Transcriptional Defect of an Inherited NKX2-5 Haplotype Comprising a SNP, a Nonsynonymous and a Synonymous Mutation, Associated with Human Congenital Heart Disease

Leonard Eisenberg,Evelyn Sattlegger,Stella Marie Reamon-Buettner,Yari Ciribilli,Alberto Inga,Jürgen Borlak,Armin Wessel

doi:10.1371/journal.pone.0083295

Abstract

Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity.

Highlights

Deciphering the exact causes of congenital heart disease (CHD) in humans is a complicated task
We investigated genomic DNA isolated from 49 heart tissues of 28 Caucasian patients with various heart abnormalities, including septal defects as well as Tetralogy of Fallot (TOF) and hypoplastic left heart syndrome (HLHS) (Table 1)
Search for mutations in NKX2-5, identified two patients who were heterozygous for three sequence alterations including two that will affect the alanine residue at position 119, i.e. c.356C.A (p.A119E) and c.355G.T, (p.A119S)

Summary

Introduction

Deciphering the exact causes of congenital heart disease (CHD) in humans is a complicated task. Germline mutations in several transcription factor genes that govern heart development have been implicated in the disease, detection frequency is low. More than 40 different NKX2-5 mutations have been identified so far, but only five were detected more than once in unrelated individuals. Families have their own ‘private’ mutation, and such mutation can lead to a variety of cardiac defects even within the same family. This suggests that CHD cannot just be explained by simple monogenic inheritance or by a single germline mutation. There is emerging evidence that CHD is a multifactorial disease in which genetic factors, environmental factors and gene-environment interactions are key events resulting in mutations, chromosomal aberrations or abnormal gene expression (see reviews [4,5])

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Conclusion