| Background: The morbidity of diabetes mellitus (DM) has been kept on increasing as the life level improved and lifestyle changed those years. WHO estimated that there was about 140,000,000 diabetic patients in the world in 1998. By the year 2025, there will be 300 millions. The increment will be mainly in Asia. Now there are 20,000,000 DM patients in China, by year 2025, there will be 50,000,000. And diabetic nephropathy (DN) is one of the main complications that occurs in 30-40% of diabetic patients. Virtually all cases of nephropathy ultimately progress to end-stage renal disease, That means that'll be 20,000,000 DM patients with DN in 2025. The medical expences will be tremendiously heavy to the government. So that how to prevent DN become a impediouly task. Recent researches indicated that the development of diabetic nephropathy shows remarkable variation among individuals and ethnics. And DN occurs in familial clusters and ethnic racial groups. All that suggested that genetic factors might be involved in the development of DN.One of the mediators implicated in the development of DN is nitric oxide (NO) system . Nitric oxide acts as a neuronal and vascular messenger that activates multiple intracellular pathways, including inhibition of platelet aggregation and adhesion, and tubuloglomerular feedback in the kidney. Nitric oxide synthases (NOS) catalyze the conversion of arginine to NO. Nitric oxidesynthase exists in three main forms: endothelial nitric oxide synthase (eNOS), neural nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). Nitric oxide is synthesized on demand for short periods by eNOS or nNOS. In contrast, iNOS gene is expressed only after cell activation which then produces a relatively large amount of NO for comparatively long periods of time.The complex promoter of human NOS gene contains multiple overlapping open reading frames, and it has been suggested that this region may have an important role in translational regulation of human NOS mRNA. Polymorphism in this region could potentially affect NOS expression. Four kinds of polymorphism have been identified in iNOS gene. Two of them concern variation in repeated sequences and each is located in the 5'-flanking region of iNOS. Both of them have been thought to play a role in the development of chronic diabetic nephropathy in Caucasian/European diabetes patients. One is in an AAAT repeat at -756 to -716 relative to the major transcription start site (+1) and involves an insertion or deletion of 1 repeat unit. The other, located 2.7 to 2.5 kb upstream, consists of CCTTT pentanucleotide repeat, is of possible relevance to hypertension and diabetic nephropathy. The third and fourth polymorphisms, haven't been reported to have any association with diabetes, variants (-969G→C and -1173C→T)) have been found to be linked with malaria infection in Africans.eNOS is encoded by a gene located on chromosome 7q35-36, comprising 26 exons that span 21kb. Polymorphisms in eNOS reduced the promoter activity and eNOS transcriptions significantly. Recently, three kinds of polymorphism in eNOS gene have been found to be linked with DN and several sequence differences were identified in this region. One is a 27bp repeat polymorphism in intron 4; the other is T-768→C single nucleotide mutation in promoter region. In persons with C allele, promoter activity is less than half of that seen in those who possess T allele. The third is a (CA)n polymorphism in intron 13 of eNOS gene.Currently, there is no available information for iNOS and eNOS polymorphisms in different ethnic T2DM patients residing in Southeast Asia.Objectivy: The aim of this study was to assess the polymorphisms in iNOS and eNOS in different ethnic groups and identify their associations with diabetic nephropathy.Methods: 258 type 2 diabetes patients (150 Chinese, 71 Malay, and 37 Indian) were randomly selected from both inpatients and outpatients of National University Hospital, age 27—80. Two kinds of iNOS and three kinds of eNOS polymorphisms were identified by PCR, cloning, microsatellite and/or nucleotide sequencing. The criteria of diagnozing clinical diabetic nephropathy is the appearance of albumin in the urine(>30mg/d or 20ug/min).Genotype: DNA was extracted from whole blood. Genotypes for the AAAT polymorphism were determined by polymerase chain reaction (PCR) with the following primers: sense 5'-TGG TGC ATG CCT GTA GTC C-3'; anti-sense 5'-CAG GCC TCT GAG ATG TTG GTC-31. The former was labeled with FAM during synthesis by Genset (QIAGEN Gmbh,Germany). The PCR mix (100 uL) consisted of 200 ng template DNA, 200ng per primer, 200 uM of each dNTP, 2.5 U Taq DNA polymerase ( QIAGEN PCR Kit, Germany). After initial denaturation at 94°C for 5 minutes, there were 10 cycles of 94°C, 65°C, and 72°C for 1 minute each, followed by 15 cycles of 94°C, 60°C, and 72°C for 1 minute each, and finally 20 cycles of 94°C, 58°C, and 72°C for 1 minute each, finishing with a step at 72°C for 2 minutes.For the CCTTT multiallelic marker, PCR primer sequences were: forward: 5'-CCT TCT CAC AGA TTG CCA CCC CTG G-31 (FAM-labeled); reverse: 5'-AGA GCC TCC AGA GAG CTA TGG TCG C-31. The final PCR mix and PCR cycles were the same as above.For the (CA)n multiallelic marker, PCR primer sequences were: forward : 5'-CGG AGA ATG GAG AGG TGA GAA C-31; reverse : 5'-GGG ACG CGC AGT TAA GCA AAT G-31. The former was labeled with FAM during synthesis by Genset (QIAGEN Gmbh,Germany). The final PCR mix and PCR cycles were the same as above.For T-786—>C mutation, PCR primer sequences were: forward: 5'-GCA TGC ACT CTG GCC TGA AGT G-31; reverse: 5'-CAG GAA GCT GCC TTCCAG TGC-3'. The PCR products were incubated with Mspl for 2 hours, after which the restricted products were separated by electrophoresis on 2% agarose gel and visualized by ethidium bromide staining with ultraviolet transillumination. The recognition sequence of Mspl is 5' C^CGG 3'. There is one recognition site in the T allele of 222bp PCR fragment that will be recognized by Mspl and be digested into two fragments (161 bp and 61bp). The mutation of T-786—>C creates another recognition site and the 161 bp fragment will be further digested into 116bp and 45bp small fragments. Cloning: The products from PCR reactions were first purified with Qiagen PCR Purification kit, then, were cloned into pCR?II-TOPO? using the TOPO TA Cloning? kit (Invitrogen, Netherlands) according to the manufacturer's instructions. DNA from randomly selected positive colonies were amplified, purified and subsequently sequenced using the ABI 377 automated sequencer with fluorescent dye terminators.All PCR products were electrophoresed on an ABI 377 automated sequencer (Applied Biosystems), and genotypes were assigned with ABI Genotyper software.Statistical Analysis: Variations of both genotype frequency and allele frequency among ethnic groups were tested with Chi-square test (SPSS 11.5, USA). A binary logistic model was used to detect the associations of gene polymorphisms with DN. Age and gender were adjusted in this model. The significance was considered when P<0.05.ResultsAAAT polymorphism in iNOS— there were significant differences among different ethnic groupsAll published studies identified that AAAT polymorphism was biallelic ((AAAT)3 and (AAAT)4 alleles). Our study found that it was triallelic, there were three kinds of alleles, (AAAT)4, (AAAT)5 and (AAAT)6. Our results showed: 1) No (AAAT)3 has been found; 2) Allele frequency of (AAAT)4 was 0.984, much higher than that Morris et al has reported (0.2); 3) Allele frequency of (AAAT)5 was 0.014 (Morris haven't reported any case with 5 repeats); 4). Anew allele with 6 AAAT repeats was first reported. The (AAAT)5 allele displayed significant differences in allele frequency among different ethnic groups (p=0.03), but the allele distribution failed to display any association with DN.CCTTT polymorphism in iNOS—there were significant differences in overall allele distribution among Singaporean, Caucasian and African and lack of association with diabetic nephropathy in Singaporean patientsTotally ten alleles were identified, nine alleles ranging from 8 to 14 and 16 to 17 (A8-A14, A16, A17) in Chinese, 7 from A9 -A15 in Indian and 6 from A10-A14 and A16 in Malay. There are significant differences in allele distribution between Asian(C, M, I) and Caucasian or African population (p=0.001 Asian vs Caucasian; P=0.00 Asian vs African). We can see from the bar chart, there are two peaks in allele distribution in African, and the most common allele was (CCTTT)13, while for Caucasian and Chinese, there were only one peak, the most common allele was (CCTTT) 12. No significant difference in overall allele distribution was observed among different ethnic groups in Asia patients (p=o.625) and between groups, with versus without nephropathy (p=0.065, Fig.3). Since (CCTTT) 14 allele (A 14) has been reported to be associated with lower risk of diabetic nephropathy and retinopathy in type 1 diabetes; (CCTTT)12 (A12) and (CCTTT)13 (A13) accounted for half of the alleles percentile. We compared these particular alleles. The most common allele, A12, was significantly different in allele frequency in Malay (24%) comparing with Chinese (61.4%; p=0.003), but lack of association with DN (p=0.66). The second most common allele was A13, had a significantly higher allele frequency in Indian T2DM with DN (58.6%) comparing with Malay group (32.6%, p=0.048; Fisher's exact test p=0.024). Six patients got A14, 5 with DN as complication, among them, 2 were Chinese, 3 Indian and 1 Malay. The frequency of A14 allele was 0.012. A14 carrier displayed no significant difference comparing these with versus without DN group (p=0.14). The overall homozygosity was 8(3.1%) in 258 patients, 2 were Chinese, 1 was Indian and 5 were Malays. The Malay T2DM had the highest homozygosity (1.9%).27bp repeat polymorphism in eNOSTotally four kinds of genotype (b/b, a/b, a/a and b/c) and three types of allele (a, b and c) were identified, whereas all previous studies reported that there were only three kinds of genotype (b/b, a/b, a/a) and two kinds of allele-insertion allele (with five 27bp-repeat, b allele) and deletion (with four 27bp-repeat, a allele). Our study found there were three kinds of alleles in this polymorphism. We identified the third alleles, allele c, had six 27bp-repeat. Totally 3 patients were found to have six 27bp repeats, two were Chinese, one was Malay. Though all the three patients with c allele and four homozygotes of a/a have got DN as complications, c allele and a/a genotype didn't display any association with DN. The overall genotype and allele frequencies were not significantly different between patients with and without nephropathy (p=0.151) and among different ethnic groups (p=0.691). the frequency of a/a, b/a, b/b and b/c were 1.6%, 16.3%, 81% and 1.1% while it were 3.0%, 26.8%, 70.2% and 0.0% in Japanese. 42 patients with a/b genotype, 23 Chinese, 11 Indian, 8 Malay; The allele frequency of the most common allele—b (5 repeats) is 0.897 in Chinese; 0.937 in Malay; and 0.82 in Indian, similar to that in Australia(0.88) and Janpane(0.86 ).Neugebauer S et al demostrated that a allele was the risk factor for DN. However, our finding failed to identify any association of allele frequencies with DN (p=0.151), which was not in consistent with Neugebauer's study but tallied with Fujita H et al's .T—>C single nucleotide mutationThe frequency of C/C, T/C and T/T was 0.0%, 16.7% and 83.3% respectively, while it was 0%, 28.6% and 71.4% in DN patients in Japan . Our finding showed:1. The overall allele distribution failed to show significant differences among different ethnic groups (p=0.42)2. No association was identified between allele frequencies and DN (p=0.175 for overall; p=0.79 within Chinese; p=0.66 within Malay; p=0.41 within Indian).3. The overall C allele frequency was 0.083, was 0.047 in groups with DN, 0.037 in groups without DN (were 0.17, 0.027and 0.092 respectively in USA T1DM, 1996)4. No C/C homozygote was found5. There was significant difference of the c allele frequency between Singaporean and Japanese (p=0.00) or American (p=0.00)(CA)n repeat polymorphismTotally 25 alleles was identified in Singapore type 2 diabetes, 23 alleles among Chinese, 22 alleles among Malay and 20 alleles among Indian. The most common allele was (CA)25 in Chinese, was (CA)22 in Malay and (CA)27 in Indian. There were significant differences in allele distribution among different ethnic groups in Singapore (p=0.00) and between Singaporean and Caucasian hypertension patients (p= 0.00). No significant difference of the allele distribution between groups with and without DN (p=0.117). Zancih A et al (2000) reported (this is the only paper about CA polym and DM) that (CA)n polym was not associated with DN.Cinclusion:The linkage of 27bp repeat and T—>C mutationAsakimori et al [11] reported that T'786—>C mutation showed strong linkage disequilibrium with 27bp repeat polymorphism in intron 4. Our study confirmed their finding. T allele in T" —>C mutation had stronge linkage with b allele of 27bp repeat polymorphism.Conclusion:Our research on iNOS gene showed:1. AAAT polymorphism was triallelic, (AAAT)4, (AAAT)5 and (AAAT)6 in our research. (AAAT)3 wasn't identified in Singaporean participants. (AAAT)5, (AAAT)6 haven't been reported in Caucasian. (AAAT)5 allele frequency showed significant differences among different ethnic groups. We first reported a new allele sequence—(AAAT)6.2. Study on CCTTT polymorphism demonstrated that there were the significant differences in the overall allele distribution between Asian(C, M, I)...
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