Association Of The SNPs In The HFE Gene With Susceptibility To Amyotrophic Lateral Sclerosis In The Population With The Han Nationality In China

Objective:Amyotrophic lateral sclerosis (ALS) is characterized by loss of motor neurons and proliferation of glial cells. All patients experience muscle weakness,atrophyand spasticity. This is the consequence of the loss of both upper and lower motor neurons. About 5% to 10% of ALS patients have a family history (FALS), and most of these patients inherit the disease in an autosomal dominant way.The large majority of patients (90% to 95%) has no clear family history and is considered as sporadic ALS (SALS). There is no effective treatment for ALS, Ultimately, patients become paralyzed and denervation of respiratory muscles causes the death of the patient. Oxidative stress may be the important cause of motor death in ALS. Nervous system is more vulnerable to oxidative damage.Iron is an essential metallic microelement for life. Oxidative damage mediated by iron overload has been thought to be the cause of cellular apoptosis and damage.Iron misregulation promotes oxidative stress and abnormally high iron levels have been found in the spinal cords of patients with neurodegenerative. Cellular iron mismanagement and oxidative stress are associated with a number of neurodegenerative diseases. Oxidative stress which caused by iron overload may be the important pathogenesis of ALS.Although the mechanism misregulation of iron homeostasis in central nervous system (CNS) is uncertain, the HFE gene mutation may be a cause. HFE gene was identified on the short arm of chromosome 6. The gene encodes a protein which contains 343 amino acids, the protein is involved in the regulation of cellular iron metabolism. HFE participants in the regulation of iron homeostasis and the main distribution is in the human gastrointestinal epithelial cells. It has been reported to interact with the transferring receptor(Tf) to regulate cellular iron uptake.Meanwhile, the HFE-TfR2 complex is proposed to trigger hepcidin activation. Therefore, when HFE is defective, the loss of iron sensing could contribute to accumulation of iron and occurrence of oxidative stress in many tissues.There are three common mutations of HFE gene (C282Y, H63D and S65C). HFE gene mutations may be a risk of ALS. Some studies about the relationship between HFE gene mutations and ALS were reported, however little was reported about the association of HFE gene with ALS in Chinese. This study was designed to investigate the association of HFE polymorphisms with the risk of ALS. In this study, case-control study, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used and the same DNA samples were sequenced to ientify the HFE polymorphism. By this way, we hope to offer some evidence for the prevention and therapy of ALS at molecular level.Methods:1 Researching objects: This case-control study included 124 ALS patients and 210 healthy controls. Diagnosis and differential diagnosis of the SALS patients were confirmed by E1 Escorial diagnostic criteria defined by the World Federation of Neurology. All the cases of ALS were sporadic.The cases of control group were selected randomly from the Physical Examina- tion individuals in our hospital matched with ALS group in age and sex. There are no blood relationships between the two groups. All the control cases should be consistent with the standard as follows: Han people with no genetic predisposition to disease and various kinds of diseases such as tumor, cerebrovascular disorder, emotional disturbance.Informed consent was obtained from all participant.2 Human blood DNA extraction: Five milliliter of venous blood from each subject was drawn and genomic DNA was extracted.3 Analyses of C282Y, H63D and S65C: PCR and restriction enzyme digestion were performed to screen for C282Y, H63D and S65C mutations of the HFE on 334 bloods. The PCR products were separated and detected by agarose gel electrophoresis. The restriction enzyme Rsa I, Mbo I and Hinf I were used to cut C282Y, H63D and S65C PCR products respective- ly.4 Sequencing the DNA samples: Ten blood DNA samples from ALS cases or normal control with HFE gene mutations were selected and the PCR products were send to Shanghai Biology Engineering Technology Corporation for sequencing.5 Statistical analysis was performed using SPSS 13.0 software package. Hardy-Weinberg analysis was performed by comparing the observed and expected genotype frequencies in each groups using Chi-square test.Between cases and controls, t test was used to examine the difference of age. The comparisons of genotype and allele frequency were performed by Chi-square test. The odds ratio(OR)and 95% confidence interval(CI) were calculated using an unconditional logistic regression model. P<0.05 was considered significant for all statistical analyses.Result:1 The distributions of HFE genotypes among healthy controls and SALS cases were compatible with those expected genotype frequencies from Hardy-Weinberg equilibrium, suggesting that the samples could represent groups.2 Results indicated that the C282Y mutation and the S65Cmutation may be absent in the population with the Han nationality in China. Twenty-four samples expressing H63D heterozygous mutation included sixteen SALS patients and eight normal people. There were no C282Y and S65C mutations. The frequencies of H63D heterozygous mutation of HFE gene in SALS patients and healthy controls were 12.90% and 3.81% respectively (P=0.02). The CG genotype significantly increased the risk of SALS(OR=3.74, 95%CI=1.55- 9.02).3 For HFE gene H63D genotypes, stratification analysis were performed by disease onset location, symptom duration and age at onset, no significant differences were found in genotype distribution (P=0.612, P=0.677, P=0.915, P=0.626 respectively).4 The loss of Mbo I site showed that there was H63D heterozygous mutation in the squenced ten samples. And the other ten samples were all CC homozygous.Conclusions:1 The risk of SALS significantly increases in subjects with HFE gene H63D genotype especially CG heterozygous mutation. Therefore H63D CG genotype may be a potential risk factor for SALS.2 No C282Y and S65C polymorphisms were found in the detected Chinese. The results indicate that the C282Y and S65C polymorphisms may be absent in the Population with the Han nationality in China. They may have no association with susceptibility to the SALS.3 In the SALS patients, HFE gene H63D genotype stratification analysis showed there were no significant differences in genotype distributions according to disease onset location, symptom duration, age at onset and gender.