LADA与CTLA-4、VDR、MIC-A基因多态性的相关性研究
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摘要
【目的】
探讨细胞毒性T淋巴细胞相关抗原4基因外显子1的49位点、维生素D受体基因BsmⅠ酶切位点、MHC相关基因A外显子5多态性与深圳市成人迟发性自身免疫性糖尿病的关系。
【方法】
糖尿病诊断和分型标准按1997年ADA标准。在深圳市第二人民医院门诊、综合科病房、内分泌科病房随机选取LADA患者及2型糖尿病,分为LADA组及2型糖尿病组,并随机从深圳市第二人民医院体检中心选取健康体检人员作为对照组。按标准确定LADA患者、2型糖尿病和对照人员。空腹血糖、餐后2h血糖、空腹胰岛素、CP及餐后2h胰岛素、CP水平测定分别由深圳市第二人民医院检验科和放免室完成用购自加拿大Beyondbio公司试剂盒测定LADA患者、2型糖尿病和对照人员GADAb、ICA。基因组DNA制备用购至美国OMEGA公司的试剂盒提取。查找Gene bank后设计引物序列,应用PCR方法扩增CTLA-4、VDR、MIC-A目的基因,然后用2%琼脂糖凝胶电泳,溴化乙锭染色,紫外灯下观察PCR扩增结果并拍照。用BbvI内切酶、Bsm I内切酶特异地结合于CTLA-4、VDR目的基因的DNA序列之内,并切割双链DNA,然后用2%琼脂糖凝胶电泳,溴化乙锭染色,紫外灯下观察酶切结果并拍照。MIC-A外显子5基因PCR产物加变性上样缓冲液,95℃变性,8%
变性聚丙烯酰胺凝胶电泳, 0.2%硝酸银染色,紫外灯下观察结果并拍照。
【结果】
成功的从血液中提取基因组DNA,扩增CTLA-4、VDR、MIC-A目的基因及用BbvI内切酶、Bsm I内切酶特异地酶切CTLA-4、VDR目的基因。MIC-A变性聚丙烯酰胺凝胶电泳,硝酸银染色。LADA患者CTLA-4基因外显子1G49等位基因频率显著高于正常对照组(P<0.01)和T2DM组(P<0.01),主要表现为基因型GG显著高于正常对照组(P=1.3×10~(-2))和T2DM组(P=3.4×10~(-2) ),基因型AG也明显高于正常对照组(P<0.01 )和T2DM组(P=3.3×10~(-2)),基因型AA显著降低正常对照组(P<0.01 )和T2DM组(P<0.01 )。T2DM患者VDR基因B等位基因频率明显高于正常对照组(P=2.7×10~(-2)),基因型Bb明显高于正常对照组(P=1.6×10~(-2) ),基因型bb明显低于正常对照组(P=1.6×10~(-2));LADA患者Bb基因型频率以及B等位基因频率与对照组比较差异无统计学意义(P>0.05)。LADA患者A5.1等位基因频率显著高于正常对照组(P<0.05);2型糖尿病MICA等位基因频率与正常组比较差异无统计学意义(P>0.05)。
【结论】
CTLA-4基因A/G49多态性、MICA5.1等位基因与深圳市LADA易感性相关。VDR基因BsmⅠ位点多态性与LADA易感性可能无关,但与T2DM的易感性密切相关。
【Objective】
To investigate the association of cytotoxic T lymphocyte-associated antigen 4 gene、vitamin D receptor gene and MICA gene exon 5 polymorphism with latent autoimmune diabetes in adults.
【Methods】
The diagnosis and classification for diabetes mellitus was referred to diagnosis standard in ADA. Patients with LADA and type 2 diabetes in clinics, the ward of general department, the Department of Endocrinology of Shenzhen Second People's Hospital were randomly selected and were divided into LADA group and type 2 diabetes group. Health Physical Examination personnel in Medical Examination Center of Hospital were randomly selected and were classified as control group.LADA patients、type 2 diabetes and control personnel were classified according to standards. Fasting blood glucose、insulin、CP and blood sugar、insulin、CP 2 hours after meals were chemical examined by Laboratory Department and the Department of Nuclear Medicine. GADAb, ICA of LADA patients、type 2 diabetes and control personnel were determined by the kit purchased from the Beyondbio company of Canada. DNA was extracted by the kit purchased from the OMEGA company of United States.Primers were designed after searching over the gene bank. The coding region of the gene of CTLA-4、VDR、MIC-A was amplified by PCR. Amplified PCR products were evaluated by 2% agarose gel electrophoresis and ethidium bromide staining,were observed and took photographs with ultra-violet transmission analysator. The DNA of CTLA-4、VDR was identified with restriction enzyme digestion. Digested products were evaluated by 2% agarose gel electrophoresis and ethidium bromide staining,were observed and took photographs with ultra-violet transmission analysator. Amplified PCR products of MIC-A gene were analyzed by denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining,were observed and took photographs with ultra-violet transmission analysator.
【Results】
DNA was successfully extracted from blood. The coding region of the gene of CTLA-4、VDR、MIC-A was amplified by PCR. The DNA of CTLA-4、VDR was identified with restriction enzyme digestion. Digested products were evaluated by 2% agarose gel electrophoresis and ethidium bromide staining. Amplified PCR products of MIC-A gene were analyzed by denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining.A highly significant increase in the frequency of the G allele was seen in patients with LADA compared with controls(P<0.01).This reflected an increase in the GG genotype in patients(P=1.3×10~(-2))and a significant decrease in the AA genotype(P<0.01).The frequency of the G allele was significantly higher in patients with LADA than in type 2 diabetes patients (P<0.01).This reflected an increase in the GG genotype in patients(P=3.4×10~(-2) )and a significant decrease in the AA genotype(P<0.01 ).A highly significant increase in the frequency of the B allele was seen in T2DM patients compared with controls(P=2.7×10~(-2)).This reflected an increase in the Bb genotype in patients(P=1.6×10~(-2))and a significant decrease in the bb genotype(P=1.6×10~(-2)).The Bb or bb frequency was not significantly higher in patients with LADA than in the control group(P>0.05),and so was the B allele.A highly significant increase in the frequency of the A5.1 allele was seen in patients with LADA compared with controls(P<0.05).The A5.1 allele frequency was not significantly higher in patients with T2DM than in the control group(P>0.05)
【Conclusion】
The polymorphism of MICA and CTLA-4 gene A/G49 confers susceptibility to LADA. The study shows that the vitamin D receptor BsmⅠpolymorphism may be not related to LADA,but may be tightly related to T2DM.
探讨细胞毒性T淋巴细胞相关抗原4基因外显子1的49位点、维生素D受体基因BsmⅠ酶切位点、MHC相关基因A外显子5多态性与深圳市成人迟发性自身免疫性糖尿病的关系。
【方法】
糖尿病诊断和分型标准按1997年ADA标准。在深圳市第二人民医院门诊、综合科病房、内分泌科病房随机选取LADA患者及2型糖尿病,分为LADA组及2型糖尿病组,并随机从深圳市第二人民医院体检中心选取健康体检人员作为对照组。按标准确定LADA患者、2型糖尿病和对照人员。空腹血糖、餐后2h血糖、空腹胰岛素、CP及餐后2h胰岛素、CP水平测定分别由深圳市第二人民医院检验科和放免室完成用购自加拿大Beyondbio公司试剂盒测定LADA患者、2型糖尿病和对照人员GADAb、ICA。基因组DNA制备用购至美国OMEGA公司的试剂盒提取。查找Gene bank后设计引物序列,应用PCR方法扩增CTLA-4、VDR、MIC-A目的基因,然后用2%琼脂糖凝胶电泳,溴化乙锭染色,紫外灯下观察PCR扩增结果并拍照。用BbvI内切酶、Bsm I内切酶特异地结合于CTLA-4、VDR目的基因的DNA序列之内,并切割双链DNA,然后用2%琼脂糖凝胶电泳,溴化乙锭染色,紫外灯下观察酶切结果并拍照。MIC-A外显子5基因PCR产物加变性上样缓冲液,95℃变性,8%
变性聚丙烯酰胺凝胶电泳, 0.2%硝酸银染色,紫外灯下观察结果并拍照。
【结果】
成功的从血液中提取基因组DNA,扩增CTLA-4、VDR、MIC-A目的基因及用BbvI内切酶、Bsm I内切酶特异地酶切CTLA-4、VDR目的基因。MIC-A变性聚丙烯酰胺凝胶电泳,硝酸银染色。LADA患者CTLA-4基因外显子1G49等位基因频率显著高于正常对照组(P<0.01)和T2DM组(P<0.01),主要表现为基因型GG显著高于正常对照组(P=1.3×10~(-2))和T2DM组(P=3.4×10~(-2) ),基因型AG也明显高于正常对照组(P<0.01 )和T2DM组(P=3.3×10~(-2)),基因型AA显著降低正常对照组(P<0.01 )和T2DM组(P<0.01 )。T2DM患者VDR基因B等位基因频率明显高于正常对照组(P=2.7×10~(-2)),基因型Bb明显高于正常对照组(P=1.6×10~(-2) ),基因型bb明显低于正常对照组(P=1.6×10~(-2));LADA患者Bb基因型频率以及B等位基因频率与对照组比较差异无统计学意义(P>0.05)。LADA患者A5.1等位基因频率显著高于正常对照组(P<0.05);2型糖尿病MICA等位基因频率与正常组比较差异无统计学意义(P>0.05)。
【结论】
CTLA-4基因A/G49多态性、MICA5.1等位基因与深圳市LADA易感性相关。VDR基因BsmⅠ位点多态性与LADA易感性可能无关,但与T2DM的易感性密切相关。
【Objective】
To investigate the association of cytotoxic T lymphocyte-associated antigen 4 gene、vitamin D receptor gene and MICA gene exon 5 polymorphism with latent autoimmune diabetes in adults.
【Methods】
The diagnosis and classification for diabetes mellitus was referred to diagnosis standard in ADA. Patients with LADA and type 2 diabetes in clinics, the ward of general department, the Department of Endocrinology of Shenzhen Second People's Hospital were randomly selected and were divided into LADA group and type 2 diabetes group. Health Physical Examination personnel in Medical Examination Center of Hospital were randomly selected and were classified as control group.LADA patients、type 2 diabetes and control personnel were classified according to standards. Fasting blood glucose、insulin、CP and blood sugar、insulin、CP 2 hours after meals were chemical examined by Laboratory Department and the Department of Nuclear Medicine. GADAb, ICA of LADA patients、type 2 diabetes and control personnel were determined by the kit purchased from the Beyondbio company of Canada. DNA was extracted by the kit purchased from the OMEGA company of United States.Primers were designed after searching over the gene bank. The coding region of the gene of CTLA-4、VDR、MIC-A was amplified by PCR. Amplified PCR products were evaluated by 2% agarose gel electrophoresis and ethidium bromide staining,were observed and took photographs with ultra-violet transmission analysator. The DNA of CTLA-4、VDR was identified with restriction enzyme digestion. Digested products were evaluated by 2% agarose gel electrophoresis and ethidium bromide staining,were observed and took photographs with ultra-violet transmission analysator. Amplified PCR products of MIC-A gene were analyzed by denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining,were observed and took photographs with ultra-violet transmission analysator.
【Results】
DNA was successfully extracted from blood. The coding region of the gene of CTLA-4、VDR、MIC-A was amplified by PCR. The DNA of CTLA-4、VDR was identified with restriction enzyme digestion. Digested products were evaluated by 2% agarose gel electrophoresis and ethidium bromide staining. Amplified PCR products of MIC-A gene were analyzed by denaturing polyacrylamide gel electrophoresis(PAGE) and silver staining.A highly significant increase in the frequency of the G allele was seen in patients with LADA compared with controls(P<0.01).This reflected an increase in the GG genotype in patients(P=1.3×10~(-2))and a significant decrease in the AA genotype(P<0.01).The frequency of the G allele was significantly higher in patients with LADA than in type 2 diabetes patients (P<0.01).This reflected an increase in the GG genotype in patients(P=3.4×10~(-2) )and a significant decrease in the AA genotype(P<0.01 ).A highly significant increase in the frequency of the B allele was seen in T2DM patients compared with controls(P=2.7×10~(-2)).This reflected an increase in the Bb genotype in patients(P=1.6×10~(-2))and a significant decrease in the bb genotype(P=1.6×10~(-2)).The Bb or bb frequency was not significantly higher in patients with LADA than in the control group(P>0.05),and so was the B allele.A highly significant increase in the frequency of the A5.1 allele was seen in patients with LADA compared with controls(P<0.05).The A5.1 allele frequency was not significantly higher in patients with T2DM than in the control group(P>0.05)
【Conclusion】
The polymorphism of MICA and CTLA-4 gene A/G49 confers susceptibility to LADA. The study shows that the vitamin D receptor BsmⅠpolymorphism may be not related to LADA,but may be tightly related to T2DM.
引文
[1] Wenying Yang,Juming Lu,Jianping Weng,et al. Prevalence of Diabetes among Men and Women in China [J].The New England Journal Of Medicine, 2010, 362: 1090- 1101.
[2] Lohmann T,Kellner K,Verlohren H J,et al.Titre and combination of ICA and artoantibodies to glutamic acid derarboxylase discriminate two clinically distinet types of latent autoimmune diabetes in adults(LADA)[J]. Diabetologia, 2001, 44(8):1005- 1010.
[3] Isomaa B, Almgren P, Henricsson M, et al. Chronic comp lications in patients with slowly progressing autoimmune type 1 diabetes (LADA) .Diabetes Care, 1999, 22: 134721353.
[4] Gottsater A, Ahmed M, Lilja B, et al. Islet cell antibodies at diagnosis, but not leanness, relate to a better cardiovascular risk factor profile 5 years after diagnosis ofN IDDM. Diabetes Care, 1996, 19: 602-63.
[5] Tuomi T, Carlsson A, Li H, et al. Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies. Diabetes, 1999,48: 1502157.
[6] Kobayashi T, Nakanishi K, Nurase T, et al. Small doses of subcutaneous insulin as a strategy for preventing slowly p rogressiveβcell failure in islet cell antibody-positive patients with clinical features of NIDDM. Diabetes, 1996, 45: 622-626.
[7] Beales PE, Giorgini A, Annovazzi A, et al. Metformin does not alter diabetes incidence in the NOD mouse. Horm Metab Res, 1997, 29:261-263.
[8] Kobayashi T , Maruyama T , Shimada A , et al . Insulin intervention to preserve beta cells in slowly progressive insulin2depecdent ( type 1) diabetes mellitus. Ann N YAcad Sci , 2002 , 958:117-130
[9] Beales PE, Pozzilli P. Thiazolidinediones for the prevention of diabetes in the non-obese diabetic (NOD) mouse: imp lications for human type 1 diabetes. DiabetesMetab Res Rev, 2002, 18: 114-117.
[10]李霞,周智广,黄干,等.罗格列酮对低滴度谷氨酸脱羧酶抗体的LADA患者胰岛β细胞功能的影响.中华内分泌代谢杂志, 2004,20: 564-567.
[11] Zhou Z, Li X, Huang G, et al. Rosiglitazone combined with insulin preserves islet beta cell function in adult2onset latent autoimmune diabetes (LADA) . DiabetesMetab Res Rev, 2005, 21: 203-208.
[12] Kobayashi T, Maruyama T, Shimada A, et al. Insulin intervention to preserveβcells in slowly progressive insulin dependent ( type 1 ) diabetes mellitus. Ann NY Acad Sci, 2002, 958:117-130.
[13] Todd M,Brusko,CliveH,etal.Functtionnal Defects and the Influence of Age on the Frequency of CD4+CD25+ T cells in Type l Diabetes[J].Diabetes,2005,54(2):1407- 1414.
[14] Nora Hosszufalusi,Agnes Vatay,Katalin Rajezy,et al.Similar genetic features and different islet cell autoantibody patten of Latent autoimmune diabetes in adults (LADA) compared with adult-onset tpye l diabctes with rapid progression[J].Diabdes Care,2003,26(3):452-459.
[15] Liang H,Yagi K,Asano A,et al.Assoeiation between CTLA-4 49A/G polytnorphism and type1B diabetes in Japanese population[J].Endocrine,2004,25(2):105-109.
[16] Caputo M,Cerrone GE,López Ap,et al.Cytotoxie T lymphoeyte antigen 4 heterozygous codon 49 A/G dimorphism is associated to latent antoimmune diabetes in adults(LADA)[J].Autoinunumty2005,38(4):277-281.
[17] Nistico L, Buzzetn R, Pntchard L E, et al. The CTLA-4 gene region of chromosome 2q33 is linked to and associated with type 1 diabetes. Hum Mol Genet, 1996, 5:1075.
[18] Donner H, Rau H, Walfish P G, et al. CTLA-4 alanine-17 confers genetic susceptibility to Graves’disease and to type 1 diabetes mellitus[ J ]. J Clin Endocrinol Metab, 1997, 82:143-146.
[19] Dipartimento di, Medicina Interma, Universitàdegli, et al. Immunologic and genetic aspects of latent autoimmune diabetes in the adult[J]. Minerva Endocrinol, 2003, 28(4): 297-312.
[20] Douroudis K,Prans E,Kisand K,et al. Cytotoxic T-lymphocyte antigen 4 gene polymorphisms are associated with latent autoimmune diabetes in adults.ClinicaChimica Acta,2009,403(1-2):226-228.
[21] Haller K,Kisand K,Pisarev H,et al.Insulin gene VNTR, CTLA-4 +49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group.Tissue Antigens ,2007,69(2):121-127.
[22] Cosentino A,Gambelunghe G,Tortoioli C,et a1.CTLA-4 gene polymorphism contributes to the genetic risk for latent autoimmune diabetes in adults.Ann N Y Acad Sci,2002,958:337-340.
[23] Yang ZF.Li XJ.CTLA-4 gene polymorphism is not associated with latent autoimmtme diabetes of the adults in Chinese population.Diabetes,2000,49(Suppl 1):A403.
[24] Douroudis K, Prans E, Uibo R.CTLA-4 promoter polymorphisms are associated with latent autoimmune diabetes in adults.Hum Immunol,2009 ,70(11):921-924.
[25] BAN Y, TANIYAMA M, YANAGAWA T, et al. Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population [J]. BMC Med Genet, 2001, 2(1):7-18.
[26] WANG SG, LIU JH, HU SQ, et al. Association of vitamin receptor genepolymorphisms with idiopathic hypercalciaria[J]. China Journal of Modern Medicine, 2003,13(4):4-7.
[27] CHIU K C, CHUANG L M, YOON C. The vitamin D receptor polymorphism in the translation initiation codon is a risk factor for insulin resistance in glucose tolerant Caucasians[J]. BMC Med Genet, 2001,2:2-11.
[28] Motohoshi T, Yamada S, Yanagawa T, et a1. Vitamin D receptor gene polymorphism affects onset pattern of type l diabetes[J].J Clin Endocrinol Metab, 2003. 88: 3137-3140.
[20] Chang T J,Lei H H, Yeh J I, et al. Vitamin D receptor gene polymorphism influence susceptibility to type 1 diabetes mellitus in the Taiwanese population[J]. Clin Endocrenol(Oxf),2000,52:575-580.
[30] McDermott M F,Ramachandran A Ogunkolade BW,et al. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians[J].Diabetologia 1997,40:971-975.
[31] Speer G,Cseh K,Winkler G,Vargha P,et.al.Vitamin D and estrogen receptor gene polymorphisms in type 2 diabetes mellitus and in android type obesity.Eur J Endoerinol.2001 APr;144(4):385-9.
[32] Niskanen LK,Tuomi T,Karjalainen J,etal.GAD antibodies in NIDDM.Ten-year follow-up from the diagnosis.Diabetes Care.1995,18(12):1557-65.
[33] Falorni A,Gambelunghe G,Forini F,etal.Autoantibody recognition of COOH-terminal epitopes of GAD65 marks the risk for insulin requirement in adult-onset diabetes mellitus.J Clin Endoerinol Metab.2000 Jan:85(l):309-16.
[34] Sanjeevi CB,Gambelunghe G,Falorni A,etal.Geneties of latent autoimmune diabetes in adlults.Ann NY Acad Sci.2002,958:107-111
[35] F Pociot,M F McDermott.Genetics of type 1diabetes mellitus.Genes and Inununity2002,3(5):235-249
[36] Torn C,Gupta M,Nikitina Zake L,etal.Heterozygosity for MICA5.0/MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent genetic risk factors for latent autoimmune diabetes in adults.Hum Immunol.2003,64(9):902-9.
[37] Borissova AM, Tankova T, Kirilov G, et a1. The effect of vitamin D3 on insulin secretion and peripheral insulin sensitivity in type 2 diabetic patients.Int J Clin Pract 2003; 57(4): 258-261
[38] Mimura T, Funatsu H, Uchigata Y, et al. Relationship between human leukocyte antigen status and proliferative diabetic retinopathy in patients with younger-onset type 1 diabetes mellitus. AmJ Ophthalmol,2003,135:844-848.
[39] Kawabata Y, Ikegami H, Kawaguchi Y,et al. Asian-specific HLA haplotypes reveal heterogeneity of contribution of HLA-DR and–DQ haplotypes to usceptibility to type 1 diabetes. Diabetes, 2002, 51:545-551.
[40]李玉钟,卢纹凯,高颖,等. HLA-DR-DQ连锁基因单倍体与成人缓慢进展型和速发型1型糖尿病的相关性研究. Chin J Endocrinol Metab, 1998,14(5): 302-304.
[41]肖建中,杨文英,刘娟,等.人类白细胞相关抗原HLADQB1基因与IDDM的关联[ J ].中华内分泌代谢杂志,1997,13 (1):7-10.
[1] Lohmann T,Kellner K,Verlohren H J,et al.Titre and combination of ICA and artoantibodies to glutamic acid derarboxylase discriminate two clinically distinet types of latent autoimmune diabetes in adults (LADA)[J].Diabetologia,2001, 44(8): 1005-1010.
[2] TuomiT,Groop LC,ZimmetP Z,et al.Antibodies to glutamic acid decarboxylaser eveal latent autoimmune diabetes mellitus in adults with a non-insulin dependent onset of disease.Diabetes.1993,42:359-362
[3] Landin-OlssonM.Latent autoimmune diabetes in adults.2002,Ann N Y Acad Sci.2002,958:112-116.
[4] Tuomi T,Carlsson A,Li H,etal.Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies.Diabetes.1999 Jan;4 8(1):150-7.
[5] Falomi A,Calcinaro F.Autoantibody profile and epitope mapping in latent autoimmune diabetes in adults.2002,Ann N Y Acad Sci.2002,958:99-106
[6] Niskanen LK,TuomiT,Karjalainen J,etal.GAD antibodies in NIDDM. Ten-year follow-up from the diagnosis.Diabetes Care.1995,18(12):1557-65
[7] Landin-OlssonM.Latent autoimmune diabetes in adults.Ann N Y Acad Sci 2002 Apr;958:112-6
[8] Tunrer R,StrattonI,Horton Vetal.UKPDS 25:autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for perdiction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group.Lancet.1997 Nov 1;350(9087):1288-93.
[9] Kanungo A,S anjeevi CB.IA-2 autoantibodies are predominant in latent Autoimmune diabetes in adults patients from eastern India. Ann N Y Acad Sci.2003 Nov;1005:390-4.
[10] Sanjeevi CB,Balaji M,Balaji Vetal.Autoantibodies to GAD65 and IA-2 antibodies are increased,but not tissue transglutaminase(TTG-Ab)in type 2 diabetes mellitus(T2DM) patients from South India.Ann NY Acad Sci.2003 Nov;1005:387-9.
[11] Kawasaki E,Abiru N,Ide A,Epitope analysis of GAD65 autoantibodies in Japanese patients with autoimmune diabetes.Ann NY Acad Sci.2003 Nov;1005:440-8.
[12] Lohmann T,Kellner K,Verlohren HJ,etal.Titre and combination of ICA and autoantibodies to glutamic acid decarboxylase discriminate two clinically distinct types of latent autoimmune diabetes inadults(LADA).Diabetologia.2001 Aug;44(8): 1005-10.
[13] L.Monetini,M.GCavallo,S.Manfrini,et al.Antibodies to bovine beta-casein in diabetes and other autoimmune diseases.Horm Metab Res 2002, 34:455-459
[14] Mimura T, Funatsu H, Uchigata Y, et al. Relationship between human leukocyte antigen status and proliferative diabetic retinopathy in patients with younger-onset type 1 diabetes mellitus. AmJ Ophthalmol,2003,135:844-848.
[15] Kawabata Y, Ikegami H, Kawaguchi Y,et al. Asian-specific HLA haplotypes reveal heterogeneity of contribution of HLA-DR and–DQ haplotypes to usceptibility to type 1 diabetes. Diabetes, 2002, 51:545-551.
[16] Torm C,Gupta M,Nikitina Zake L,et al. Heerozygosity for MICA5.0/MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent gentetic risk factors for latent autoimmune diabetes in adults. Hum Immunol, 2003,64(9):902-909.
[17] Hosszu falusi N, Vatay A, Rajczy K, et al. Similar genetic features and different islet cell autoantibody pattern of latent autoimmune diabetes in adults(LADA) compared with adult-onset type 1 diabetes with rapid progression. Diabetes Care, 2003,26(2):452-457.
[18]李玉钟,卢纹凯,高颖,等. HLA-DR-DQ连锁基因单倍体与成人缓慢进展型和速发型1型糖尿病的相关性研究. Chin J Endocrinol Metab, 1998,14(5): 302-304.
[19]肖建中,杨文英,刘娟,等.人类白细胞相关抗原HLADQB1基因与IDDM的关联[ J ].中华内分泌代谢杂志,1997,13 (1):7-10.
[20] Nistico L, Buzzetn R, Pntchard L E, et al. The CTLA-4 gene region of chromosome 2q33 is linked to and associated with type 1 diabetes. Hum Mol Genet, 1996, 5:1075.
[21] Douroudis K,Prans E,Kisand K,et al. Cytotoxic T-lymphocyte antigen 4 gene polymorphisms are associated with latent autoimmune diabetes in adults.Clinica Chimica Acta,2009,403(1-2):226-228.
[22] Haller K,Kisand K,Pisarev H,et al.Insulin gene VNTR, CTLA-4 +49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group.Tissue Antigens ,2007,69(2):121-127.
[23] Cosentino A,Gambelunghe G,Tortoioli C,et a1.CTLA-4 gene polymorphism contributes to the genetic risk for latent autoimmune diabetes in adults.Ann N Y Acad Sci,2002,958:337-340.
[24] Sanjeevi CB,Gambelunghe G,Falorni A,etal.Geneties of latent autoimmune diabetes in adlults.Ann NY Acad Sci.2002,958:107-111
[25] F Pociot,M F McDermott.Genetics of type 1diabetes mellitus.Genes and Inununity2002,3(5):235-249
[26] Torn C,Gupta M,Nikitina Zake L,etal.Heterozygosity for MICA5.0/MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent genetic risk factors for latent autoimmune diabetes in adults.Hum Immunol.2003,64(9):902-9.
[27] Borissova AM, Tankova T, Kirilov G, et a1. The effect of vitamin D3 on insulin secretion and peripheral insulin sensitivity in type 2 diabetic patients.Int J Clin Pract 2003; 57(4): 258-261
[28] Torn C, Gupta M, Nikitina Zake L,etal. Heterozygosity for MICA 5.0 /MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent genetic risk factors for latent autoimmune diabetes in adults.HumImmunol.2003, 64 (9):902-9.
[29] IsomaaB ,AlmgernP,Henricsson M,etal.Chronic complications in patients with slowly progressing autoimmune type 1 diabetes( LADA).Diabetes Care.1999 Aug;22(8): 1347-53
[30] Hosszúfalusi N,Vatay A,Rajczy K,et al. Similar Genetic Features and Diferent Islet Cell Autoantibody Pattern of Latent Autoimmune Diabetes in Adults(LADA)Compared with Adult-Onset Type1 Diabetes With Rapid Progression.Diabetes Care2003,26(2):452-457
[31] Isomaa B, Almgren P, Henricsson M, et al. Chronic comp lications in patients with slowly progressing autoimmune type 1 diabetes (LADA) .Diabetes Care, 1999, 22: 134721353.
[32] Gottsater A, Ahmed M, Lilja B, et al. Islet cell antibodies at diagnosis, but not leanness, relate to a better cardiovascular risk factor profile 5 years after diagnosis ofN IDDM. Diabetes Care, 1996, 19: 602-63.
[33] Tuomi T, Carlsson A, Li H, et al. Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies. Diabetes, 1999,48: 1502157.
[34] Kobayashi T, Nakanishi K, Nurase T, et al. Small doses of subcutaneous insulin as a strategy for preventing slowly p rogressiveβcell failure in islet cell antibody-positive patients with clinical features of NIDDM. Diabetes, 1996, 45: 622-626.
[35] Beales PE, Giorgini A, Annovazzi A, et al. Metformin does not alter diabetes incidence in the NOD mouse. Horm Metab Res, 1997, 29:261-263.
[36] Kobayashi T , Maruyama T , Shimada A , et al . Insulin intervention to preserve beta cells in slowly progressive insulin2depecdent ( type 1) diabetes mellitus. Ann N YAcad Sci , 2002 , 958:117-130
[37] Beales PE, Pozzilli P. Thiazolidinediones for the prevention of diabetes in the non-obese diabetic (NOD) mouse: imp lications for human type 1 diabetes. DiabetesMetab Res Rev, 2002, 18: 114-117.
[38]李霞,周智广,黄干,等.罗格列酮对低滴度谷氨酸脱羧酶抗体的LADA患者胰岛β细胞功能的影响.中华内分泌代谢杂志, 2004,20: 564-567.
[39] Zhou Z, Li X, Huang G, et al. Rosiglitazone combined with insulin preserves islet beta cell function in adult2onset latent autoimmune diabetes (LADA) . DiabetesMetab Res Rev, 2005, 21: 203-208.
[40] Kobayashi T, Maruyama T, Shimada A, et al. Insulin intervention to preserveβcells in slowly progressive insulin dependent ( type 1 ) diabetes mellitus. Ann NY Acad Sci, 2002, 958:117-130.
[2] Lohmann T,Kellner K,Verlohren H J,et al.Titre and combination of ICA and artoantibodies to glutamic acid derarboxylase discriminate two clinically distinet types of latent autoimmune diabetes in adults(LADA)[J]. Diabetologia, 2001, 44(8):1005- 1010.
[3] Isomaa B, Almgren P, Henricsson M, et al. Chronic comp lications in patients with slowly progressing autoimmune type 1 diabetes (LADA) .Diabetes Care, 1999, 22: 134721353.
[4] Gottsater A, Ahmed M, Lilja B, et al. Islet cell antibodies at diagnosis, but not leanness, relate to a better cardiovascular risk factor profile 5 years after diagnosis ofN IDDM. Diabetes Care, 1996, 19: 602-63.
[5] Tuomi T, Carlsson A, Li H, et al. Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies. Diabetes, 1999,48: 1502157.
[6] Kobayashi T, Nakanishi K, Nurase T, et al. Small doses of subcutaneous insulin as a strategy for preventing slowly p rogressiveβcell failure in islet cell antibody-positive patients with clinical features of NIDDM. Diabetes, 1996, 45: 622-626.
[7] Beales PE, Giorgini A, Annovazzi A, et al. Metformin does not alter diabetes incidence in the NOD mouse. Horm Metab Res, 1997, 29:261-263.
[8] Kobayashi T , Maruyama T , Shimada A , et al . Insulin intervention to preserve beta cells in slowly progressive insulin2depecdent ( type 1) diabetes mellitus. Ann N YAcad Sci , 2002 , 958:117-130
[9] Beales PE, Pozzilli P. Thiazolidinediones for the prevention of diabetes in the non-obese diabetic (NOD) mouse: imp lications for human type 1 diabetes. DiabetesMetab Res Rev, 2002, 18: 114-117.
[10]李霞,周智广,黄干,等.罗格列酮对低滴度谷氨酸脱羧酶抗体的LADA患者胰岛β细胞功能的影响.中华内分泌代谢杂志, 2004,20: 564-567.
[11] Zhou Z, Li X, Huang G, et al. Rosiglitazone combined with insulin preserves islet beta cell function in adult2onset latent autoimmune diabetes (LADA) . DiabetesMetab Res Rev, 2005, 21: 203-208.
[12] Kobayashi T, Maruyama T, Shimada A, et al. Insulin intervention to preserveβcells in slowly progressive insulin dependent ( type 1 ) diabetes mellitus. Ann NY Acad Sci, 2002, 958:117-130.
[13] Todd M,Brusko,CliveH,etal.Functtionnal Defects and the Influence of Age on the Frequency of CD4+CD25+ T cells in Type l Diabetes[J].Diabetes,2005,54(2):1407- 1414.
[14] Nora Hosszufalusi,Agnes Vatay,Katalin Rajezy,et al.Similar genetic features and different islet cell autoantibody patten of Latent autoimmune diabetes in adults (LADA) compared with adult-onset tpye l diabctes with rapid progression[J].Diabdes Care,2003,26(3):452-459.
[15] Liang H,Yagi K,Asano A,et al.Assoeiation between CTLA-4 49A/G polytnorphism and type1B diabetes in Japanese population[J].Endocrine,2004,25(2):105-109.
[16] Caputo M,Cerrone GE,López Ap,et al.Cytotoxie T lymphoeyte antigen 4 heterozygous codon 49 A/G dimorphism is associated to latent antoimmune diabetes in adults(LADA)[J].Autoinunumty2005,38(4):277-281.
[17] Nistico L, Buzzetn R, Pntchard L E, et al. The CTLA-4 gene region of chromosome 2q33 is linked to and associated with type 1 diabetes. Hum Mol Genet, 1996, 5:1075.
[18] Donner H, Rau H, Walfish P G, et al. CTLA-4 alanine-17 confers genetic susceptibility to Graves’disease and to type 1 diabetes mellitus[ J ]. J Clin Endocrinol Metab, 1997, 82:143-146.
[19] Dipartimento di, Medicina Interma, Universitàdegli, et al. Immunologic and genetic aspects of latent autoimmune diabetes in the adult[J]. Minerva Endocrinol, 2003, 28(4): 297-312.
[20] Douroudis K,Prans E,Kisand K,et al. Cytotoxic T-lymphocyte antigen 4 gene polymorphisms are associated with latent autoimmune diabetes in adults.ClinicaChimica Acta,2009,403(1-2):226-228.
[21] Haller K,Kisand K,Pisarev H,et al.Insulin gene VNTR, CTLA-4 +49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group.Tissue Antigens ,2007,69(2):121-127.
[22] Cosentino A,Gambelunghe G,Tortoioli C,et a1.CTLA-4 gene polymorphism contributes to the genetic risk for latent autoimmune diabetes in adults.Ann N Y Acad Sci,2002,958:337-340.
[23] Yang ZF.Li XJ.CTLA-4 gene polymorphism is not associated with latent autoimmtme diabetes of the adults in Chinese population.Diabetes,2000,49(Suppl 1):A403.
[24] Douroudis K, Prans E, Uibo R.CTLA-4 promoter polymorphisms are associated with latent autoimmune diabetes in adults.Hum Immunol,2009 ,70(11):921-924.
[25] BAN Y, TANIYAMA M, YANAGAWA T, et al. Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population [J]. BMC Med Genet, 2001, 2(1):7-18.
[26] WANG SG, LIU JH, HU SQ, et al. Association of vitamin receptor genepolymorphisms with idiopathic hypercalciaria[J]. China Journal of Modern Medicine, 2003,13(4):4-7.
[27] CHIU K C, CHUANG L M, YOON C. The vitamin D receptor polymorphism in the translation initiation codon is a risk factor for insulin resistance in glucose tolerant Caucasians[J]. BMC Med Genet, 2001,2:2-11.
[28] Motohoshi T, Yamada S, Yanagawa T, et a1. Vitamin D receptor gene polymorphism affects onset pattern of type l diabetes[J].J Clin Endocrinol Metab, 2003. 88: 3137-3140.
[20] Chang T J,Lei H H, Yeh J I, et al. Vitamin D receptor gene polymorphism influence susceptibility to type 1 diabetes mellitus in the Taiwanese population[J]. Clin Endocrenol(Oxf),2000,52:575-580.
[30] McDermott M F,Ramachandran A Ogunkolade BW,et al. Allelic variation in the vitamin D receptor influences susceptibility to IDDM in Indian Asians[J].Diabetologia 1997,40:971-975.
[31] Speer G,Cseh K,Winkler G,Vargha P,et.al.Vitamin D and estrogen receptor gene polymorphisms in type 2 diabetes mellitus and in android type obesity.Eur J Endoerinol.2001 APr;144(4):385-9.
[32] Niskanen LK,Tuomi T,Karjalainen J,etal.GAD antibodies in NIDDM.Ten-year follow-up from the diagnosis.Diabetes Care.1995,18(12):1557-65.
[33] Falorni A,Gambelunghe G,Forini F,etal.Autoantibody recognition of COOH-terminal epitopes of GAD65 marks the risk for insulin requirement in adult-onset diabetes mellitus.J Clin Endoerinol Metab.2000 Jan:85(l):309-16.
[34] Sanjeevi CB,Gambelunghe G,Falorni A,etal.Geneties of latent autoimmune diabetes in adlults.Ann NY Acad Sci.2002,958:107-111
[35] F Pociot,M F McDermott.Genetics of type 1diabetes mellitus.Genes and Inununity2002,3(5):235-249
[36] Torn C,Gupta M,Nikitina Zake L,etal.Heterozygosity for MICA5.0/MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent genetic risk factors for latent autoimmune diabetes in adults.Hum Immunol.2003,64(9):902-9.
[37] Borissova AM, Tankova T, Kirilov G, et a1. The effect of vitamin D3 on insulin secretion and peripheral insulin sensitivity in type 2 diabetic patients.Int J Clin Pract 2003; 57(4): 258-261
[38] Mimura T, Funatsu H, Uchigata Y, et al. Relationship between human leukocyte antigen status and proliferative diabetic retinopathy in patients with younger-onset type 1 diabetes mellitus. AmJ Ophthalmol,2003,135:844-848.
[39] Kawabata Y, Ikegami H, Kawaguchi Y,et al. Asian-specific HLA haplotypes reveal heterogeneity of contribution of HLA-DR and–DQ haplotypes to usceptibility to type 1 diabetes. Diabetes, 2002, 51:545-551.
[40]李玉钟,卢纹凯,高颖,等. HLA-DR-DQ连锁基因单倍体与成人缓慢进展型和速发型1型糖尿病的相关性研究. Chin J Endocrinol Metab, 1998,14(5): 302-304.
[41]肖建中,杨文英,刘娟,等.人类白细胞相关抗原HLADQB1基因与IDDM的关联[ J ].中华内分泌代谢杂志,1997,13 (1):7-10.
[1] Lohmann T,Kellner K,Verlohren H J,et al.Titre and combination of ICA and artoantibodies to glutamic acid derarboxylase discriminate two clinically distinet types of latent autoimmune diabetes in adults (LADA)[J].Diabetologia,2001, 44(8): 1005-1010.
[2] TuomiT,Groop LC,ZimmetP Z,et al.Antibodies to glutamic acid decarboxylaser eveal latent autoimmune diabetes mellitus in adults with a non-insulin dependent onset of disease.Diabetes.1993,42:359-362
[3] Landin-OlssonM.Latent autoimmune diabetes in adults.2002,Ann N Y Acad Sci.2002,958:112-116.
[4] Tuomi T,Carlsson A,Li H,etal.Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies.Diabetes.1999 Jan;4 8(1):150-7.
[5] Falomi A,Calcinaro F.Autoantibody profile and epitope mapping in latent autoimmune diabetes in adults.2002,Ann N Y Acad Sci.2002,958:99-106
[6] Niskanen LK,TuomiT,Karjalainen J,etal.GAD antibodies in NIDDM. Ten-year follow-up from the diagnosis.Diabetes Care.1995,18(12):1557-65
[7] Landin-OlssonM.Latent autoimmune diabetes in adults.Ann N Y Acad Sci 2002 Apr;958:112-6
[8] Tunrer R,StrattonI,Horton Vetal.UKPDS 25:autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for perdiction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group.Lancet.1997 Nov 1;350(9087):1288-93.
[9] Kanungo A,S anjeevi CB.IA-2 autoantibodies are predominant in latent Autoimmune diabetes in adults patients from eastern India. Ann N Y Acad Sci.2003 Nov;1005:390-4.
[10] Sanjeevi CB,Balaji M,Balaji Vetal.Autoantibodies to GAD65 and IA-2 antibodies are increased,but not tissue transglutaminase(TTG-Ab)in type 2 diabetes mellitus(T2DM) patients from South India.Ann NY Acad Sci.2003 Nov;1005:387-9.
[11] Kawasaki E,Abiru N,Ide A,Epitope analysis of GAD65 autoantibodies in Japanese patients with autoimmune diabetes.Ann NY Acad Sci.2003 Nov;1005:440-8.
[12] Lohmann T,Kellner K,Verlohren HJ,etal.Titre and combination of ICA and autoantibodies to glutamic acid decarboxylase discriminate two clinically distinct types of latent autoimmune diabetes inadults(LADA).Diabetologia.2001 Aug;44(8): 1005-10.
[13] L.Monetini,M.GCavallo,S.Manfrini,et al.Antibodies to bovine beta-casein in diabetes and other autoimmune diseases.Horm Metab Res 2002, 34:455-459
[14] Mimura T, Funatsu H, Uchigata Y, et al. Relationship between human leukocyte antigen status and proliferative diabetic retinopathy in patients with younger-onset type 1 diabetes mellitus. AmJ Ophthalmol,2003,135:844-848.
[15] Kawabata Y, Ikegami H, Kawaguchi Y,et al. Asian-specific HLA haplotypes reveal heterogeneity of contribution of HLA-DR and–DQ haplotypes to usceptibility to type 1 diabetes. Diabetes, 2002, 51:545-551.
[16] Torm C,Gupta M,Nikitina Zake L,et al. Heerozygosity for MICA5.0/MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent gentetic risk factors for latent autoimmune diabetes in adults. Hum Immunol, 2003,64(9):902-909.
[17] Hosszu falusi N, Vatay A, Rajczy K, et al. Similar genetic features and different islet cell autoantibody pattern of latent autoimmune diabetes in adults(LADA) compared with adult-onset type 1 diabetes with rapid progression. Diabetes Care, 2003,26(2):452-457.
[18]李玉钟,卢纹凯,高颖,等. HLA-DR-DQ连锁基因单倍体与成人缓慢进展型和速发型1型糖尿病的相关性研究. Chin J Endocrinol Metab, 1998,14(5): 302-304.
[19]肖建中,杨文英,刘娟,等.人类白细胞相关抗原HLADQB1基因与IDDM的关联[ J ].中华内分泌代谢杂志,1997,13 (1):7-10.
[20] Nistico L, Buzzetn R, Pntchard L E, et al. The CTLA-4 gene region of chromosome 2q33 is linked to and associated with type 1 diabetes. Hum Mol Genet, 1996, 5:1075.
[21] Douroudis K,Prans E,Kisand K,et al. Cytotoxic T-lymphocyte antigen 4 gene polymorphisms are associated with latent autoimmune diabetes in adults.Clinica Chimica Acta,2009,403(1-2):226-228.
[22] Haller K,Kisand K,Pisarev H,et al.Insulin gene VNTR, CTLA-4 +49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group.Tissue Antigens ,2007,69(2):121-127.
[23] Cosentino A,Gambelunghe G,Tortoioli C,et a1.CTLA-4 gene polymorphism contributes to the genetic risk for latent autoimmune diabetes in adults.Ann N Y Acad Sci,2002,958:337-340.
[24] Sanjeevi CB,Gambelunghe G,Falorni A,etal.Geneties of latent autoimmune diabetes in adlults.Ann NY Acad Sci.2002,958:107-111
[25] F Pociot,M F McDermott.Genetics of type 1diabetes mellitus.Genes and Inununity2002,3(5):235-249
[26] Torn C,Gupta M,Nikitina Zake L,etal.Heterozygosity for MICA5.0/MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent genetic risk factors for latent autoimmune diabetes in adults.Hum Immunol.2003,64(9):902-9.
[27] Borissova AM, Tankova T, Kirilov G, et a1. The effect of vitamin D3 on insulin secretion and peripheral insulin sensitivity in type 2 diabetic patients.Int J Clin Pract 2003; 57(4): 258-261
[28] Torn C, Gupta M, Nikitina Zake L,etal. Heterozygosity for MICA 5.0 /MICA5.1 and HLA-DR3-DQ2/DR4-DQ8 are independent genetic risk factors for latent autoimmune diabetes in adults.HumImmunol.2003, 64 (9):902-9.
[29] IsomaaB ,AlmgernP,Henricsson M,etal.Chronic complications in patients with slowly progressing autoimmune type 1 diabetes( LADA).Diabetes Care.1999 Aug;22(8): 1347-53
[30] Hosszúfalusi N,Vatay A,Rajczy K,et al. Similar Genetic Features and Diferent Islet Cell Autoantibody Pattern of Latent Autoimmune Diabetes in Adults(LADA)Compared with Adult-Onset Type1 Diabetes With Rapid Progression.Diabetes Care2003,26(2):452-457
[31] Isomaa B, Almgren P, Henricsson M, et al. Chronic comp lications in patients with slowly progressing autoimmune type 1 diabetes (LADA) .Diabetes Care, 1999, 22: 134721353.
[32] Gottsater A, Ahmed M, Lilja B, et al. Islet cell antibodies at diagnosis, but not leanness, relate to a better cardiovascular risk factor profile 5 years after diagnosis ofN IDDM. Diabetes Care, 1996, 19: 602-63.
[33] Tuomi T, Carlsson A, Li H, et al. Clinical and genetic characteristics of type 2 diabetes with and without GAD antibodies. Diabetes, 1999,48: 1502157.
[34] Kobayashi T, Nakanishi K, Nurase T, et al. Small doses of subcutaneous insulin as a strategy for preventing slowly p rogressiveβcell failure in islet cell antibody-positive patients with clinical features of NIDDM. Diabetes, 1996, 45: 622-626.
[35] Beales PE, Giorgini A, Annovazzi A, et al. Metformin does not alter diabetes incidence in the NOD mouse. Horm Metab Res, 1997, 29:261-263.
[36] Kobayashi T , Maruyama T , Shimada A , et al . Insulin intervention to preserve beta cells in slowly progressive insulin2depecdent ( type 1) diabetes mellitus. Ann N YAcad Sci , 2002 , 958:117-130
[37] Beales PE, Pozzilli P. Thiazolidinediones for the prevention of diabetes in the non-obese diabetic (NOD) mouse: imp lications for human type 1 diabetes. DiabetesMetab Res Rev, 2002, 18: 114-117.
[38]李霞,周智广,黄干,等.罗格列酮对低滴度谷氨酸脱羧酶抗体的LADA患者胰岛β细胞功能的影响.中华内分泌代谢杂志, 2004,20: 564-567.
[39] Zhou Z, Li X, Huang G, et al. Rosiglitazone combined with insulin preserves islet beta cell function in adult2onset latent autoimmune diabetes (LADA) . DiabetesMetab Res Rev, 2005, 21: 203-208.
[40] Kobayashi T, Maruyama T, Shimada A, et al. Insulin intervention to preserveβcells in slowly progressive insulin dependent ( type 1 ) diabetes mellitus. Ann NY Acad Sci, 2002, 958:117-130.