Haploinsufficiency of TAB2 causes congenital heart defects in humans

Abstract

Congenital heart defects (CHDs) are the most common major developmental anomalies and the most frequent cause for perinatal mortality, but their etiology remains often obscure. We identified a locus for CHDs on 6q24-q25. Genotype-phenotype correlations in 12 patients carrying a chromosomal deletion on 6q delineated a critical 850 kb region on 6q25.1 harboring five genes. Bioinformatics prioritization of candidate genes in this locus for a role in CHDs identified the TGF-beta-activated kinase 1/MAP3K7 binding protein 2 gene (TAB2) as the top-ranking candidate gene. A role for this candidate gene in cardiac development was further supported by its conserved expression in the developing human and zebrafish heart. Moreover, a critical, dosage-sensitive role during development was demonstrated by the cardiac defects observed upon titrated knockdown of tab2 expression in zebrafish embryos. To definitively confirm the role of this candidate gene in CHDs, we performed mutation analysis of TAB2 in 402 patients with a CHD, which revealed two evolutionarily conserved missense mutations. Finally, a balanced translocation was identified, cosegregating with familial CHD. Mapping of the breakpoints demonstrated that this translocation disrupts TAB2. Taken together, these data clearly demonstrate a role for TAB2 in human cardiac development.

Figure 1

Fine Mapping of Chromosome Aberrations in 6q24-q25, and Candidate-Gene Prioritization (A) Position of deletions found in patients with a CHD (red bars) or without a CHD (black bars). Deletion reference letters are shown on the bars. Regions containing deletion breakpoints are shown in gray. Phenotypes shown as in Table S1. Deletion F was found in a male and his mother, both affected by CHDs. The critically deleted region for CHDs is demarcated by a light red box. (B) Position of deletions found in the region deleted in all CHD patients (commonly deleted region) with respect to the 11 annotated genes encoded in this region. The deletion found in patient H (black, no CHD) does not affect the TAB2 gene, and it demarcates the critically deleted region, which contains five genes. The arrow indicates the position of the translocation breakpoint on chromosome 6 found in family N.

Figure 2

TAB2 Expression in Human Embryonic Hearts (A) Section of a 5.5 wk embryo. TAB2 expression (brown color) is prominent in the ventricular trabeculae and in the endocardial cushions of the outflow tract. (B) Magnification of endocardial cushions. (C) Frontal section of a 7.5 wk embryo. (D) Magnification showing cytoplasmic expression of TAB2 in the endothelial cells lining the developing aortic valves. Abbreviations are as follows: At, atrium; Ao, aorta; EC, endocardial cushions; Tr, trabeculae; Ve, ventricle.

Figure 3

Gene Expression of tab2 in the Developing Zebrafish mRNA expression of tab2 in the developing zebrafish at distinct developmental stages (indicated in the lower left corner). Boxed parts of embryos at 30 hpf and 55 hpf are repeated in (A) and (C″′), respectively. (A): Magnification showing expression of tab2 in the dorsal aorta (arrow) and posterior cardinal vein (arrowhead). (B) Transverse section through the hindbody of a zebrafish at 30 hpf depicted in (A), showing restricted expression in the dorsal aorta (arrowhead) and posterior cardinal vein (arrow). (C): Bright-field image of the expression of tab2 in the cardiac outflow tract at 55 hpf (arrow). (C′) DAPI staining of the section depicted in (C) (pseudocolored in blue). (C″) The section depicted in (C), immunostained with Alexa-647-labeled GFP antibody (pseudocolored in red), displaying the position of the cardiac outflow tract (arrow). (C″′) Image of a 55 hpf, whole-mount-stained embryo, presenting the orientation of the section shown in (C), (C′), and (C″) (white line).

Figure 4

tab2 Knockdown in Zebrafish (A) Schematic organization of the tab2 gene in the zebrafish genome. Two alternative splice isoforms as detected by PCR on the reverse-transcribed mRNA are shown. Introns are dotted lines, exons full boxes (coding sequences in black, noncoding sequences in orange). The target sites of the sbMO) and tbMO are indicated by double arrowheads. The lower schema indicates the effect of sbMO injection on the mature tab2 mRNA (isoforms 1 and 2). (B) Gel electrophoresis of the product of PCR on cDNA extracted from nonmorphant (WT) or 2 ng sbMO-injected zebrafish embryos 24 hpf and a DNA marker for size comparison, showing one band around 2000 bp in the wild-type situation, and two additional fragments in the morphant situation. (C) Effect of TAB2 sbMO injection on the amount of correctly spliced mRNA. (D) Wild-type AB embryo at 48 hpf. (D′) Noninjected embryo at 48 hpf expressing GFP in vascular endothelial cells under the influence of an flk promoter (flk-GFP). (E) Morphant embryo at 48 hpf. Note the enlarged pericardial sac (arrow). (E′) Morphant flk-GFP embryo at 48 hpf. Note the thin and elongated heart. (F) Phenotypical classification of sbMO-injected zebrafish embryos at 12 hpf. Defects in epiboly progression are evident, and progression of the yolk-syncytial layer is schematically displayed below. Phenotypes are ordered from severe to normal. Class I: anterior-posterior gradient is not evident. Class II: the yolk-syncytial layer has not progresses until the vegetal pole; two different embryos are shown to illustrate the continuum in phenotypes in class II. Class III: wild-type. Left, posterior; up, dorsal. (G) Distribution of phenotypes at 12 hpf dependent upon the sbMO dose injected. Y axis: percentage of embryos in each class as indicated by the color code. X axis: amount of TAB2 sbMO injected at the one-cell stage. At least 42 embryos were successfully injected for each dose. In total, over 230 embryos were successfully injected. (H) Phenotypical classification of sbMO-injected zebrafish embryos at 24 hpf. Phenotypes are ordered from severe to normal. Class I: death. Other classes are as described in the main text. Lateral images: up, anterior. (I) Distribution of phenotypes at 24 hpf dependent upon the sbMO dose injected. Y axis: percentage of embryos in this class. X axis: amount of TAB2 sbMO injected at the one-cell stage. At least 42 embryos were successfully injected for each dose. In total, over 230 embryos were successfully injected. Injection of 4 ng of cMO did not increase the frequency of abnormal phenotypes in comparison to noninjected or 0 ng-injected control embryos (not shown).

Figure 5

TAB2 Is Mutated and Disrupted in CHD Patients (A and B) Partial TAB2 reference-sequence-read traces and corresponding traces of missense mutations as identified in patients L and M (phenotypes detailed in the main text). (C) Conservation of mutated residues in several tetrapod and fish lineages. TAB2 is not found in lower lineages; the mutated residues are not conserved in the paralogous TAB3. (D) Pedigree of family N. Translocation carriers are marked, and the breakpoint on chromosome 6 disrupts TAB2. Phenotypes are as annotated in the insert and are detailed in the main text.