Expanding the phenotypic spectrum of ARID1B-mediated disorders and identification of altered cell-cycle dynamics due to ARID1B haploinsufficiency

Abstract

Background: Mutations in genes encoding components of the Brahma-associated factor (BAF) chromatin remodeling complex have recently been shown to contribute to multiple syndromes characterised by developmental delay and intellectual disability. ARID1B mutations have been identified as the predominant cause of Coffin-Siris syndrome and have also been shown to be a frequent cause of nonsyndromic intellectual disability. Here, we investigate the molecular basis of a patient with an overlapping but distinctive phenotype of intellectual disability, plantar fat pads and facial dysmorphism.

Methods/results: High density microarray analysis of the patient demonstrated a heterozygous deletion at 6q25.3, which resulted in the loss of four genes including AT Rich Interactive Domain 1B (ARID1B). Subsequent quantitative real-time PCR analysis revealed ARID1B haploinsufficiency in the patient. Analysis of both patient-derived and ARID1B knockdown fibroblasts after serum starvation demonstrated delayed cell cycle re-entry associated with reduced cell number in the S1 phase. Based on the patient's distinctive phenotype, we ascertained four additional patients and identified heterozygous de novo ARID1B frameshift or nonsense mutations in all of them.

Conclusions: This study broadens the spectrum of ARID1B associated phenotypes by describing a distinctive phenotype including plantar fat pads but lacking the hypertrichosis or fifth nail hypoplasia associated with Coffin-Siris syndrome. We present the first direct evidence in patient-derived cells that alterations in cell cycle contribute to the underlying pathogenesis of syndromes associated with ARID1B haploinsufficiency.

Figure 1

Clinical phenotype of patient 1. The facial photographs show dysmorphism including a broad face, narrow palpebral fissures, thin upper lip, full lower lip and low-set ears (A). The plantar fat pads anteromedial to the heel and fetal toe pads are prominent (B) and the hands show fetal finger pads and pillowing over the metacarpal heads (C).

Figure 2

Molecular characterisation of patient 1. High density SNP chip array and CNV analysis identified a heterozygous de novo 4;6 reciprocal translocation that resulted in the deletion of four genes encoding sorting nexin-9 (SNX9), zinc finger DHHC-type containing 14 (ZDHHC14), transmembrane protein 242 (TMEM242) and AT rich interactive domain 1B (ARID1B) at 6q25.3.

Figure 3

ARID1B haploinsufficiency results in delayed S₁ Phase entry. Real Time Quantitative PCR analysis of patient-derived (Patient 1) and control (90087 and 90090) fibroblasts was performed using Taqman probes directed against ARID1B and the housekeeping control RPLO. Samples were normalised to control 90090, which was assigned a value of 1 and analysed by the Pfaffl relative quantification method. The expression of ARID1B was significantly reduced in both Patient 1 and the control 90090 after shRNAmir mediated knockdown of ARID1B (90090-KD), whereas the non-silencing control did not alter ARID1B expression (90090-NS) (A). Cell cycle dynamics of serum-starved cells undergoing S₁ phase after serum replenishment was analysed using ModFit LT. The percentage of cells undergoing S₁ phase at 20 hours post serum replenishment was significantly lower in both patient 1 (P = 0.0002) and 90090-KD (P = 0.005) compared to control (B).

Figure 4

Analysis of ARID1B in the patient cohort. The protein domain organisation of ARID1B and the position of the four mutations identified by sequencing are shown. The forward and reverse sequencing traces demonstrate the NM_020732.3(ARID1B):c.3208_3209delAA (p.(Lys1070Alafs*47)) mutation in patient 2 (A), the NM_020732.3(ARID1B):c.2306_2308delCCGinsTCCGCAGCCACTCC (p.(Pro769Leufs*17)) mutation in patient 3 (B), the NM_020732.3(ARID1B):c.4273dupT (p.(Tyr1425Leufs*34)) mutation in patient 4 (C) and the NM_020732.3(ARID1B):c.2941C > T (p.(Gln981*)) mutation in patient 5 (D). Where available, the forward sequencing trace for the parents is also shown. Mutation co-ordinates are derived from refseq NM_020732.3.