ABSTRACT
Background Age-related macular degeneration (AMD) is the main cause of irreversible loss of central vision in old population.The incidence of AMD is increasing year by year,but the mechanism is not clearly understood.Objective This study was to investigate the association between genetic variants and the risk of AMD in Ningxia population.Methods This study was approved by Ethic Committee of Ningxia People's Hospital and complied with the Helsinki Declaration.Written informed consent was obtained from each subject.One hundred and fifty patients with AMD and 145 ethnicity-and gender-matched controls were recruited in Ningxia Eye Hospital from January 2012 to March 2013.All individuals underwent comprehensive eye examinations and genomic DNA was prepared from peripheral blood.The single nucleotide polymorphisms (SNPs) of 8 susceptibility loci in four candidate genes,including complement factor H (CFH),complement factor B (CFB),age-related maculopathy susceptibility 2 (ARMS2) and high temperature required factor A1 (HTRA1),were genotyped with Mass Array and MALDI-TOF technique by Sequenom platform.The distribution of genotype was tested for Hardy-Weinberge equilibrium (HWE).The differences of genotype distribution of allele and haplotype frequencies were compared between patients and controls using chi-squared test and the P value was significant at ＜ 0.006 level after correction of age,and the relationship of genotype distribution with AMD was evaluated by Logistic regression analysis.Measures of linkage disequilibrium (LD) was carried out by Haploview.Results All the genetypes met HWE.Seven SNPs were found to be different in the genotypic distributions and allele frequencies between patients and normal controls (all at P＜ 0.05),however,after Bonferroni correction,the differences of only four SNPs were significant between the patients and controls in the genotype and allele distributions,including the SNPs of rs10737680 and rs1410996 in CFH gene,the SNP of rs10490924 in ARMS2 gene and SNP of rs11200638 in HTRA1 gene.The allele distributions of rs800292 (Pallele =0.006,OR =1.643,95 ％ CI:1.155-2.336) in CFH and rs641153 (Pallele =0.002,OR =0.273,95 ％ CI:0.120-0.620) in CFB were significantly associated with AMD.In addition,five SNPs in CFH gene were consisted of two blocks after analysis by Haploview.In addition,five SNPs in CFH were consisted of two blocks after analysis by Haploview.The first one SNPs (including rs551397 and rs800292) and another one SNPs (including rs12124794,rs10737680) and rs1410996 were in strong linkage disequilibrium (D'=1.00).After 50 000 permutations,the GC and AT haplotypes of the first block and the AAC,TCT and ACT haplotypes in the second block were significantly different between AMD patients and controls (P =0.010,0.010,0.001,0.041 and 0.033,respectively).The allel T of rs641153 was a protective factor of AMD (P=0.002,OR =0.273,95％ CI:0.120-0.620).Conclusions The SNPs rs10737680 and rs1410996 in CFH,rs10490924 of ARMS2 gene and rs11200638 of HTRA1 gene are associated with AMD in Ningxia population.
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Background Researches determined that ELOVL4 gene is a disease-causing gene of Stargard tmacular dystrophy and is a elongation enzyme of very long chain fatty acids.Stargardt type Ⅲ(STGD3) may be associated with the metabolism of extensive enzyme of very long chain fatty acids.To explore the effect of ELOVL4 in STGD3 and its relationship with the metabolism of extensive enzyme of very long chain fatty acids is of important clinical significance.Objective This study was to determine the role of ELOVL4 gene for the pathogenesis of STGD3.Methods Recombinant adenovirus type 5 carrying mouse ELOVL4 gene and green fluorescent protein (GFP) was transfected into pheochromocytoma cells (PC12 cells),and then the cells were divided into PC12 group,PC12+Ad5-GFP group and PC12+AdS-ELOVL4 group.Ad-GFP or Ad-ELOVL4 was added into the culture medium for 24 hours with the virus plasmid 1 × 104-2× 104 pfu/ml.Expression of ELOVL4 mRNA in the PC12 was quantified by quantitative real time PCR(qRT-PCR) and was described as relative value to the expression of RPL19.ELOVL4 protein was assayed by Western blot.The transfected cells were treated with arachidonic acid (AA,20:4n6),eicosapentaenoic acid (EPA,20:5n3) and docosahexaenoic acid (DHA,22:6n3) individually for 48 hours.The cells were collected,and total lipids were extracted,and fatty acid methyl esters were prepared and analyzed by gas chromatography-mass spectrometry (GC-MS).Results The relative expression levels of ELOVL4 mRNA in PC12 cells in the PC12+Ad5-ELOVL4 group,PC12+Ad5-GFP group and PC12 group were 0.833± O.138,0.027t±0.002 and 0.024 ±0.002,with a significant difference among the 3 groups (F =102.700,P =0.000),and relative expression levels of PC12+Ad-ELOVL4 were 30-40 folds more than those in the PC12 group and the PC12+Ad-GFP group.Western blot assay showed a stronger response band for ELOVL4 protein in the PC12+Ad-ELOVL4 group.GC-MS found that abundant polyunsaturated fatty acids (C28-C38) were synthesized by PC12 cells in the PC12+Ad-ELOVL4 group,with the more levels in C34 and C36.Conclusions ELOVL4 can promote the synthesis of C28-C38 polyunsatured fatty acid in PC12 cells,which offers a novel clue for the treatment of STGD3.
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ObjectiveTo investigate the correlation between single nucleotide polymorphism (SNP)of complement factor H (CFH) gene and exudative age-related macular degeneration (AMD) susceptibility.MethodsThis is a retrospective case control study. 136 exudative AMD patients (AMD group) and 140age and sex-matched normal subjects (control group) were enrolled in this study. The peripheral blood was collected,polymorphism genotypes and frequency of CFH Y402H(rs1061170),CFH-257C ＞ T (rs3753394) and CFH IVS15 (rs1329428)were measured by polymerase chain reaction (PCR) and allelespecific restriction endonuclease digestion. The SHEsis software was performed on haplotype construction to analyze the frequency.ResultsThere are TT, TC, CC genotypes and T, C allele in CFH Y402H (rs1061170); CC, CT, TT genotypes and C, T allele in CFH-257C＞T (rs3753394); AA, AG, GG genotypes and A, G allele in CFH IVS15 (rs1329428). The differences of genotypes and allele frequency between 2 groups were statistically significant (P＜0. 05). The TC genotype in CFH Y402H, TT genotype in CFH-257C＞T (rs3753394) and GG genotype in CFH IVS15 (rs1329428) were associated with exudative AMD susceptibility (OR=4. 11,2. 55,3.11;P＜0.05). The T, C and G allele were the risk alleles (OR=3.14,1.72,1.79;P＜0. 05). The differences of frequency between TCG, CTG and CTA haplotype were statistically significant(X2 =10.53,6.60, 32.82;P＜0.05). ConclusionThere is correlation between SNPs of CFH gene and exudative AMD susceptibility.
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Objective To investigate the disease causing gene of Stargardt disease. Method Fifteen patients with Stargardt disease were analyzed with 11 primers of the 11 exons of ABCR gene by using PCR SSCP and DNA direct sequencing techniques. Results Three newly detected disease causing mutations were found. Among those mutations, one is a frameshift mutation and others are single base transition. Conclusion This research confirmed that ABCR gene is associated with Stargardt disease, and 3 new mutations of ABCR gene were found.