Disruption of MeCP2-TCF20 complex underlies distinct neurodevelopmental disorders.

MeCP2 is associated with Rett syndrome (RTT), MECP2 duplication syndrome, and a number of conditions with isolated features of these diseases, including autism, intellectual disability, and motor dysfunction. MeCP2 is known to broadly bind methylated DNA, but the precise molecular mechanism driving disease pathogenesis remains to be determined. Using proximity-dependent biotinylation (BioID), we identified a transcription factor 20 (TCF20) complex that interacts with MeCP2 at the chromatin interface. Importantly, RTT-causing mutations in MECP2 disrupt this interaction. TCF20 and MeCP2 are highly coexpressed in neurons and coregulate the expression of key neuronal genes. Reducing Tcf20 partially rescued the behavioral deficits caused by MECP2 overexpression, demonstrating a functional relationship between MeCP2 and TCF20 in MECP2 duplication syndrome pathogenesis. We identified a patient exhibiting RTT-like neurological features with a missense mutation in the PHF14 subunit of the TCF20 complex that abolishes the MeCP2-PHF14-TCF20 interaction. Our data demonstrate the critical role of the MeCP2-TCF20 complex for brain function.

Heterozygous ANKRD17 loss-of-function variants cause a syndrome with intellectual disability, speech delay, and dysmorphism.

ANKRD17 is an ankyrin repeat-containing protein thought to play a role in cell cycle progression, whose ortholog in Drosophila functions in the Hippo pathway as a co-factor of Yorkie. Here, we delineate a neurodevelopmental disorder caused by de novo heterozygous ANKRD17 variants. The mutational spectrum of this cohort of 34 individuals from 32 families is highly suggestive of haploinsufficiency as the underlying mechanism of disease, with 21 truncating or essential splice site variants, 9 missense variants, 1 in-frame insertion-deletion, and 1 microdeletion (1.16 Mb). Consequently, our data indicate that loss of ANKRD17 is likely the main cause of phenotypes previously associated with large multi-gene chromosomal aberrations of the 4q13.3 region. Protein modeling suggests that most of the missense variants disrupt the stability of the ankyrin repeats through alteration of core structural residues. The major phenotypic characteristic of our cohort is a variable degree of developmental delay/intellectual disability, particularly affecting speech, while additional features include growth failure, feeding difficulties, non-specific MRI abnormalities, epilepsy and/or abnormal EEG, predisposition to recurrent infections (mostly bacterial), ophthalmological abnormalities, gait/balance disturbance, and joint hypermobility. Moreover, many individuals shared similar dysmorphic facial features. Analysis of single-cell RNA-seq data from the developing human telencephalon indicated ANKRD17 expression at multiple stages of neurogenesis, adding further evidence to the assertion that damaging ANKRD17 variants cause a neurodevelopmental disorder.

De novo variants predicting haploinsufficiency for DIP2C are associated with expressive speech delay.

The disconnected (disco)-interacting protein 2 (DIP2) gene was first identified in D. melanogaster and contains a DNA methyltransferase-associated protein 1 (DMAP1) binding domain, Acyl-CoA synthetase domain and AMP-binding sites. DIP2 regulates axonal bifurcation of the mushroom body neurons in D. melanogaster and is required for axonal regeneration in the neurons of C. elegans. The DIP2 homologues in vertebrates, Disco-interacting protein 2 homolog A (DIP2A), Disco-interacting protein 2 homolog B (DIP2B), and Disco-interacting protein 2 homolog C (DIP2C), are highly conserved and expressed widely in the central nervous system. Although there is evidence that DIP2C plays a role in cognition, reports of pathogenic variants in these genes are rare and their significance is uncertain. We present 23 individuals with heterozygous DIP2C variants, all manifesting developmental delays that primarily affect expressive language and speech articulation. Eight patients had de novo variants predicting loss-of-function in the DIP2C gene, two patients had de novo missense variants, three had paternally inherited loss of function variants and six had maternally inherited loss-of-function variants, while inheritance was unknown for four variants. Four patients had cardiac defects (hypertrophic cardiomyopathy, atrial septal defects, and bicuspid aortic valve). Minor facial anomalies were inconsistent but included a high anterior hairline with a long forehead, broad nasal tip, and ear anomalies. Brainspan analysis showed elevated DIP2C expression in the human neocortex at 10-24 weeks after conception. With the cases presented herein, we provide phenotypic and genotypic data supporting the association between loss-of-function variants in DIP2C with a neurocognitive phenotype.

ARF1-related disorder: phenotypic and molecular spectrum.

PURPOSE: ARF1 was previously implicated in periventricular nodular heterotopia (PVNH) in only five individuals and systematic clinical characterisation was not available. The aim of this study is to provide a comprehensive description of the phenotypic and genotypic spectrum of ARF1-related neurodevelopmental disorder. METHODS: We collected detailed phenotypes of an international cohort of individuals (n=17) with ARF1 variants assembled through the GeneMatcher platform. Missense variants were structurally modelled, and the impact of several were functionally validated. RESULTS: De novo variants (10 missense, 1 frameshift, 1 splice altering resulting in 9 residues insertion) in ARF1 were identified among 17 unrelated individuals. Detailed phenotypes included intellectual disability (ID), microcephaly, seizures and PVNH. No specific facial characteristics were consistent across all cases, however microretrognathia was common. Various hearing and visual defects were recurrent, and interestingly, some inflammatory features were reported. MRI of the brain frequently showed abnormalities consistent with a neuronal migration disorder. CONCLUSION: We confirm the role of ARF1 in an autosomal dominant syndrome with a phenotypic spectrum including severe ID, microcephaly, seizures and PVNH due to impaired neuronal migration.

Non-coding and Loss-of-Function Coding Variants in TET2 are Associated with Multiple Neurodegenerative Diseases.

We conducted genome sequencing to search for rare variation contributing to early-onset Alzheimer's disease (EOAD) and frontotemporal dementia (FTD). Discovery analysis was conducted on 435 cases and 671 controls of European ancestry. Burden testing for rare variation associated with disease was conducted using filters based on variant rarity (less than one in 10,000 or private), computational prediction of deleteriousness (CADD) (10 or 15 thresholds), and molecular function (protein loss-of-function [LoF] only, coding alteration only, or coding plus non-coding variants in experimentally predicted regulatory regions). Replication analysis was conducted on 16,434 independent cases and 15,587 independent controls. Rare variants in TET2 were enriched in the discovery combined EOAD and FTD cohort (p = 4.6 × 10-8, genome-wide corrected p = 0.0026). Most of these variants were canonical LoF or non-coding in predicted regulatory regions. This enrichment replicated across several cohorts of Alzheimer's disease (AD) and FTD (replication only p = 0.0029). The combined analysis odds ratio was 2.3 (95% confidence interval [CI] 1.6-3.4) for AD and FTD. The odds ratio for qualifying non-coding variants considered independently from coding variants was 3.7 (95% CI 1.7-9.4). For LoF variants, the combined odds ratio (for AD, FTD, and amyotrophic lateral sclerosis, which shares clinicopathological overlap with FTD) was 3.1 (95% CI 1.9-5.2). TET2 catalyzes DNA demethylation. Given well-defined changes in DNA methylation that occur during aging, rare variation in TET2 may confer risk for neurodegeneration by altering the homeostasis of key aging-related processes. Additionally, our study emphasizes the relevance of non-coding variation in genetic studies of complex disease.

De novo variants in CNOT9 cause a neurodevelopmental disorder with or without epilepsy.

PURPOSE: The study aimed to clinically and molecularly characterize the neurodevelopmental disorder associated with heterozygous de novo variants in CNOT9. METHODS: Individuals were clinically examined. Variants were identified using exome or genome sequencing. These variants were evaluated using in silico predictions, and their functional relevance was further assessed by molecular models and research in the literature. The variants have been classified according to the criteria of the American College of Medical Genetics. RESULTS: We report on 7 individuals carrying de novo missense variants in CNOT9, p.(Arg46Gly), p.(Pro131Leu), and p.(Arg227His), and, recurrent in 4 unrelated individuals, p.(Arg292Trp). All affected persons have developmental delay/intellectual disability, with 5 of them showing seizures. Other symptoms include muscular hypotonia, facial dysmorphism, and behavioral abnormalities. Molecular modeling predicted that the variants are damaging and would lead to reduced protein stability or impaired recognition of interaction partners. Functional analyses in previous studies showed a pathogenic effect of p.(Pro131Leu) and p.(Arg227His). CONCLUSION: We propose CNOT9 as a novel gene for neurodevelopmental disorder and epilepsy.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

PURPOSE: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze "poison exons" (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. METHODS: We curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. RESULTS: Across 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. CONCLUSION: With a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Deleterious Variation in BRSK2 Associates with a Neurodevelopmental Disorder.

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 × 10-6). 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.

Inherited variants in CHD3 show variable expressivity in Snijders Blok-Campeau syndrome.

PURPOSE: Common diagnostic next-generation sequencing strategies are not optimized to identify inherited variants in genes associated with dominant neurodevelopmental disorders as causal when the transmitting parent is clinically unaffected, leaving a significant number of cases with neurodevelopmental disorders undiagnosed. METHODS: We characterized 21 families with inherited heterozygous missense or protein-truncating variants in CHD3, a gene in which de novo variants cause Snijders Blok-Campeau syndrome. RESULTS: Computational facial and Human Phenotype Ontology-based comparisons showed that the phenotype of probands with inherited CHD3 variants overlaps with the phenotype previously associated with de novo CHD3 variants, whereas heterozygote parents are mildly or not affected, suggesting variable expressivity. In addition, similarly reduced expression levels of CHD3 protein in cells of an affected proband and of healthy family members with a CHD3 protein-truncating variant suggested that compensation of expression from the wild-type allele is unlikely to be an underlying mechanism. Notably, most inherited CHD3 variants were maternally transmitted. CONCLUSION: Our results point to a significant role of inherited variation in Snijders Blok-Campeau syndrome, a finding that is critical for correct variant interpretation and genetic counseling and warrants further investigation toward understanding the broader contributions of such variation to the landscape of human disease.

Genome sequencing as a first-line diagnostic test for hospitalized infants.

PURPOSE: SouthSeq is a translational research study that undertook genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern United States, which are historically underrepresented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants, and 14% of infants harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing, suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results show the utility of GS because it provides early-in-life detection of clinically relevant genetic variations not detected by current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.

Identifying rare, medically relevant variation via population-based genomic screening in Alabama: opportunities and pitfalls.

PURPOSE: To evaluate the effectiveness and specificity of population-based genomic screening in Alabama. METHODS: The Alabama Genomic Health Initiative (AGHI) has enrolled and evaluated 5369 participants for the presence of pathogenic/likely pathogenic (P/LP) variants using the Illumina Global Screening Array (GSA), with validation of all P/LP variants via Sanger sequencing in a CLIA-certified laboratory before return of results. RESULTS: Among 131 variants identified by the GSA that were evaluated by Sanger sequencing, 67 (51%) were false positives (FP). For 39 of the 67 FP variants, a benign/likely benign variant was present at or near the targeted P/LP variant. Variants detected within African American individuals were significantly enriched for FPs, likely due to a higher rate of nontargeted alternative alleles close to array-targeted P/LP variants. CONCLUSION: In AGHI, we have implemented an array-based process to screen for highly penetrant genetic variants in actionable disease genes. We demonstrate the need for clinical validation of array-identified variants in direct-to-consumer or population testing, especially for diverse populations.

A state-based approach to genomics for rare disease and population screening.

PURPOSE: The Alabama Genomic Health Initiative (AGHI) is a state-funded effort to provide genomic testing. AGHI engages two distinct cohorts across the state of Alabama. One cohort includes children and adults with undiagnosed rare disease; a second includes an unselected adult population. Here we describe findings from the first 176 rare disease and 5369 population cohort AGHI participants. METHODS: AGHI participants enroll in one of two arms of a research protocol that provides access to genomic testing results and biobank participation. Rare disease cohort participants receive genome sequencing to identify primary and secondary findings. Population cohort participants receive genotyping to identify pathogenic and likely pathogenic variants for actionable conditions. RESULTS: Within the rare disease cohort, genome sequencing identified likely pathogenic or pathogenic variation in 20% of affected individuals. Within the population cohort, 1.5% of individuals received a positive genotyping result. The rate of genotyping results corroborated by reported personal or family history varied by gene. CONCLUSIONS: AGHI demonstrates the ability to provide useful health information in two contexts: rare undiagnosed disease and population screening. This utility should motivate continued exploration of ways in which emerging genomic technologies might benefit broad populations.

PPA2-associated sudden cardiac death: extending the clinical and allelic spectrum in 20 new families.

PURPOSE: Biallelic hypomorphic variants in PPA2, encoding the mitochondrial inorganic pyrophosphatase 2 protein, have been recently identified in individuals presenting with sudden cardiac death, occasionally triggered by alcohol intake or a viral infection. Here we report 20 new families harboring PPA2 variants. METHODS: Synthesis of clinical and molecular data concerning 34 individuals harboring five previously reported PPA2 variants and 12 novel variants, 11 of which were functionally characterized. RESULTS: Among the 34 individuals, only 6 remain alive. Twenty-three died before the age of 2 years while five died between 14 and 16 years. Within these 28 cases, 15 died of sudden cardiac arrest and 13 of acute heart failure. One case was diagnosed prenatally with cardiomyopathy. Four teenagers drank alcohol before sudden cardiac arrest. Progressive neurological signs were observed in 2/6 surviving individuals. For 11 variants, recombinant PPA2 enzyme activities were significantly decreased and sensitive to temperature, compared to wild-type PPA2 enzyme activity. CONCLUSION: We expand the clinical and mutational spectrum associated with PPA2 dysfunction. Heart failure and sudden cardiac arrest occur at various ages with inter- and intrafamilial phenotypic variability, and presentation can include progressive neurological disease. Alcohol intake can trigger cardiac arrest and should be strictly avoided.

Defining the phenotype of FHF1 developmental and epileptic encephalopathy.

Fibroblast growth-factor homologous factor (FHF1) gene variants have recently been associated with developmental and epileptic encephalopathy (DEE). FHF1 encodes a cytosolic protein that modulates neuronal sodium channel gating. We aim to refine the electroclinical phenotypic spectrum of patients with pathogenic FHF1 variants. We retrospectively collected clinical, genetic, neurophysiologic, and neuroimaging data of 17 patients with FHF1-DEE. Sixteen patients had recurrent heterozygous FHF1 missense variants: 14 had the recurrent p.Arg114His variant and two had a novel likely pathogenic variant p.Gly112Ser. The p.Arg114His variant is associated with an earlier onset and more severe phenotype. One patient carried a chromosomal microduplication involving FHF1. Twelve patients carried a de novo variant, five (29.5%) inherited from parents with gonadic or somatic mosaicism. Seizure onset was between 1 day and 41 months; in 76.5% it was within 30 days. Tonic seizures were the most frequent seizure type. Twelve patients (70.6%) had drug-resistant epilepsy, 14 (82.3%) intellectual disability, and 11 (64.7%) behavioral disturbances. Brain magnetic resonance imaging (MRI) showed mild cerebral and/or cerebellar atrophy in nine patients (52.9%). Overall, our findings expand and refine the clinical, EEG, and imaging phenotype of patients with FHF1-DEE, which is characterized by early onset epilepsy with tonic seizures, associated with moderate to severe ID and psychiatric features.

MYT1L mutations cause intellectual disability and variable obesity by dysregulating gene expression and development of the neuroendocrine hypothalamus.

Deletions at chromosome 2p25.3 are associated with a syndrome consisting of intellectual disability and obesity. The smallest region of overlap for deletions at 2p25.3 contains PXDN and MYT1L. MYT1L is expressed only within the brain in humans. We hypothesized that single nucleotide variants (SNVs) in MYT1L would cause a phenotype resembling deletion at 2p25.3. To examine this we sought MYT1L SNVs in exome sequencing data from 4, 296 parent-child trios. Further variants were identified through a genematcher-facilitated collaboration. We report 9 patients with MYT1L SNVs (4 loss of function and 5 missense). The phenotype of SNV carriers overlapped with that of 2p25.3 deletion carriers. To identify the transcriptomic consequences of MYT1L loss of function we used CRISPR-Cas9 to create a knockout cell line. Gene Ontology analysis in knockout cells demonstrated altered expression of genes that regulate gene expression and that are localized to the nucleus. These differentially expressed genes were enriched for OMIM disease ontology terms "mental retardation". To study the developmental effects of MYT1L loss of function we created a zebrafish knockdown using morpholinos. Knockdown zebrafish manifested loss of oxytocin expression in the preoptic neuroendocrine area. This study demonstrates that MYT1L variants are associated with syndromic obesity in humans. The mechanism is related to dysregulated expression of neurodevelopmental genes and altered development of the neuroendocrine hypothalamus.

Variants in MED12L, encoding a subunit of the mediator kinase module, are responsible for intellectual disability associated with transcriptional defect.

PURPOSE: Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation. MED12L is a subunit of the kinase module, which is one of the four subcomplexes of the mediator complex. Other subunits of the kinase module have been already implicated in intellectual disability, namely MED12, MED13L, MED13, and CDK19. METHODS: We describe an international cohort of seven affected individuals harboring variants involving MED12L identified by array CGH, exome or genome sequencing. RESULTS: All affected individuals presented with intellectual disability and/or developmental delay, including speech impairment. Other features included autism spectrum disorder, aggressive behavior, corpus callosum abnormality, and mild facial morphological features. Three individuals had a MED12L deletion or duplication. The other four individuals harbored single-nucleotide variants (one nonsense, one frameshift, and two splicing variants). Functional analysis confirmed a moderate and significant alteration of RNA synthesis in two individuals. CONCLUSION: Overall data suggest that MED12L haploinsufficiency is responsible for intellectual disability and transcriptional defect. Our findings confirm that the integrity of this kinase module is a critical factor for neurological development.

Correction: Variants in MED12L, encoding a subunit of the Mediator kinase module, are responsible for intellectual disability associated with transcriptional defect.

In the Acknowledgements section of the paper the authors neglected to mention that the study was supported by a grant from the National Human Genome Research Institute (NHGRI) UM1HG007301 (S.H., M.L.T.). In addition, the award of MD was associated with the authors Michelle L. Thompson and Susan Hiatt instead of PhD. The PDF and HTML versions of the Article have been modified accordingly.

Correction: Secondary findings from clinical genomic sequencing: prevalence, patient perspectives, family history assessment, and health-care costs from a multisite study.

The originally published version of this Article contained errors in Fig. 2. The numbers below the black arrowheads were incorrect; please see incorrect Figure in associated Correction. These errors have now been corrected in the PDF and HTML versions of the Article.

Secondary findings from clinical genomic sequencing: prevalence, patient perspectives, family history assessment, and health-care costs from a multisite study.

PURPOSE: Clinical sequencing emerging in health care may result in secondary findings (SFs). METHODS: Seventy-four of 6240 (1.2%) participants who underwent genome or exome sequencing through the Clinical Sequencing Exploratory Research (CSER) Consortium received one or more SFs from the original American College of Medical Genetics and Genomics (ACMG) recommended 56 gene-condition pair list; we assessed clinical and psychosocial actions. RESULTS: The overall adjusted prevalence of SFs in the ACMG 56 genes across the CSER consortium was 1.7%. Initially 32% of the family histories were positive, and post disclosure, this increased to 48%. The average cost of follow-up medical actions per finding up to a 1-year period was $128 (observed, range: $0-$678) and $421 (recommended, range: $141-$1114). Case reports revealed variability in the frequency of and follow-up on medical recommendations patients received associated with each SF gene-condition pair. Participants did not report adverse psychosocial impact associated with receiving SFs; this was corroborated by 18 participant (or parent) interviews. All interviewed participants shared findings with relatives and reported that relatives did not pursue additional testing or care. CONCLUSION: Our results suggest that disclosure of SFs shows little to no adverse impact on participants and adds only modestly to near-term health-care costs; additional studies are needed to confirm these findings.

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

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.