Association Of PAX4 And NeuroD1 Gene Polymorphism With Islet Autoantibody-negtive Ketosis-prone Diabetes

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.