A Study of Birth Defects: Genetic and Epigenetic Determinants

Abstract

Background : Both structural and functional anomalies in newborn babies are termed as "congenital malformation" by World Health Organization (WHO). Congenital malformation or birth defect is a serious health issue of present time that has burdened the globe with a significant morbidity and mortality of children. With recent awareness and advancement in diagnostic imaging techniques, the detection of birth defect has increased. Consequently, the elective abortion of fetus detected with congenital anomaly has also been increased along with spontaneous abortion. The global prevalence of birth defect ranges from 3-4% whereas, the prevalence is as high as 6-7% in countries like India. The organogenesis of the fetus happens during 3rd to 8th week of gestation. This is a very sensitive period where the birth anomalies are formed. It can be a complete absence of anatomical part or a malformation of the same. Down syndrome (DS), a consequence of chromosomal aneuploidy, is a severe form of the genetic defect resulting in intellectual disabilities and other anatomical and physiological defects in children. The presence of all or part of the third copy of chromosome 21 instead of two is known as Trisomy 21 which is the major cause of DS. Similarly, any perturbation in cardiac development may give rise to another group of significant birth anomaly called congenital heart defects (CHD) which is also a clinical feature found in most cases of DS. This includes Ventricular Septum Defects (VSD), Tetralogy of Fallot (TOF) etc. The association of DS and CHD is a significant topic of research worldwide as both the conditions shares common etiology and risk factors. However, still, there is a lacuna in understanding the exact pathogenesis of these two conditions. It is known that both genetic and environmental factors independently and/or concurrently play an important role in pathoprogression of these two defects. Moreover, existing literature indicates that age of the parent is a significant risk factor for developing DS in the new born. Recent evidence also indicates that along with environmental and epigenetic factors, the lifestyle and occupational factors also play a major role in the formation of birth anomalies. Aim : The present study aims at studying the occurrence of Trisomy 21 by constitutional karyotyping in a given population in Gujarat. In addition, the study also attempts at revealing the parental and meiotic origin based on STR marker study by techniques like QF-PCR (Quantitative Fluorescent Polymerase Chain Reaction). Keeping in mind the epigenetic influence on the pathophysiology of CHD, the study aims to determine genome-wide DNA methylation profiles associated with the VSD and TOF. Moreover, the study focuses on the validation of candidate pathogenic mutations and their genetic susceptibility factors to CHD. Finally, the work attempts to integrate the genetic and network analysis to underlying etiology of the CHD phenotype. Method: A total of 122 DS patients from Gujarat (Western India) were enrolled in the study with parents following informed consent. The history of the patients was recorded by a detailed questionnaire. The various possible confounding factors like paternal age, consanguineous marriage, and environmental exposure history by occupation or lifestyle was also recorded. Complete clinical assessment of the subjects and information related to parental age during conception, habits, religion, medical history, health status, pedigree analysis, reproductive history was recorded. Total 360 blood samples were collected including DS patients (N=120), and their parents (Mother N=120, Father N=120) for DNA Isolation. The study was primarily aimed at investigating the occurrence of a major etiology of Down syndrome, Trisomy 21 by constitutional karyotyping. Moreover, the parental and meiotic origin was assessed by QF-PCR by using STR markers. The epigenetic variations and the genes involved in novel signaling pathways in the pathogenesis of CHD was done by detecting the methylation pattern from dried blood spots. The CpG-level methylation status of genetic samples from subjects with TOF and VSD including ethnicity and age-matched controls were obtained. The Infinium Human Methylation450 Bead Chips were used to identify methylation status. Data were analyzed with Illumina’s Genome Studio methylation analysis package program. Literature data mining for co-occurrence of gene names and keywords of interest were performed using Chilibot. Validation of methylation status of the identified by CpG sites was done by bisulfite sequencing. We have done a detailed analysis of the GDAC FIREHOSE database and the result indicated a strong correlation of expression of genes involved in the pathogenesis of TOF and VSD with the differential methylation status. Further, Each CpG site was also examined in the ENCODE databases using the UCSC genome browser for its location (e.g., promoter, intron, etc.), the presence or lack of H3K27AC laying, and transcription factor occupancy (e.g., PolR2a, PHF8 etc.). Results and Conclusion: Among the 122 DS patients, free Trisomy 21 was present in 110 DS patients (90.2%). Out of the remaining 12 patients, 10 were detected with Mosaic DS and 2 with Robertsonian Translocation (ROB) (1.6%). Moreover, an analysis of STR markers by QF-PCR determined the aneuploidy accurately by relative allele dose quantitation. The result showed the 95% of aneuploidy is of maternal origin and out of which 92% of the nondisjunction occurs due to the maternal meiotic error I (MMI). The risk of occurrence of DS was independent of parental age, however, the present occupation and environmental exposure played a pivotal role in risk calculation. We further investigated the phenotypic characteristics of patients with DS and correlated these anatomical defects with physiological impairment of functions. Among clinical features, thyroid disorder was the most abundant one and was reported in 32% of the cases. Other two major disorders found in DS patients were sight disorder (24%), CHD (21%), and the digestive disorder (12%). Among the rest, skin disorder and ear disorder occurred in an equal number of patients. The genome-wide DNA methylation profiles associated with non syndromic CHD revealed 64 loci whose degree of methylation is possibly related to the development of Tetralogy of Fallot (TOF) whereas, 80 loci with VSD. In VSD, we also found changes in levels of 8 miRNAs independently or in combination affecting 160 genes. To our knowledge this is the first report in which placental analysis has been used for determining the pathogenesis of and predicting VSD.