Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

Abstract

Purpose: Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 and has not been implicated in human disease.

Methods: We identify 5 unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and 2 missense variants were identified in probands with neurodevelopmental symptoms, including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models.

Results: Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles.

Conclusion: Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder.

Keywords: Epigenetic; H3K4 methylation; Microcephaly; Neurodevelopmental disorder; RBBP5.

Figure 1. Human subjects with RBBP5 de novo variants exhibit a range of clinical features.

Dysmorphic features in individual 1, including hypertelorism, high arched eyebrow, long eyelashes, synophrys, and board nasal tip as shown in (A); retrognathia, large ear, and a preauricular ear tag as shown in (B); bilateral 5th finger clinodactyly and prominent fingertip pad in (C). Dysmorphic features in individual 3 as shown in (D) with midface hypoplasia and cupped ears, clinodacyly in (E), and supernumerary teeth in (F). Dysmorphic features in individual 4 as shown in (G) with short and upslanting palpebral fissures, high forehead, anteverted nostrils, and sparse eyebrows. (H) and (I) shows the dysmorphic facial features, including sparse eyebrows, short nose, long philtrum, small and squared ears, and small mouth with thin lips, in individual 5. Common phenotypes are illustrated in (J).

Figure 2. Bioinformatic and structural variant analysis.

The functional domains of RBBP5 and position of variants in MetaDome are shown in (A). The evolutionarily conserved residues affected by variants are shown in (B). The protein expression of FLAG-tagged human RBBP5 reference and variants were shown in (C). Structure analysis of T232 and E296 were performed in RBBP5^WD40 of the cryo-EM structure of MLL3-ubNCP complex. The overall structure of RBBP5^WD40 complexed with a nucleosome core particle mono-ubiquitinated at the Lys 120 of histone H2B (ubNCP). The RBBP5 WD40 repeat 4 is sandwiched between ubiquitin and core histones. The RBBP5^WD40 is shown in orange and ubiquitin in blue (D). Detailed view of the recognition interface of RBBP5^WD40-ubiquitin. Residue p.(T232I), which is located on the α-helix-containing loop of RBBP5^WD40 blade 5, lies close to residues L8, T9, and H68 of ubiquitin (E). All these residues are shown in stick model. Detailed view of the interaction interface between RBBP5^WD40 and histone H2B-H4. Two loops (loop 1 and loop 2), which connect the WD40 propeller blades 5, 6, and 7, interact with nucleosome directly. Residue p.(E296D) is located on loop 2 and shown in stick model (F).

Figure 3. Ubiquitous expression of RBBP5 in flies induces lethality and results in microcephaly in the larval developmental stage.

The Drosophila life cycle is approximately 10 days long at 25°C. Expression of the human RBBP5 or either missense variant with a strong ubiquitous driver (Actin^GAL4) is larval lethal (Actin^GAL4 / RBBP5^Ref observed in 0/128 F1 progeny, o/e 0.0; Actin^GAL4/RBBP5^T232I observed in 0/144 F1 progeny, o/e 0.0; Actin^GAL4/RBBP5^E296D observed in 0/277, o/e 0.0). With a weak ubiquitous driver (da^GAL4), expression of the human reference is larval lethal, but expression of p.(T232I) or p.(E296D) is pupal lethal. Expression with tissue specific (ey-, elav-, or repo^GAL4) drivers does not affect viability in (A). Representative developmentally staged control late L3 brains (UAS-lacZ; da^GAL4) with Deadpan staining of neuroblasts and intermediate progenitor cells in green and Prospero staining of neural progenitors in red in (B). Experimental RBBP5 reference (RBBP5^Ref; da^GAL4) brain shown in (C), RBBP5^T232I (RBBP5^T232I; da^GAL4) in (D) and RBBP5^E296D (RBBP5^E296D; da^GAL4) in (E). Quantification of ubiquitous overexpression (da^GAL4) of RBBP5^Ref, RBBP5^T232I, and RBBP5^E296D compared with UAS-lacZ (one-way ANOVA, ns, P > .05, *P < .05, **P < .01, ***P < .001, ****P < .0001) in (F). Created with Biorender.com.

Figure 4. Human RBBP5 expression induces a small eye phenotype not recapitulated by the p.(T232I) variant.

Overexpression with eyeless-GAL4 (ey^GAL4) in a control line (ey^GAL4 / UAS-lacZ) as shown in (A), ey^GAL4 / RBBP5^Ref in (B), ey^GAL4 / RBBP5^T232I in (C), and ey^GAL4 / RBBP5^E296D in (D). Expression of RBBP5^Ref and RBBP5^E296D results in a small eye phenotype compared with UAS-lacZ. Expression of RBBP5^T232I does not induce a small eye phenotype and eye size is not significantly different than controls (one-way ANOVA, ns P > .05, *P < .05, **P < .01, ***P < .001, ****P < .0001) in (E).

Figure 5. Rbbp5 is expressed in a subset of neurons and glia in the Drosophila brain.

Rbbp5^{Kozak GAL4}/UAS mCherry.NLS expression pattern in the dorsal larval brain shown in (A). Elav expression in (B), and merge in (C) with co-localization in the ventral nerve cord and optic lobes of the central brain. Rbbp5^{Kozak GAL4}/UAS mCherry.NLS expression pattern in the ventral larval brain shown in (D). Repo expression in (E), and merge in (F) with co-localization in the ventral nerve cord and optic lobes.

Figure 6. RBBP5 human transgenes fail to rescue loss of Drosophila Rbbp5.

Heterozygous L3 Rbbp5 loss-of-function controls (Rbbp5^{Kozak GAL4}/+) with Deadpan-positive neuroblasts and intermediate progenitor cells in green and Prospero-positive neural progenitor cells in red in (A). Homozygous loss-of-function with Rbbp5^{Kozak GAL4} crossed to a deficiency line Df(3L)BSC447 that includes the Rbbp5 locus (Rbbp5^{Kozak GAL4} /Df(3L)BSC447) in (B). Attempted rescue with the human RBBP5^Ref (RBBP5^Ref; Rbbp5^{Kozak GAL4}/Df(3L)BSC447) in (C), RBBP5^T232I (RBBP5^T232I; Rbbp5^{Koza kGAL4}/Df(3L)BSC447) in (D), RBBP5^E296D (RBBP5^E296D; Rbbp5^{Kozak GAL4}/Df(3L)BSC447) in (E). Quantification of the microcephaly phenotype (brain area) by genotype (one-way ANOVA, ns P > .05, *P < .05, **P < .01, ***P < .001, ****P < .0001) in (F). RBBP5^Ref, RBBP5^T232I, and RBBP5^E296D fail to rescue loss of the Drosophila Rbbp5. RBBP5^Ref expression induces a significantly more severe microcephaly phenotype than Rbbp5^{Kozak GAL4}/Df(3L)BSC447, and brain size of Rbbp5^T232I; Rbbp5^KozakGAL4/Df(3L)BSC447 or Rbbp5^E296D; Rbbp5^KozakGAL4/Df(3L)BSC447 larvae is not significantly different than Rbbp5^KozakGAL4/Df(3L)BSC447.