Introduction
Congenital heart defects (CHD) are among the most common congenital malformations in newborns with approximately 1 million children born each year worldwide. Although the mortality of CHD has decreased in the past decade(1), the quality of life of these patients is often compromised due to severe physical and psychological problems.(2) Several studies have shown a multifactorial origin of birth defects like CHD, where interactions between genetic predispositions and environmental exposures in early pregnancy are strongly involved in the pathogenesis.(3) The most common CHD are cardiac outflow defects (COD), with ventricular septal defect (VSD) as the most prevalent phenotype, accounting for approximately 35% of CHD.(1) COD share the same multifactorial origin and pathogenesis as neural tube defects where derangements in neural crest cells due to induced excessive oxidative stress and inflammation, are involved in the pathophysiology.
The extracardiac contribution of neural crest cells together with the secondary heart field, both located in the pharynx, play an essential role in the septation process of the outflow tracts of the cardiac ventricle.(4, 5)
Maternal hyperhomocysteinemia is a risk factor for the development of neural crest related anomalies in offspring, like spina bifida, cleft lip and/or palate and congenital outflow defects in offspring.(6, 7) Hyperhomocysteinemia disturbs apoptosis and myocardialization of the cardiac outflow tract, by affecting the cardiac neural crest cells.(8) Maternal health conditions, such as maternal age, obesity, tobacco and alcohol use, poor nutrition and lifestyle enhances oxidative stress and inflammation and are identified as risk factors for COD.(9) Telomeres are nucleoprotein structures that cap the end of chromosomes and thereby protect against unwanted recombination and degradation.(10) In humans, TL shortens in somatic cells with advancing age due to the increased number of cellular divisions. Excessive TL shortening is an index of senescence, causes genomic instability and is associated with a higher risk of age-related diseases. Recent findings showed that newborn TL determines the adult TL(11). It has been suggested that TL is a long term biomarker of chronic oxidative stress compared to hyperhomocysteinemia as short term biomarker.(12, 13) Primary prevention of COD in offspring might be possible when we have more insight into the role of TL as a biomarker in the pathogenesis to be used as future predictor of COD, and the associations with maternal health conditions. Therefore, as a first step we aim to study the association between periconception maternal TL and the risk of having a child with a neural-crest related COD.
Materials and Methods Study populationData and blood samples were used from the HAVEN-study (Acronym for Heart Defects, Vascular Status, Genetic Factors and Nutrition), a multicenter case control triad study on COD, conducted at the Department of Obstetrics and Gynaecology, Division of Obstetrics and Foetal Medicine of the Erasmus MC, University Medical Centre Rotterdam, The Netherlands. The study was designed to investigate parental health, environmental and genetic determinants in the pathogenesis and prevention of COD offspring. Data has been collected between 2003-2010 and the study design has previously been described in detail(14). The HAVEN study, enrolled 904 case and control mothers (Figure 1). Mothers pregnant at the study moment (n=77), missing folate or homocysteine status (n=94) and missing TL assessment (n=3) were excluded from the analysis. In total, 306 case mothers of a child with COD and 424 control mothers of a child without congenital malformations were selected and included for analysis. In summary, all cases were diagnosed with COD by ultrasound and/or cardiac catheterization and/or surgery by a paediatric cardiologist. Control children and their parents were eligible if congenital malformations or chromosomal abnormalities were not present. The VSD group consist of perimembranous ventricular septal defects and atrioventricular septal defects. Only singletons, biological parents with no familial relationship and whom are familiar with the Dutch language (writing and reading), could participate. Venous blood samples were drawn from all mothers, approximately 1 year after the periconception period of the index-pregnancy, i.e., around 15 months. The same interval was used to obtain general information lifestyle behaviours and demographic data by questionnaires. This interval was chosen to mimic the periconceptional health status of the mother and to minimize the risk of undiagnosed less severe COD in the control group. The study protocol was approved by The Central Committee on Research involving Human Subjects and the institutional review boards of all participating hospitals. All parents gave their written informed consent, as well as on behalf of their participating child.
DNA-isolation and Telomere length measurement Genomic DNA from case and control mothers was extracted from EDTA blood samples with the Reliaprep kit (Promega, Leiden, Netherlands) on a Tecan Evo robot. DNA concentrations were measured with the Nanodrop (ThermoFisher, Waltham, United States of America) and normalized to 50 ng/ul. Relative TL (TS ratio) was measured using a qPCR assay based on the method described by Cawthon(15) with minor modifications. For each sample the telomere and 36B4 assay were run in the same well position but in different 384 wells PCR plates. Each reaction contained 2 ng DNA, 1 uM of each of the telomere primers (tel1b-forward: GGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT, tel2b-reverse: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCTT) or 250 nM of the 34B4 primers (36B4u-forward: CAGCAAGTGGGAAGGTGTAATCC, 36B4d-reverse: CCCATTCTATCATCAACGGGTACAA) and 1x Quantifast SYBR green PCR Mastermix (Qiagen, Hilden, Germany). The reactions for both assays were performed in duplicate for each sample in a QuantStudio Flex 7 real time PCR machine (Applied Biosystems, Waltham, United States of America). The cycle threshold (Ct) values and PCR efficiencies were calculated per plate using the MINER algorithm.(16) Duplicate Ct values with a Coefficient of Variance (CV) of more than 1% were repeated a second time in a different run. The average Ct values (of the duplicate measurements) per sample were adjusted for PCR efficiency using the formula Q=1/(1+PCR eff)^Ct. The TS ratio was calculated by dividing the Q of the telomere assay by the Q of the 34B4 assay. Each 384 wells PCR plate contained a set of 7 control samples. The average TS ratio of these 7 samples was used to normalize for plate batch effects. To validate the measured TS ratios, 170 random samples were run twice and the CV of that experiment was 4.5%.