Publications by authors named "Michael E Talkowski"

108 Publications

Dystonia-specific mutations in THAP1 alter transcription of genes associated with neurodevelopment and myelin.

Am J Hum Genet 2021 11 20;108(11):2145-2158. Epub 2021 Oct 20.

Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; The Collaborative Center for X-Linked Dystonia-Parkinsonism, Department of Neurology, Massachusetts General Hospital, Charlestown, MA 02129, USA. Electronic address:

Dystonia is a neurologic disorder associated with an increasingly large number of genetic variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is critical in understanding the pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations. We used this strategy to investigate the molecular consequences of genetic variation in THAP1, which encodes a transcription factor linked to neural differentiation. Multiple pathogenic mutations associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter DNA-binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight alterations in a common induced pluripotent stem cell background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling followed by joint analysis of the most robust signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. On the basis of these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.
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http://dx.doi.org/10.1016/j.ajhg.2021.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8595948PMC
November 2021

Prevalence and phenotypic impact of rare potentially damaging variants in autism spectrum disorder.

Mol Autism 2021 Oct 6;12(1):65. Epub 2021 Oct 6.

Seaver Autism Center for Research and Treatment, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

Background: The Autism Sequencing Consortium identified 102 high-confidence autism spectrum disorder (ASD) genes, showing that individuals with ASD and with potentially damaging single nucleotide variation (pdSNV) in these genes had lower cognitive levels and delayed age at walking, when compared to ASD participants without pdSNV. Here, we made use of a Swedish sample of individuals with ASD (called PAGES, for Population-Based Autism Genetics & Environment Study) to evaluate the frequency of pdSNV and their impact on medical and psychiatric phenotypes, using an epidemiological frame and universal health reporting. We then combine findings with those for potentially damaging copy number variation (pdCNV).

Methods: SNV and CNV calls were generated from whole-exome sequencing and chromosome microarray data, respectively. Birth and medical register data were used to collect phenotypes.

Results: Of 808 individuals assessed by sequencing, 69 (9%) had pdSNV in the 102 ASC genes, and 144 (18%) had pdSNV in the 102 ASC genes or in a larger set of curated neurodevelopmental genes (from the Deciphering Developmental Disorders study, the gene2phenotype database, and the Radboud University gene lists). Three or more individuals had pdSNV in GRIN2B, POGZ, SATB1, DYNC1H1, SCN8A, or CREBBP. In comparison, out of the 996 individuals from whom CNV were called, 105 (11%) carried one or more pdCNV, including four or more individuals with CNV in the recurrent 15q11q13, 22q11.2, and 16p11.2 loci. Carriers of pdSNV were more likely to have intellectual disability (ID) and epilepsy, while carriers of pdCNV showed increased rates of congenital anomalies and scholastic skill disorders. Carriers of either pdSNV or pdCNV were more likely to have ID, scholastic skill disorders, and epilepsy.

Limitations: The cohort only included individuals with autistic disorder, the more severe form of ASD, and phenotypes are defined from medical registers. Not all genes studied are definitively ASD genes, and we did not have de novo information to aid in classification.

Conclusions: In this epidemiological sample, rare pdSNV were more common than pdCNV and the combined yield of potentially damaging variation was substantial at 27%. The results provide compelling rationale for the use of high-throughout sequencing as part of routine clinical workup for ASD and support the development of precision medicine in ASD.
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http://dx.doi.org/10.1186/s13229-021-00465-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8495954PMC
October 2021

Physiological Characterization and Transcriptomic Properties of GnRH Neurons Derived From Human Stem Cells.

Endocrinology 2021 09;162(9)

Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USA.

Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus play a key role in the regulation of reproductive function. In this study, we sought an efficient method for generating GnRH neurons from human embryonic and induced pluripotent stem cells (hESC and hiPSC, respectively). First, we found that exposure of primitive neuroepithelial cells, rather than neuroprogenitor cells, to fibroblast growth factor 8 (FGF8), was more effective in generating GnRH neurons. Second, addition of kisspeptin to FGF8 further increased the efficiency rates of GnRH neurogeneration. Third, we generated a fluorescent marker mCherry labeled human embryonic GnRH cell line (mCh-hESC) using a CRISPR-Cas9 targeting approach. Fourth, we examined physiological characteristics of GnRH (mCh-hESC) neurons: similar to GnRH neurons in vivo, they released the GnRH peptide in a pulsatile manner at ~60 min intervals; GnRH release increased in response to high potassium, kisspeptin, estradiol, and neurokinin B challenges; and injection of depolarizing current induced action potentials. Finally, we characterized developmental changes in transcriptomes of GnRH neurons using hESC, hiPSC, and mCh-hESC. The developmental pattern of transcriptomes was remarkably similar among the 3 cell lines. Collectively, human stem cell-derived GnRH neurons will be an important tool for establishing disease models to understand diseases, such as idiopathic hypothalamic hypogonadism, and testing contraceptive drugs.
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http://dx.doi.org/10.1210/endocr/bqab120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294693PMC
September 2021

A deep learning approach to identify gene targets of a therapeutic for human splicing disorders.

Nat Commun 2021 06 7;12(1):3332. Epub 2021 Jun 7.

Center for Genomic Medicine, Massachusetts General Hospital Research Institute, Boston, MA, USA.

Pre-mRNA splicing is a key controller of human gene expression. Disturbances in splicing due to mutation lead to dysregulated protein expression and contribute to a substantial fraction of human disease. Several classes of splicing modulator compounds (SMCs) have been recently identified and establish that pre-mRNA splicing represents a target for therapy. We describe herein the identification of BPN-15477, a SMC that restores correct splicing of ELP1 exon 20. Using transcriptome sequencing from treated fibroblast cells and a machine learning approach, we identify BPN-15477 responsive sequence signatures. We then leverage this model to discover 155 human disease genes harboring ClinVar mutations predicted to alter pre-mRNA splicing as targets for BPN-15477. Splicing assays confirm successful correction of splicing defects caused by mutations in CFTR, LIPA, MLH1 and MAPT. Subsequent validations in two disease-relevant cellular models demonstrate that BPN-15477 increases functional protein, confirming the clinical potential of our predictions.
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http://dx.doi.org/10.1038/s41467-021-23663-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185002PMC
June 2021

16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.

Nat Commun 2021 05 18;12(1):2897. Epub 2021 May 18.

Department of Neurology, F.M. Kirby Neurobiology Center, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.
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http://dx.doi.org/10.1038/s41467-021-23113-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131375PMC
May 2021

Familial thrombocytopenia due to a complex structural variant resulting in a WAC-ANKRD26 fusion transcript.

J Exp Med 2021 06;218(6)

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, MA.

Advances in genome sequencing have resulted in the identification of the causes for numerous rare diseases. However, many cases remain unsolved with standard molecular analyses. We describe a family presenting with a phenotype resembling inherited thrombocytopenia 2 (THC2). THC2 is generally caused by single nucleotide variants that prevent silencing of ANKRD26 expression during hematopoietic differentiation. Short-read whole-exome and genome sequencing approaches were unable to identify a causal variant in this family. Using long-read whole-genome sequencing, a large complex structural variant involving a paired-duplication inversion was identified. Through functional studies, we show that this structural variant results in a pathogenic gain-of-function WAC-ANKRD26 fusion transcript. Our findings illustrate how complex structural variants that may be missed by conventional genome sequencing approaches can cause human disease.
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http://dx.doi.org/10.1084/jem.20210444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056752PMC
June 2021

Expectations and blind spots for structural variation detection from long-read assemblies and short-read genome sequencing technologies.

Am J Hum Genet 2021 05 30;108(5):919-928. Epub 2021 Mar 30.

Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Program in Medical and Population Genetics and Stanley Center for Psychiatric Disorders, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA 02142, USA; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and medical genetic initiatives are reliant upon short-read whole-genome sequencing (srWGS), which presents challenges for the detection of structural variants (SVs) relative to emerging long-read WGS (lrWGS) technologies. Given this ubiquity of srWGS in large-scale genomics initiatives, we sought to establish expectations for routine SV detection from this data type by comparison with lrWGS assembly, as well as to quantify the genomic properties and added value of SVs uniquely accessible to each technology. Analyses from the Human Genome Structural Variation Consortium (HGSVC) of three families captured ~11,000 SVs per genome from srWGS and ~25,000 SVs per genome from lrWGS assembly. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplication (SD) and simple repeat (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of reference sequence, we observed extremely high (93.8%) concordance between technologies for deletions in these datasets. In contrast, lrWGS was superior for detection of insertions across all genomic contexts. Given that non-SD/SR sequences encompass 95.9% of currently annotated disease-associated exons, improved sensitivity from lrWGS to discover novel pathogenic deletions in these currently interpretable genomic regions is likely to be incremental. However, these analyses highlight the considerable added value of assembly-based lrWGS to create new catalogs of insertions and transposable elements, as well as disease-associated repeat expansions in genomic sequences that were previously recalcitrant to routine assessment.
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http://dx.doi.org/10.1016/j.ajhg.2021.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206509PMC
May 2021

De novo structural mutation rates and gamete-of-origin biases revealed through genome sequencing of 2,396 families.

Am J Hum Genet 2021 04 5;108(4):597-607. Epub 2021 Mar 5.

Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA; Department of Biomedical Informatics, University of Utah, Salt Lake City, UT 84112, USA; Utah Center for Genetic Discovery, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

Each human genome includes de novo mutations that arose during gametogenesis. While these germline mutations represent a fundamental source of new genetic diversity, they can also create deleterious alleles that impact fitness. Whereas the rate and patterns of point mutations in the human germline are now well understood, far less is known about the frequency and features that impact de novo structural variants (dnSVs). We report a family-based study of germline mutations among 9,599 human genomes from 33 multigenerational CEPH-Utah families and 2,384 families from the Simons Foundation Autism Research Initiative. We find that de novo structural mutations detected by alignment-based, short-read WGS occur at an overall rate of at least 0.160 events per genome in unaffected individuals, and we observe a significantly higher rate (0.206 per genome) in ASD-affected individuals. In both probands and unaffected samples, nearly 73% of de novo structural mutations arose in paternal gametes, and we predict most de novo structural mutations to be caused by mutational mechanisms that do not require sequence homology. After multiple testing correction, we did not observe a statistically significant correlation between parental age and the rate of de novo structural variation in offspring. These results highlight that a spectrum of mutational mechanisms contribute to germline structural mutations and that these mechanisms most likely have markedly different rates and selective pressures than those leading to point mutations.
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http://dx.doi.org/10.1016/j.ajhg.2021.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8059337PMC
April 2021

Xenopus models suggest convergence of gene signatures on neurogenesis in autism.

Neuron 2021 03;109(5):743-745

Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research and Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address:

Willsey et al. dissect phenotypes associated with in vivo disruption of ten ASD-associated genes using a hypothesis-free, parallelized approach in Xenopus tropicalis. These studies continue to implicate cortical neurons in ASD pathogenesis and suggest a convergence on functions related to neurogenesis.
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http://dx.doi.org/10.1016/j.neuron.2021.02.017DOI Listing
March 2021

Haplotype-resolved diverse human genomes and integrated analysis of structural variation.

Science 2021 04 25;372(6537). Epub 2021 Feb 25.

New York Genome Center, New York, NY 10013, USA.

Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average minimum contig length needed to cover 50% of the genome: 26 million base pairs) integrate all forms of genetic variation, even across complex loci. We identified 107,590 structural variants (SVs), of which 68% were not discovered with short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterized 130 of the most active mobile element source elements and found that 63% of all SVs arise through homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.
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http://dx.doi.org/10.1126/science.abf7117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026704PMC
April 2021

Evidence for secondary-variant genetic burden and non-random distribution across biological modules in a recessive ciliopathy.

Nat Genet 2020 11 12;52(11):1145-1150. Epub 2020 Oct 12.

Center for Human Disease Modeling, Duke University Medical Center, Durham, NC, USA.

The influence of genetic background on driver mutations is well established; however, the mechanisms by which the background interacts with Mendelian loci remain unclear. We performed a systematic secondary-variant burden analysis of two independent cohorts of patients with Bardet-Biedl syndrome (BBS) with known recessive biallelic pathogenic mutations in one of 17 BBS genes for each individual. We observed a significant enrichment of trans-acting rare nonsynonymous secondary variants in patients with BBS compared with either population controls or a cohort of individuals with a non-BBS diagnosis and recessive variants in the same gene set. Strikingly, we found a significant over-representation of secondary alleles in chaperonin-encoding genes-a finding corroborated by the observation of epistatic interactions involving this complex in vivo. These data indicate a complex genetic architecture for BBS that informs the biological properties of disease modules and presents a model for secondary-variant burden analysis in recessive disorders.
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http://dx.doi.org/10.1038/s41588-020-0707-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8272915PMC
November 2020

Histone deacetylase knockouts modify transcription, CAG instability and nuclear pathology in Huntington disease mice.

Elife 2020 09 29;9. Epub 2020 Sep 29.

Center for Genomic Medicine, Harvard Medical School, Boston, United States.

Somatic expansion of the Huntington's disease (HD) CAG repeat drives the rate of a pathogenic process ultimately resulting in neuronal cell death. Although mechanisms of toxicity are poorly delineated, transcriptional dysregulation is a likely contributor. To identify modifiers that act at the level of CAG expansion and/or downstream pathogenic processes, we tested the impact of genetic knockout, in mice, of or in medium-spiny striatal neurons that exhibit extensive CAG expansion and exquisite disease vulnerability. Both knockouts moderately attenuated CAG expansion, with knockout decreasing nuclear huntingtin pathology. knockout resulted in a substantial transcriptional response that included modification of transcriptional dysregulation elicited by the allele, likely via mechanisms unrelated to instability suppression. Our results identify novel modifiers of different aspects of HD pathogenesis in medium-spiny neurons and highlight a complex relationship between the expanded allele and with implications for targeting transcriptional dysregulation in HD.
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http://dx.doi.org/10.7554/eLife.55911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581428PMC
September 2020

Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production.

JCI Insight 2020 09 3;5(17). Epub 2020 Sep 3.

Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient-derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.
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http://dx.doi.org/10.1172/jci.insight.138576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526459PMC
September 2020

New gene discoveries highlight functional convergence in autism and related neurodevelopmental disorders.

Curr Opin Genet Dev 2020 12 23;65:195-206. Epub 2020 Aug 23.

Center for Genomic Medicine, Massachusetts General Hospital, Boston MA, United States; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston MA, United States; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge MA, United States; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, United States. Electronic address:

Over the last two years, remarkable gene discovery efforts have implicated disruption of pathways involving gene regulatory functions and neuronal processes in autism spectrum disorder (ASD), and more broadly defined neurodevelopmental disorders (NDDs). Functional studies in the developing brain and across cell types demonstrate that the spatiotemporal expression patterns of many of these genes coalesce on subnetworks with distinct developmental trajectories. Here, we review the convergent biological processes derived from gene discovery and functional genomics in ASD and NDD from 2018-2020. We further probe the mechanistic insights that suggest these frequently perturbed pathways are interconnected and, ultimately, converge on specific functional deficits in human neurodevelopment.
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http://dx.doi.org/10.1016/j.gde.2020.07.001DOI Listing
December 2020

Whole exome sequencing analyses reveal gene-microbiota interactions in the context of IBD.

Gut 2021 02 10;70(2):285-296. Epub 2020 Jul 10.

Department of Gastroenterology and Hepatology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands

Objective: Both the gut microbiome and host genetics are known to play significant roles in the pathogenesis of IBD. However, the interaction between these two factors and its implications in the aetiology of IBD remain underexplored. Here, we report on the influence of host genetics on the gut microbiome in IBD.

Design: To evaluate the impact of host genetics on the gut microbiota of patients with IBD, we combined whole exome sequencing of the host genome and whole genome shotgun sequencing of 1464 faecal samples from 525 patients with IBD and 939 population-based controls. We followed a four-step analysis: (1) exome-wide microbial quantitative trait loci (mbQTL) analyses, (2) a targeted approach focusing on IBD-associated genomic regions and protein truncating variants (PTVs, minor allele frequency (MAF) >5%), (3) gene-based burden tests on PTVs with MAF <5% and exome copy number variations (CNVs) with site frequency <1%, (4) joint analysis of both cohorts to identify the interactions between disease and host genetics.

Results: We identified 12 mbQTLs, including variants in the IBD-associated genes , , and . For example, the decrease of the pathway acetyl-coenzyme A biosynthesis, which is involved in short chain fatty acids production, was associated with variants in the gene (false discovery rate <0.05). Changes in functional pathways involved in the metabolic potential were also observed in participants carrying rare PTVs or CNVs in , and genes. These genes are known for their function in the immune system. Moreover, interaction analyses confirmed previously known IBD disease-specific mbQTLs in .

Conclusion: This study highlights that both common and rare genetic variants affecting the immune system are key factors in shaping the gut microbiota in the context of IBD and pinpoints towards potential mechanisms for disease treatment.
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http://dx.doi.org/10.1136/gutjnl-2019-319706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815889PMC
February 2021

Transcriptional consequences of MBD5 disruption in mouse brain and CRISPR-derived neurons.

Mol Autism 2020 06 5;11(1):45. Epub 2020 Jun 5.

Molecular Neurogenetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.

Background: MBD5, encoding the methyl-CpG-binding domain 5 protein, has been proposed as a necessary and sufficient driver of the 2q23.1 microdeletion syndrome. De novo missense and protein-truncating variants from exome sequencing studies have directly implicated MBD5 in the etiology of autism spectrum disorder (ASD) and related neurodevelopmental disorders (NDDs). However, little is known concerning the specific function(s) of MBD5.

Methods: To gain insight into the complex interactions associated with alteration of MBD5 in individuals with ASD and related NDDs, we explored the transcriptional landscape of MBD5 haploinsufficiency across multiple mouse brain regions of a heterozygous hypomorphic Mbd5 mouse model, and compared these results to CRISPR-mediated mutations of MBD5 in human iPSC-derived neuronal models.

Results: Gene expression analyses across three brain regions from Mbd5 mice showed subtle transcriptional changes, with cortex displaying the most widespread changes following Mbd5 reduction, indicating context-dependent effects. Comparison with MBD5 reduction in human neuronal cells reinforced the context-dependence of gene expression changes due to MBD5 deficiency. Gene co-expression network analyses revealed gene clusters that were associated with reduced MBD5 expression and enriched for terms related to ciliary function.

Limitations: These analyses included a limited number of mouse brain regions and neuronal models, and the effects of the gene knockdown are subtle. As such, these results will not reflect the full extent of MBD5 disruption across human brain regions during early neurodevelopment in ASD, or capture the diverse spectrum of cell-type-specific changes associated with MBD5 alterations.

Conclusions: Our study points to modest and context-dependent transcriptional consequences of Mbd5 disruption in the brain. It also suggests a possible link between MBD5 and perturbations in ciliary function, which is an established pathogenic mechanism in developmental disorders and syndromes.
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http://dx.doi.org/10.1186/s13229-020-00354-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275313PMC
June 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

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

Whole-Genome and RNA Sequencing Reveal Variation and Transcriptomic Coordination in the Developing Human Prefrontal Cortex.

Cell Rep 2020 04;31(1):107489

Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA 94158, USA.

Gene expression levels vary across developmental stage, cell type, and region in the brain. Genomic variants also contribute to the variation in expression, and some neuropsychiatric disorder loci may exert their effects through this mechanism. To investigate these relationships, we present BrainVar, a unique resource of paired whole-genome and bulk tissue RNA sequencing from the dorsolateral prefrontal cortex of 176 individuals across prenatal and postnatal development. Here we identify common variants that alter gene expression (expression quantitative trait loci [eQTLs]) constantly across development or predominantly during prenatal or postnatal stages. Both "constant" and "temporal-predominant" eQTLs are enriched for loci associated with neuropsychiatric traits and disorders and colocalize with specific variants. Expression levels of more than 12,000 genes rise or fall in a concerted late-fetal transition, with the transitional genes enriched for cell-type-specific genes and neuropsychiatric risk loci, underscoring the importance of cataloging developmental trajectories in understanding cortical physiology and pathology.
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http://dx.doi.org/10.1016/j.celrep.2020.03.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295160PMC
April 2020

Large-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism.

Cell 2020 02 23;180(3):568-584.e23. Epub 2020 Jan 23.

Department of Psychiatry, School of Medicine, Trinity College Dublin, Dublin, Ireland.

We present the largest exome sequencing study of autism spectrum disorder (ASD) to date (n = 35,584 total samples, 11,986 with ASD). Using an enhanced analytical framework to integrate de novo and case-control rare variation, we identify 102 risk genes at a false discovery rate of 0.1 or less. Of these genes, 49 show higher frequencies of disruptive de novo variants in individuals ascertained to have severe neurodevelopmental delay, whereas 53 show higher frequencies in individuals ascertained to have ASD; comparing ASD cases with mutations in these groups reveals phenotypic differences. Expressed early in brain development, most risk genes have roles in regulation of gene expression or neuronal communication (i.e., mutations effect neurodevelopmental and neurophysiological changes), and 13 fall within loci recurrently hit by copy number variants. In cells from the human cortex, expression of risk genes is enriched in excitatory and inhibitory neuronal lineages, consistent with multiple paths to an excitatory-inhibitory imbalance underlying ASD.
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http://dx.doi.org/10.1016/j.cell.2019.12.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250485PMC
February 2020

SYCP2 Translocation-Mediated Dysregulation and Frameshift Variants Cause Human Male Infertility.

Am J Hum Genet 2020 01 19;106(1):41-57. Epub 2019 Dec 19.

Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA; Medical and Population Genetics Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA; Manchester Academic Health Science Centre, University of Manchester, Manchester M13 9NT, UK. Electronic address:

Unexplained infertility affects 2%-3% of reproductive-aged couples. One approach to identifying genes involved in infertility is to study subjects with this clinical phenotype and a de novo balanced chromosomal aberration (BCA). While BCAs may reduce fertility by production of unbalanced gametes, a chromosomal rearrangement may also disrupt or dysregulate genes important in fertility. One such subject, DGAP230, has severe oligozoospermia and 46,XY,t(20;22)(q13.3;q11.2). We identified exclusive overexpression of SYCP2 from the der(20) allele that is hypothesized to result from enhancer adoption. Modeling the dysregulation in budding yeast resulted in disrupted structural integrity of the synaptonemal complex, a common cause of defective spermatogenesis in mammals. Exome sequencing of infertile males revealed three heterozygous SYCP2 frameshift variants in additional subjects with cryptozoospermia and azoospermia. In sum, this investigation illustrates the power of precision cytogenetics for annotation of the infertile genome, suggests that these mechanisms should be considered as an alternative etiology to that of segregation of unbalanced gametes in infertile men harboring a BCA, and provides evidence of SYCP2-mediated male infertility in humans.
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http://dx.doi.org/10.1016/j.ajhg.2019.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042487PMC
January 2020

Prioritization of genes driving congenital phenotypes of patients with de novo genomic structural variants.

Genome Med 2019 12 4;11(1):79. Epub 2019 Dec 4.

Center for Molecular Medicine and Oncode Institute, University Medical Center Utrecht, 3584 CX, Utrecht, the Netherlands.

Background: Genomic structural variants (SVs) can affect many genes and regulatory elements. Therefore, the molecular mechanisms driving the phenotypes of patients carrying de novo SVs are frequently unknown.

Methods: We applied a combination of systematic experimental and bioinformatic methods to improve the molecular diagnosis of 39 patients with multiple congenital abnormalities and/or intellectual disability harboring apparent de novo SVs, most with an inconclusive diagnosis after regular genetic testing.

Results: In 7 of these cases (18%), whole-genome sequencing analysis revealed disease-relevant complexities of the SVs missed in routine microarray-based analyses. We developed a computational tool to predict the effects on genes directly affected by SVs and on genes indirectly affected likely due to the changes in chromatin organization and impact on regulatory mechanisms. By combining these functional predictions with extensive phenotype information, candidate driver genes were identified in 16/39 (41%) patients. In 8 cases, evidence was found for the involvement of multiple candidate drivers contributing to different parts of the phenotypes. Subsequently, we applied this computational method to two cohorts containing a total of 379 patients with previously detected and classified de novo SVs and identified candidate driver genes in 189 cases (50%), including 40 cases whose SVs were previously not classified as pathogenic. Pathogenic position effects were predicted in 28% of all studied cases with balanced SVs and in 11% of the cases with copy number variants.

Conclusions: These results demonstrate an integrated computational and experimental approach to predict driver genes based on analyses of WGS data with phenotype association and chromatin organization datasets. These analyses nominate new pathogenic loci and have strong potential to improve the molecular diagnosis of patients with de novo SVs.
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http://dx.doi.org/10.1186/s13073-019-0692-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894143PMC
December 2019

Biallelic mutation of FBXL7 suggests a novel form of Hennekam syndrome.

Am J Med Genet A 2020 01 21;182(1):189-194. Epub 2019 Oct 21.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts.

Hennekam lymphangiectasia-lymphedema syndrome is an autosomal recessive disorder characterized by congenital lymphedema, intestinal lymphangiectasia, facial dysmorphism, and variable intellectual disability. Known disease genes include CCBE1, FAT4, and ADAMTS3. In a patient with clinically diagnosed Hennekam syndrome but without mutations or copy-number changes in the three known disease genes, we identified a homozygous single-exon deletion affecting FBXL7. Specifically, exon 3, which encodes the F-box domain and several leucine-rich repeats of FBXL7, is eliminated. Our analyses of databases representing >100,000 control individuals failed to identify biallelic loss-of-function variants in FBXL7. Published studies in Drosophila indicate Fbxl7 interacts with Fat, of which human FAT4 is an ortholog, and mutation of either gene yields similar morphological consequences. These data suggest that FBXL7 may be the fourth gene for Hennekam syndrome, acting via a shared pathway with FAT4.
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http://dx.doi.org/10.1002/ajmg.a.61392DOI Listing
January 2020

Role of the Chromosome Architectural Factor SMCHD1 in X-Chromosome Inactivation, Gene Regulation, and Disease in Humans.

Genetics 2019 10 16;213(2):685-703. Epub 2019 Aug 16.

Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an architectural factor critical for X-chromosome inactivation (XCI) and the repression of select autosomal gene clusters. In mice, homozygous nonsense mutations in cause female-specific embryonic lethality due to an XCI defect. However, although human mutations in are associated with congenital arhinia and facioscapulohumeral muscular dystrophy type 2 (FSHD2), the diseases do not show a sex-specific bias, despite the essential nature of XCI in humans. To investigate whether there is a dosage imbalance for the sex chromosomes, we here analyze transcriptomic data from arhinia and FSHD2 patient blood and muscle cells. We find that -linked dosage compensation is maintained in these patients. In mice, SMCHD1 controls not only protocadherin () gene clusters, but also genes critical for craniofacial development. Ablating results in aberrant expression of these genes, coinciding with altered chromatin states and three-dimensional (3D) topological organization. In a subset of FSHD2 and arhinia patients, we also found dysregulation of clustered , but not genes. Overall, our study demonstrates preservation of XCI in arhinia and FSHD2, and implicates SMCHD1 in the regulation of the 3D organization of select autosomal gene clusters.
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http://dx.doi.org/10.1534/genetics.119.302600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781896PMC
October 2019

Primary cilia defects causing mitral valve prolapse.

Sci Transl Med 2019 05;11(493)

Center for Genomic Medicine, Department of Neurology, Massachusetts General Hospital Research Institute, Harvard Medical School, 185 Cambridge St., Boston, MA 02114, USA.

Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.
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http://dx.doi.org/10.1126/scitranslmed.aax0290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331025PMC
May 2019

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

Nat Commun 2019 04 16;10(1):1784. Epub 2019 Apr 16.

Pacific Biosciences, Menlo Park, CA, 94025, USA.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.
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http://dx.doi.org/10.1038/s41467-018-08148-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467913PMC
April 2019

ELP1 Splicing Correction Reverses Proprioceptive Sensory Loss in Familial Dysautonomia.

Am J Hum Genet 2019 04 21;104(4):638-650. Epub 2019 Mar 21.

Center for Genomic Medicine, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA 02114, USA; Department of Neurology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP); this mutation leads to variable skipping of exon 20 and to a drastic reduction of ELP1 in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception; this loss leads to severe gait ataxia, spinal deformities, and respiratory insufficiency due to neuromuscular incoordination. There is currently no effective treatment for FD, and the disease is ultimately fatal. The development of a drug that targets the underlying molecular defect provides hope that the drastic peripheral neurodegeneration characteristic of FD can be halted. We demonstrate herein that the FD mouse TgFD9;Ikbkap recapitulates the proprioceptive impairment observed in individuals with FD, and we provide the in vivo evidence that postnatal correction, promoted by the small molecule kinetin, of the mutant ELP1 splicing can rescue neurological phenotypes in FD. Daily administration of kinetin starting at birth improves sensory-motor coordination and prevents the onset of spinal abnormalities by stopping the loss of proprioceptive neurons. These phenotypic improvements correlate with increased amounts of full-length ELP1 mRNA and protein in multiple tissues, including in the peripheral nervous system (PNS). Our results show that postnatal correction of the underlying ELP1 splicing defect can rescue devastating disease phenotypes and is therefore a viable therapeutic approach for persons with FD.
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http://dx.doi.org/10.1016/j.ajhg.2019.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451698PMC
April 2019
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