SNP and DNA methylation analyses of a monozygotic twins discordant for complete endocardial cushion defect: a case report

Abstract

The exact cause of complete endocardial cushion defect (ECD) is still unknown. This report describes a unique pair of monozygotic twins (MZ twins) discordant for ECD. The chromosome karyotyping analysis revealed normal karyotype of 46, XY, 16qh+ and mat in both MZ twins. A genome-wide analysis of DNA using the Affymetrix SNP 6.0 revealed identical genotyping of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). An extensive methylation assay was carried out by NimbleGen 3 × 720 K CpG Island Plus RefSeq Promoter Arrays to analyze the potential epigenetic differences. The DNA methylation profiles of the affected twin seemed increased compared with that of the unaffected twin. However, further validation of Notch1 promoter hypermethylation and six top-ranked differentially methylated CpG sites by sodium bisulfate modification and methylation-specific PCR, failed to reveal consistent methylation differences between the twins. Other relevant factors, such as heritability and penetrance of the condition that place the MZ twins near to a threshold for ECD or variations in local epigenetic events in the twins' heart tissues, are probably responsible for the phenotypic discordance.

Keywords: DNA polymorphism; Monozygotic twins; complete endocardial cushion defects; discordant phenotype; epigenetic difference.

Figure 1

Imaging characteristics of color doppler echocardiography, which revealed the complete endocardial cushion defect (A), transposition of great artery (B) and blood flow (C) of the twin.

Figure 2

DNA CNV analyses roughly revealed no differences among C1, C2, F1 and M2. C1-the ECD twin; C2-the normal twin; F1-the father; M2-the mother. CNV: Copy Number Variation; ECD: Endocardial Cushion Defect.

Figure 3

The twins’ (C1, C2) DNA copy number deletion inherit from the father (F1), which means structural features of DNA polymorphism.

Figure 4

SignalMap software graph of differential DNA methylation in CpG islands of Notch1 gene using a Human Meth 3 × 720 K microarray. C1-the ECD twin, C2-the normal twin. The hypermethylated region in chromosome 9 occurred in C1 (the second row green strips for the Log2-ratio) when compared with C2 (the fourth row red strips). The first yellow bars were the outline of Log2-ratio and region features of the Notch 1 gene between C1 and C2. The third purple strips and the fifth yellow strips demonstrate the methylation peak score respectively corresponding C1 and C2 that showed significant methylation differences. The black bars, brown lines, blue bars and the point line below indicate CpG islands, primary transcripts, tiled regions and transcription start sites, respectively. The light blue box highlights the differentially methylated region between the cases and the control. ECD: Endocardial Cushion Defect.

Figure 5

DNA sequencing in one of the differentially methylated CpG sites on the promoter regions of Notch1 gene between the twins after methylation-specific PCR. Promoter methylation of Notch1 in C1-the ECD twin was not significantly changed than that in C2-the normal twin with the MZ twins phenotype discordance. ECD: Endocardial Cushion Defect.