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. 2012 Jul 15;21(14):3255-63.
doi: 10.1093/hmg/dds165. Epub 2012 Apr 27.

Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease (V体育官网入口)

Scott Smemo  1 , Luciene C Campos, Ivan P Moskowitz, José E Krieger, Alexandre C Pereira, Marcelo A Nobrega
Affiliations

Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease

Scott Smemo et al. Hum Mol Genet. .

Abstract

Recent studies have identified the genetic underpinnings of a growing number of diseases through targeted exome sequencing. However, this strategy ignores the large component of the genome that does not code for proteins, but is nonetheless biologically functional. To address the possible involvement of regulatory variation in congenital heart diseases (CHDs), we searched for regulatory mutations impacting the activity of TBX5, a dosage-dependent transcription factor with well-defined roles in the heart and limb development that has been associated with the Holt-Oram syndrome (heart-hand syndrome), a condition that affects 1/100 000 newborns. Using a combination of genomics, bioinformatics and mouse genetic engineering, we scanned ∼700 kb of the TBX5 locus in search of cis-regulatory elements. We uncovered three enhancers that collectively recapitulate the endogenous expression pattern of TBX5 in the developing heart. We re-sequenced these enhancer elements in a cohort of non-syndromic patients with isolated atrial and/or ventricular septal defects, the predominant cardiac defects of the Holt-Oram syndrome, and identified a patient with a homozygous mutation in an enhancer ∼90 kb downstream of TBX5. Notably, we demonstrate that this single-base-pair mutation abrogates the ability of the enhancer to drive expression within the heart in vivo using both mouse and zebrafish transgenic models VSports手机版. Given the population-wide frequency of this variant, we estimate that 1/100 000 individuals would be homozygous for this variant, highlighting that a significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the Holt-Oram syndrome. .

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Figure 1.
Figure 1.
Regulatory landscape of the TBX5 locus. Whole mount and histological sections through the heart of embryonic day 11.5 (E11.5) embryos. Top row: In situ hybridizations showing endogenous expression (purple) of (A) RBM19, (B) TBX5 and (C) TBX3. Bottom row: β-Galactosidase staining (blue) captures the regulatory landscape of enhancers within BACs (D) RP23-173F14-Tn7, (E) RP23-267B15-LacZ and (F) RP23-235J6-Tn7. The genes probed for in A, B and C are contained, respectively, within the BACs tested in D, E and F. There is a 46 bp gap between the BACs containing RBM19 and TBX5, encompassing nucleotides 120 424 690–120 424 646. There is 1140 bp of overlap between the BACs containing TBX5 and TBX3 encompassing nucleotides 120 216 612–120 217 754. Panels B, D–F, forelimb is outlined. A–E, transverse sections; F, sagittal section. A, atrium; RA, right atrium; LA, left atrium; AS, atrial septum; V, ventricle; RV, right ventricle; LV, left ventricle; VS, ventricular septum; AVC, atrioventricular canal.
Figure 2.
Figure 2.
Location of and β-galactosidase expression driven by TBX5 enhancers. (A and B) The TBX5 locus. (C) Individual elements tested for enhancer activity. Sequences with enhancer activity consistent with the expression pattern of TBX5 are dark blue. (D) Pairwise alignments of the mouse genome to human and chicken identify evolutionarily conserved regions (ECRs), which are displayed as peaks. (E–G) Whole mount, isolated hearts and transverse histological sections of transgenic animals carrying individual TBX5 enhancers at E11.5. Consistent expression, across multiple lines, was observed for all three enhancers in the ventricular myocardium, and although it is evident here for ECRs 2 and 16, atrial expression was not recapitulated in independent lines. Only enhancer 2 reproducibly drove expression outside the heart, in the eye. See Fig. 1 for abbreviations and Supplementary Material, Figs S1–S6 for additional lines and time points.
Figure 3.
Figure 3.
Enhancer 9 genomic context and nucleotide conservation. (A) Human alignment to chicken, opossum and mouse at the enhancer 9 region (black bars, top) shows that this enhancer contains two short segments of highly conserved nucleotides (red peaks). (B) One patient possessed a homozygous G→T sequence alteration (hg19 chr12:114 704 515), with two heterozygous parents. (C) The variant nucleotide (boxed) is highly conserved and overlaps a predicted, conserved binding site for TAL1 (D).
Figure 4.
Figure 4.
Reduced binding to mutant DNA. Radioactive DNA probe containing wild-type G allele (WT) is bound to a greater extent than probe containing mutant T allele (mut) (A, lanes 1, 2 versus 6, 7; B, 20 and 30 μg). Cold WT probe competitively displaces mutant probe faster than WT (A, lanes 8–10 versus 3–5; B, ×5, ×10 and ×20). Statistical analyses were performed with two-way ANOVA. Each bar represents median ± standard error (n= 3). *P< 0.05 versus wild-type.
Figure 5.
Figure 5.
A regulatory variant abrogates enhancer function. (A) A single-base-pair mutation in enhancer 9 abrogates its ability to drive cardiac expression. (B) Enhancer 9B, containing a 515 bp ECR within enhancer 9, drives cardiac expression in zebrafish. When mutated similarly, it too loses enhancer activity.
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