Identification of over 500 epigenetic regulators in humans raises an interesting question regarding how chromatin dysregulation contributes to different diseases. Bromodomain and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator possessing three histone-binding domains, one non-specific DNA-binding module, and several motifs for interacting with and activating three lysine acetyltransferases. Genetic analyses of fish brpf1 and mouse Brpf1 have uncovered an important role in skeletal, hematopoietic, and brain development, but it remains unclear how BRPF1 is linked to human development and disease. Here, we describe an intellectual disability disorder in ten individuals with inherited or de novo monoallelic BRPF1 mutations. Symptoms include infantile hypotonia, global developmental delay, intellectual disability, expressive language impairment, and facial dysmorphisms. Central nervous system and spinal abnormalities are also seen in some individuals. These clinical features overlap with but are not identical to those reported for persons with KAT6A or KAT6B mutations, suggesting that BRPF1 targets these two acetyltransferases and additional partners in humans. Functional assays showed that the resulting BRPF1 variants are pathogenic and impair acetylation of histone H3 at lysine 23, an abundant but poorly characterized epigenetic mark. We also found a similar deficiency in different lines of Brpf1-knockout mice. These data indicate that aberrations in the chromatin regulator gene BRPF1 cause histone H3 acetylation deficiency and a previously unrecognized intellectual disability syndrome.

SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. For SOX6, these processes include, but are not limited to, neurogenesis and skeletogenesis. Variants in half of the SOX genes have been shown to cause severe developmental and adult syndromes, referred to as SOXopathies. We here provide evidence that SOX6 variants also cause a SOXopathy. Using clinical and genetic data, we identify 19 individuals harboring various types of SOX6 alterations and exhibiting developmental delay and/or intellectual disability; the individuals are from 17 unrelated families. Additional, inconstant features include attention-deficit/hyperactivity disorder (ADHD), autism, mild facial dysmorphism, craniosynostosis, and multiple osteochondromas. All variants are heterozygous. Fourteen are de novo, one is inherited from a mosaic father, and four offspring from two families have a paternally inherited variant. Intragenic microdeletions, balanced structural rearrangements, frameshifts, and nonsense variants are predicted to inactivate the SOX6 variant allele. Four missense variants occur in residues and protein regions highly conserved evolutionarily. These variants are not detected in the gnomAD control cohort, and the amino acid substitutions are predicted to be damaging. Two of these variants are located in the HMG domain and abolish SOX6 transcriptional activity in vitro. No clear genotype-phenotype correlations are found. Taken together, these findings concur that SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy that often includes ADHD and abnormal skeletal and other features.

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development.

Autosomal-recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is mainly characterized by recurrent, often fatal, respiratory and gastrointestinal infections. About 50% of patients carry mutations in the DNA methyltransferase 3B gene (DNMT3B) (ICF1). The remaining patients carry unknown genetic defects (ICF2) but share with ICF1 patients the same immunological and epigenetic features, including hypomethylation of juxtacentromeric repeat sequences. We performed homozygosity mapping in five unrelated ICF2 patients with consanguineous parents and then performed whole-exome sequencing in one of these patients and Sanger sequencing in all to identify mutations in the zinc-finger- and BTB (bric-a-bric, tramtrack, broad complex)-domain-containing 24 (ZBTB24) gene in four consanguineously descended ICF2 patients. Additionally, we found ZBTB24 mutations in an affected sibling pair and in one patient for whom it was not known whether his parents were consanguineous. ZBTB24 belongs to a large family of transcriptional repressors that include members, such as BCL6 and PATZ1, with prominent regulatory roles in hematopoietic development and malignancy. These data thus indicate that ZBTB24 is involved in DNA methylation of juxtacentromeric DNA and in B cell development and/or B and T cell interactions. Because ZBTB24 is a putative DNA-binding protein highly expressed in the lymphoid lineage, we predict that by studying the molecular function of ZBTB24, we will improve our understanding of the molecular pathophysiology of ICF syndrome and of lymphocyte biology in general.

The immunoglobulin heavy-chain locus (IGH) encodes variable (IGHV), diversity (IGHD), joining (IGHJ), and constant (IGHC) genes and is responsible for antibody heavy-chain biosynthesis, which is vital to the adaptive immune response. Programmed V-(D)-J somatic rearrangement and the complex duplicated nature of the locus have impeded attempts to reconcile its genomic organization based on traditional B-lymphocyte derived genetic material. As a result, sequence descriptions of germline variation within IGHV are lacking, haplotype inference using traditional linkage disequilibrium methods has been difficult, and the human genome reference assembly is missing several expressed IGHV genes. By using a hydatidiform mole BAC clone resource, we present the most complete haplotype of IGHV, IGHD, and IGHJ gene regions derived from a single chromosome, representing an alternate assembly of ∼1 Mbp of high-quality finished sequence. From this we add 101 kbp of previously uncharacterized sequence, including functional IGHV genes, and characterize four large germline copy-number variants (CNVs). In addition to this germline reference, we identify and characterize eight CNV-containing haplotypes from a panel of nine diploid genomes of diverse ethnic origin, discovering previously unmapped IGHV genes and an additional 121 kbp of insertion sequence. We genotype four of these CNVs by using PCR in 425 individuals from nine human populations. We find that all four are highly polymorphic and show considerable evidence of stratification (Fst = 0.3-0.5), with the greatest differences observed between African and Asian populations. These CNVs exhibit weak linkage disequilibrium with SNPs from two commercial arrays in most of the populations tested.

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD." All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations.

Genomic imprinting is a mechanism in which gene expression varies depending on parental origin. Imprinting occurs through differential epigenetic marks on the two parental alleles, with most imprinted loci marked by the presence of differentially methylated regions (DMRs). To identify sites of parental epigenetic bias, here we have profiled DNA methylation patterns in a cohort of 57 individuals with uniparental disomy (UPD) for 19 different chromosomes, defining imprinted DMRs as sites where the maternal and paternal methylation levels diverge significantly from the biparental mean. Using this approach we identified 77 DMRs, including nearly all those described in previous studies, in addition to 34 DMRs not previously reported. These include a DMR at TUBGCP5 within the recurrent 15q11.2 microdeletion region, suggesting potential parent-of-origin effects associated with this genomic disorder. We also observed a modest parental bias in DNA methylation levels at every CpG analyzed across ∼1.9 Mb of the 15q11-q13 Prader-Willi/Angelman syndrome region, demonstrating that the influence of imprinting is not limited to individual regulatory elements such as CpG islands, but can extend across entire chromosomal domains. Using RNA-seq data, we detected signatures consistent with imprinted expression associated with nine novel DMRs. Finally, using a population sample of 4,004 blood methylomes, we define patterns of epigenetic variation at DMRs, identifying rare individuals with global gain or loss of methylation across multiple imprinted loci. Our data provide a detailed map of parental epigenetic bias in the human genome, providing insights into potential parent-of-origin effects.

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.