【作者】 周敏；

【导师】 周智广；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 酮症倾向糖尿病(ketosis-prone diabetes,KPD)是一种以无诱因的酮症或酮症酸中毒起病而临床表现异质性很大的综合症,它包括自身免疫性1型糖尿病(1A型)和胰岛自身抗体阴性的特发1型糖尿病(1B型)。目前对于胰岛自身抗体阴性的KPD的病因知之甚少,由于该类患者体内缺乏自身免疫破坏p细胞的证据,因此,遗传和环境因素可能是其主要病因,在遗传性因素中影响p细胞分化成熟和胰岛素分泌的转录因子如PAX4、NeuroD1、PDX1等近年来受到研究者的关注。但在已有的相关研究中缺乏中国人群的研究,有关多态性的关联分析手段也相对简单。因此本研究选择与p细胞分化成熟和胰岛素分泌有关的转录因子PAX4和NeuroD1基因作为候选基因,设计一个两步的病例-对照关联分析,即首先在小样本人群中应用变性高效液相色谱技术(DHPLC)进行PAX4基因突变筛查,从检测出的单核苷酸多态性(single nucleotide polymorphism, SNP)中选择等位基因频率>1%的4个SNP,加上我们在另一研究中已对NeuroD1基因筛查出的一个SNP,在所有样本中对两基因的5个SNP进行基因分型及关联分析。通过采用单点分析,单倍型分析与多点联合作用分析相结合的关联分析方法,探讨这些基因的多态性对中国汉族自身抗体阴性KPD易感性的影响。第一部分胰岛自身抗体阴性酮症倾向糖尿病的PAX4基因突变筛查目的：建立变性高效液相色谱技术(DHPLC)的检测方法,筛查中国汉族胰岛自身抗体阴性的酮症倾向糖尿病(KPD)的PAX4基因突变设计：病例-对照研究方法：采用变性高效液相色谱技术(DHPLC)结合直接测序法,检测141例胰岛自身抗体GAD-Ab和IA-2A阴性的酮症起病糖尿病患者和112例非糖尿病正常对照者的PAX4基因的九个外显子区的突变情况。结果：1.成功建立了DHPLC检测PAX4基因的方法。2.DHPLC检测到PAX4基因的外显子1、4、5、9分别有2、137、41和86例出现异常峰型,经测序证实均有碱基改变,检出符合率100%。3.共检测到PAX4基因9个外显子区的7种碱基改变,它们分别是：外显子1的错义突变Arg31Gln (G298A),外显子4的同义突变Gln173Gln (A725G, rs327517)和错义突变Lys147Arg (A649G),外显子5的错义突变Arg183Cys (C753T)、Arg192Ser (C780A, rs3824004)和Arg192His(G781A, rs2233580),外显子9的错义突变His321Pro (A1168C, rs712701)。其中外显子4的错义突变Lys147Arg (A649G)为新发现的突变位点。结论：在中国汉族人群中,PAX4基因有6种常见多态性改变,分别为Arg31Gln、Gln173Gln、Arg183Cys、Arg192Ser、Arg192His、His321Pro。第二部分自身抗体阴性KPD的PAX4基因多态性关联研究目的：探讨PAX4基因多态性与中国汉族胰岛自身抗体阴性酮症倾向糖尿病(KPD)的关系。设计：病例-对照研究方法：采用多聚酶链反应限制性片段长度多态性(PCR-RFLP)技术检测296例胰岛自身抗体GAD-Ab和IA-2A阴性的KPD患者、399例正常对照者的PAX4基因外显子9的多态性His321Pro的基因型。采用PCR-直接测序法检测PAX4基因外显子5的多态性Arg183Cys、Arg192Ser、Arg192His的基因型。分析PAX4基因各多态性与KPD的关系,同时分析各多态性位点所构成的单体型与KPD的关系。结果：1.自身抗体阴性的KPD患者PAX4 Arg183Cys的CT基因型和T等位基因频率与正常对照者比较,差异均无显著性。CT基因型患者与CC基因型患者的BMI(P=0.025)和FCP水平(P=0.024)比较差异均有显著性。2.自身抗体阴性的KPD患者与正常对照组比较,Arg192Ser的AA基因型(0.7%vs0%,χ2=8.183,P=0.015)和A等位基因频率(6.6%VS 3.4%,OR=2.014,95%CI=1.218～3.329,P=0.005)明显增高。其中,男性(6.7%vs3.5%,P=0.03)和起病年龄<20岁(9.6% vs 3.4%,P=0.003)的KPD患者A等位基因频率高于正常对照。3.自身抗体阴性的KPD患者PAX4基因Arg192His的AA基因型(16.9％vs10.1％,P=0.04)和A等位基因频率(10.6% VS6.8％,OR=1.641,95％CI=1.122～2.400,P=0.01)高于正常对照者。女性(10.8% vs 5.6%,P=0.04)和起病年龄≥20岁(11.1% vs 6.8%,P=0.007)的KPD患者的A等位基因频率高于正常对照。4.自身抗体阴性的KPD患者PAX4基因His321Pro各基因型频率和等位基因频率与对照组比较,差异均无显著性(P>0.05)。男性KPD患者的CC基因型(15％vs 12.1％,χ2=6.74,P=0.03)和C等位基因频率(40.3％vs 31.9％,χ2=4.25,P=0.04)均高于女性KPD患者。5、自身抗体阴性的KPD患者PAX4基因单体型AACC的频率(8.8％vs 5.2％,X2=7.199,OR=1.778,95％CI=1.164～2.717,P=0.007)和单体型CGAC的频率(4.0％vs2.0％,χ2=4.611,OR=2.065, 95％CI=1.087～3.923, P=0.032)均高于正常对照。而单体型CGCC在正常对照组中的频率高于起病年龄≥35岁KPD患者(35.7% vs28.7％,χ2=4.64,OR=0.725,95％CI=0.54～0.975,P=0.031).结论：1.PAX4基因Arg192Ser和Arg192His多态性与中国汉族胰岛自身抗体阴性的KPD有关联。2.PAX4基因单体型AACC和单体型CGAC是中国汉族胰岛自身抗体阴性的KPD的易感单体型,单体型CGCC是起病年龄≥35岁KPD患者的保护性单体型。第三部分NeuroD1基因A45T多态性与自身抗体阴性KPD的关系目的：探讨NeuroD1基因A45T多态性与中国汉族胰岛自身抗体阴性酮症倾向糖尿病(KPD)的关系。设计：病例-对照研究。方法：采用PCR-测序法检测NeuroD1基因A45T基因型,分析NeuroD1基因A45T多态性与KPD的关系,同时分析NeuroD1基因A45T多态性与PAX4基因各多态性位点的交互作用对KPD的影响。结果：1.自身抗体阴性的KPD患者NeuroD1基因A45T的从基因型频率和A等位基因频率与NC组比较差异均无显著性(P=0.34)。对患者进行年龄和性别分层后仍没有发现NeuroD1基因A45T基因型频率与对照组有差别。2.选择PAX4基因的3个多态位点C780A、G781A、C1168A与NeuroD1基因的Ala45Thr多态性位点进行对KPD的交互作用分析。结果显示,Ala45Thr多态性与PAX4的各多态性位点对KPD没有交互作用。结论：1. NeuroD1基因的Ala45Thr多态性与中国汉族自身抗体阴性的KPD没有相关性。2. NeuroD1基因的Ala45Thr多态性位点与PAX4基因的C780A、-G781A、C1168A多态性位点在对KPD的易感性中没有基因-基因之间的交互作用。更多还原

【Abstract】 Part 1 Screening and detection of Pax4 gene mutations in islet autoantibody-negative ketosis-prone diabetic patientsObjective:To establish a denaturing high performance liquid chromatography (dHPLC) method for detection mutations of paired box gene 4 (PAX4) in Chinese Han population with islet autoantibody-negative ketosis-prone diabetes (KPD).Design:Case-control studyMethods:With dHPLC and direct sequencing, nine exons of PAX4 gene were screened for mutations in 141 patients with islet autoantibody-negative KPD, and 112 nondiabetic unrelated controls.Results:1. A dHPLC method for detection gene mutations of paired box gene 4 was established.2. The dHPLC system detected 2、137、41 and 86 samples abnormal peak at exons 1、4、5、9 of PAX4 gene, and was confirmed by sequencing. The coincidence is 100%.3. Seven single nucleotide variations were detected in nine exons of PAX4 gene. They are the missense mutation Arg31 Gln (G298A) in exon 1, synonymous mutation Gln173Gln (A725G, rs327517) and missense mutation Lys147Arg(A649g) in exon 4,missense mutations Arg183Cys (C753T)、Arg192Ser (C780A, rs3824004) and Arg192His (G781A, rs2233580)in exon 5; missense mutation His321Pro(A1168C, rs712701) in exon 9. Among these, the missense mutation Lys147Arg(A649g) in exon 4 was a new mutation.Conclusion:1. In Chinese Han population, six polymorphisms had been found, they are Arg31Gln、Gln173Gln、Arg183Cys、Arg192Ser、Arg192His and His321Pro. Part2 Association of PAX4 gene polymorphism with islet autoantibody-negtive ketosis-prone diabetesObjective:To investigate the association of PAX4 gene polymorphism with islet autoantibody-negative ketosis-prone diabetes(KPD) in Chinese Han population.Design:case-control study.Methods:A total of 296 patients with islet autoantibody-negative ketosis-prone diabetes and 399 nondiabetic unrelated controls were typed for the PAX4 gene excon 9 polymorphisms Pro321His by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method, and the PAX4 gene excon 5 polymorphisms were typed by PCR sequencing-based typing method. Association of PAX4 gene polymorphism with islet autoantibody-negative KPD and PAX4 gene haplotypes were analyzed in KPD group and NC group.Result:1. There was no significant difference in CT genotype frequency or T allele frequency between islet autoantibody-negative KPD subjects and normal controls. The BMI (P=0.025)and FCP(P=0.024) had significant difference in CT or CC patients.2. Compared with normal controls, AA genotypic frequency(0.7% vs 0%,μ2=8.183, P=0.015) and A allele freuency (6.6% vs 3.4%, OR= 2.014, 95%CI=1.218～3.329,P=0.005) of Arg192Ser were higher in islet autoantibody-negtive KPD subjects. The A allele frequency in male(6.7% vs 3.5%,P=0.03) or onset age<20yr (9.6% vs 3.4%,P=0.003)in KPD subjects were significant higher than in normal controls.3. AA genotypic frequency(16.9% vs 10%,P=0.04) and A allele frequency (10.6% vs 6.8%, OR=1.641,95%CI=1.122～2.400, P=0.01) of PAX4 Arg192His were significant different between islet autoantibody-negtive KPDs and normal controls. The A allele frequency in female (10.8% vs 5.6%, P=0.04) or onset age≥20 yr (11.1% vs 6.8%, P=0.007) in KPD subjects were significant higher than that in normal controls.4. There were no significant difference(p>0.05)of PAX4 His321Pro between islet autoantibody-negative KPDs and normal controls. CC genotype frequency(15% vs 12.1%,χ2=6.74, P=0.03) and C allele frequency (40.3% vs 31.9%,χ2=4.25,P=0.04) in male were significant higher than that in females.5. Haplotype AACC frequency (8.8%vs5.2%,χ2=7.199, OR=1.778, 95%CI=1.164～2.717,P=0.007) and haplotype CGAC freuency (4.0%vs2.0%,χ2=4.611, OR=2.065,95%CI=1.087～3.923,P=0.032) of PAX4 were significant higher in islet autoantibody-negtive KPD patients than those in normal controls, while the haplotype CGCC frequency in KPD patients at onset age≥35yr is significantly lower than that in normal controls (35.7% vs 28.7%,χ2=4.64, P=0.031, OR=0.725,95%CI=0.54～0.975)Conclusions:1. In Chinese Han population, PAX4 gene polymorphism Arg192Ser and Arg192His may be associated with islet autoantibody negative KPD.2. In Chinese Han population, haplotypes AACC and CGAC may be susceptive haplotypes for islet autoantibody-negative KPD, and haplotypes CGCC was a protective haplotype for islet autoantibody-negtive KPD at onset age≥35yr. Part 3 Relationship of NeuroDl polymorphism A45T with islet autoantibody-negative kerosis-prone diabetesObjective:To study the relationship between the polymorphism A45T of NeuroDl and islet autoantibody-negative KPD in Chinese Han population, and investigate the interactive effects of NeuroD1 A45T and PAX4 gene polymorphism C780A、G781A and C1168A on islet autoantibody-negtive KPD.Design:Case-control study.Methods:A total of 296 patients with islet autoantibody-negative ketosis-prone diabetes and 399 nondiabetic unrelated controls were typed for the NeuroD A45T polymorphisms by PCR sequencing-based typing method. The distribution and characteristics of NeuroDl genotypes were studied in KPD group and NC group. The interaction of NeuroDl A45T and PAX4 C780A,G781A,C1168A in islet autoantibody-negative KPD were also investigated.Results:1. There was no significant difference in frequency of AA genotypes and A allele of NeruoDl polymorphism A45T between autoantibody-negative KPD patients and control subjects (p=0.34). No significant difference was been found in genotypic frequency of NeuroDl A45T in KPD patients and control subjects when patients were stratified according to age or sex.2. Three polymorphisms of PAX4 gene, C780A, G781A and C1168A, had been selected to analyze the correlation with NeuroD1 Ala45Thr. The result showed there was no interaction of Ala45Thr and the PAX4 polymorphisms.Conclusion:1. There was no associated NeuroDl polymorphism Ala45Thr with islet auto-antibodies negative KPD in Chinese Han population.2. There was no gene-gene interaction of NeuroD1 polymorphism Ala45Thr and the PAX4 polymorphisms C780A, G781A and C1168A in susceptive of KPD.更多还原