Novel NKX2-5 mutations in diseased heart tissues of patients with cardiac malformations

Abstract

NKX2-5 is a homeodomain-containing transcription factor important in cardiac development. Familial mutations in the NKX2-5 gene are associated with cardiac abnormalities, but mutations are rare in sporadic cases. We studied the pathology and molecular genetics of NKX2-5 in diseased heart tissues of 68 patients with complex congenital heart disease (CHD), particularly atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD). We also studied DNA extracted from 16 normal hearts, as well as lymphocytic DNA from 50 healthy volunteers, 7 families, and 4 unrelated individuals with CHD. Direct sequencing revealed 53 NKX2-5 mutations in the diseased heart tissues, including nonsynonymous substitutions in the homeodomain of NKX2-5. We found common mutations among unrelated patients, but certain mutations were specific to VSDs and AVSDs. Many patients had multiple NKX2-5 mutations, up to 14 nonsynonymous mutations per patient in VSDs. Importantly, these nonsynonymous mutations were mainly absent in normal heart tissues of the same CHD patients, thus indicating somatic origin and mosaicism of mutations. Further, observed mutations were completely absent in normal hearts and lymphocytic DNA of healthy individuals. Our findings provide new insights for somatic NKX2-5 mutations to be of importance in congenital heart disease.

Figure 1

Classification of cardiac malformations in patients. A: Diagram showing classification of defects with interatrial shunt. 1, ASD at fossa ovalis (ostium secundum type); 2a, ASD at superior vena cava ostium (sinus venosus type); 2b, ASD at inferior vena cava ostium (sinus venosus type); 3, ASD at coronary sinus ostium (sinus coronarius type); 4, atrioventricular septum defect (ostium primum type). B: Diagram showing classification of defects with interventricular shunt. Inlet, trabecular, and infundibular septal components are separated by dotted lines. 1, VSD at membranous septum, subaortic (perimembranous septal defect); 2a, VSD of outlet septum, subarterial, subvalvar; 2b, VSD of outlet septum not related to arterial valves; 3, VSD of inlet septum (central muscular septal defect); 4a, VSD of apical septum, central (centroapical muscular septal defect); 4b, VSD of apical septum, marginal. C, D: Large atrioventricular septal defects (arrows). E: Muscular ventricular septal defect (arrows). F: Arrows, atrial septal defect (ostium secundum type).

Figure 2

Amplification of NKX2–5 fragments from formalin-fixed tissues. A: Location of PCR primers on the genomic sequence of NKX2.5. B: Quality and concentration of genomic DNA isolated from formalin-fixed heart tissues in 1% ethidium bromide gel, M (kb plus ladder); 50, 100, 150 ng (lambda DNA standard). C: Amplified 1F24/1AR fragments (489 bp) in VSDs, ASDs, AVSDs (2 failed PCR). D: 2F/2R24 fragments (472 bp) in 29 VSDs (1 failed PCR). E: 3F/3R24 fragments (573 bp) in 29 VSDs (2 failed PCR).

Figure 3

Detection and confirmation of NKX2–5 mutations. Heterozygous loci as detected by direct sequencing (electropherograms) are confirmed by PCR-RFLP assay or cloning and re-sequencing of clones allowing detection of two alleles. A: Analysis of two patients (E10VSD, F04AVSD) who are compound heterozygous for mutations in the third helix of the homeodomain. BfmI and BsgI are PCR-RFLP assays for Lys192Arg and Lys194Arg, respectively; while TaqI is for Lys183Glu. B: Analysis of a patient (D03VSD) showing multiple known mutations and more than two haplotypes. Clone 1: C/A/C (all reference alleles); clone 2: C/A/T (recombinant type); clone 3: T/C/T (all mutant alleles).

Figure 4

Evidence of somatic nature of identified NKX2–5 mutations in diseased heart tissues. A: Multiple nonsynonymous mutations in VSDs, ASDs, and AVSDs. B: Mutations are mainly absent in matched normal and diseased cardiac tissues of the same patient. C: No difference in NKX2–5 mutation spectrum in Down and non-Down syndrome AVSD.