Defining heterochromatin in C. elegans through genome-wide analysis of the heterochromatin protein 1 homolog HPL-2

Formation of heterochromatin serves a critical role in organizing the genome and regulating gene expression. In most organisms, heterochromatin flanks centromeres and telomeres. To identify heterochromatic regions in the heavily studied model C. elegans, which possesses holocentric chromosomes with dispersed centromeres, we analyzed the genome-wide distribution of the heterochromatin protein 1 (HP1) ortholog HPL-2 and compared its distribution to other features commonly associated with heterochromatin. HPL-2 binding highly correlates with histone H3 mono- and dimethylated at lysine 9 (H3K9me1 and H3K9me2) and forms broad domains on autosomal arms. Although HPL-2, like other HP1 orthologs, binds H3K9me peptides in vitro, the distribution of HPL-2 in vivo appears relatively normal in mutant embryos that lack H3K9me, demonstrating that the chromosomal distribution of HPL-2 can be achieved in an H3K9me-independent manner. Consistent with HPL-2 serving roles independent of H3K9me, hpl-2 mutant worms display more severe defects than mutant worms lacking H3K9me. HPL-2 binding is enriched for repetitive sequences, and on chromosome arms is anticorrelated with centromeres. At the genic level, HPL-2 preferentially associates with well-expressed genes, and loss of HPL-2 results in up-regulation of some binding targets and down-regulation of others. Our work defines heterochromatin in an important model organism and uncovers both shared and distinctive properties of heterochromatin relative to other systems.Eukaryotic genomes are packaged into two general types of chromatin: euchromatin and heterochromatin. This packaging is important for the regulation of gene expression and organization of the genome. Initially, heterochromatin was cytologically defined as the condensed, dark-staining chromatin that remains visible throughout the cell cycle (Heitz 1928). Since then, numerous molecular characteristics of heterochromatin have been identified. These include an enrichment of repetitive DNA elements, such as satellite DNA and sequences derived from transposable elements, and enrichment of histone H3 methylated at lysine 9 (H3K9me) (Grewal and Elgin 2002). Another hallmark of heterochromatin is the enrichment of heterochromatin protein 1 (HP1), a highly conserved, small nonhistone protein first identified in Drosophila (James and Elgin 1986). Heterochromatin is typically concentrated at pericentric and subtelomeric regions. How heterochromatin is distributed in an organism with numerous centromeres distributed along the length of each chromosome (i.e., holocentric) is not known. This paper defines the distribution of an HP1 protein and heterochromatin in the nematode C. elegans, which possesses holocentric chromosomes and is a valuable model for genome organization, chromosome segregation, gene expression, and development.It has been shown through a variety of methods that the chromo domain (CD) of metazoan HP1 proteins specifically recognizes H3K9me2 and H3K9me3 (Bannister et al. 2001; Jacobs et al. 2001; Lachner et al. 2001). Evidence that this interaction is important for proper HP1 protein localization comes from the observation that loss or reduction of H3K9me results in loss or reduction of HP1 binding in vivo (Bannister et al. 2001; Lachner et al. 2001; Schotta et al. 2002; Seum et al. 2007; Tzeng et al. 2007). Other interactions besides the CD-H3K9me interaction are involved in HP1 localization as well, as Drosophila SU(VAR)205 (also known as HP1A) is able to associate with promoter regions of genes independently of H3K9me (Figueiredo et al. 2012), and HP1A lacking its CD is able to associate with heterochromatin (Smothers and Henikoff 2001). Furthermore, in vitro studies have shown that mouse CBX1, CBX3, and CBX5 (also known as HP1β, HP1γ, and HP1α, respectively) bind the histone-fold domain of histone H3 (Nielsen et al. 2001) and that fly HP1A binds DNA in a sequence-independent manner (Zhao et al. 2000). Interestingly, studies in fission yeast, flies, and mammals have demonstrated that the RNAi machinery and RNA itself contribute to HP1 protein localization (Pal-Bhadra et al. 2004; Verdel et al. 2004; Maison et al. 2011). Taken together, these studies implicate interactions between HP1 and methylated histone tails, histone cores, DNA, and RNA as contributing to the recruitment and retention of HP1 at particular DNA regions in vivo. In this study, we specifically tested whether H3K9me is required for proper HP1 localization in C. elegans.The nematode C. elegans has two HP1 paralogous proteins: HP1 Like (heterochromatin protein) 1 and 2 (HPL-1 and HPL-2) (Couteau et al. 2002). HPL-2 serves more roles and/or more important roles than HPL-1, as hpl-2 mutants display diverse defects while hpl-1 mutants generally lack observable mutant phenotypes. HPL-2 is an important factor for germline health, as hpl-2 mutants display maternal-effect sterility at elevated temperature (25°C) (Coustham et al. 2006) and a reduced ability to silence exogenous “non-self” sequences in the germline (Couteau et al. 2002; Robert et al. 2005; Ashe et al. 2012; Shirayama et al. 2012). HPL-2 is also important in somatic development, as hpl-2 mutants show larval, somatic gonad, and vulval developmental defects (Schott et al. 2006). Comparisons of hpl-2 hpl-1 double mutants and hpl-2 single mutants suggest that HPL-2 and HPL-1 have some overlapping roles, as double mutant worms display more severe phenotypes than hpl-2 alone (Schott et al. 2006; Shirayama et al. 2012). Because HPL-2 is the more important of the two C. elegans HP1 homologs and is the only HP1 homolog in C. briggsae, a close relative of C. elegans (Vermaak and Malik 2009), we focused our current study on HPL-2.Here, we show that HPL-2 binding to chromatin highly correlates with H3K9me1 and H3K9me2 throughout the genome and that HPL-2-enriched regions form domains that are also enriched for repetitive DNA elements. These observations suggest that HPL-2 indeed has functions associated with heterochromatin and that HPL-2-enriched domains represent the distribution of heterochromatin in C. elegans. Surprisingly, H3K9me is not necessary for the normal distribution of HPL-2, as the genome-wide pattern of HPL-2 is largely unchanged in met-2 set-25 mutant embryos, which Towbin et al. reported and we verified to lack H3K9me (Towbin et al. 2012). Consistent with HPL-2 having roles independent of H3K9me, met-2 set-25 mutants display less sterility at elevated temperature than hpl-2. Interestingly, worm heterochromatin has a unique distribution relative to other organisms: enrichment on the autosomal “arms” and on the leftmost region of the X chromosome. On autosomal arms, elevated HPL-2 levels flank centromeric chromatin, creating regions that resemble pericentric heterochromatin. HPL-2 shows a bias toward association with well-expressed genes, where it seems to repress the expression of some genes it binds and promote the expression of others. Our studies uncover both shared and unique properties of worm heterochromatin compared to other organisms, and reveal how heterochromatin is distributed in an organism with holocentric chromosomes.     

De novo variants in the PABP domain of PABPC1 lead to developmental delay

PurposeThe study aimed to investigate the role of PABPC1 in developmental delay (DD).MethodsChildren were examined by geneticists and pediatricians. Variants were identified using exome sequencing and standard downstream bioinformatics pipelines. We performed in silico molecular modeling and coimmunoprecipitation to test if the variants affect the interaction between PABPC1 and PAIP2. We performed in utero electroporation of mouse embryo brains to enlighten the function of PABPC1.ResultsWe describe 4 probands with an overlapping phenotype of DD, expressive speech delay, and autistic features and heterozygous de novo variants that cluster in the PABP domain of PABPC1. Further symptoms were seizures and behavioral disorders. Molecular modeling predicted that the variants are pathogenic and would lead to decreased binding affinity to messenger RNA metabolism-related proteins, such as PAIP2. Coimmunoprecipitation confirmed this because it showed a significant weakening of the interaction between mutant PABPC1 and PAIP2. Electroporation of mouse embryo brains showed that Pabpc1 knockdown decreases the proliferation of neural progenitor cells. Wild-type Pabpc1 could rescue this disturbance, whereas 3 of the 4 variants did not.ConclusionPathogenic variants in the PABP domain lead to DD, possibly because of interference with the translation initiation and subsequently an impaired neurogenesis in cortical development.     

Expanding SPTAN1 monoallelic variant associated disorders: From epileptic encephalopathy to pure spastic paraplegia and ataxia

PurposeNonerythrocytic αII-spectrin (SPTAN1) variants have been previously associated with intellectual disability and epilepsy. We conducted this study to delineate the phenotypic spectrum of SPTAN1 variants.MethodsWe carried out SPTAN1 gene enrichment analysis in the rare disease component of the 100,000 Genomes Project and screened 100,000 Genomes Project, DECIPHER database, and GeneMatcher to identify individuals with SPTAN1 variants. Functional studies were performed on fibroblasts from 2 patients.ResultsStatistically significant enrichment of rare (minor allele frequency < 1 × 10^–5) probably damaging SPTAN1 variants was identified in families with hereditary ataxia (HA) or hereditary spastic paraplegia (HSP) (12/1142 cases vs 52/23,847 controls, p = 2.8 × 10^–5). We identified 31 individuals carrying SPTAN1 heterozygous variants or deletions. A total of 10 patients presented with pure or complex HSP/HA. The remaining 21 patients had developmental delay and seizures. Irregular αII-spectrin aggregation was noted in fibroblasts derived from 2 patients with p.(Arg19Trp) and p.(Glu2207del) variants.ConclusionWe found that SPTAN1 is a genetic cause of neurodevelopmental disorder, which we classified into 3 distinct subgroups. The first comprises developmental epileptic encephalopathy. The second group exhibits milder phenotypes of developmental delay with or without seizures. The final group accounts for patients with pure or complex HSP/HA.     

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay,seizures, and hypotonia

PurposeNeurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene.MethodsThrough large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual.ResultsWe identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and 1 was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy.ConclusionThis collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.     

The pleiotropy associated with de novo variants in CHD4, CNOT3, and SETD5 extends to moyamoya angiopathy

PurposeMoyamoya angiopathy (MMA) is a cerebrovascular disease characterized by occlusion of large arteries, which leads to strokes starting in childhood. Twelve altered genes predispose to MMA but the majority of cases of European descent do not have an identified genetic trigger.MethodsExome sequencing from 39 trios were analyzed.ResultsWe identified four de novo variants in three genes not previously associated with MMA: CHD4, CNOT3, and SETD5. Identification of additional rare variants in these genes in 158 unrelated MMA probands provided further support that rare pathogenic variants in CHD4 and CNOT3 predispose to MMA. Previous studies identified de novo variants in these genes in children with developmental disorders (DD), intellectual disability, and congenital heart disease.ConclusionThese genes encode proteins involved in chromatin remodeling, and taken together with previously reported genes leading to MMA-like cerebrovascular occlusive disease (YY1AP1, SMARCAL1), implicate disrupted chromatin remodeling as a molecular pathway predisposing to early onset, large artery occlusive cerebrovascular disease. Furthermore, these data expand the spectrum of phenotypic pleiotropy due to alterations of CHD4, CNOT3, and SETD5 beyond DD to later onset disease in the cerebrovascular arteries and emphasize the need to assess clinical complications into adulthood for genes associated with DD.     

Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder,apraxia, and insensitivity to pain

PurposeWe characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1β subunit of the cyclic AMP-dependent protein kinase A (PKA).MethodsVariants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development.ResultsRecent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs.ConclusionOur study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.     

Haploinsufficiency of ARFGEF1 is associated with developmental delay,intellectual disability,and epilepsy with variable expressivity

PurposeADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized.MethodsInternational data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing.ResultsWe identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity.ConclusionWe provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.     

Exome reports A de novo GNB2 variant associated with global developmental delay,intellectual disability,and dysmorphic features

Heterotrimeric G proteins are composed of α, β, and γ subunits and are involved in integrating signals between receptors and effector proteins. The 5 human Gβ proteins (encoded by GNB1, GNB2, GNB3, GNB4, and GNB5) are highly similar. Variants in GNB1 were identified as a genetic cause of developmental delay. De novo variant in GNB2 has recently been reported as a cause of sinus node dysfunction and atrioventricular block but not as a cause of developmental delay. Trio-based whole-exome sequencing was performed on an individual with global developmental delay, muscle hypotonia, multiple congenital joint contractures and dysmorphism such as brachycephalus, thick eyebrows, thin upper lip, micrognathia, prominent chin, and bilateral tapered fingers. We identified a de novo GNB2 variant c.229G>A, p.(Gly77Arg). Notably, pathogenic substitutions of the homologous Gly77 residue including an identical variant (p.Gly77Arg, p.Gly77Val, p.Gly77Ser, p.Gly77Ala) of GNB1, a paralog of GNB2, was reported in individuals with global developmental delay and hypotonia. Clinical features of our case overlap with those of GNB1 variants. Our study suggests that a GNB2 variant may be associated with syndromic global developmental delay.     

The CHD4-related syndrome: a comprehensive investigation of the clinical spectrum,genotype–phenotype correlations,and molecular basis

PurposeSifrim–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.MethodsWe 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.ResultsThe 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.ConclusionThe 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.     

Biallelic variants in OGDH encoding oxoglutarate dehydrogenase lead to a neurodevelopmental disorder characterized by global developmental delay,movement disorder,and metabolic abnormalities

PurposeThis study aimed to establish the genetic cause of a novel autosomal recessive neurodevelopmental disorder characterized by global developmental delay, movement disorder, and metabolic abnormalities.MethodsWe performed a detailed clinical characterization of 4 unrelated individuals from consanguineous families with a neurodevelopmental disorder. We used exome sequencing or targeted-exome sequencing, cosegregation, in silico protein modeling, and functional analyses of variants in HEK293 cells and Drosophila melanogaster, as well as in proband-derived fibroblast cells.ResultsIn the 4 individuals, we identified 3 novel homozygous variants in oxoglutarate dehydrogenase (OGDH) (NM_002541.3), which encodes a subunit of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase. In silico homology modeling predicts that c.566C>T:p.(Pro189Leu) and c.890C>A:p.(Ser297Tyr) variants interfere with the structure and function of OGDH. Fibroblasts from individual 1 showed that the p.(Ser297Tyr) variant led to a higher degradation rate of the OGDH protein. OGDH protein with p.(Pro189Leu) or p.(Ser297Tyr) variants in HEK293 cells showed significantly lower levels than the wild-type protein. Furthermore, we showed that expression of Drosophila Ogdh (dOgdh) carrying variants homologous to p.(Pro189Leu) or p.(Ser297Tyr), failed to rescue developmental lethality caused by loss of dOgdh. SpliceAI, a variant splice predictor, predicted that the c.935G>A:p.(Arg312Lys)/p.(Phe264_Arg312del) variant impacts splicing, which was confirmed through a mini-gene assay in HEK293 cells.ConclusionWe established that biallelic variants in OGDH cause a neurodevelopmental disorder with metabolic and movement abnormalities.