Association Study Of DNAJC12 And GCH1 Gene Mutations With Sporadic Parkinson’s Disease In Chinese Han Population

Background:Parkinson’s disease(PD)is a common neurodegenerative disease mainly occurs in middle-aged and elderly people.The clinical characteristics of PD are bradykinesia,rest tremor,muscular rigidity and abnormal posture and gait.The pathological manifestations are extensive loss of dopaminergic neurons in substantia nigra and formation of Lewy bodies.The prevalence of PD in Chinese people is about 1.7%in the population of 65 years old or elder.With the aging of the our population,our country will have more PD patients in the future,which will bring greater burden.The pathogenesis of PD is not fully elucidated,but genetic factors are definitely involved in PD onset.Recently,two studies have reported several patients from different families carrying DNAJC12 gene mutations,which are characterized by progressive neurodevelopmental retardation and dystonia due to the lack of dopamine and serotonin.Another study showed that DNAJC12 gene mutations were also confirmed in two unrelated families with early-onset Parkinson’s disease symptoms.DNAJC12 gene was involved in dopamine biosynthesis and transportation,and also plays an important role in vesicle regeneration and protein folding.In addition to DNAJC12 gene,the GCH1 gene mutations may also affect the synthesis of monoamine neurotransmitters,especially the dopamine,by affecting the synthesis of tetrahydrobiobutterfly(BH4)and tyrosine hydroxylase(TH).Previous studies have reported that the rare GCH1 gene mutations in Caucasians increase the risk of PD development by seven times.Subsequently,several groups explored the relationship between rare mutations in the GCH1 gene and PD risk in Canadians and Norwegians,but the findings of these studies were controversial.Similarly,on the GCH1 gene,a single nucleotide polymorphism rs11158026,located in the intron region,was confirmed to be associated with an increased risk of PD by a large-scale meta-analysis of European genome-wide association data.Several groups in China have also investigated the relationship between this SNP locus and the risk of PD in Chinese population,but the conclusions are controversial either.Based on the results above mentioned and the remained controversy,this case-control study was conducted to further clarify the relationship between DNAJC12,GCH1 gene mutations and SNP rs11158026 in intron region of GCH1 gene and the risk of sporadic PD in Chinese Han population.Objective:To explore the frequency of DNAJC12 and GCH1 gene mutations and SNP rs11158026 of GCH1 gene in Chinese Han population,and analyze the relationship between the frequency of these gene mutations and SNP locus rs11158026 and the risk of sporadic PD in Chinese Han population.Methods:This study was a case-control study.The case group consisted of 702 sporadic PD patients who visited the Department of Neurology,the First Affiliated Hospital of Zhengzhou University.All patients in the PD group were diagnosed by two neurologists according to the diagnostic criteria of the United Kingdom PD Society Brain Bank.The control group consisted of 728 health controls matching the age and sex with the PD group who visited the same hospital physical examination center.All the 1430 subjects were from the Chinese Han population.This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University,and all the subjects understood and signed the informed consent.Genomic DNA was extracted from peripheral venous blood samples of all subjects through standard steps provided by the kit.The concentration and purity of DNA were detected by the instrument,and the qualified DNA was stored in the refrigerator at–80℃until it was taken.The primers of all exons of DNAJC12 gene,all exons of GCH1gene and SNP rs11158026 of GCH1 intron were designed by Primer Premier 6.0software,and all target areas were amplified by polymerase chain reaction.After amplification,the quality and length of the products were detected by gel electrophoresis,and then Sanger sequenced.The allele frequencies and genotype frequencies of gene mutation loci and SNP locus in the PD group and the control group were calculated from the sequencing results.Statistical analysis was carried out using SPSS23.0 software.The distribution of allele and genotype frequencies of the target mutations in the population was verified byχ~2 test to see if it follows the Hardy-Weinberg(H-W)equilibrium.Pearsonχ2 and Fisher’s exat test were used to calculate the difference of allele and genotype frequencies between the PD group and the control group.Meta-analysis was used to further explore the relationship between the SNP rs11158026 in the GCH1 gene intron region and the risk of sporadic PD in Chinese population.Result:1.There was no significant difference in age and sex composition between the PD group and the control group(p>0.05).The distribution of genotype frequencies of the mutations in the PD group and the control group was in accordance with Hardy-Weinberg(H-W)equilibrium(p>0.05),indicating that the genotype distribution of the samples in this study was uniform and representative.2.By sequencing all exon regions and exon-intron binding regions of DNAJC12gene,we did not find the pathogenic mutations of DNAJC12 gene reported previously,but we found 7 SNPs in the exon-intron binding region of DNAJC12 gene.However,for these 7 SNPs,we found that there were no differences in allele frequency and genotype frequency between the PD group and the control group,or between the early-onset PD group and late-onset PD group.3.By sequencing all the exon regions,exon-intron binding regions and SNP locus rs11158026 of GCH1 gene,we found that 5 PD patients and 1 healthy control carried3 rare heterozygous mutations(c.239G>A,c.68C>T,c.196C>T).2 mutations(c.239G>A,C.68C>T)have been reported previously,and 1 mutation(c.196C>T)is a newly discovered mutation by our study.The total frequency of GCH1 gene mutations was 0.71%(5/702)in PD group and 0.14%(1/728)in control group.There was no significant difference(Fisher’s exact test p=0.117836)either.As for the SNP locus rs11158026 located in the intron region of GCH1 gene,it was found that the distribution of the gene frequency and genotype frequency of this SNP locus in the PD group and the control group was significantly different(p=0.032089 and p=0.014946,respectively).PD patients carried more C alleles(OR=1.2,95%CI=1.04-1.39)than that of the normal control group.A Meta-analysis composed of 5 studies(including our study)was conducted to explore the relationship between the frequency of this SNP and the risk of PD in Chinese population.A total of3946 PD patients and 3666 normal controls were included in this Meta-analysis.The results of this Meta-analysis showed that the SNP rs11158026 variant was also significantly associated with the increased risk of PD.The Meta-analysis results were consistent with the results of our study.Conclusion:The present study suggested that:1.DNAJC12 gene mutations may not a risk factor for sporadic PD in Chinese Han population;2.GCH1 gene mutations may not be associated with an increased risk of sporadic PD in Chinese Han population either,but the result may be attributed to a relatively small sample size;3.SNP locus rs11158026 in intron region of GCH1 may be associated with an increased risk of sporadic PD in Chinese Han population.This conclusion was also confirmed by our large sample size Meta-analysis.