CUI Hong-gang,TIAN Xiao-fei,LUO Xiao-guang,LI Feng-rui,ZHU Lan-hui,ZHOU Yi-shu,REN Yan,PANG Hao

（1.School of Forensic Medicine,China Medical University,Shenyang 110001,China;2.Department of Forensic Medicine,Hebei North University,Zhangjiakou 075000,China;3.Department of Neurology,First Affiliated Hospital of China Medical University,Shenyang 110001,China）

Association of Five SNPs at the PARK16 locus as a Susceptibility Locus with Parkinson’s Disease for Forensic Application

CUI Hong-gang1,TIAN Xiao-fei2,LUO Xiao-guang3,LI Feng-rui1,ZHU Lan-hui1,ZHOU Yi-shu1,REN Yan3,PANG Hao1

（1.School of Forensic Medicine,China Medical University,Shenyang 110001,China;2.Department of Forensic Medicine,Hebei North University,Zhangjiakou 075000,China;3.Department of Neurology,First Affiliated Hospital of China Medical University,Shenyang 110001,China）

To investigate the association of five SNPs（rs823083,rs708723,rs4951261,rs823076 and rs16856110）at the PARK16 locus with Parkinson’s disease（PD）,and to potentiate its forensic application.The genomic DNAs of 215 PD patients and 212 matched controls from the northern Han Chinese population were amplified in two independent PCR systems and subsequently genotyped by digestion with the three endonucleases（HinfⅠ,NcoⅠand MspⅠ）.The genetic parameters and association studies were carried out with SPSS 13.0,Haploview version 4.2 and PLINK 1.07 softwares.We detected accurately all genotypes in the five SNPs with multiplex PCR-RFLP and mismatched multiplex PCR-RFLP techniques.The genotypes of four SNPs,except for rs823083,were in Hardy-Weinberg equilibrium.The four SNPs,rs16856110,rs4951261,rs708723 and rs823076,which were in linkage equilibrium,should not be associated with PD（P-values ranging from 0.077 to 0.544）.The SNPs investigated at the PARK16 locus were not found to be involved in PD-associated blocks in the northern Han Chinese population. The allele distributions of rs708723,rs4951261,rs823076 and rs16856110 in the northern Han Chinese population can be highly polymorphic,which can be applied to genetic analysis and forensic practices.

forensic genetics;polymorphism,single nucleotide;Parkinson disease;PARK16

Article IC:1004-5619（2013）03-0185-05

Parkinson’s disease（PD）,one of the most common age-related neurodegenerative movement disorders,affects 1%-2%of individuals older than 65, which is characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta （SNpc）.The main clinical features of PD are restingtremor,bradykinesia,posturalinstabilityand rigidity[1].Although the exact etiology of PD remains unclear,the genetic factor has a potential function in the pathological progress[2].In the past years, genome-wide association studies（GWAS）have been employed to identify new loci for PD.The PARK16 region,containing five genes（SLC45A3,NUCKS1, RAB7L1,SLC41A1 and PM20D1）,was identified as a susceptibility locus for PD in a Japanese study[3],in which seven SNPs（rs16856139,rs823128,rs823122, rs947211,rs823156,rs708730 and rs11240572）were shown to be significant.The rs823128 was later reported to be associated with PD in a European GWAS[4].In an Asian study,the rs823128 was also found to be related to PD in ethnic Han Chinese subjects[5].The association between rs823128 and PD has also been supported by Chilean and Taiwanese studies[6-7].The most strongly SNP associated with PD,rs823114,located in an inter-genic region proximal to NUCKS1,was identified in an Ashkenazi Jewish population[8].In an association study[9],a novel RAB7L1 mutation,c.379-12insT,was identified to be related to PD（P=0.032 5）in a cohort and casecontrol study from the United Kingdom.Recently,a two-stage meta-analysis provided further support for the findings that PARK16 region was a PD risk locus[10].Using a case-control methodology,two studies were conducted to investigate the association of the GWAS-linked SNPs at the PARK16 locus with the risk of PD in Mainland China[11-12],one showing that the minor alleles could significantly reduce the risk ofdevelopingPDatthethreeSNPs,rs823156, rs823128 and rs16856139;the other indicating that the two SNPs,rs16856139 and rs11240572,conferred a protective effect against PD.

Such studies confirmed the PARK16 region as a candidate locus for PD.It is unknown,however, whether there are more SNPs at this locus associated with PD or whether there are refined PD-related regionsinthePARK16locus.Sinceautosomal SNPs have been of great interest for the potential advantages in paternity testing because of the low mutation rates,and especially in the analysis of degraded samples by use of short amplicons,the polymorphic information of the SNPs in the PARK16 locus may be useful to forensic practices.In the current study,we selected five SNPs（rs823083, rs708723,rs4951261,rs823076 and rs16856110）in five genes,respectively,to explore their potential association with PD in northern Han Chinese population and its application in forensic medicine.

Materials and methods

Patients and controls

A total of 215 ethnic Han Chinese patients（mean age,61.00±11.66,ranging from 24 to 85;male by 53.02%）as case group were recruited from northern China,all fulfilling the criteria for the clinical diagnosis of PD with at least 2 of 3 clinical signs （bradykinesia,tremor,and rigidity）and a positive response to levodopa therapy.They were diagnosed with idiopathic PD by movement disorder neurologists at the First Affiliated Hospital of China Medical University in Liaoning province of P.R.China. In ethnicity,age and gender,212 matched controls as control group（mean age,62.87±10.44,ranging from 34 to 86;male by 58.96%）,were healthy withnodiagnosesofneurodegenerativediseases. There was no statistical difference in ethnicity,age or gender between these two groups（P＞0.05）,from whom was obtained the informed consent,with the approval obtained from the local ethics committee.

Genotyping

AllgenomicDNAswereisolatedfromthe leukocytes collected from the subjects’peripheral blood samples,and extracted using the SDS-proteinase K-phenol-chloroform method.According to thepublishedsequencesofSNPsrs4951261in NUCKS1,rs823083 in PM20D1,we synthetically generated a HinfⅠrestriction endonuclease site in each amplified product using mismatched PCR primers （Sangon,Shanghai,China）.A natural HinfⅠendonuclease site exists in the sequence around rs708723 in RAB7L1;a natural NcoⅠendonuclease site,in the sequence around rs823076 in SLC41A1;and a natural MspⅠendonuclease site,in the sequence around rs16856110 in SLC45A3.The sequences of the primers were shown in Table 1.

Table 1Information about the five SNPs and parameters for multiplex PCR amplification

MultiplexPCR-RFLPwasusedtoidentify polymorphisms in the SNPs.Two different reaction mixes for PCR amplification were carried out in a final volume of 20 μL PCR buffer containing 1×Es Taq MasterMix（CWBIO,Beijing,China）,50-100ng genomicDNA,andprimerconcentrationsshown in Table 1.The first reaction included rs823083, rs708723 and rs4951261 loci,and the second reaction contained rs823076 and rs16856110 loci.PCR amplification was performed under the same conditions:denaturation at 94℃for 1 min;94℃for 30 s, 60℃for 30s,and 72℃for 30s for 35 cycles.For restriction enzyme digestion,about 2 μL PCR product from the first reaction and 1.5 U HinfⅠ（TaKaRa, Dalian,China）were added to 10 μL 1×H buffer, and about 1.5 μL PCR product from the second re action and 0.5 U NcoⅠ（TaKaRa,Dalian,China）, 0.5U MspⅠ（TaKaRa,Dalian,China）were added to 10 μL 2×K buffer with 0.02%BSA.The reaction mixtures were incubated at 37℃for more than 2 h. The digested products were separated by polyacrylamide gel electrophoresis（T=6%,C=5%）.The gels were dyed with 10 000×GeneFinderTM（Bio-V,Xiamen,China）,and observed under ultraviolet.To confirm the formation of synthetically mismatched sequences,directDNAsequencing of eachPCR product was performed with a BigDye Terminator Cycle Sequencing Kit（Applied Biosystems,USA）.

Statistical analysis

The allelic and genotypic frequencies of the five SNPs in 215 PD cases were investigated and compared with that in 212 controls.The genetic parameters and association studies,which including polymorphism information content（PIC）,probability of discrimination power（DP）,excluding probability of paternity（PE）,and expected heterozygosity（He）were analyzed with SPSS 13.0（SPSS Inc.,USA）, Haploview version 4.2（Daly lab at the Broad Institute Cambridge,MA 02141,USA）[13]and PLINK for Windows version 1.07（Free Software Foundation, Inc.,USA）[14].A two-tailed P value less than 0.05 was considered statistically significant.

Results

Twomismatchedsequences（rs823083and rs4951261）containing the HinfⅠrecognition site were correctly generated and confirmed by DNA sequencing.The sizes of PCR products were 258 bp （rs823083）,176 bp（rs708723）,144 bp（rs4951261）（Fig.1A）and 282bp（rs823076）,202bp（rs16856110）（Fig.1B）.The fragments shorter than 32 bp ran off the gel under the electrophoresis conditions.These results indicated that the mismatched multiplex PCRRFLP assay was successful and the genotypes of the five SNPs were correctly identified using this method.

The genetic parameters for each SNP site were shown in Table 2.Among the five SNPs,rs823083 was not in Hardy-Weinberg equilibrium via Fisher’s exact test.The frequency of the g.205717823AA plus CA genotype in the rs4951261 locus and the g.205631767 AA plus AG genotype in the rs16856110 locus were over 90%in both PD cases and controls. The linkage disequilibrium（LD）results from the haplotype analysis showed that the four SNPs in Hardy-Weinberg equilibrium were in linkage equilibrium.From the association analysis,the odds ratio （OR）values of the five SNPs ranged from 0.781 4 to 0.903 9（Table 2）,showing a lack of association between the SNPs’genotypes and PD in the northern Han Chinese population.In addition,the forensic parameters were shown in Table 3.

Fig.1Genotyping of the five SNPs by multiplex PCR-RFLP

Table 2Allele and genotype frequencies of the five SNPs at the PARK16 locus in 215 PD cases and 212 controls

Continued

Table 3The polymorphic parameters for the four SNPs in the northern Han Chinese population

Discussion

In the previous studies[15-16],we had analyzed gene polymorphisms by mismatch and mismatched multiplex PCR-RFLP techniques,which was an efficient,rapid,and economical way to augment genotyping output.We could accurately determine all genotypes in the previous investigation.In the current study,we performed the same strategies to investigate the allelic frequencies of the five SNPs in the PD-associated PARK16 locus.As expected,the genotyping technique might effectively detect these SNPs both in the PD patients and normal subjects. GWAS applied high-throughput genotyping technologies to assaying thousands of SNPs and related them to clinical conditions and measurable traits.Undoubtedly,GWAS was an important advance in discovering geneticvariants influencing disease[17],which indeedproduced more invaluable information.To date,many GWAS have been conducted in PD. However,it has not been realistic for all laboratories to perform GWAS to display complete PD-related genetic background because of condition restriction.To duplicate and validate the GWAS involved in susceptible PD genes,different genotyping methods were reasonably employed,which might effectively complement those lacks of genotypic data in multiple races and areas.The current strategy not only provided multiplex PCR amplification and restriction enzyme digestion（artificially induced recognizable site）in an independent system,but also identified various genotypes by gel electrophoresis. Thus,this straightforward assay will allow for more SNPs in more PD-associated genes to be detected as easily as possible.

With the use of GWAS,many susceptible SNPs for PD at the PARK16 locus were identified,suggesting that there might be more independent association signals at the locus.Indeed,the analysis of GWAS-linked SNPs reaffirmed PARK16 as a susceptibility region that there were many PD-related blocks at the locus[4-8,11-12].The PARK16 locus spans 5 transcripts,SLC41A1,RAB7L1,NUCKS1,SLC45A3 and PM20D1.SLC41A1 was a magnesium（Mg2+）transporter[18].Intriguingly,Mg2+deficiency was reported to be an environmental risk factor for the amyotrophic lateral sclerosis（ALS）-parkinsonism/dementia complex（MIM105500）[19].RAB7L1 was a small GTP-binding protein that played an important role in regulation of exocytotic and endocytotic pathways[20]. NUCKS1 was a nuclear protein containing several consensus phosphorylation sites for casein kinaseⅡand cyclin-dependent kinases of unknown function[21]. SLC45A3 was originally found to be associated with prostate cancer[22]and PM20D1 was linked to metal ion binding and peptidase activity.Numerous biochemically functional metals,such as iron,copper, zinc and manganese,had been found to be associated with PD[23-25].The gene expressions interacting with metal ions indicated a genetic basis for a potential etiopathological pathway associated with PD. All these data showed that the PARK16 locus might be involved,playing an important role in the pathological progress of PD.Thus,accumulating data including more susceptible SNPs identified in the region and association analyses of the SNPs among different transcripts might help systemically understand potential significance between the PARK16 locus and PD.

The PARK16 locus can be of scientific interest since it contains a few candidate genes on 1q32 associated with PD,some SNPs of them showing a significant association with a lower risk of PD.Further LD analysis revealed that these SNPs lay within several LD blocks.Thus,we respectively selected one SNP for each gene to observe allelic or haplotypic association with PD.This analysis was to determine whether more SNPs in the five genes would indicate an independent association or whether the associations with the SNPs would be primarily due to high LD.In this current study,however,association analysis indicated no evidence for the five SNPs as genetic risk factors for PD in the northern Chinesepopulation.In agreement with the European study, the SNP rs708723 lacked the unequivocal evidence of association either[10].The results might be related to the location of the selected SNPs,which were located in the intron of each gene and did not play a regulatory role in gene expression and/or link to causal variants within the same LD block.As yet,it remained unknown which of the SNPs and which of the genes within the susceptibility region could exert a pathogenic effect.Undoubtedly,the current study accumulated meaningful data for the systemic analysis of the PARK16 region.

The SNP markers will serve an important role in analyzing challenging forensic samples and complex kinship analysis[26].In the current study,the investigated SNPs showed good polymorphic information in the northern Han Chinese population through genetic parameters estimation,which might be applied to forensic practices.

Acknowledgments

ThiscurrentstudywasfundedbyNational Natural Science Foundation of China（No.81172713）.

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（Received date:2012-12-19）

（Editor:LI Cheng-tao）

DF795.2Document code:A

10.3969/j.issn.1004-5619.2013.03.007

Author:CUI Hong-gang（1987—）,postgraduate in forensic serology;E-mail:cmu062208@163.com

PANG Hao,professor,Ph.D,tutor in forensic genetics;E-mail:panghao8@gmail.com