Identification and functional analysis ofCITED2 mutations in patients with congenital heart defects

Abstract

Recent reports have demonstrated that mice lacking the transcription factor Cited2 die in utero showing various cardiac malformations. We present for the first time functionally relevant mutations of CITED2 in patients with congenital heart defects (CHDs). CITED2 encodes a CREBBP/EP300 interacting transcriptional modulator of HIF1A and TFAP2. To study the potential impact of sequence variations in CITED2 for CHDs in humans, we screened a cohort of 392 well-characterized patients and 192 control individuals using DHPLC, sequencing, and Amplifluor genotyping techniques. We identified 15 CITED2 nucleotide alterations. Seven of these alterations were found only in CHD patients and were not detected in controls, including three mutations leading to alterations of the amino acid sequence (p.Ser170_Gly178del, p.Gly178_Ser179ins9, and p.Ser198_Gly199del). All three of these amino acid changing mutations cluster in the serine-glycine-rich junction of the protein, to which no functionality had heretofore been assigned. Here we show that these mutations significantly reduce the capacity of CITED2 to transrepress HIF1A, and that the p.Ser170_Gly178del mutation significantly diminishes TFAP2C coactivation. This reveals a modifying role for the serine-glycine-rich region in CITED2 function. In summary, the observation of these mutations in patients with septal defects indicates that CITED2 has a causative impact in the development of CHD in humans.

Specific transcriptional changes in human fetuses with autosomal trisomies

“…Thus it is interesting that CITED2 showed significant down-regulation in trisomy 18 samples. Mutations in CITED2 have been reported in patients with CHD supplying further evidence for a regulatory effect of CITED2 in the heart development in humans (Sperling et al, 2005). However, we could not ascertain a correlation between CITED2 down-regulation and CHD because of the small number of subjects studied and due to missing postnatal pathoanatomical findings confirming a CHD.…”

MicroRNAs in the Myocyte Enhancer Factor 2 (MEF2)-regulated Gtl2-Dio3 Noncoding RNA Locus Promote Cardiomyocyte Proliferation by Targeting the Transcriptional Coactivator Cited2

“…Cardiomyocyte-specific Cited2 knockout mice revealed a requirement specifically in cardiomyocytes with defects in normal myocardial thickening and ventricular septation (32). Furthermore, mutations in Cited2 are associated with congenital heart disease in humans, pointing to an important role for this transcriptional coactivator in cardiac muscle (48,49).…”

Dominant-Negative ALK2 Allele Associates With Congenital Heart Defects

“…Such approaches, when coupled with kindred linkage and/or detailed functional analyses, can identify novel causative mutations in genes previously suspected to function in AVS development. [3][4][5] In an effort to identify genetic lesions that may cause CHD, we used a candidate approach and sequenced the coding regions of 32 candidate genes in DNA from patients with defects associated with improper EC development. We focused on the functional characterization of a number of coding single-nucleotide polymorphisms (cSNPs) in the ALK2 gene.…”

“…Embryos exhibited an intermediate dorsalizing effect (C2 in 50% of embryos; Figure 3b and 3d), confirming the dominant nature of the L343P allele. To investigate downstream signaling, Western blot analysis for phosphorylated SMAD 1,5,8 (pSmad1) proteins, the downstream target of ALK2, was performed on RNAinjected embryos. pSmad1 levels were higher in wtALK2-injected embryos and higher still in embryos injected with a constitutively active form of ALK2, 28 whereas injection of ALK2 DN resulted in significantly lower pSmad1 levels compared with wtALK2-injected embryos (Figure 3e and 3f; PϽ0.05).…”