Publications by authors named "Susan M Hiatt"

26 Publications

  • Page 1 of 1

Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy.

Am J Hum Genet 2021 05;108(5):857-873

GeneDx, Gaithersburg, MD 20877, USA.

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206167PMC
May 2021

Long-read genome sequencing for the molecular diagnosis of neurodevelopmental disorders.

HGG Adv 2021 Apr 16;2(2). Epub 2021 Jan 16.

HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA.

Exome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches. Here, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads. We identified variants and created assemblies in each trio, with global metrics indicating these datasets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), L1-mediated insertion in that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large structural variants, including insertion-translocations affecting and , which we show disrupt transcript levels. We consider this extensive structural variation likely pathogenic. The breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs, support the hypothesis that long-read genome sequencing can substantially improve rare disease genetic discovery rates.
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http://dx.doi.org/10.1016/j.xhgg.2021.100023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087252PMC
April 2021

The Therapeutic Odyssey: Positioning Genomic Sequencing in the Search for a Child's Best Possible Life.

AJOB Empir Bioeth 2021 Jul-Sep;12(3):179-189. Epub 2021 Apr 12.

Division of Pediatric Clinical and Translational Research, University of Louisville, Louisville, Kentucky, USA.

The desire of parents to obtain a genetic diagnosis for their child with intellectual disability and associated symptoms has long been framed as a diagnostic odyssey, an arduous and sometimes perilous journey focused on the goal of identifying a cause for the child's condition. Semi-structured interviews (N = 60) were conducted with parents of children (N = 59, aged 2-24 years) with intellectual disability and/or developmental delay (IDD) who underwent genome sequencing at a single pediatric multispecialty clinic. Interviews were conducted after parents received their child's sequencing result (positive findings, negative findings, or variants of unknown significance). Thematic analysis was performed on all interviews. Parents reported that obtaining a genetic diagnosis was one important step in their overall goal of helping their child live their best life possible life. They intended to use the result as a tool to help their child by seeking the correct school placement and obtaining benefits and therapeutic services. For the parents of children with IDD, the search for a genetic diagnosis is best conceptualized as a part of parents' ongoing efforts to leverage various diagnoses to obtain educational and therapeutic services for their children. Cleaving parents' search for a genetic diagnosis from these broader efforts obscures the value that some parents place on a sequencing result in finding and tailoring therapies and services beyond the clinic. Interviews with parents reveal, therefore, that genomic sequencing is best understood as one important stage of an ongoing that largely takes place outside the clinic. Findings suggest the need to expand translational research efforts to contextualize a genetic diagnosis within parents' broader efforts to obtain educational and therapeutic services outside clinical contexts.
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http://dx.doi.org/10.1080/23294515.2021.1907475DOI Listing
October 2021

De novo TBR1 variants cause a neurocognitive phenotype with ID and autistic traits: report of 25 new individuals and review of the literature.

Eur J Hum Genet 2020 06 31;28(6):770-782. Epub 2020 Jan 31.

Department of Pediatrics, The Barbara Bush Children's Hospital, Maine Medical Center, Portland, OR, USA.

TBR1, a T-box transcription factor expressed in the cerebral cortex, regulates the expression of several candidate genes for autism spectrum disorders (ASD). Although TBR1 has been reported as a high-confidence risk gene for ASD and intellectual disability (ID) in functional and clinical reports since 2011, TBR1 has only recently been recorded as a human disease gene in the OMIM database. Currently, the neurodevelopmental disorders and structural brain anomalies associated with TBR1 variants are not well characterized. Through international data sharing, we collected data from 25 unreported individuals and compared them with data from the literature. We evaluated structural brain anomalies in seven individuals by analysis of MRI images, and compared these with anomalies observed in TBR1 mutant mice. The phenotype included ID in all individuals, associated to autistic traits in 76% of them. No recognizable facial phenotype could be identified. MRI analysis revealed a reduction of the anterior commissure and suggested new features including dysplastic hippocampus and subtle neocortical dysgenesis. This report supports the role of TBR1 in ID associated with autistic traits and suggests new structural brain malformations in humans. We hope this work will help geneticists to interpret TBR1 variants and diagnose ASD probands.
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http://dx.doi.org/10.1038/s41431-020-0571-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253452PMC
June 2020

BAZ2B haploinsufficiency as a cause of developmental delay, intellectual disability, and autism spectrum disorder.

Hum Mutat 2020 05 7;41(5):921-925. Epub 2020 Feb 7.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.

The bromodomain adjacent to zinc finger 2B gene (BAZ2B) encodes a protein involved in chromatin remodeling. Loss of BAZ2B function has been postulated to cause neurodevelopmental disorders. To determine whether BAZ2B deficiency is likely to contribute to the pathogenesis of these disorders, we performed bioinformatics analyses that demonstrated a high level of functional convergence during fetal cortical development between BAZ2B and genes known to cause autism spectrum disorder (ASD) and neurodevelopmental disorder. We also found an excess of de novo BAZ2B loss-of-function variants in exome sequencing data from previously published cohorts of individuals with neurodevelopmental disorders. We subsequently identified seven additional individuals with heterozygous deletions, stop-gain, or de novo missense variants affecting BAZ2B. All of these individuals have developmental delay (DD), intellectual disability (ID), and/or ASD. Taken together, our findings suggest that haploinsufficiency of BAZ2B causes a neurodevelopmental disorder, whose cardinal features include DD, ID, and ASD.
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http://dx.doi.org/10.1002/humu.23992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262739PMC
May 2020

Pathogenic WDFY3 variants cause neurodevelopmental disorders and opposing effects on brain size.

Brain 2019 09;142(9):2617-2630

GeneDx, Clinical Genomics, 207 Perry Parkway Gaithersburg, MD, USA.

The underpinnings of mild to moderate neurodevelopmental delay remain elusive, often leading to late diagnosis and interventions. Here, we present data on exome and genome sequencing as well as array analysis of 13 individuals that point to pathogenic, heterozygous, mostly de novo variants in WDFY3 (significant de novo enrichment P = 0.003) as a monogenic cause of mild and non-specific neurodevelopmental delay. Nine variants were protein-truncating and four missense. Overlapping symptoms included neurodevelopmental delay, intellectual disability, macrocephaly, and psychiatric disorders (autism spectrum disorders/attention deficit hyperactivity disorder). One proband presented with an opposing phenotype of microcephaly and the only missense-variant located in the PH-domain of WDFY3. Findings of this case are supported by previously published data, demonstrating that pathogenic PH-domain variants can lead to microcephaly via canonical Wnt-pathway upregulation. In a separate study, we reported that the autophagy scaffolding protein WDFY3 is required for cerebral cortical size regulation in mice, by controlling proper division of neural progenitors. Here, we show that proliferating cortical neural progenitors of human embryonic brains highly express WDFY3, further supporting a role for this molecule in the regulation of prenatal neurogenesis. We present data on Wnt-pathway dysregulation in Wdfy3-haploinsufficient mice, which display macrocephaly and deficits in motor coordination and associative learning, recapitulating the human phenotype. Consequently, we propose that in humans WDFY3 loss-of-function variants lead to macrocephaly via downregulation of the Wnt pathway. In summary, we present WDFY3 as a novel gene linked to mild to moderate neurodevelopmental delay and intellectual disability and conclude that variants putatively causing haploinsufficiency lead to macrocephaly, while an opposing pathomechanism due to variants in the PH-domain of WDFY3 leads to microcephaly.
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http://dx.doi.org/10.1093/brain/awz198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736092PMC
September 2019

A YWHAZ Variant Associated With Cardiofaciocutaneous Syndrome Activates the RAF-ERK Pathway.

Front Physiol 2019 8;10:388. Epub 2019 Apr 8.

Department of Cell, Developmental and Integrative Biology, The University of Alabama at Birmingham, Birmingham, AL, United States.

Cardiofaciocutaneous (CFC) syndrome is a genetic disorder characterized by distinctive facial features, congenital heart defects, and skin abnormalities. Several germline gain-of-function mutations in the RAS/RAF/MEK/ERK pathway are associated with the disease, including , and . CFC syndrome thus belongs to a group of disorders known as RASopathies, which are all caused by pathogenic mutations in various genes encoding components of the RAS pathway. We recently identified novel variants in , a 14-3-3 family member, in individuals with a phenotype consistent with CFC that may potentially be deleterious and disease-causing. In the current study, we take advantage of the vertebrate model to analyze the functional consequence of a particular variant, S230W, and investigate the molecular mechanisms underlying its activity. We show that compared with wild type , the S230W variant induces severe embryonic defects when ectopically expressed in early embryos. The S230W variant also rescues the defects induced by a dominant negative FGF receptor more efficiently and enhances Raf-stimulated Erk phosphorylation to a higher level than wild type . Although neither nor the variant promotes membrane recruitment of Raf proteins, the variant binds to more Raf and escapes phosphorylation by casein kinase 1a. Our data provide strong support to the hypothesis that the S230W variant of is a gain-of-function mutation in the RAS-ERK pathway and may underlie a CFC phenotype.
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http://dx.doi.org/10.3389/fphys.2019.00388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465419PMC
April 2019

Deleterious Variation in BRSK2 Associates with a Neurodevelopmental Disorder.

Am J Hum Genet 2019 04 14;104(4):701-708. Epub 2019 Mar 14.

HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA. Electronic address:

Developmental delay and intellectual disability (DD and ID) are heterogeneous phenotypes that arise in many rare monogenic disorders. Because of this rarity, developing cohorts with enough individuals to robustly identify disease-associated genes is challenging. Social-media platforms that facilitate data sharing among sequencing labs can help to address this challenge. Through one such tool, GeneMatcher, we identified nine DD- and/or ID-affected probands with a rare, heterozygous variant in the gene encoding the serine/threonine-protein kinase BRSK2. All probands have a speech delay, and most present with intellectual disability, motor delay, behavioral issues, and autism. Six of the nine variants are predicted to result in loss of function, and computational modeling predicts that the remaining three missense variants are damaging to BRSK2 structure and function. All nine variants are absent from large variant databases, and BRSK2 is, in general, relatively intolerant to protein-altering variation among humans. In all six probands for whom parents were available, the mutations were found to have arisen de novo. Five of these de novo variants were from cohorts with at least 400 sequenced probands; collectively, the cohorts span 3,429 probands, and the observed rate of de novo variation in these cohorts is significantly higher than the estimated background-mutation rate (p = 2.46 × 10). We also find that exome sequencing provides lower coverage and appears less sensitive to rare variation in BRSK2 than does genome sequencing; this fact most likely reduces BRSK2's visibility in many clinical and research sequencing efforts. Altogether, our results implicate damaging variation in BRSK2 as a source of neurodevelopmental disease.
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http://dx.doi.org/10.1016/j.ajhg.2019.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451696PMC
April 2019

Variants in TCF20 in neurodevelopmental disability: description of 27 new patients and review of literature.

Genet Med 2019 09 11;21(9):2036-2042. Epub 2019 Feb 11.

Spectrum Health Medical Genetics, Grand Rapids, MI, USA.

Purpose: To define the clinical characteristics of patients with variants in TCF20, we describe 27 patients, 26 of whom were identified via exome sequencing. We compare detailed clinical data with 17 previously reported patients.

Methods: Patients were ascertained through molecular testing laboratories performing exome sequencing (and other testing) with orthogonal confirmation; collaborating referring clinicians provided detailed clinical information.

Results: The cohort of 27 patients all had novel variants, and ranged in age from 2 to 68 years. All had developmental delay/intellectual disability. Autism spectrum disorders/autistic features were reported in 69%, attention disorders or hyperactivity in 67%, craniofacial features (no recognizable facial gestalt) in 67%, structural brain anomalies in 24%, and seizures in 12%. Additional features affecting various organ systems were described in 93%. In a majority of patients, we did not observe previously reported findings of postnatal overgrowth or craniosynostosis, in comparison with earlier reports.

Conclusion: We provide valuable data regarding the prognosis and clinical manifestations of patients with variants in TCF20.
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http://dx.doi.org/10.1038/s41436-019-0454-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171701PMC
September 2019

ZMIZ1 Variants Cause a Syndromic Neurodevelopmental Disorder.

Am J Hum Genet 2019 02 10;104(2):319-330. Epub 2019 Jan 10.

Cook Children's Medical Center, Fort Worth, TX 76102, USA.

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.
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http://dx.doi.org/10.1016/j.ajhg.2018.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369415PMC
February 2019

De novo mutations in the GTP/GDP-binding region of RALA, a RAS-like small GTPase, cause intellectual disability and developmental delay.

PLoS Genet 2018 11 30;14(11):e1007671. Epub 2018 Nov 30.

HudsonAlpha Institute for Biotechnology, Huntsville, AL, United States of America.

Mutations that alter signaling of RAS/MAPK-family proteins give rise to a group of Mendelian diseases known as RASopathies. However, among RASopathies, the matrix of genotype-phenotype relationships is still incomplete, in part because there are many RAS-related proteins and in part because the phenotypic consequences may be variable and/or pleiotropic. Here, we describe a cohort of ten cases, drawn from six clinical sites and over 16,000 sequenced probands, with de novo protein-altering variation in RALA, a RAS-like small GTPase. All probands present with speech and motor delays, and most have intellectual disability, low weight, short stature, and facial dysmorphism. The observed rate of de novo RALA variants in affected probands is significantly higher (p = 4.93 x 10(-11)) than expected from the estimated random mutation rate. Further, all de novo variants described here affect residues within the GTP/GDP-binding region of RALA; in fact, six alleles arose at only two codons, Val25 and Lys128. The affected residues are highly conserved across both RAL- and RAS-family genes, are devoid of variation in large human population datasets, and several are homologous to positions at which disease-associated variants have been observed in other GTPase genes. We directly assayed GTP hydrolysis and RALA effector-protein binding of the observed variants, and found that all but one tested variant significantly reduced both activities compared to wild-type. The one exception, S157A, reduced GTP hydrolysis but significantly increased RALA-effector binding, an observation similar to that seen for oncogenic RAS variants. These results show the power of data sharing for the interpretation and analysis of rare variation, expand the spectrum of molecular causes of developmental disability to include RALA, and provide additional insight into the pathogenesis of human disease caused by mutations in small GTPases.
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http://dx.doi.org/10.1371/journal.pgen.1007671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291162PMC
November 2018

Mutations in MAST1 Cause Mega-Corpus-Callosum Syndrome with Cerebellar Hypoplasia and Cortical Malformations.

Neuron 2018 12 15;100(6):1354-1368.e5. Epub 2018 Nov 15.

Centre de référence des Malformations et Maladies Congénitales du Cervelet et Département de Génétique et Embryologie Médicale, APHP, Hôpital Trousseau, 75012 Paris, France.

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.
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http://dx.doi.org/10.1016/j.neuron.2018.10.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436622PMC
December 2018

NBEA: Developmental disease gene with early generalized epilepsy phenotypes.

Ann Neurol 2018 11 25;84(5):788-795. Epub 2018 Oct 25.

Department of Pediatrics, University of Washington, Seattle, WA.

NBEA is a candidate gene for autism, and de novo variants have been reported in neurodevelopmental disease (NDD) cohorts. However, NBEA has not been rigorously evaluated as a disease gene, and associated phenotypes have not been delineated. We identified 24 de novo NBEA variants in patients with NDD, establishing NBEA as an NDD gene. Most patients had epilepsy with onset in the first few years of life, often characterized by generalized seizure types, including myoclonic and atonic seizures. Our data show a broader phenotypic spectrum than previously described, including a myoclonic-astatic epilepsy-like phenotype in a subset of patients. Ann Neurol 2018;84:796-803.
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http://dx.doi.org/10.1002/ana.25350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249120PMC
November 2018

Approaches to carrier testing and results disclosure in translational genomics research: The clinical sequencing exploratory research consortium experience.

Mol Genet Genomic Med 2018 11 21;6(6):898-909. Epub 2018 Aug 21.

Treuman Katz Center for Pediatric Bioethics, Seattle Children's Research Institute, Seattle, Washington.

Background: Clinical genome and exome sequencing (CGES) is primarily used to address specific clinical concerns by detecting risk of future disease, clarifying diagnosis, or directing treatment. Additionally, CGES makes possible the disclosure of autosomal recessive and X-linked carrier results as additional secondary findings, and research about the impact of carrier results disclosure in this context is needed.

Methods: Representatives from 11 projects in the clinical sequencing exploratory research (CSER) consortium collected data from their projects using a structured survey. The survey focused on project characteristics, which variants were offered and/or disclosed to participants as carrier results, methods for carrier results disclosure, and project-specific outcomes. We recorded quantitative responses and report descriptive statistics with the aim of describing the variability in approaches to disclosing carrier results in translational genomics research projects.

Results: The proportion of participants with carrier results was related to the number of genes included, ranging from 3% (three genes) to 92% (4,600 genes). Between one and seven results were disclosed to those participants who received any positive result. Most projects offered participants choices about whether to receive some or all of the carrier results. There were a range of approaches to communicate results, and many projects used separate approaches for disclosing positive and negative results.

Conclusion: Future translational genomics research projects will need to make decisions regarding whether and how to disclose carrier results. The CSER consortium experience identifies approaches that balance potential participant interest while limiting impact on project resources.
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http://dx.doi.org/10.1002/mgg3.453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305639PMC
November 2018

Genomic sequencing identifies secondary findings in a cohort of parent study participants.

Genet Med 2018 12 12;20(12):1635-1643. Epub 2018 Apr 12.

HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.

Purpose: Clinically relevant secondary variants were identified in parents enrolled with a child with developmental delay and intellectual disability.

Methods: Exome/genome sequencing and analysis of 789 "unaffected" parents was performed.

Results: Pathogenic/likely pathogenic variants were identified in 21 genes within 25 individuals (3.2%), with 11 (1.4%) participants harboring variation in a gene defined as clinically actionable by the American College of Medical Genetics and Genomics. These 25 individuals self-reported either relevant clinical diagnoses (5); relevant family history or symptoms (13); or no relevant family history, symptoms, or clinical diagnoses (7). A limited carrier screen was performed yielding 15 variants in 48 (6.1%) parents. Parents were also analyzed as mate pairs (n = 365) to identify cases in which both parents were carriers for the same recessive disease, yielding three such cases (0.8%), two of which had children with the relevant recessive disease. Four participants had two findings (one carrier and one noncarrier variant). In total, 71 of the 789 enrolled parents (9.0%) received secondary findings.

Conclusion: We provide an overview of the rates and types of clinically relevant secondary findings, which may be useful in the design and implementation of research and clinical sequencing efforts to identify such findings.
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http://dx.doi.org/10.1038/gim.2018.53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185813PMC
December 2018

De novo mutations in MED13, a component of the Mediator complex, are associated with a novel neurodevelopmental disorder.

Hum Genet 2018 May 8;137(5):375-388. Epub 2018 May 8.

Language and Genetics Department, Max Planck Institute for Psycholinguistics, Nijmegen, The Netherlands.

Many genetic causes of developmental delay and/or intellectual disability (DD/ID) are extremely rare, and robust discovery of these requires both large-scale DNA sequencing and data sharing. Here we describe a GeneMatcher collaboration which led to a cohort of 13 affected individuals harboring protein-altering variants, 11 of which are de novo, in MED13; the only inherited variant was transmitted to an affected child from an affected mother. All patients had intellectual disability and/or developmental delays, including speech delays or disorders. Other features that were reported in two or more patients include autism spectrum disorder, attention deficit hyperactivity disorder, optic nerve abnormalities, Duane anomaly, hypotonia, mild congenital heart abnormalities, and dysmorphisms. Six affected individuals had mutations that are predicted to truncate the MED13 protein, six had missense mutations, and one had an in-frame-deletion of one amino acid. Out of the seven non-truncating mutations, six clustered in two specific locations of the MED13 protein: an N-terminal and C-terminal region. The four N-terminal clustering mutations affect two adjacent amino acids that are known to be involved in MED13 ubiquitination and degradation, p.Thr326 and p.Pro327. MED13 is a component of the CDK8-kinase module that can reversibly bind Mediator, a multi-protein complex that is required for Polymerase II transcription initiation. Mutations in several other genes encoding subunits of Mediator have been previously shown to associate with DD/ID, including MED13L, a paralog of MED13. Thus, our findings add MED13 to the group of CDK8-kinase module-associated disease genes.
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http://dx.doi.org/10.1007/s00439-018-1887-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973976PMC
May 2018

High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies.

Am J Hum Genet 2017 Nov;101(5):664-685

Manchester Centre for Genomic Medicine, St. Mary's Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester M13 9WL, UK.

Developmental and epileptic encephalopathy (DEE) is a group of conditions characterized by the co-occurrence of epilepsy and intellectual disability (ID), typically with developmental plateauing or regression associated with frequent epileptiform activity. The cause of DEE remains unknown in the majority of cases. We performed whole-genome sequencing (WGS) in 197 individuals with unexplained DEE and pharmaco-resistant seizures and in their unaffected parents. We focused our attention on de novo mutations (DNMs) and identified candidate genes containing such variants. We sought to identify additional subjects with DNMs in these genes by performing targeted sequencing in another series of individuals with DEE and by mining various sequencing datasets. We also performed meta-analyses to document enrichment of DNMs in candidate genes by leveraging our WGS dataset with those of several DEE and ID series. By combining these strategies, we were able to provide a causal link between DEE and the following genes: NTRK2, GABRB2, CLTC, DHDDS, NUS1, RAB11A, GABBR2, and SNAP25. Overall, we established a molecular diagnosis in 63/197 (32%) individuals in our WGS series. The main cause of DEE in these individuals was de novo point mutations (53/63 solved cases), followed by inherited mutations (6/63 solved cases) and de novo CNVs (4/63 solved cases). De novo missense variants explained a larger proportion of individuals in our series than in other series that were primarily ascertained because of ID. Moreover, these DNMs were more frequently recurrent than those identified in ID series. These observations indicate that the genetic landscape of DEE might be different from that of ID without epilepsy.
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http://dx.doi.org/10.1016/j.ajhg.2017.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673604PMC
November 2017

Genomic diagnosis for children with intellectual disability and/or developmental delay.

Genome Med 2017 05 30;9(1):43. Epub 2017 May 30.

HudsonAlpha Institute for Biotechnology, 601 Genome Way, Huntsville, AL, 35806, USA.

Background: Developmental disabilities have diverse genetic causes that must be identified to facilitate precise diagnoses. We describe genomic data from 371 affected individuals, 309 of which were sequenced as proband-parent trios.

Methods: Whole-exome sequences (WES) were generated for 365 individuals (127 affected) and whole-genome sequences (WGS) were generated for 612 individuals (244 affected).

Results: Pathogenic or likely pathogenic variants were found in 100 individuals (27%), with variants of uncertain significance in an additional 42 (11.3%). We found that a family history of neurological disease, especially the presence of an affected first-degree relative, reduces the pathogenic/likely pathogenic variant identification rate, reflecting both the disease relevance and ease of interpretation of de novo variants. We also found that improvements to genetic knowledge facilitated interpretation changes in many cases. Through systematic reanalyses, we have thus far reclassified 15 variants, with 11.3% of families who initially were found to harbor a VUS and 4.7% of families with a negative result eventually found to harbor a pathogenic or likely pathogenic variant. To further such progress, the data described here are being shared through ClinVar, GeneMatcher, and dbGaP.

Conclusions: Our data strongly support the value of large-scale sequencing, especially WGS within proband-parent trios, as both an effective first-choice diagnostic tool and means to advance clinical and research progress related to pediatric neurological disease.
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http://dx.doi.org/10.1186/s13073-017-0433-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448144PMC
May 2017

Mutations in EBF3 Disturb Transcriptional Profiles and Cause Intellectual Disability, Ataxia, and Facial Dysmorphism.

Am J Hum Genet 2017 Jan 22;100(1):117-127. Epub 2016 Dec 22.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

From a GeneMatcher-enabled international collaboration, we identified ten individuals affected by intellectual disability, speech delay, ataxia, and facial dysmorphism and carrying a deleterious EBF3 variant detected by whole-exome sequencing. One 9-bp duplication and one splice-site, five missense, and two nonsense variants in EBF3 were found; the mutations occurred de novo in eight individuals, and the missense variant c.625C>T (p.Arg209Trp) was inherited by two affected siblings from their healthy mother, who is mosaic. EBF3 belongs to the early B cell factor family (also known as Olf, COE, or O/E) and is a transcription factor involved in neuronal differentiation and maturation. Structural assessment predicted that the five amino acid substitutions have damaging effects on DNA binding of EBF3. Transient expression of EBF3 mutant proteins in HEK293T cells revealed mislocalization of all but one mutant in the cytoplasm, as well as nuclear localization. By transactivation assays, all EBF3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene CDKN1A, and in situ subcellular fractionation experiments demonstrated that EBF3 mutant proteins were less tightly associated with chromatin. Finally, in RNA-seq and ChIP-seq experiments, EBF3 acted as a transcriptional regulator, and mutant EBF3 had reduced genome-wide DNA binding and gene-regulatory activity. Our findings demonstrate that variants disrupting EBF3-mediated transcriptional regulation cause intellectual disability and developmental delay and are present in ∼0.1% of individuals with unexplained neurodevelopmental disorders.
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http://dx.doi.org/10.1016/j.ajhg.2016.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5223027PMC
January 2017

Germline and somatic mutations in the gene in focal cortical dysplasia and epilepsy.

Neurol Genet 2016 Dec 31;2(6):e118. Epub 2016 Oct 31.

The Danish Epilepsy Centre Filadelfia (R.S.M., G.R.), Dianalund, Denmark; Institute for Regional Health Services (R.S.M.), University of Southern Denmark, Odense; Sorbonne Universités (S.W., E.M., V.L., E.L., S.B.), UPMC Univ Paris 06 UMR S 1127, Inserm U1127, CNRS UMR 7225, AP-HP, Institut du cerveau et la moelle (ICM)-Hôpital Pitié-Salpêtrière, Paris, France; Epilepsy Unit (S.W., V.L.), AP-HP Groupe hospitalier Pitié-Salpêtrière, Paris, France; Neurogenetics Group (S.W.), VIB-Department of Molecular Genetics; Laboratory of Neurogenetics (S.W.), Institute Born-Bunge, University of Antwerp, Belgium; Department of Neurology (S.W.), University Hospital Antwerp, Belgium; Department of Pediatric Neurosurgery (M.C., S.F.-S., G.D.), Fondation Rothschild, Paris, France; Université Paris Sorbonne Cité (V.T.), INSERM UMR-S1147 MEPPOT, CNRS SNC5014, Centre Universitaire des Saints-Pères, Paris, France; Department of Neurology (E.M.B.), University of Alabama at Birmingham; HudsonAlpha Institute for Biotechnology (S.M.H., J.W.P., K.M.B., G.M.C.), Huntsville, AL; Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories (D.M., V.C., R.G.), Neuroscience Department, A Meyer Children's Hospital, University of Florence, Florence, Italy; Genosplice (P.d.l.G.), Institut du Cerveau et de la Moelle épinière, ICM, Paris, France; Amplexa Genetics (L.H.G.L.), Odense, Denmark; Department of Genetics and Cytogenetics (E.L., S.B.), AP-HP Groupe hospitalier Pitié-Salpêtrière, Paris, France; and University of Copenhagen (G.R.), Denmark.

Objective: To assess the prevalence of somatic mutations in focal cortical dysplasia (FCD) and of germline mutations in a broad range of epilepsies.

Methods: We collected 20 blood-brain paired samples from patients with FCD and searched for somatic variants using deep-targeted gene panel sequencing. Germline mutations in were assessed in a French research cohort of 93 probands with focal epilepsies and in a diagnostic Danish cohort of 245 patients with a broad range of epilepsies. Data sharing among collaborators allowed us to ascertain additional germline variants in .

Results: We detected recurrent somatic variants (p.Ser2215Phe, p.Ser2215Tyr, and p.Leu1460Pro) in the gene in 37% of participants with FCD II and showed histologic evidence for activation of the mTORC1 signaling cascade in brain tissue. We further identified 5 novel de novo germline missense variants in 6 individuals with a variable phenotype from focal, and less frequently generalized, epilepsies without brain malformations, to macrocephaly, with or without moderate intellectual disability. In addition, an inherited variant was found in a mother-daughter pair with nonlesional autosomal dominant nocturnal frontal lobe epilepsy.

Conclusions: Our data illustrate the increasingly important role of somatic mutations of the gene in FCD and germline mutations in the pathogenesis of focal epilepsy syndromes with and without brain malformation or macrocephaly.
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http://dx.doi.org/10.1212/NXG.0000000000000118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089441PMC
December 2016

RefSeq: an update on mammalian reference sequences.

Nucleic Acids Res 2014 Jan 19;42(Database issue):D756-63. Epub 2013 Nov 19.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, MD 20894, USA.

The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database is a collection of annotated genomic, transcript and protein sequence records derived from data in public sequence archives and from computation, curation and collaboration (http://www.ncbi.nlm.nih.gov/refseq/). We report here on growth of the mammalian and human subsets, changes to NCBI's eukaryotic annotation pipeline and modifications affecting transcript and protein records. Recent changes to NCBI's eukaryotic genome annotation pipeline provide higher throughput, and the addition of RNAseq data to the pipeline results in a significant expansion of the number of transcripts and novel exons annotated on mammalian RefSeq genomes. Recent annotation changes include reporting supporting evidence for transcript records, modification of exon feature annotation and the addition of a structured report of gene and sequence attributes of biological interest. We also describe a revised protein annotation policy for alternatively spliced transcripts with more divergent predicted proteins and we summarize the current status of the RefSeqGene project.
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http://dx.doi.org/10.1093/nar/gkt1114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965018PMC
January 2014

Current status and new features of the Consensus Coding Sequence database.

Nucleic Acids Res 2014 Jan 11;42(Database issue):D865-72. Epub 2013 Nov 11.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Building 38A, 8600 Rockville Pike, Bethesda, MD 20894, USA, Center for Biomolecular Science and Engineering, University of California Santa Cruz (UCSC), Santa Cruz, CA 95064, USA, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK and Howard Hughes Medical Institute, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

The Consensus Coding Sequence (CCDS) project (http://www.ncbi.nlm.nih.gov/CCDS/) is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies by the National Center for Biotechnology Information (NCBI) and Ensembl genome annotation pipelines. Identical annotations that pass quality assurance tests are tracked with a stable identifier (CCDS ID). Members of the collaboration, who are from NCBI, the Wellcome Trust Sanger Institute and the University of California Santa Cruz, provide coordinated and continuous review of the dataset to ensure high-quality CCDS representations. We describe here the current status and recent growth in the CCDS dataset, as well as recent changes to the CCDS web and FTP sites. These changes include more explicit reporting about the NCBI and Ensembl annotation releases being compared, new search and display options, the addition of biologically descriptive information and our approach to representing genes for which support evidence is incomplete. We also present a summary of recent and future curation targets.
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http://dx.doi.org/10.1093/nar/gkt1059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965069PMC
January 2014

Caenorhabditis elegans FOS-1 and JUN-1 regulate plc-1 expression in the spermatheca to control ovulation.

Mol Biol Cell 2009 Sep 1;20(17):3888-95. Epub 2009 Jul 1.

Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Purdue University, West Lafayette, IN 47907, USA.

Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecal-specific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.
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http://dx.doi.org/10.1091/mbc.e08-08-0833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735487PMC
September 2009

Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans.

Methods 2008 Jul 27;45(3):185-91. Epub 2008 Jun 27.

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue Cancer Center, Purdue University, 575 Stadium Mall Drive, RHPH224D, West Lafayette, Indiana, IN 47907-2091, USA.

Protein interactions are essential components of signal transduction in cells. With the progress in genome-wide yeast two hybrid screens and proteomics analyses, many protein interaction networks have been generated. These analyses have identified hundreds and thousands of interactions in cells and organisms, creating a challenge for further validation under physiological conditions. The bimolecular fluorescence complementation (BiFC) assay is such an assay that meets this need. The BiFC assay is based on the principle of protein fragment complementation, in which two non-fluorescent fragments derived from a fluorescent protein are fused to a pair of interacting partners. When the two partners interact, the two non-fluorescent fragments are brought into proximity and an intact fluorescent protein is reconstituted. Hence, the reconstituted fluorescent signals reflect the interaction of two proteins under study. Over the past six years, the BiFC assay has been used for visualization of protein interactions in living cells and organisms, including our application of the BiFC assay to the transparent nematode Caenorhabditis elegans. We have demonstrated that BiFC analysis in C. elegans provides a direct means to identify and validate protein interactions in living worms and allows visualization of temporal and spatial interactions. Here, we provide a guideline for the implementation of BiFC analysis in living worms and discuss the factors that are critical for BiFC analysis.
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http://dx.doi.org/10.1016/j.ymeth.2008.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570267PMC
July 2008

Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis.

Nat Protoc 2008 ;3(4):588-96

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-2091, USA.

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.
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http://dx.doi.org/10.1038/nprot.2008.16DOI Listing
October 2008
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