Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease characterized by the loss of motor neurons in the spinal cord, brainstem, and cerebral cortex, which results in muscle weakness, atrophy, and finally medulla paralysis and death in 3-5 years after disease onset due to respiratory failure. About 5%-10% of patients with ALS are familial ALS (FALS), which are associated with the mutations of Cu/Zn superoxide dismutase (SOD1) gene and alsin, vesicle-associated membrane protein B gene (VAPB), and angiogenin gene (ANG).1,2 Sporadic ALS accounts for about 90% of ALS cases, but the aetiology is largely unknown. Most of the researchers consider it to be a complex disease,3,4 which is not typically Mendelian monogenic inheritance but can be affected by several genes and environmental factors.5,6 The research strategy of its susceptible factors is to figure out the relationship between single nucleotide polymorphisms (SNPs) and the disease,7,8 which can figure out the biological process of the motor neuron degeneration and develop drugs that may cure this lethal disease. In order to identify SALS susceptibility genes, carrying out a genome-wide association (GWA) study to detect the related SNP has been a hot issue in the last two years. There have been several GWA studies reporting some ALS susceptible SNPs.9-14 The variant rs10260404 in DPP6 gene is strongly associated with susceptibility to ALS across different populations of European ancestry (from The Netherlands, Belgium and Sweden) as well as in a cohort of American patients, but no data of Asians (including Chinese) yet. We investigate whether the polymorphism of rs10260404 in DPP6 gene in Chinese Han origin is associated with sporadic amyotrophic lateral sclerosis (SALS) to compare the ethnic differences between Chinese and other populations.
A total of 58 Chinese subjects affected by ALS (male/female: 34/24, mean age of (46.4±14.0) years) were recruited for this study from the outpatient clinic and in-patient at Department of Neurology in Peking Union Medical College Hospital, Chinese Academy of Medical Sciences. All patients met revised E1 Escorial criteria for probable or definite ALS (1998).15 All of lower motor neuron dysfunction established by clinical and electromyographic examination including single fiber electromyography16 and familiar cases were excluded. The control group consisted of 52 healthy volunteers (male/female: 28/24, mean age of (45.3±15.1) years).
PCR and high resolution melting (HRM) genotyping and sequencing analysis
Inquire the date of rs10260404 from PubMed, design the primers and probe using Primer 5, the sequence of forward primer is 5′-AGCCCTGCACTGATTCCACCA-3′, the sequence of reverse primer is 5′-GAAACTGTCCTCATACAAAG-3′, the sequence of probe is 5′-TGACATACGAGGCCCAGA-amino modified-3′. Genomic DNA was isolated from peripheral blood according to EDTA anti-coagulating blood salting out method, asymmetric PCR was performed to amplify the target sequence of rs10260404, the PCR reaction system is 25 μl which consisted of PCR buffer 2.5 μl, dNTP 2 μl, forward primer (20 μm) 0.5 μl, reverse primer (4 μm) 0.5 μl, probe P (20 μm) 0.5 μl, Tag enzyme 0.125 μl, LC-Green 2.5 μl, templet DNA 2 μl, ddH2O 14.375 μl, paraffin oil 25 μl. The program: 45 seconds, 94°C denaturing; 45 seconds, 64.5°C annealing; 30 seonds, 72°C extending, entrained for 40 cycles. The PCR products were denatured and scanned using a Light Scanner, and the temperature melting curve was created, then the genotype of SNP was automatically analyzed and precisely differentiated by the software according to the curve. Some of the products were confirmed by sequencing.
The μ test was used to calculate the congruousness of Hardy-Weinberg equilibrium of allele frequencies and genotype frequencies between two groups. The χ2 test was used to compare the difference of allele frequencies and genotype frequencies between ALS group and control group. P <0.05 is considered to be statistically significant.
Genotype and the allele frequency
The HRM genotyping of rs10260404 in patients with ALS and in healthy controls were shown in Figures 1 and 2. Several PCR products were selected to be sequenced randomly. The results were shown in Figure 3.
Hardy-Weinberg equilibrium test
We analyzed genotype frequencies and allele frequencies between the ALS patients and the controls statistically. Genotype frequencies and allele frequencies can be calculated by direct gene counting method (Table 1). Several tests were used (control group Pearson test P=0.540, likelihood-ratio (Llr) test P=0.570, Exact test P=0.445: case group Pearson test P=0.230, Llr test P=0.273, Exact test P=0.223), all of which P >0.05, indicating that rs10260404 genotype was in Hardy-Weinberg equilibrium in ALS patients and controls, rs10260404 was linkage equilibrium on the population of the subjects of the study, and the control group was representative. The control group matched for gender and age to ALS cases, and the differences were no statistically significant.
Comparison of genotype and allele frequencies between two groups
The genotype frequencies and the odds ratio (OR) analysis of ALS case group and control group use the four-fold table materials, χ2 test and OR were calculated by specific software, the results were shown in Table 2.
It was demonstrated that there was no statistical significance of the rs10260404 allele frequencies in a cohort of Chinese Han origin between ALS cases and controls (χ2=0.29, OR=1.26, 95% CI 0.55-2.87, P >0.05). And the others like heterozygote, homozygote and genotype frequencies had no statistical significances between ALS group and control group. Cochran-Armitage trend test had no statistical significance either between ALS group and control group (common OR=1.263, χ2=0.26, P=0.611).
So far there have been several GWA reports releasing the association between SALS and SNPs.17 It has been demonstrated that the variant rs10260404 within the DPP6 gene is strongly associated with susceptibility to SALS across different populations of Switzerland, Dutch, Belgium, USA and Ireland. DPP6 gene encodes a dipeptidylpeptidase-like protein which can regulate the biological activity of neuropeptides, it binds special potassium channels altering their expression and biophysical properties.18 The mechanism of how DPP6 gene and protein cause ALS is still unclear yet, further study may provide insight into genetic variation at this locus.
Schymick et al10 had no positive findings from a genome-wide association study of the United States. Cronin et al11 combined data from their own study of the Irish population and data from the study of Shymick et al,10 found ALS associated SNP, rs10260404 (linkage to gene DPP6). van Es et al12 combined their first-stage data and the accomplished Dutch data, finally found 15 candidate SNPs. After selecting a new case and control groups (the samples from Dutch, Belgium and Switzerland population), they confirmed that variant rs10260404 is associated with susceptibility to SALS. Del Bo et al19 reached the same conclusion from the Italian population. But Cronin and his colleagues20 recently reported a joint analysis of GWA data from Ireland and the publicly available data sets from the United States and the Netherlands, 27 SNPs were commonly associated on joint analysis of the Irish, US and Dutch GWA. These 27 SNPs were genotyped in an expanded Irish cohort (312 patients with SALS; 259 controls) and an additional Polish cohort (218 patients; 356 controls). Pooling of data for 1267 patients with ALS and 1336 control subjects did not identify any association between rs10260404 and susceptibility to SALS after Bonferroni correction.
In our research, we did not find significant difference of rs10260404 between Chinese ALS group and control group. Rs10260404 was in linkage equilibrium on the population of the subjects of the study, and the control group was representative. In order to avoid the statistical error, we apply several test methods.21 There may be several reasons that we did not reach any significance. First it may be related with the genetic background, samples from different ethnics often have different SNPs. And secondly the results from the several researches before on rs10260404 may be false positive. In the association analysis of 100 variants, assuming significant level is 0.05, there may be chances of more than 99% that we can observe significant association between at least 1 variant and the disease. Therefore, multiple test correction should be used during statistical analysis, Bonferroni method is commonly applied which can assure the chance of false positive never passes the expected significant level. In the already-done research, we should consider whether the strict correction had already kicked out the SNP which did have a weak association with the Chinese Han people. Therefore, it is necessary to select some SNPs from the publicly available data and start the second-stage association research in the Chinese Han people and the related SNPs may be identified.
The method we used to identify SNP is not the traditional way that first amplify the target segment and then sequence it, but amplifying the target, then adding some LC-Green saturated fluorescent dye and denaturizing and renaturizing during appropriate circumstances, using HRM Scanning to obtain the genotype and sequence.22 This method is economy, convenient and sensitive, any change of the base from the target segment can be detected from the melting curve. The sample whose genotype had been confirmed by sequencing was set as a reference in every group of our research.
Comparing with GWA design, the number of our sample is not big, but as a single SNP case-control research, it can satisfy the request of statistic.23 Further study with more cases in Chinese population is really necessary, especially when Cronin et al20 got an opposite result when increasing the sample size by adding Poland population. Our research is the first research on the association between the susceptibility of SALS and SNP in china. We believe that the more advanced of the SNP technology and the less cost of it, the more researches will be carried out on the association between the susceptibility of SALS and SNP in Chinese population.
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