Identification of a Novel Mutation in <i>BRD4</i> that Causes Autosomal Dominant Syndromic Congenital Cataracts Associated with Other Neuro-Skeletal Anomalies

<div><p>Congenital cataracts can occur as a non-syndromic isolated ocular disease or as a part of genetic syndromes accompanied by a multi-systemic disease. Approximately 50% of all congenital cataract cases have a heterogeneous genetic basis. Here, we describe three generations of a family with an autosomal dominant inheritance pattern and common complex phenotypes, including bilateral congenital cataracts, short stature, macrocephaly, and minor skeletal anomalies. We did not find any chromosomal aberrations or gene copy number abnormalities using conventional genetic tests; accordingly, we conducted whole-exome sequencing (WES) to identify disease-causing genetic alterations in this family. Based on family WES data, we identified a novel <i>BRD4</i> missense mutation as a candidate causal variant and performed cell-based experiments by ablation of endogenous <i>BRD4</i> expression in human lens epithelial cells. The protein expression levels of connexin 43, p62, LC3BII, and p53 differed significantly between control cells and cells in which endogenous <i>BRD4</i> expression was inhibited. We inferred that a <i>BRD4</i> missense mutation was the likely disease-causing mutation in this family. Our findings may improve the molecular diagnosis of congenital cataracts and support the use of WES to clarify the genetic basis of complex diseases.</p></div

Clinical findings of the proband and preoperative eyes of the father.

<p>(A) In the upper panel, both of the father’s eyes showed white congenital cataracts before preoperative slit-lamp images were obtained, and the lower panel shows slit-lamp images for both of the proband’s eyes, showing postoperative bilateral phacoemulsification with posterior chamber lens implantations. (B) Brachydactyly on both hands was observed and hand X-rays revealed short 3rd and 4th left metacarpals and a short right 3rd metacarpal. (C) Macrocephaly without definite brain anomalies on 1.5-tesla magnetic resonance imaging. (D) Foot radiographs showing flat feet and both accessory navicular bones.</p

Ablation of endogenous BRD4 expression in HLE-B3 cells using shRNA.

<p>HLE-B3 cells were not transfected (wild-type), mock-transfected (shNT), or transfected with shRNA targeting <i>BRD4</i> (shBRD4) for 48 h, and were then subjected to RT-PCR (A) and western blotting (B) to detect BRD4 mRNA and protein levels. β-Actin and GAPDH served as loading controls. ShBRD4 significantly ablated endogenous BRD4 at both the mRNA and protein levels. Decreases in p53, p62, LC3BI, and CX43 protein levels, and an increase in LC3BII were detected after BRD4 knockdown. Protein levels were analyzed by western blotting. Western blots from three independent experiments were quantified and normalized against β-actin. The quantities were determined using ImageJ (National Institutes of Health) and all data were normalized to β-actin. ***<i>p</i> < 0.001 vs. wild type.</p

Summary of five candidate variants with a consistent inheritance pattern detected by whole exome sequencing analyses.

<p>Summary of five candidate variants with a consistent inheritance pattern detected by whole exome sequencing analyses.</p

Summary of clinical data for the affected patients in the study family.

<p>Summary of clinical data for the affected patients in the study family.</p

BRD4 protein expression profile in mouse tissues obtained from two male mice.

<p>Protein levels of BRD4 and β-actin were analyzed by western blotting and protein quantities were determined using ImageJ (National Institutes of Health, Bethesda, MD, USA) by densitometry. Relative ratios of the Brd4/β-actin proteins are shown.</p

Pedigree and Sanger sequencing of the family with congenital cataracts and amino acid sequence conservation.

<p>(A) Pedigree of the family. The three generations of the family comprised four affected members (I-2, II-2, III-1, and III-2) and six unaffected members. The black arrow indicates the proband (III-2). Darkened symbols represent affected members. Stars indicate sampled subjects. Genotypes at the <i>BRD4</i> mutation site are indicated below each examined family member. (B) Sequencing chromatograms, vertical arrows indicate the mutation site. Sanger sequencing confirmed the <i>BRD4</i> heterozygous mutation (c.910C>T, p.His304Tyr) in all patients (II-3, III-2, and III-1) and the wild-type genotype in the unaffected family members (II-5 and III-3). (C) Conservation analysis of the amino acid sequence; the mutation site was highly conserved across vertebrate species.</p

Comparison of the amino acid sequences in the polymorphism sites of the four genes among the species tested.

<p>Multiple sequence alignments of the amino acid sequences of the <i>OR10P1</i> (A), <i>SQRDL</i> gene (B), and <i>NLRP8</i> (C) are shown.</p

List of top 10 nonsynonymous SNPs associated with osteoporosis in Korean postmenopausal women by logistic regression analysis.

<p>Age, BMI, and residential area were included as covariates in the additive genetic models. Sample size for 80% power at α = 0.05 is based on KARE parameters, including minor allele frequency of cases, odds ratio and the osteoporosis prevalence of Korean women. The SNP positions are based on the NCBI Build 36 human genome assembly. Abbreviations: A1, minor allele; A2, major allele; Add <i>p</i>, <i>p</i>-value in the additive genetic model; BP, base pair; Chr., chromosome; CI, confidence interval; MAF, minor allele frequency; OR, odds ratio; and SNP, single nucleotide polymorphism.</p><p>List of top 10 nonsynonymous SNPs associated with osteoporosis in Korean postmenopausal women by logistic regression analysis.</p

Plots of the association results of SNPs in the four osteoporosis-associated genes in the Korean postmenopausal women of the KARE cohort.

<p>The regional association plots of the <i>OR10P1</i> (A), <i>SQRDL</i> gene (B), and <i>NLRP8</i> (C) were generated using the SNAP database (<a href="http://www.broadinstitute.org/mpg/snap/" target="_blank">http://www.broadinstitute.org/mpg/snap/</a>). The statistical significance (–log₁₀<i>p</i>-value) of the analyzed SNPs is plotted. The red diamond with a SNP number and <i>p</i>-value represents the SNP most strongly associated with osteoporosis, and its correlated SNPs are shown in the indicated colors in accordance with the levels of linkage disequilibrium (LD) (<i>r</i>²). The recombination rate estimated from the HapMap CHB and JPT population data is shown by a blue line. The position (Mb) of each gene on human chromosomes (NCBI build 36) is shown at the bottom.</p