better understand disease etiology.

Methods: Exome sequencing was applied to 1001 undiagnosed patients recruited from more than 40 neuromuscular disease referral centers; standardized phenotypic information was collected for each patient. Exomes were examined for variants in 429 genes associated with muscle conditions.

Results: We identified suspected pathogenic variants in 52% of patients across 87 genes. We detected 401 novel variants, 116 of which were recurrent. Variants in CAPN3, DYSF, ANO5, DMD, RYR1, TTN, COL6A2, and SGCA collectively accounted for over half of the solved cases; while variants in newer disease genes, such as BVES and POGLUT1, were also found. The remaining well-characterized unsolved patients (48%) need further investigation.

Conclusion: Using our unique infrastructure, we developed a pathway to expedite muscle disease diagnoses. Our data suggest that exome sequencing should be used for pathogenic variant detection in patients with suspected genetic muscle diseases, focusing first on the most common disease genes described here, and subsequently in rarer and newly characterized disease genes.
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http://dx.doi.org/10.1038/s41436-020-0840-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462745PMC
September 2020

The effect of LRRK2 loss-of-function variants in humans.

Nat Med 2020 06 27;26(6):869-877. Epub 2020 May 27.

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Human genetic variants predicted to cause loss-of-function of protein-coding genes (pLoF variants) provide natural in vivo models of human gene inactivation and can be valuable indicators of gene function and the potential toxicity of therapeutic inhibitors targeting these genes. Gain-of-kinase-function variants in LRRK2 are known to significantly increase the risk of Parkinson's disease, suggesting that inhibition of LRRK2 kinase activity is a promising therapeutic strategy. While preclinical studies in model organisms have raised some on-target toxicity concerns, the biological consequences of LRRK2 inhibition have not been well characterized in humans. Here, we systematically analyze pLoF variants in LRRK2 observed across 141,456 individuals sequenced in the Genome Aggregation Database (gnomAD), 49,960 exome-sequenced individuals from the UK Biobank and over 4 million participants in the 23andMe genotyped dataset. After stringent variant curation, we identify 1,455 individuals with high-confidence pLoF variants in LRRK2. Experimental validation of three variants, combined with previous work, confirmed reduced protein levels in 82.5% of our cohort. We show that heterozygous pLoF variants in LRRK2 reduce LRRK2 protein levels but that these are not strongly associated with any specific phenotype or disease state. Our results demonstrate the value of large-scale genomic databases and phenotyping of human loss-of-function carriers for target validation in drug discovery.
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http://dx.doi.org/10.1038/s41591-020-0893-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303015PMC
June 2020

Transcript expression-aware annotation improves rare variant interpretation.

Nature 2020 05 27;581(7809):452-458. Epub 2020 May 27.

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

The acceleration of DNA sequencing in samples from patients and population studies has resulted in extensive catalogues of human genetic variation, but the interpretation of rare genetic variants remains problematic. A notable example of this challenge is the existence of disruptive variants in dosage-sensitive disease genes, even in apparently healthy individuals. Here, by manual curation of putative loss-of-function (pLoF) variants in haploinsufficient disease genes in the Genome Aggregation Database (gnomAD), we show that one explanation for this paradox involves alternative splicing of mRNA, which allows exons of a gene to be expressed at varying levels across different cell types. Currently, no existing annotation tool systematically incorporates information about exon expression into the interpretation of variants. We develop a transcript-level annotation metric known as the 'proportion expressed across transcripts', which quantifies isoform expression for variants. We calculate this metric using 11,706 tissue samples from the Genotype Tissue Expression (GTEx) project and show that it can differentiate between weakly and highly evolutionarily conserved exons, a proxy for functional importance. We demonstrate that expression-based annotation selectively filters 22.8% of falsely annotated pLoF variants found in haploinsufficient disease genes in gnomAD, while removing less than 4% of high-confidence pathogenic variants in the same genes. Finally, we apply our expression filter to the analysis of de novo variants in patients with autism spectrum disorder and intellectual disability or developmental disorders to show that pLoF variants in weakly expressed regions have similar effect sizes to those of synonymous variants, whereas pLoF variants in highly expressed exons are most strongly enriched among cases. Our annotation is fast, flexible and generalizable, making it possible for any variant file to be annotated with any isoform expression dataset, and will be valuable for the genetic diagnosis of rare diseases, the analysis of rare variant burden in complex disorders, and the curation and prioritization of variants in recall-by-genotype studies.
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http://dx.doi.org/10.1038/s41586-020-2329-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334198PMC
May 2020

The mutational constraint spectrum quantified from variation in 141,456 humans.

Nature 2020 05 27;581(7809):434-443. Epub 2020 May 27.

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.
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http://dx.doi.org/10.1038/s41586-020-2308-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334197PMC
May 2020

Evaluating drug targets through human loss-of-function genetic variation.

Nature 2020 05 27;581(7809):459-464. Epub 2020 May 27.

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Naturally occurring human genetic variants that are predicted to inactivate protein-coding genes provide an in vivo model of human gene inactivation that complements knockout studies in cells and model organisms. Here we report three key findings regarding the assessment of candidate drug targets using human loss-of-function variants. First, even essential genes, in which loss-of-function variants are not tolerated, can be highly successful as targets of inhibitory drugs. Second, in most genes, loss-of-function variants are sufficiently rare that genotype-based ascertainment of homozygous or compound heterozygous 'knockout' humans will await sample sizes that are approximately 1,000 times those presently available, unless recruitment focuses on consanguineous individuals. Third, automated variant annotation and filtering are powerful, but manual curation remains crucial for removing artefacts, and is a prerequisite for recall-by-genotype efforts. Our results provide a roadmap for human knockout studies and should guide the interpretation of loss-of-function variants in drug development.
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http://dx.doi.org/10.1038/s41586-020-2267-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272226PMC
May 2020

A structural variation reference for medical and population genetics.

Nature 2020 05 27;581(7809):444-451. Epub 2020 May 27.

Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Structural variants (SVs) rearrange large segments of DNA and can have profound consequences in evolution and human disease. As national biobanks, disease-association studies, and clinical genetic testing have grown increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD) have become integral in the interpretation of single-nucleotide variants (SNVs). However, there are no reference maps of SVs from high-coverage genome sequencing comparable to those for SNVs. Here we present a reference of sequence-resolved SVs constructed from 14,891 genomes across diverse global populations (54% non-European) in gnomAD. We discovered a rich and complex landscape of 433,371 SVs, from which we estimate that SVs are responsible for 25-29% of all rare protein-truncating events per genome. We found strong correlations between natural selection against damaging SNVs and rare SVs that disrupt or duplicate protein-coding sequence, which suggests that genes that are highly intolerant to loss-of-function are also sensitive to increased dosage. We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than all noncoding effects. Finally, we identified very large (over one megabase), rare SVs in 3.9% of samples, and estimate that 0.13% of individuals may carry an SV that meets the existing criteria for clinically important incidental findings. This SV resource is freely distributed via the gnomAD browser and will have broad utility in population genetics, disease-association studies, and diagnostic screening.
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http://dx.doi.org/10.1038/s41586-020-2287-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334194PMC
May 2020

Characterising the loss-of-function impact of 5' untranslated region variants in 15,708 individuals.

Nat Commun 2020 05 27;11(1):2523. Epub 2020 May 27.

National Heart and Lung Institute and MRC London Institute of Medical Sciences, Imperial College London, Du Cane Road, London, W12 0NN, UK.

Upstream open reading frames (uORFs) are tissue-specific cis-regulators of protein translation. Isolated reports have shown that variants that create or disrupt uORFs can cause disease. Here, in a systematic genome-wide study using 15,708 whole genome sequences, we show that variants that create new upstream start codons, and variants disrupting stop sites of existing uORFs, are under strong negative selection. This selection signal is significantly stronger for variants arising upstream of genes intolerant to loss-of-function variants. Furthermore, variants creating uORFs that overlap the coding sequence show signals of selection equivalent to coding missense variants. Finally, we identify specific genes where modification of uORFs likely represents an important disease mechanism, and report a novel uORF frameshift variant upstream of NF2 in neurofibromatosis. Our results highlight uORF-perturbing variants as an under-recognised functional class that contribute to penetrant human disease, and demonstrate the power of large-scale population sequencing data in studying non-coding variant classes.
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http://dx.doi.org/10.1038/s41467-019-10717-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253449PMC
May 2020

Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes.

Nat Commun 2020 05 27;11(1):2539. Epub 2020 May 27.

Program in Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA, 02142, USA.

Multi-nucleotide variants (MNVs), defined as two or more nearby variants existing on the same haplotype in an individual, are a clinically and biologically important class of genetic variation. However, existing tools typically do not accurately classify MNVs, and understanding of their mutational origins remains limited. Here, we systematically survey MNVs in 125,748 whole exomes and 15,708 whole genomes from the Genome Aggregation Database (gnomAD). We identify 1,792,248 MNVs across the genome with constituent variants falling within 2 bp distance of one another, including 18,756 variants with a novel combined effect on protein sequence. Finally, we estimate the relative impact of known mutational mechanisms - CpG deamination, replication error by polymerase zeta, and polymerase slippage at repeat junctions - on the generation of MNVs. Our results demonstrate the value of haplotype-aware variant annotation, and refine our understanding of genome-wide mutational mechanisms of MNVs.
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http://dx.doi.org/10.1038/s41467-019-12438-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253413PMC
May 2020

Expanding the disease phenotype of ADSSL1-associated myopathy in non-Korean patients.

Neuromuscul Disord 2020 04 14;30(4):310-314. Epub 2020 Feb 14.

John Walton Muscular Dystrophy Research Centre, Translational and Clinical Research Institute, Newcastle University and Newcastle Hospitals NHS Foundation Trust, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK. Electronic address:

Adenylosuccinate synthase (ADSSL1) is a muscle specific enzyme involved in the purine nucleotide cycle and responsible for the conversion of inosine monophosphate to adenosine monophosphate. Since 2016, when mutations in the ADSSL1 gene were first described to be associated with an adult onset distal myopathy, nine patients with compound heterozygous variants in the ADSSL1 gene, all of Korean origin, have been identified. Here we report a novel ADSSL1 mutation and describe two sporadic cases of Turkish and Indian origin. Many of the clinical features of both patients and muscle histopathology and muscle MRI findings, were in accordance with previously reported findings in the adult onset distal myopathy individuals. However, one of our patients presented with progressive, proximally pronounced weakness, severe muscle atrophy and early contractures. Thus, mutations in ADSSL1 have to be considered in patients with both distal and proximal muscle weakness and across various ethnicities.
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http://dx.doi.org/10.1016/j.nmd.2020.02.006DOI Listing
April 2020

Characterising a healthy adult with a rare HAO1 knockout to support a therapeutic strategy for primary hyperoxaluria.

Elife 2020 03 24;9. Epub 2020 Mar 24.

Blizard Institute and Institute for Population Health Sciences, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.

By sequencing autozygous human populations, we identified a healthy adult woman with lifelong complete knockout of (expected ~1 in 30 million outbred people). (glycolate oxidase) silencing is the mechanism of lumasiran, an investigational RNA interference therapeutic for primary hyperoxaluria type 1. Her plasma glycolate levels were 12 times, and urinary glycolate 6 times, the upper limit of normal observed in healthy reference individuals (n = 67). Plasma metabolomics and lipidomics (1871 biochemicals) revealed 18 markedly elevated biochemicals (>5 sd outliers versus n = 25 controls) suggesting additional HAO1 effects. Comparison with lumasiran preclinical and clinical trial data suggested she has <2% residual glycolate oxidase activity. Cell line p.Leu333SerfsTer4 expression showed markedly reduced HAO1 protein levels and cellular protein mis-localisation. In this woman, lifelong knockout is safe and without clinical phenotype, de-risking a therapeutic approach and informing therapeutic mechanisms. Unlocking evidence from the diversity of human genetic variation can facilitate drug development.
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http://dx.doi.org/10.7554/eLife.54363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108859PMC
March 2020

The genetic and clinical landscape of nanophthalmos and posterior microphthalmos in an Australian cohort.

Clin Genet 2020 05 5;97(5):764-769. Epub 2020 Mar 5.

Department of Ophthalmology, Flinders University, Adelaide, Australia.

Nanophthalmos and posterior microphthalmos are ocular abnormalities in which both eyes are abnormally small, and typically associated with extreme hyperopia. We recruited 40 individuals from 13 kindreds with nanophthalmos or posterior microphthalmos, with 12 probands subjected to exome sequencing. Nine probands (69.2%) were assigned a genetic diagnosis, with variants in MYRF, TMEM98, MFRP, and PRSS56. Two of four PRSS56 families harbored the previously described c.1066dupC variant implicated in over half of all reported PRSS56 kindreds, with different surrounding haplotypes in each family suggesting a mutational hotspot. Individuals with a genetic diagnosis had shorter mean axial lengths and higher hyperopia than those without, with recessive forms associated with the most extreme phenotypes. These findings detail the genetic architecture of nanophthalmos and posterior microphthalmos in a cohort of predominantly European ancestry, their relative clinical phenotypes, and highlight the shared genetic architecture of rare and common disorders of refractive error.
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http://dx.doi.org/10.1111/cge.13722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811993PMC
May 2020

A brief history of human disease genetics.

Nature 2020 01 8;577(7789):179-189. Epub 2020 Jan 8.

Wellcome Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, UK.

A primary goal of human genetics is to identify DNA sequence variants that influence biomedical traits, particularly those related to the onset and progression of human disease. Over the past 25 years, progress in realizing this objective has been transformed by advances in technology, foundational genomic resources and analytical tools, and by access to vast amounts of genotype and phenotype data. Genetic discoveries have substantially improved our understanding of the mechanisms responsible for many rare and common diseases and driven development of novel preventative and therapeutic strategies. Medical innovation will increasingly focus on delivering care tailored to individual patterns of genetic predisposition.
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http://dx.doi.org/10.1038/s41586-019-1879-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405896PMC
January 2020

Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome.

J Am Soc Nephrol 2019 12 15;30(12):2338-2353. Epub 2019 Nov 15.

Renal Division, Department of Medicine, Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany;

Background: Mutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology.

Methods: We used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene and in podocyte-like nephrocytes.

Results: We identified hemizygous missense mutations in the gene in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of in impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the ortholog of nephrin. Expression of RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas overexpression revealed a partial phenotypic overlap to loss of function.

Conclusions: Novel mutations in are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.
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http://dx.doi.org/10.1681/ASN.2019040414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900796PMC
December 2019

Recurrent TTN metatranscript-only c.39974-11T>G splice variant associated with autosomal recessive arthrogryposis multiplex congenita and myopathy.

Hum Mutat 2020 02 3;41(2):403-411. Epub 2019 Dec 3.

Kids Neuroscience Centre, Kids Research, Children's Hospital at Westmead, Westmead, New South Wales, Australia.

We present eight families with arthrogryposis multiplex congenita and myopathy bearing a TTN intron 213 extended splice-site variant (NM_001267550.1:c.39974-11T>G), inherited in trans with a second pathogenic TTN variant. Muscle-derived RNA studies of three individuals confirmed mis-splicing induced by the c.39974-11T>G variant; in-frame exon 214 skipping or use of a cryptic 3' splice-site effecting a frameshift. Confounding interpretation of pathogenicity is the absence of exons 213-217 within the described skeletal muscle TTN N2A isoform. However, RNA-sequencing from 365 adult human gastrocnemius samples revealed that 56% specimens predominantly include exons 213-217 in TTN transcripts (inclusion rate ≥66%). Further, RNA-sequencing of five fetal muscle samples confirmed that 4/5 specimens predominantly include exons 213-217 (fifth sample inclusion rate 57%). Contractures improved significantly with age for four individuals, which may be linked to decreased expression of pathogenic fetal transcripts. Our study extends emerging evidence supporting a vital developmental role for TTN isoforms containing metatranscript-only exons.
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http://dx.doi.org/10.1002/humu.23938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306402PMC
February 2020

Genetic regulatory variation in populations informs transcriptome analysis in rare disease.

Science 2019 10 10;366(6463):351-356. Epub 2019 Oct 10.

New York Genome Center, New York, NY, USA.

Transcriptome data can facilitate the interpretation of the effects of rare genetic variants. Here, we introduce ANEVA (analysis of expression variation) to quantify genetic variation in gene dosage from allelic expression (AE) data in a population. Application of ANEVA to the Genotype-Tissues Expression (GTEx) data showed that this variance estimate is robust and correlated with selective constraint in a gene. Using these variance estimates in a dosage outlier test (ANEVA-DOT) applied to AE data from 70 Mendelian muscular disease patients showed accuracy in detecting genes with pathogenic variants in previously resolved cases and led to one confirmed and several potential new diagnoses. Using our reference estimates from GTEx data, ANEVA-DOT can be incorporated in rare disease diagnostic pipelines to use RNA-sequencing data more effectively.
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http://dx.doi.org/10.1126/science.aay0256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814274PMC
October 2019

Severe neurodevelopmental disease caused by a homozygous TLK2 variant.

Eur J Hum Genet 2020 03;28(3):383-387

John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.

A distinct neurodevelopmental phenotype characterised mainly by mild motor and language delay and facial dysmorphism, caused by heterozygous de novo or dominant variants in the TLK2 gene has recently been described. All cases reported carried either truncating variants located throughout the gene, or missense changes principally located at the C-terminal end of the protein mostly resulting in haploinsufficiency of TLK2. Through whole exome sequencing, we identified a homozygous missense variant in TLK2 in a patient showing more severe symptoms than those previously described, including cerebellar vermis hypoplasia and West syndrome. Both parents are heterozygous for the variant and clinically unaffected highlighting that recessive variants in TLK2 can also be disease causing and may act through a different pathomechanism.
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http://dx.doi.org/10.1038/s41431-019-0519-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028915PMC
March 2020

Identification of a Novel Deep Intronic Mutation in CAPN3 Presenting a Promising Target for Therapeutic Splice Modulation.

J Neuromuscul Dis 2019 ;6(4):475-483

Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.

Calpainopathy, also known as limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) or LGMD R1 Calpain3-related, is one of the most common genetically characterized forms of limb-girdle muscular dystrophy with a wide range of phenotypic severity. We evaluated a consanguineous family with a clinical phenotype consistent with calpainopathy in whom conventional sequencing did not detect any mutations in the CAPN3 gene. Using whole exome sequencing paired with haplotype analysis, we identified a homozygous deep intronic single base pair deletion in CAPN3 (c.946-29delT). Familial segregation studies were consistent with recessive inheritance. Immunoblotting of muscle tissue from the patient showed complete absence of calpain 3. In silico analysis predicted the deletion to disrupt the branch point and subsequently alter splicing of exon 7. Studies of patient fibroblasts and muscle tissue confirmed altered splicing, resulting in an inclusion of a 389-bp intronic sequence upstream of exon 7, originating from a cryptic splice acceptor site in intron 6. This out-of-frame insertion results in a premature stop codon, leading to an apparent absence of protein likely due to degradation of the transcript via nonsense-mediated decay. We then designed phosphorodiamidate morpholino oligomers (PMOs) as splice modulators to block the new splice acceptor site. This approach successfully prevented the aberrant splicing - reverting the majority of the splice to the wildtype transcript. These results confirm the pathogenicity of this novel deep intronic mutation and provide a mutation-specific therapeutic strategy. Thus, deep intronic mutations in CAPN3 may be pathogenic and should be considered in the appropriate clinical setting. The identification of mutations which may be missed by traditional Sanger sequencing is essential as they may be excellent targets for individualized therapeutic strategies using RNA-directed splice modulation.
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http://dx.doi.org/10.3233/JND-190414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522968PMC
April 2020