Pediatric Cerebral Palsy Susceptibility Gene Polymorphism, Copy Number Variation And DNA Methylation Studies

Section 1 Genetic association study of adaptor protein complex 4 with cerebral palsy in Han Chinese populationAdaptor protein complex 4 (AP-4) plays a key role in vesicle formation, trafficking, and sorting processes, which are critical for the development and function of the brain. AP-4 consists of four subunits encoded by AP4E1, AP4B1, AP4M1 and AP4S1 genes respectively. A number of studies have identified the involvement of AP-4-mediated vesicular trafficking pathway in the etiology of cerebral palsy, the most notable of which are the causative mutations identified in each of them that have recently been reported in different CP families. We therefore postulated that variations in AP-4 coding genes may play an important role in the susceptibility to CP. In the present study, sixteen SNPs were geneotyped among 517 CP patients and 502 healthy controls from the Chinese Han population. We systematically analyzed the association of the AP4E1, AP4B1, AP4M1 and AP4S1 genes with CP on the basis of clinical characters. No significant associations were found between these variants and the risk of overall CP. Subgroup analyses showed that rs1217401 of AP4B1 was significantly associated with CP as a sequela of hypoxic-ischemic encephalopathy (HIE) (HIE+CP) (allele:p= 0.042151; genotype:p= 4.46×10-6). The current results indicate that 16 variants studied in four units of AP-4 have no detected effects on the susceptibility to overall CP, but AP4B1 is a susceptible gene for HIE+CP in the Chinese population.Section 2 Genetic association study of COL4A1 with cerebral palsy in Han Chinese populationCOL4A1 gene, that encodes α1 chain of type IV collagen, is mapped on the telomeric region of 13q (13q34). Type IV collagen is the major structural component of basement membranes structure and are present in all metazoans. Collagen IV networks not only provide structural support to the cells and tissues, but also affect the biological fate during and after the development. COL4A1 is the most abundant component of type IV collagen in basement membrane and is widely expressed in all tissues, including the brain. COL4A1 mutations cause highly penetrant multi-system disorders. More recently, Harteman and colleagues found two COL4A1 mutations in atypical periventricular haemorrhagic infarction (PVHI) patients, and these two patients were also diagnosed with spastic unilateral CP. Thus, we speculate that the COL4A1 may be a susceptibility gene for cerebral palsy. In the present study, seven SNPs of COL4A1 were geneotyped among 351 CP patients and 220 healthy controls from the Chinese Han population based on a Sequenom iPLEX Gold massARRAY platform. Results show that there were statistically significant differences of allele frequencies between cases and controls at rs1961495 and rs1411040. GMDR analysis was also conducted to assess the impacts of combinations between the seven SNPs of COL4A1 and the four-locus model had the highest level of testing accuracy (59.77%). So, four-locus model was the best GMDR model. These results confirmed our hypothesis that COL4A1 gene had interactive effects on the risk of CP, and suggested that use of multiple statistical approaches could be a better strategy to elucidate complex gene interaction.Section 3 Association study of copy number variation with cerebral palsy in Han Chinese populationCopy number variation (CNV) is a form of genetic variation. CNVs were once considered benign or may have unknown clinical significance. However, more and more clinical phenotype-genotype association studies have shown that CNVs is related to polygenic disease. At present, researchers in different populations have identified many CNVs and CNVs area were associated with diseases. CNV was found to be the major risk factor for many mental diseases, which is the complication of cerebral palsy. Therefore, we speculate that CNV is also the cause of cerebral palsy. In this study, we use MLPA and aCGH technology to investigate the relationship of several CNVs and cerebral palsy susceptibility in the Han Chinese population. We found four CNV loci in 702 cases of children with cerebral palsy. These four CNVs were Chr 17p11.2 del 5.57Mb, chr22 q13.31-ql3.33 del 2.1Mb, chr19 p13.3 dup 4.7Mb, and Chr15p11.2-q13.1 dup 20.5Mb. Among these CNVs, chr22 q13.31-q13.33 del 2.1Mb, and chr19 p13.3 dup 4.7Mb were found in the same case. The results show that, CNV is the cause of cerebral palsy and high-throughput technologies may help verify this.Section 4 DNA methylation study of cerebral palsy twinsA growing number of studies have shown that genome epigenetics plays an important role in the growth, differentiation and development process of human individuals. Cerebral palsy is a complex disease with genetic heterogeneity and clinical heterogeneity, while epigenetics may play an important role in the pathogenesis of cerebral palsy. As the most common form of epigenetics, DNA methylation should contribute to the pathogenic mechanism of cerebral palsy. Compared to the irrelevant population, twin pairs discordant for disease are an ideal model for DNA methylation research. In this study we performed DNA methylation profiling using the DNA extracted from whole blood of 6 twin pairs (2 dizygotic twins and 4 monozygotic twins) and the Infinium DNA methylation BeadChip technology covering more than 450,000 CpG sites genome wide. By analyzing the datasets of all 6 twin pairs, we found that differentially methylated CpG sites in many genes are associated with inflammatory disease and Inflammation-related pathways. This result suggest that a link between inflammatory response and cerebral palsy.