De novo variants in RYBP are associated with a severe neurodevelopmental disorder and congenital anomalies

Abstract

Purpose: Polycomb group proteins are key epigenetic transcriptional regulators. Multiple neurodevelopmental disorders are associated with pathogenic variants of the genes encoding Polycomb group proteins. RYBP is a core component of the noncanonical Polycomb Repressor Complex 1; however, its role in disease is unclear.

Methods: Functional consequences of RYBP variants were assessed using in vitro cellular and in vivo Drosophila melanogaster studies.

Results: We described 7 individuals with heterozygous de novo variants of RYBP and their clinical findings, including severe developmental delay, dysmorphisms, and multiple congenital anomalies. We showed that all single-nucleotide variants in RYBP localize to the N-terminal domain of the gene, which encodes the zinc-finger domain and ubiquitin-binding moiety. In vitro studies have demonstrated that the RYBP c.132C>G p.(Cys44Trp) variant causes reduced protein expression but does not affect the binding of YY1, RING1B, or ubiquitin. In vivo overexpression studies in Drosophila melanogaster showed a dramatic functional difference between human RYBP and its variant forms, affecting the C44 amino acid residue. DNA methylation studies suggested a possible episignature associated with RYBP-related disorder.

Conclusion: Heterozygous de novo variants in RYBP are associated with an identifiable syndromic neurodevelopmental disorder with multiple congenital anomalies.

Keywords: Epigenetic; Neurodevelopment; Polycomb; RYBP; Ubiquitin.

Figure 1:

De novo variants in RYBP are associated with facial dysmorphism. A. Proband 1 at age 15y (left 2 panels), proband 2 at 9 months of age (middle upper panel), proband 3 at age 4 years (middle lower panel), proband 4 at age 9 years (right panel). Common dysmorphic features include a broad forehead, downslanting palpebral fissures, prominent nose, and small mouth with downturned corners. B. Sanger sequencing results for proband 1 showing heterozygous base changes in proband (upper panel noted with black arrows). Parents’ results are shown in the 2 lower panels. C. Exome sequencing (ES) showing heterozygous variants in proband 1. The lower panel shows the expression of the variants on RNA sequencing (RNAseq) in fibroblasts from proband 1.

Figure 2:

RYBP protein domains and conservation between different species. A. Schematic depiction of RYBP protein domains and variants identified in the probands. Protein residues encoding the NZF domain are denoted in cyan blue. The zinc-coordinating cysteines are in red, and critical residues for ubiquitin binding are designated by stars. The RING1 and YY1 binding domain is denoted in yellow. B. Protein alignment (CLUSTAL O (1.2.4)) between human, mouse and Drosophila Melanogaster RYBP. The zinc-coordinating cysteines are noted in red.

Figure 3:

Overexpression of human and variant RYBP protein in Drosophila melanogaster models. A. Wing-specific overexpression of human reference RYBP in nubbin GAL4 line causes a wing margin defect phenotype that is not seen with overexpression of the C44 RYBP variants. B. Overexpression of human reference and variant RYBP in the dorsal-midline-specific pannier GAL4. LacZ overexpressing flies serve as controls. Dashed rectangle indicates dorsal-midline region. Female flies on the left, male flies on the right. Flies overexpressing hRYBP^Ref show midline defect with ridge formation in the thoraco-abdominal region. There is increased midline abdominal pigmentation. Wings show significant blistering. The animals overexpressing the hRYBP^C44W or hRYBP^C44S variants show less significant midline ridges and less wing abnormalities.

Figure 4:

Methylation episignature related to RYBP. A. MDS (multidimensional scaling) clustering of control samples (green) and Gabriele-de Vries syndrome (GADEVS, blue). Proband 1’s sample is designated in red. B. MDS plots of control samples (green) and PRC2-associated disorder samples (blue). Proband 1 sample designated in red.

Figure 5:

RYBP C44W variant protein level is decreased in vitro. Left panel: 293T cells were transfected with either wild type RYBP or C44W variant RYBP, and protein level assessed with anti-RYBP antibody. Tubulin levels are shown for loading control. Right panel: graph showing ratio between wild type RYBP protein and C44W variant protein level after normalization to tubulin. SD and mean shown in graph (n=4).

Figure 6:

RYBP protein ubiquitination is not affected by RYBP C44W variant. 293T cells were transfected with wild type or variant Myc-tagged RYBP alone or in combination with His-tagged ubiquitin. His-tagged ubiquitinated proteins were pulled down with anti-His, and ubiquitinated RYBP was identified with anti-Myc antibody. Input (IN) shown for each pull down. IP-immunoprecipitation.

Figure 7:

RYBP and YY1 protein interaction is not affected by RYBP C44W variant. Pull down of YY1 with wild type RYBP or variant RYBP in 293T cells. Immunoprecipitation performed with anti-HA antibody and western blot with anti-Myc antibody for RYBP. Inp-input, IgG- immunoglobulin G (control), α-HA-anti HA.