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The purpose of this study is to gain insights into potential genetic factors contributing to the infant's vulnerability to Sudden Unexpected Infant Death (SUID). Whole Genome Sequencing (WGS) was performed on 144 infants that succumbed to SUID, and 573 healthy adults. Variants were filtered by gnomAD allele frequencies and predictions of functional consequences. Variants of interest were identified in 88 genes, in 64.6% of our cohort. Seventy-three of these have been previously associated with SIDS/SUID/SUDP. Forty-three can be characterized as cardiac genes and are related to cardiomyopathies, arrhythmias, and other conditions. Variants in 22 genes were associated with neurologic functions. Variants were also found in 13 genes reported to be pathogenic for various systemic disorders and in two genes associated with immunological function. Variants in eight genes are implicated in the response to hypoxia and the regulation of reactive oxygen species (ROS) and have not been previously described in SIDS/SUID/SUDP. Seventy-two infants met the triple risk hypothesis criteria. Our study confirms and further expands the list of genetic variants associated with SUID. The abundance of genes associated with heart disease and the discovery of variants associated with the redox metabolism have important mechanistic implications for the pathophysiology of SUID.
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As the number of genes associated with various germline disorders continues to grow, it is becoming more difficult for clinical laboratories to maintain separate assays for interrogating disease-focused gene panels. One solution to this challenge is termed slice testing, where capture backbone is used to analyze data specific to a set of genes, and for this article, we will focus on exome. A key advantage to this strategy is greater flexibility by adding genes as they become associated with disease or the ability to accommodate specific provider requests. Here, we provide expert consensus recommendations and results from an Association for Molecular Pathology-sponsored survey of clinical laboratories performing exome sequencing to compare a slice testing approach with traditional static gene panels and comprehensive exome analysis. We explore specific considerations for slices, including gene selection, analytic performance, coverage, quality, and interpretation. Our goal is to provide comprehensive guidance for clinical laboratories interested in designing and using slice tests as a diagnostic.
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Consejeros , Patología Molecular , Humanos , Estados Unidos , Patólogos , Encuestas y CuestionariosRESUMEN
CONTEXT.: Next-generation sequencing (NGS)-based assays are used for diagnosis of diverse inherited disorders. Limited data are available pertaining to interlaboratory analytical performance of these assays. OBJECTIVE.: To report on the College of American Pathologists (CAP) NGS Germline Program, which is methods based, and explore the evolution in laboratory testing practices. DESIGN.: Results from the NGS Germline Program from 2016-2020 were analyzed for interlaboratory analytical performance. Self-reported laboratory testing practices were also evaluated. RESULTS.: From 2016-2020, a total of 297 laboratories participated in at least 1 program mailing. Of the 289 laboratories that provided information on tests offered, 138 (47.8%) offered only panel testing throughout their enrollment, while 35 (12.1%) offered panels and exome testing, 30 (10.4%) offered only exomes, 9 (3.1%) offered only genomes, and 15 (5.2%) offered panels, exomes, and genomes. The remainder (62 laboratories, 21.4%) changed their test offerings during the 2016-2020 timeframe. Considering each genomic position/interval, the median detection percentage at variant positions across the 2016-2020 mailings ranged from 94.3% to 100%, while at reference positions (no variant detected), the median correct response percentage was 100% across all mailings. When considering performance of individual laboratories, 89.5% (136 of 152) to 98.0% (149 of 152) of laboratories successfully met the detection threshold (≥90% of the variants present), while 94.6% (87 of 92) to 100% (163 of 163) of laboratories met the 95% specificity threshold across mailings. CONCLUSIONS.: Since the inception of this program, laboratories have consistently performed well. The median sensitivity and specificity of detection of sequence variants included in this program (eg, single nucleotide variants, insertions, and deletions) were 100.0%.
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Purpose: To gain insights into potential genetic factors contributing to the infant's vulnerability to Sudden Unexpected Infant Death (SUID). Methods: Whole Genome Sequencing (WGS) was performed on 145 infants that succumbed to SUID, and 576 healthy adults. Variants were filtered by gnomAD allele frequencies and predictions of functional consequences. Results: Variants of interest were identified in 86 genes, 63.4% of our cohort. Seventy-one of these have been previously associated with SIDS/SUID/SUDP. Forty-three can be characterized as cardiac genes and are related to cardiomyopathies, arrhythmias, and other conditions. Variants in 22 genes were associated with neurologic functions. Variants were also found in 13 genes reported to be pathogenic for various systemic disorders. Variants in eight genes are implicated in the response to hypoxia and the regulation of reactive oxygen species (ROS) and have not been previously described in SIDS/SUID/SUDP. Seventy-two infants met the triple risk hypothesis criteria (Figure 1). Conclusion: Our study confirms and further expands the list of genetic variants associated with SUID. The abundance of genes associated with heart disease and the discovery of variants associated with the redox metabolism have important mechanistic implications for the pathophysiology of SUID.
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Clinical laboratory implementation of next-generation sequencing (NGS)-based constitutional genetic testing has been rapid and widespread. In the absence of widely adopted comprehensive guidance, there remains substantial variability among laboratories in the practice of NGS. One issue of sustained discussion in the field is whether and to what extent orthogonal confirmation of genetic variants identified by NGS is necessary or helpful. The Association for Molecular Pathology Clinical Practice Committee convened the NGS Germline Variant Confirmation Working Group to assess current evidence regarding orthogonal confirmation and to establish recommendations for standardizing orthogonal confirmation practices to support quality patient care. On the basis of the results of a survey of the literature, a survey of laboratory practices, and subject expert matter consensus, eight recommendations are presented, providing a common framework for clinical laboratory professionals to develop or refine individualized laboratory policies and procedures regarding orthogonal confirmation of germline variants detected by NGS.
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Consejeros , Patología Molecular , Humanos , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Células GerminativasRESUMEN
Exome reanalysis is useful for providing molecular diagnoses for previously uninformative samples. However, challenges exist in implementing a practical solution for clinicians and laboratories. This study complements the current literature by providing practical considerations for patient-level and cohort-level reanalyses. The Clinical and Laboratory Standards Institute assembled the Document Development Committee and an interpretation working group that developed the framework for reevaluation of exome-based data. We describe two distinct but complementary approaches toward exome reanalyses: clinician-initiated patient-level reanalysis, and laboratory-initiated cohort-level reanalysis. We highlight the advantages and constraints for both approaches, and provide a high-level conceptual guide for ordering clinicians and laboratories through the critical decision pathways. Because clinical exome sequencing continues to be the standard of care in genetics, exome reanalysis would be critical in increasing the overall diagnostic yield. A systematic guide will facilitate the efficient adoption of reevaluation of exome data for laboratories, health care professionals, genetic counselors, and clinicians.
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Servicios de Laboratorio Clínico , Exoma , Exoma/genética , Humanos , Laboratorios , Laboratorios Clínicos , Secuenciación del ExomaRESUMEN
Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.
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Exoma , Patología Molecular , Niño , Exoma/genética , Humanos , Mutación , Enfermedades Raras/genética , Estudios Retrospectivos , Secuenciación del Exoma/métodosRESUMEN
Novel gene-disease discoveries, rapid advancements in technology, and improved bioinformatics tools all have the potential to yield additional molecular diagnoses through the reanalysis of exome sequencing data. Collaborations between clinical laboratories, ordering physicians, and researchers are also driving factors that can contribute to these new insights. Automation in ongoing natural history collection, evolving phenotype updates, advancements in processing next-generation sequencing data, and up-to-date variant-gene-disease databases are increasingly needed for systematic exome reanalysis. Here, we review some of the advantages and challenges for clinician-initiated and laboratory-initiated exome reanalysis, and we propose a model for the future that could potentially maximize the clinical utility of exome reanalysis by integrating information from electronic medical records and knowledge databases into routine clinical workflows.
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Exoma , Laboratorios Clínicos , Biología Computacional , Exoma/genética , Humanos , Fenotipo , Secuenciación del ExomaRESUMEN
PURPOSE: CACNA1C encodes the alpha-1-subunit of a voltage-dependent L-type calcium channel expressed in human heart and brain. Heterozygous variants in CACNA1C have previously been reported in association with Timothy syndrome and long QT syndrome. Several case reports have suggested that CACNA1C variation may also be associated with a primarily neurological phenotype. METHODS: We describe 25 individuals from 22 families with heterozygous variants in CACNA1C, who present with predominantly neurological manifestations. RESULTS: Fourteen individuals have de novo, nontruncating variants and present variably with developmental delays, intellectual disability, autism, hypotonia, ataxia, and epilepsy. Functional studies of a subgroup of missense variants via patch clamp experiments demonstrated differential effects on channel function in vitro, including loss of function (p.Leu1408Val), neutral effect (p.Leu614Arg), and gain of function (p.Leu657Phe, p.Leu614Pro). The remaining 11 individuals from eight families have truncating variants in CACNA1C. The majority of these individuals have expressive language deficits, and half have autism. CONCLUSION: We expand the phenotype associated with CACNA1C variants to include neurodevelopmental abnormalities and epilepsy, in the absence of classic features of Timothy syndrome or long QT syndrome.
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Trastorno Autístico , Canales de Calcio Tipo L , Síndrome de QT Prolongado , Sindactilia , Trastorno Autístico/genética , Canales de Calcio Tipo L/genética , Humanos , FenotipoRESUMEN
Retinoic acid exposures as well as defects in the retinoic acid-degrading enzyme CYP26B1 have teratogenic effects on both limb and craniofacial skeleton. An initial report of four individuals described a syndrome of fetal and infantile lethality with craniosynostosis and skeletal anomalies caused by homozygous pathogenic missense variants in CYP26B1. In contrast, a 22-year-old female was reported with a homozygous missense pathogenic variant in CYP26B1 with complex multisuture craniosynostosis and intellectual disability, suggesting that in some cases, biallelic pathogenic variants of CYP26B1 may be compatible with life. Here we describe four additional living individuals from two families with compound heterozygous pathogenic missense variants in CYP26B1. Structural assessment of these additional missense variants places them further from the catalytic site and supports a model consistent with milder nonlethal disease. In addition to previously reported findings of multisuture craniosynostosis, conductive hearing loss, joint contractures, long slender fingers, camptodactly, broad fingertips, and developmental delay/intellectual disability, skeletal imaging in our cases also revealed gracile long bones, gracile ribs, radioulnar synostosis, and carpal and/or tarsal fusions. These individuals broaden the phenotypic range of biallelic pathogenic variants in CYPB26B1 and most significantly clarify that mortality can range from perinatal lethality to survival into adulthood.
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Anomalías Múltiples/patología , Homocigoto , Mutación Missense , Radio (Anatomía)/anomalías , Ácido Retinoico 4-Hidroxilasa/genética , Sinostosis/patología , Cúbito/anomalías , Anomalías Múltiples/genética , Niño , Familia , Femenino , Humanos , Lactante , Masculino , Fenotipo , Radio (Anatomía)/patología , Sinostosis/genética , Cúbito/patologíaRESUMEN
EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.
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Proteínas de Unión al Calcio/genética , Trastornos Congénitos de Glicosilación/genética , Retículo Endoplásmico/genética , alfa-Manosidasa/genética , Adolescente , Alelos , Proteínas de Unión al Calcio/deficiencia , Línea Celular , Niño , Preescolar , Trastornos Congénitos de Glicosilación/sangre , Discapacidades del Desarrollo/genética , Femenino , Glicoproteínas/sangre , Glicosilación , Humanos , Lactante , Discapacidad Intelectual/genética , Masculino , Mutación , Linaje , Polisacáridos/sangre , Deficiencias en la Proteostasis/genética , alfa-Manosidasa/deficienciaRESUMEN
The RASopathies are a group of similar genetic syndromes with cardiovascular abnormalities, characteristic facial features, short stature, abnormalities of the skin and musculoskeletal system, and variable neurodevelopmental challenges. The most common cardiovascular abnormalities include pulmonary valvular stenosis and hypertrophic cardiomyopathy. Congenital polyvalvular disease (CPVD) refers to congenital dysplasia of two or more cardiac valves. We diagnosed a RASopathy in two individuals with CPVD and noted that CPVD in RASopathies has rarely been reported in the literature. Thus, we performed a retrospective chart review and literature review to investigate the association and characterize the phenotype of CPVD in the RASopathies. CPVD was present in 2.5% (n = 6/243) of individuals in our RASopathy cohort. Involvement of two cardiac valves, commonly the aortic and pulmonic valves, was seen in the majority of individuals (6/8; 75%) in our cohort, but only 27% (3/11) of reported CPVD and RASopathy cases in the literature. CPVD should be considered an associated cardiovascular phenotype of the RASopathies, which has implications for diagnosis and management.
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Cardiomiopatía Hipertrófica/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteínas Proto-Oncogénicas B-raf/genética , Estenosis de la Válvula Pulmonar/genética , Adolescente , Válvula Aórtica/patología , Cardiomiopatía Hipertrófica/epidemiología , Cardiomiopatía Hipertrófica/patología , Anomalías Cardiovasculares/epidemiología , Anomalías Cardiovasculares/genética , Anomalías Cardiovasculares/patología , Niño , Preescolar , Enanismo/genética , Enanismo/patología , Facies , Femenino , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Humanos , Lactante , Recién Nacido , Masculino , Anomalías Musculoesqueléticas/epidemiología , Anomalías Musculoesqueléticas/genética , Anomalías Musculoesqueléticas/patología , Síndrome de Noonan , Fenotipo , Estenosis de la Válvula Pulmonar/epidemiología , Estenosis de la Válvula Pulmonar/patología , Anomalías Cutáneas/genética , Anomalías Cutáneas/patología , Proteínas ras/genéticaRESUMEN
Carrier screening involves detection of carrier status for genes associated with recessive conditions. A negative carrier screening test result bears a nonzero residual risk (RR) for the individual to have an affected child. The RR depends on the prevalence of specific conditions and the detection rate (DR) of the test itself. Herein, we provide a detailed approach for calculating DR and RR. DR was calculated on the basis of the sum of disease allele frequencies (DAFs) of pathogenic variants found in published literature. As a proof of concept, DAF data for cystic fibrosis were compared with society guidelines. The DAF data calculated by this method were consistent with the published cystic fibrosis guideline. In addition, we compared DAF for four genes (ABCC8, ASPA, GAA, and MMUT) across three laboratories, and outlined the likely reasons for discrepancies between these laboratories. The utility of carrier screening is to support couples with information while making reproductive choices. Accurate development of DR and RR is therefore critical. The method described herein provides an unbiased and transparent process to collect, calculate, and report these data.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/prevención & control , Frecuencia de los Genes , Tamización de Portadores Genéticos/métodos , Amidohidrolasas/genética , Consanguinidad , Familia , Asesoramiento Genético/métodos , Humanos , Tamizaje Masivo/métodos , Receptores de Sulfonilureas/genética , alfa-Glucosidasas/genéticaRESUMEN
Congenital heart defects (CHD) are the most common birth defect and are both clinically and genetically heterogeneous. Truncus arteriosus (TA), characterized by a single arterial vessel arising from both ventricles giving rise to the coronary, pulmonary and systemic arteries, is rare and only responsible for 1% of all CHD. Two consanguineous families with TA were previously identified to have homozygous nonsense variants within the gene NKX2-6. NKX2-6 is a known downstream target of TBX1, an important transcriptional regulator implicated in the cardiac phenotype of 22q11.2 microdeletion syndrome. Herein, we report two siblings with TA presumably caused by compound heterozygous NKX2-6 variants without a history of consanguinity. Two in-house cohorts with conotruncal defects (CTD) were sequenced for variants in NKX2-6 and no additional cases of biallelic NKX2-6 variants were identified. The similar phenotype of these cases, and the clustering of variants that likely result in a truncated protein that disrupts the homeobox domain, suggest that biallelic loss of function for NKX2-6 is a rare genetic etiology for TA in particular, and possibly other types of CHD.
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Cardiopatías Congénitas/genética , Proteínas de Homeodominio/genética , Proteínas de Dominio T Box/genética , Tronco Arterial/fisiopatología , Alelos , Arterias/anomalías , Arterias/crecimiento & desarrollo , Niño , Preescolar , Codón sin Sentido/genética , Anomalías Congénitas/genética , Anomalías Congénitas/fisiopatología , Consanguinidad , Femenino , Cardiopatías Congénitas/complicaciones , Cardiopatías Congénitas/fisiopatología , Humanos , Lactante , Masculino , Linaje , Fenotipo , Dominios Proteicos/genética , Tronco Arterial/metabolismoRESUMEN
Human imprinting disorders cause a range of dysmorphic and neurocognitive phenotypes, and they may elude traditional molecular diagnosis such exome sequencing. The discovery of novel disorders related to imprinted genes has lagged behind traditional Mendelian disorders because current diagnostic technology, especially unbiased testing, has limited utility in their discovery. To identify novel imprinting disorders, we reviewed data for every human gene hypothesized to be imprinted, identified each mouse ortholog, determined its imprinting status in the mouse, and analyzed its function in humans and mice. We identified 17 human genes that are imprinted in both humans and mice, and have functional data in mice or humans to suggest that dysregulated expression would lead to an abnormal phenotype in humans. These 17 genes, along with known imprinted genes, were preferentially flagged 538 clinical exome sequencing tests. The identified genes were: DIRAS3 [1p31.3], TP73 [1p36.32], SLC22A3 [6q25.3], GRB10 [7p12.1], DDC [7p12.2], MAGI2 [7q21.11], PEG10 [7q21.3], PPP1R9A [7q21.3], CALCR [7q21.3], DLGAP2 [8p23.3], GLIS3 [9p24.2], INPP5F [10q26.11], ANO1 [11q13.3], SLC38A4 [12q13.11], GATM [15q21.1], PEG3 [19q13.43], and NLRP2 [19q13.42]. In the 538 clinical cases, eight cases (1.7%) reported variants in a causative known imprinted gene. There were 367/758 variants (48.4%) in imprinted genes that were not known to cause disease, but none of those variants met the criteria for clinical reporting. Imprinted disorders play a significant role in human disease, and additional human imprinted disorders remain to be discovered. Therefore, evolutionary conservation is a potential tool to identify novel genes involved in human imprinting disorders and to identify them in clinical testing.
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Secuencia Conservada , Exoma , Enfermedades Genéticas Congénitas/genética , Sitios Genéticos , Impresión Genómica , Animales , Bases de Datos Genéticas , Humanos , Ratones , Mutación , Secuenciación Completa del Genoma/métodosRESUMEN
Cystic fibrosis (CF) is one of the most common genetic diseases worldwide with high carrier frequencies across different ethnicities. Next generation sequencing of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has proven to be an effective screening tool to determine carrier status with high detection rates. Here, we evaluate the performance of the Swift Biosciences Accel-Amplicon CFTR Capture Panel using CFTR-positive DNA samples. This assay is a one-day protocol that allows for one-tube reaction of 87 amplicons that span all coding regions, 5' and 3'UTR, as well as four intronic regions. In this study, we provide the FASTQ, BAM, and VCF files on seven unique CFTR-positive samples and one normal control sample (14 samples processed including repeated samples). This method generated sequencing data with high coverage and near 100% on-target reads. We found that coverage depth was correlated with the GC content of each exon. This dataset is instrumental for clinical laboratories that are evaluating this technology as part of their carrier screening program.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Tamización de Portadores Genéticos , Composición de Base , Humanos , Análisis de Secuencia de ADNRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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PURPOSE: Pediatric cardiomyopathy is rare, has a broad differential diagnosis, results in high morbidity and mortality, and has suboptimal diagnostic yield using next-generation sequencing panels. Exome sequencing has reported diagnostic yields ranging from 30% to 57% for neonates in intensive care units. We aimed to characterize the clinical utility of exome sequencing in infantile heart failure. METHODS: Infants diagnosed with acute heart failure prior to 1 year old over a period of 34 months at a large tertiary children's hospital were recruited. Demographic and diagnostic information was obtained from medical records. Fifteen eligible patients were enrolled. RESULTS: Dilated cardiomyopathy was the predominant cardiac diagnosis, seen in 60% of patients. A molecular diagnosis was identified in 66.7% of patients (10/15). Of those diagnoses, 70% would not have been detected using multigene next-generation sequencing panels focused on cardiomyopathy or arrhythmia disease genes. Genetic testing changed medical decision-making in 53% of all cases and 80% of positive cases, and was especially beneficial when testing was expedited. CONCLUSION: Given the broad differential diagnosis and critical status of infants with heart failure, rapid exome sequencing provides timely diagnoses, changes medical management, and should be the first-tier molecular test.
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Secuenciación del Exoma/tendencias , Pruebas Genéticas/ética , Insuficiencia Cardíaca/diagnóstico , Cardiomiopatías/genética , Cardiomiopatía Dilatada/genética , Exoma/genética , Femenino , Pruebas Genéticas/tendencias , Insuficiencia Cardíaca/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante , Recién Nacido , Masculino , Resultado del Tratamiento , Secuenciación del Exoma/métodosRESUMEN
PURPOSE: Sifrim-Hitz-Weiss syndrome (SIHIWES) is a recently described multisystemic neurodevelopmental disorder caused by de novo variants inCHD4. In this study, we investigated the clinical spectrum of the disorder, genotype-phenotype correlations, and the effect of different missense variants on CHD4 function. METHODS: We collected clinical and molecular data from 32 individuals with mostly de novo variants in CHD4, identified through next-generation sequencing. We performed adenosine triphosphate (ATP) hydrolysis and nucleosome remodeling assays on variants from five different CHD4 domains. RESULTS: The majority of participants had global developmental delay, mild to moderate intellectual disability, brain anomalies, congenital heart defects, and dysmorphic features. Macrocephaly was a frequent but not universal finding. Additional common abnormalities included hypogonadism in males, skeletal and limb anomalies, hearing impairment, and ophthalmic abnormalities. The majority of variants were nontruncating and affected the SNF2-like region of the protein. We did not identify genotype-phenotype correlations based on the type or location of variants. Alterations in ATP hydrolysis and chromatin remodeling activities were observed in variants from different domains. CONCLUSION: The CHD4-related syndrome is a multisystemic neurodevelopmental disorder. Missense substitutions in different protein domains alter CHD4 function in a variant-specific manner, but result in a similar phenotype in humans.