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%.