Association Study Of Genetic Variants In PARD3with Neural Tube Defects In A Chinese Han Population

BackgroudNeural tube defects (NTDs) are one of the most common human birth defects arising from an incomplete closure of the neural tube during human embryonic development. The three common types of NTDs are anencephaly, spina bifida and encephalocele. Infants with anencephaly are stillborn or die shortly after birth, whereas infants with spina bifida and encephalocele can survive but frequently result in severe life-long handicap. So NTDs are severe and costly congenital malformations. China is a high prevalence country of NTDs. According to the latest research, Shanxi province of China has an NTD prevalence of199.38per10,000pregnancies, which is one of the highest in the world by nearly an order of magnitude. Therefore, it remains a major public health problem in this aera and China. Up to now, there is considerable evidence that genetics and environmental factors contribute to the etiology of NTDs. However, the exact genetic causes are unknown. Some recent studies showed that establishing polarization has been implicated as an important event during neural tube formation. Studies in zebrafish embryos have demonstrated that establishment of cell apicobasal polarity was an essential prerequisite for neural tube formation. The polarity protein partitioning defective3homolog (PARD3), a regulator of cell polarity, played a key role in mirror-symmetry polarized cell division and neural tube formation. Another study showed that loss of Pard3blocked cell migration across the midline and ectopic localization of Pard3led to an ectopic neural tube, which was more severe than that in Wnt planar cell polarity (Wnt/PCP) defective embryos. More severely, depleting PARD3resulted in intracellular cilia and hydrocephalus, which are frequent phenotypes in human NTDs. Given the accumulating evidence that Pard3is essential for neural tube morphogenesis in zebrafish and in the development of mammalian embryos, it deserves to be investigated for its role in human neurulation. It is well recognized that single nucleotide polymorphism (SNP) and copy number variation (CNV) are the two most common forms of genomic variation and make important contributions to disease susceptibility. Exciting data have recently emerged on the role of submicroscopic genomic imbalance or copy number variants as frequent causes of birth defects. So we hypothesized tgat PARD3was a potential candidate gene for human neurulation and that genetic variants in PARD3might be risk factors for human NTDs. ObjectiveThe aim of the study was to evaluate the association between sequence variants (including SNP and CNV) of PARD3gene and the risk of NTDs.MethodsWe conducted a case-control study in Shanxi province of China. Total53single SNPs in PARD3gene were genotyped among224embryos with NTDs and253normal embryos. CNV in25PARD3exons were genotyped among168embryos with NTDs and231normal embryos. Fetal spinal cord tissue was used for DNA extraction. iPLEX Gold SNP Genotyping Analysis was performed to determine PARD3genotypes using the Sequenom MassARRAY(?) platform. We adapted iPLEX Gold SNP Genotyping Analysis to screen for CNV. Direct sequencing after PCR and real-time PCR were performed to validate the results from the MassARRAY platform. Chi-square analysis was performed to compare the general characteristics, allele/genotype frequencies and copy numbers between case and control groups. The haplotype frequencies were estimated using online SHEsis software.ResultsThe minor allele frequencies (MAFs) of6polymorphisms (rs2496720, rs2252655, rs3851068, rs118153230, rs10827337and rs12218196) showed statistically associated with NTDs (P<0.05). After stratifying subjects by PARD3genotype and NTD phenotype, we observed a skewed distribution of this association. This significant association only existed in cases with anencephaly rather than spina bifida. Further haplotype analyses confirmed the association between PARD3polymorphism and NTDs risk (global test P=6.82e-009).CNVs were evident in21and25coding exons in both case and control groups. A novel heterozygote microdelation in exon14was identified in one anencephaly case and was absent from all controls analyzed. We determinated the exact boundaris of this heterozygote microdelation by sequencing after traditional PCR amplification. We detected the tissue-specific of the heterozygote microdelation in exon14in spinal cord, liver, heart, lung, kidney and skin. However, the results provide no evidence of tissue-specific heterozygore microdelation. Western blotting analysis with heart and kidney showed that the heterozygore microdelation disrupted the expression of isoform of PARD3at100kDa.We also reported here the adaptation of the multiplex comparative PCR combined with Sequenom MassARRAY plarform to measure the copy number for any gene or DNA fragment and compared the results with multiplex ligation-dependent probe amplication (MLPA) and real-time quantitative PCR. The results showed that this new method was comparable in accuracy to those alternative methods and it was easy to perform.ConclusionOur results suggested that PARD3polymorphisms were risk factors for NTDs in Shanxi province of China, and this association was affected by NTD phenotypes. Microdelation at the PARD3was likely to predispose to NTDs and heterozygote microdelation in exon14in PARD3had risk effect during in utero development precludes its presence in living person with less severe defects. This study provided further evidence supporting the role of sequence variants at PARD3as risk factors in the development of NTDs.