Ultra High-Resolution Gene Centric Genomic Structural Analysis of a Non-Syndromic Congenital Heart Defect, Tetralogy of Fallot

<div><p>Tetralogy of Fallot (TOF) is one of the most common severe congenital heart malformations. Great progress has been made in identifying key genes that regulate heart development, yet approximately 70% of TOF cases are sporadic and nonsyndromic with no known genetic cause. We created an ultra high-resolution gene centric comparative genomic hybridization (gcCGH) microarray based on 591 genes with a validated association with cardiovascular development or function. We used our gcCGH array to analyze the genomic structure of 34 infants with sporadic TOF without a deletion on chromosome 22q11.2 (n _male = 20; n _female = 14; age range of 2 to 10 months). Using our custom-made gcCGH microarray platform, we identified a total of 613 copy number variations (CNVs) ranging in size from 78 base pairs to 19.5 Mb. We identified 16 subjects with 33 CNVs that contained 13 different genes which are known to be directly associated with heart development. Additionally, there were 79 genes from the broader list of genes that were partially or completely contained in a CNV. All 34 individuals examined had at least one CNV involving these 79 genes. Furthermore, we had available whole genome exon arrays from right ventricular tissue in 13 of our subjects. We analyzed these for correlations between copy number and gene expression level. Surprisingly, we could detect only one clear association between CNVs and expression (<i>GSTT1</i>) for any of the 591 focal genes on the gcCGH array. The expression levels of <i>GSTT1</i> were correlated with copy number in all cases examined (r = 0.95, p = 0.001). We identified a large number of small CNVs in genes with varying associations with heart development. Our results illustrate the complexity of human genome structural variation and underscore the need for multifactorial assessment of potential genetic/genomic factors that contribute to congenital heart defects.</p></div

Correlation between copy number and gene expression of <i>GSTT1</i>.

<p><i>GSTT1</i> expression in tissue from the right ventricle of infants with TOF relative to controls. Pearson correlation coefficient; r = 0.95, p = 0.001.</p

Donor Demographics.

<p>KU Path, University of Kansas Pathology; BTB, National Institute of Childhood Diseases Brain and Tissue bank for Developmental Disorders at the University of Maryland, Baltimore MD; n/a, not available.</p

Quantification of Nampt mRNA

<p>. Total RNA was isolated from <b>A.</b> human pancreatic tissue or <b>B.</b> isolated human islets. NAMPT mRNA was quantified using a Nampt specific Taqman assay (Applied Biosystems/Life Technologies, Carlsbad, CA.) according to the manufacturer's instructions and normalized with GUSB. *Note: The Y axis uses C_T values (C_T is the threshold cycle of detection), thus increased target mRNA results in earlier detection by qRT-PCR (i.e., a smaller C_T).</p

Protein levels of Nampt in human islets are greater with age.

<p>Examples of Nampt (blue) and insulin (green) immunofluorescence co-staining in islets from donors varying from 19 weeks gestation to 72 years old. <b>A–D:</b> In fetus and young children Nampt staining was weak with little co-localization with insulin in beta cells. <b>E–H:</b> In adults, Nampt staining was stronger and more localized to beta cells. <b>I:</b> Analysis of the Nampt pixel intensity illustrates the change with age.</p

Co-localization of insulin, glucagon, and Nampt.

<p>Immunofluorescence image of an islet from an adult male stained for insulin, glucagon and Nampt. <b>A:</b> insulin staining (beta cells) using anti-insulin antibody (green). <b>B:</b> Glucagon was identified in the same islet (alpha cells) using anti-glucagon antibody (blue). <b>C:</b> Nampt was identified in the same islet using anti-Nampt antibody (red) and is found in both the islet and surrounding exocrine tissue. <b>D:</b> Overlap of all 3 images shows that the majority of Nampt co-localizes with insulin in beta cells.</p

The effects of glucose on Nampt gene and protein expression levels in human islets.

<p>Total RNA or protein was isolated from islets treated for 1 hour in 20 mM glucose. <b>A:</b> Nampt gene expression was upregulated in the presence of 20 mM glucose compared to control (2.2 mM glucose) by qRT-PCR. Each bar represents the mean fold change normalized to β-actin from five separate experiments.*P<0.05; student T test <b>B:</b> Nampt protein content did not increase in the presence of 20 mM compared to 2.2 mM glucose by western blot analysis. Shown is a representative blot from 6 separate experiments.</p

Nampt in situ binding assay – summary of conditions tested.

<p>PFA, paraformaldehye; HCL, hydrochloric acid; RT, room temperature.</p

Protein pattern of Nampt in human islets changes with age.

<p>The difference in Nampt staining in endocrine and exocrine cells is clear. A: Fetal pancreatic tissues showed nearly equal Nampt staining levels in endocrine (within white circled regions) and exocrine tissue. B: In contrast, tissue from a 39 year old shows bright Nampt staining within the islet. C: The ratio of endocrine to exocrine pixel intensity illustrates the change with age. Of note, total image brightness was increased by 20% for every pancreatic image analyzed for figure C in order to visualize the low levels of Nampt staining in the fetal tissues.</p