Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

With advances in genomic sequencing technology, the number of reported gene-disease relationships has rapidly expanded. However, the evidence supporting these claims varies widely, confounding accurate evaluation of genomic variation in a clinical setting. Despite the critical need to differentiate clinically valid relationships from less well-substantiated relationships, standard guidelines for such evaluation do not currently exist. The NIH-funded Clinical Genome Resource (ClinGen) has developed a framework to define and evaluate the clinical validity of gene-disease pairs across a variety of Mendelian disorders. In this manuscript we describe a proposed framework to evaluate relevant genetic and experimental evidence supporting or contradicting a gene-disease relationship and the subsequent validation of this framework using a set of representative gene-disease pairs. The framework provides a semiquantitative measurement for the strength of evidence of a gene-disease relationship that correlates to a qualitative classification: "Definitive," "Strong," "Moderate," "Limited," "No Reported Evidence," or "Conflicting Evidence." Within the ClinGen structure, classifications derived with this framework are reviewed and confirmed or adjusted based on clinical expertise of appropriate disease experts. Detailed guidance for utilizing this framework and access to the curation interface is available on our website. This evidence-based, systematic method to assess the strength of gene-disease relationships will facilitate more knowledgeable utilization of genomic variants in clinical and research settings.

Chromatin remodeling through histone acetyltransferase (HAT) and histone deactylase (HDAC) enzymes affects fundamental cellular processes including the cell-cycle, cell differentiation, metabolism, and apoptosis. Nonsense mutations in genes that are involved in histone acetylation and deacetylation result in multiple congenital anomalies with most individuals displaying significant developmental delay, microcephaly and dysmorphism. Here, we report a syndrome caused by de novo heterozygous nonsense mutations in KAT6A (a.k.a., MOZ, MYST3) identified by clinical exome sequencing (CES) in four independent families. The same de novo nonsense mutation (c.3385C>T [p.Arg1129∗]) was observed in three individuals, and the fourth individual had a nearby de novo nonsense mutation (c.3070C>T [p.Arg1024∗]). Neither of these variants was present in 1,815 in-house exomes or in public databases. Common features among all four probands include primary microcephaly, global developmental delay including profound speech delay, and craniofacial dysmorphism, as well as more varied features such as feeding difficulties, cardiac defects, and ocular anomalies. We further demonstrate that KAT6A mutations result in dysregulation of H3K9 and H3K18 acetylation and altered P53 signaling. Through histone and non-histone acetylation, KAT6A affects multiple cellular processes and illustrates the complex role of acetylation in regulating development and disease.

The short-rib polydactyly (SRP) syndromes are a heterogeneous group of perinatal lethal skeletal disorders with polydactyly and multisystem organ abnormalities. Homozygosity by descent mapping in a consanguineous SRP family identified a genomic region that contained DYNC2H1, a cytoplasmic dynein involved in retrograde transport in the cilium. Affected individuals in the family were homozygous for an exon 12 missense mutation that predicted the amino acid substitution R587C. Compound heterozygosity for one missense and one null mutation was identified in two additional nonconsanguineous SRP families. Cultured chondrocytes from affected individuals showed morphologically abnormal, shortened cilia. In addition, the chondrocytes showed abnormal cytoskeletal microtubule architecture, implicating an altered microtubule network as part of the disease process. These findings establish SRP as a cilia disorder and demonstrate that DYNC2H1 is essential for skeletogenesis and growth.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

We delineated a syndromic recessive preaxial brachydactyly with partial duplication of proximal phalanges to 16.8 Mb over 4 chromosomes. High-throughput sequencing of all 177 candidate genes detected a truncating frameshift mutation in the gene CHSY1 encoding a chondroitin synthase with a Fringe domain. CHSY1 was secreted from patients' fibroblasts and was required for synthesis of chondroitin sulfate moieties. Noticeably, its absence triggered massive production of JAG1 and subsequent NOTCH activation, which could only be reversed with a wild-type but not a Fringe catalytically dead CHSY1 construct. In vitro, depletion of CHSY1 by RNAi knockdown resulted in enhanced osteogenesis in fetal osteoblasts and remarkable upregulation of JAG2 in glioblastoma cells. In vivo, chsy1 knockdown in zebrafish embryos partially phenocopied the human disorder; it increased NOTCH output and impaired skeletal, pectoral-fin, and retinal development. We conclude that CHSY1 is a secreted FRINGE enzyme required for adjustment of NOTCH signaling throughout human and fish embryogenesis and particularly during limb patterning.

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.

Deletions of 17p13.3, including the LIS1 gene, result in the brain malformation lissencephaly, which is characterized by reduced gyration and cortical thickening; however, the phenotype can vary from isolated lissencephaly sequence (ILS) to Miller-Dieker syndrome (MDS). At the clinical level, these two phenotypes can be differentiated by the presence of significant dysmorphic facial features and a more severe grade of lissencephaly in MDS. Previous work has suggested that children with MDS have a larger deletion than those with ILS, but the precise boundaries of the MDS critical region and causative genes other than LIS1 have never been fully determined. We have completed a physical and transcriptional map of the 17p13.3 region from LIS1 to the telomere. Using fluorescence in situ hybridization, we have mapped the deletion size in 19 children with ILS, 11 children with MDS, and 4 children with 17p13.3 deletions not involving LIS1. We show that the critical region that differentiates ILS from MDS at the molecular level can be reduced to 400 kb. Using somatic cell hybrids from selected patients, we have identified eight genes that are consistently deleted in patients classified as having MDS. In addition, deletion of the genes CRK and 14-3-3 epsilon delineates patients with the most severe lissencephaly grade. On the basis of recent functional data and the creation of a mouse model suggesting a role for 14-3-3 epsilon in cortical development, we suggest that deletion of one or both of these genes in combination with deletion of LIS1 may contribute to the more severe form of lissencephaly seen only in patients with MDS.

Autism is a heritable but genetically complex disorder characterized by deficits in language and in reciprocal social interactions, combined with repetitive and stereotypic behaviors. As with many genetically complex disorders, numerous genome scans reveal inconsistent results. A genome scan of 345 families from the Autism Genetic Resource Exchange (AGRE) (AGRE_1), gave the strongest evidence of linkage at 17q11-17q21 in families with no affected females. Here, we report a full-genome scan of an independent sample of 91 AGRE families with 109 affected sibling pairs (AGRE_2) that also shows the strongest evidence of linkage to 17q11-17q21 in families with no affected females. Taken together, these samples provide a replication of linkage to this chromosome region that is, to our knowledge, the first such replication in autism. Fine mapping at 2-centimorgan (cM) intervals in the combined sample of families with no affected females reveals a linkage peak at 66.85 cM, which places this locus at 17q21.

Acrodysostosis is a dominantly-inherited, multisystem disorder characterized by skeletal, endocrine, and neurological abnormalities. To identify the molecular basis of acrodysostosis, we performed exome sequencing on five genetically independent cases. Three different missense mutations in PDE4D, which encodes cyclic AMP (cAMP)-specific phosphodiesterase 4D, were found to be heterozygous in three of the cases. Two of the mutations were demonstrated to have occurred de novo, providing strong genetic evidence of causation. Two additional cases were heterozygous for de novo missense mutations in PRKAR1A, which encodes the cAMP-dependent regulatory subunit of protein kinase A and which has been recently reported to be the cause of a form of acrodysostosis resistant to multiple hormones. These findings demonstrate that acrodysostosis is genetically heterogeneous and underscore the exquisite sensitivity of many tissues to alterations in cAMP homeostasis.

Neurodevelopmental disorders (NDD) are genetically and phenotypically heterogeneous conditions due to defects in genes involved in development and function of the nervous system. Individuals with NDD, in addition to their primary neurodevelopmental phenotype, may also have accompanying syndromic features that can be very helpful diagnostically especially those with recognizable facial appearance. In this study, we describe ten similarly affected individuals from six unrelated families of different ethnic origins having bi-allelic truncating variants in TMEM94, which encodes for an uncharacterized transmembrane nuclear protein that is highly conserved across mammals. The affected individuals manifested with global developmental delay/intellectual disability, and dysmorphic facial features including triangular face, deep set eyes, broad nasal root and tip and anteverted nostrils, thick arched eye brows, hypertrichosis, pointed chin, and hypertelorism. Birthweight in the upper normal range was observed in most, and all but one had congenital heart defects (CHD). Gene expression analysis in available cells from affected individuals showed reduced expression of TMEM94. Global transcriptome profiling using microarray and RNA sequencing revealed several dysregulated genes essential for cell growth, proliferation and survival that are predicted to have an impact on cardiotoxicity hematological system and neurodevelopment. Loss of Tmem94 in mouse model generated by CRISPR/Cas9 was embryonic lethal and led to craniofacial and cardiac abnormalities and abnormal neuronal migration pattern, suggesting that this gene is important in craniofacial, cardiovascular, and nervous system development. Our study suggests the genetic etiology of a recognizable dysmorphic syndrome with NDD and CHD and highlights the role of TMEM94 in early development.

Attention-deficit/hyperactivity disorder (ADHD [MIM 143465]) is a common, highly heritable neurobehavioral disorder of childhood onset, characterized by hyperactivity, impulsivity, and/or inattention. As part of an ongoing study of the genetic etiology of ADHD, we have performed a genomewide linkage scan in 204 nuclear families comprising 853 individuals and 270 affected sibling pairs (ASPs). Previously, we reported genomewide linkage analysis of a "first wave" of these families composed of 126 ASPs. A follow-up investigation of one region on 16p yielded significant linkage in an extended sample. The current study extends the original sample of 126 ASPs to 270 ASPs and provides linkage analyses of the entire sample, using polymorphic microsatellite markers that define an approximately 10-cM map across the genome. Maximum LOD score (MLS) analysis identified suggestive linkage for 17p11 (MLS=2.98) and four nominal regions with MLS values >1.0, including 5p13, 6q14, 11q25, and 20q13. These data, taken together with the fine mapping on 16p13, suggest two regions as highly likely to harbor risk genes for ADHD: 16p13 and 17p11. Interestingly, both regions, as well as 5p13, have been highlighted in genomewide scans for autism.

Autism is a genetically complex neurodevelopmental syndrome in which language deficits are a core feature. We describe results from two complimentary approaches used to identify risk variants on chromosome 7 that likely contribute to the etiology of autism. A two-stage association study tested 2758 SNPs across a 10 Mb 7q35 language-related autism QTL in AGRE (Autism Genetic Resource Exchange) trios and found significant association with Contactin Associated Protein-Like 2 (CNTNAP2), a strong a priori candidate. Male-only containing families were identified as primarily responsible for this association signal, consistent with the strong male affection bias in ASD and other language-based disorders. Gene-expression analyses in developing human brain further identified CNTNAP2 as enriched in circuits important for language development. Together, these results provide convergent evidence for involvement of CNTNAP2, a Neurexin family member, in autism, and demonstrate a connection between genetic risk for autism and specific brain structures.