Recurrent de novo missense variants across multiple histone H4 genes underlie a neurodevelopmental syndrome

Abstract

Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.

Keywords: histone H4; intellectual disability; microcephaly; neurodevelopmental disorder; nucleosome; zebrafish.

Figure 1

H4 variants identified in the cohort (A) Highly recurrent variants were found in six different H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11), which all encode an identical protein. Aggregate prevalence of disease-causing amino acid changes is also shown. The N-terminal methionine is cleaved from histone H4, and therefore all numbering is relative to the mature polypeptide, in keeping with the protein literature. (B) The affected residues of H4 (orange ribbon) either cluster to the N-terminal α-helix facing toward DNA (cluster 1, purple spheres) or are located in regions buried within the nucleosome core (cluster 2, orange spheres). Size of sphere indicates the relative prevalence of substitutions affecting that residue.

Figure 2

Clinical characteristics of individuals with histone H4 gene variants (A) Individuals with variants in histone H4 genes demonstrate a reduction in height, weight, and brain growth (OFC, occipitofrontal circumference); the latter significantly progresses as the individuals age. There are no detectable genotype-phenotype patterns separating by the specific histone H4 gene or variant cluster. ^∗∗∗∗p < 0.0001. (B) Facial dysmorphism affecting midline structures is noticeable among the cohort, but highly variable, with no obvious genotype-phenotype correlation. (C) Individuals can present with abnormalities in the appearance and position of teeth (for example, P5, P25). A recurring feature present in several individuals is a noticeable gap between the upper central incisors. (D) Individuals with variants in histone H4 genes also show a spectrum of toe anomalies, ranging from no anomalies present (for example, P21) through to severe 2–3 toe (P1, P28) or 3–4 toe (P25) syndactyly, which can be bilateral. Toes can also be short (P19, P28).

Figure 3

H4 variants induce developmental defects in zebrafish embryos (A) Phenotypical characterization in 28 hpf embryos. Representative images of observed phenotypes in zebrafish embryos 28 hpf microinjected with mRNA encoding either wild-type or identified variants at the one-cell stage. The different classes are defined on general development and necrosis. Arrowhead, cephalic necrosis; arrow, curved tail. (B and C) High magnification examples of cephalic necrosis (B) and curved tail (C) phenotypes. (D and E) Quantification of the phenotypical classification as described in (A). Variants reported in (D) were microinjected with 50 pg/embryo, and additional testing with 100 pg/embryo is reported in (E). Data marked with a hash symbol was previously published in Tessadori et al. Fisher’s exact test: ns, not significant; p > 0.05; ^∗p < 0.05; ^∗∗∗∗p < 0.0001. Scale bars: 100 μm (A); 50 μm (B and C).