ESHG Posters 6

Polymorphism of APOE gene and its relationship to diabetic neuropathy in type 1 diabetes

N. S. Shcherbak;
St.Petersburg State Medical University, St.Petersburg, RUSSIAN FEDERATION.

Neuropathy is a common complication of type 1 diabetes mellitus. These conditions usually result from diabetic microvascular injury involving small blood vessels that supply nerves (vasa nervorum). A number of facts suggest that diabetic neuropathy may involve genetic susceptibility. Recently, it is reported that APOE polymorphism may influence to development of diabetic neuropathy. The gene encoding apolipoprotein E (APOE) has been proposed as a candidate gene for vascular complication in type 1 diabetes. Apolipoprotein E was discovered as a plasma protein involved in the metabolism of lipoproteins. There are three common alleles, E2, E3, and E4, which code for three major isoforms, resulting in six common genotypes. The aim of this study was to investigate the influence of APOE gene polymorphism in the development of diabetic neuropathy in type 1 diabetes patients. The study consists of 51 patients with diabetic neuropathy and 150 without diabetic neuropathy matched to the patients by age, gender and diabetes duration. APOE polymorphism was detected by the restriction fragment length polymorphism method after a polymerase chain reaction. The distribution of APOE genotypes and alleles frequency showed no difference between the patients with diabetic neuropathy and without this complication (p>0.05). The difference between the groups was tested by Fisher’s exact test. These results suggest that APOE gene polymorphism is no associated with diabetic neuropathy in type 1 diabetes patients.

A hypothesis about two kind of function of the CC-chemokine receptor CCR5 in the case of diabetes.

I. Kalev¹, A. Mikelsaar¹, T. Podar²;
¹Tartu University, Tartu, ESTONIA, ²Tartu University, Department of Internal Medicine, Tartu, ESTONIA.

We have investigated 38 diabetes Type I and 111 diabetes Type II Estonian patients. No statistical differences were found in CCR5delta32 allele frequencies in diabetes Type I and Type II patients (0,141 and 0,108, respectively)compared with the control group of native Estonian subjects (0,148). However, we have found that in diabetes Type I age of onset was 5,4 years later in patients carrying the CCR5delta32 mutation and they have less late complications (p<0,03). In diabetes Type II there was a difference in the frequency of concomitant diseases (p=0,0006), including obesity, thyroid diseases, neoplasias ect., being higher in CCR5 wild-type genotype. From these data we can draw a conclusion, that reduced CCR5 concentration may be useful in hyperglycemic conditions. We propose, that CCR5 has two types of functions: the well-known is to bind chemokines and activate of immune cells, and the second is, as we propose, regulation of functional state of cell membrane, in some conditions also cell contacts by activating Ras and Rho proteins of target-cells. Chemokine binding selectivity is mediated by the second extracellular loop, while affinity-sensitive binding is dependent on the NH2-terminus and the first extracellular loop of CCR5.In the hyperglycemic conditions an unspecific glycosylation takes place, which changes the affinity of ligand binding. It could make the ligand-receptor complex more persistent and extend the time of Gi receptor activated state. During chronic hyperglycemic conditions the altered functional state of cell membrane might lead to different expression of late complications and/or concomitant diseases depending on the CCR5 genotype.

Analysis of triplet repeat polymorphism in the transmembrane region of the MHC class I chain-related A (MICA) gene in siblings of diabetes with high ICA level.

O. S. Glotov¹, N. L. Vartanyan², M. V. Aseev¹, T. E. Ivaschenko¹, V. S. Baranov¹;
¹Ott's Institute of Obstetrics and Gynecology RAMS, St-Petersburg, RUSSIAN FEDERATION, ²Institute of Influenza, St-Petersburg, RUSSIAN FEDERATION.

Insulin-dependent diabetes mellitus is autoimmune disorder of multifactorial etiology with a strong genetic component. The frequency of IDDM in North-West region of Russia is 1-3 : 1000 newborn. IDDM primarily develops due to selective autoimmune destruction of the insulin-producing pancreatic b cell, which leads to severe insulin deficiency. Recent studies have been demonstrated that polymorphism of MICA gene (major histocompatibility complex class 1 chain-related genes) are associated with supceptibility to type 1 diabetes. A total of 34 initially unaffected siblings with the level of cytoplasmatic islet cell antibodies (ICA) titer above 28 Juvenile Diabetes Foundation Units (JDFU) and 42 unrelated non-diabetic peoples from North-West region of Russia were analyzed for exon 5 polymorphism of MICA gene by DNA heteroduplex analysis. MICA gene has a triplet repeat polymorphism in the transmembrane region consisting of five alleles. The frequencies alleles 4, 5, 5.1, 6, 9 were 13.3%, 8.3%, 43.3%, 16.7%, 18.3% in study group and 8.3%, 16.7%, 31%, 17.8%, 26.2% in control group accordingly. More then 80% siblings were homozygote or heterozygote for A5.1 allele compared to only 57% of these subject in the control group (c2= 4,11, p<0,05).

Gene polymorphisms of the angiotensin I-converting enzyme and angiotensin II type 1 receptor A1166C in non-insulin dependent diabetes mellitus

M. Zivkovic¹, A. Stankovic¹, M. Sumarac-Dumanovic², S. Glisic¹, D. Micic², D. Alavantic¹;
¹Institute of Nuclear Sciences "Vinca", Belgrade, YUGOSLAVIA, ²Institute of Endocrinology, Diabetes and Diseases of Metabolism, Clinical Center of Serbia, Belgrade, YUGOSLAVIA.

Background: Patients with non-insulin dependent diabetes mellitus (NIDDM) have high risk of developing micro and macro vascular complications and hypertension. ACE gene polymorphism is associated with an increased risk of vascular disease and AT1 gene polymorphism with hypertension. The aim of this case/control study was to determine genotypes for gene polymorphisms of the angiotensin I-converting enzyme (ACE-ID) and angiotensin II type 1 receptor (AT1-A1166C) in NIDDM patients and control group. Methods: We examined 40 patients (preliminary study) with NIDDM and 100 healthy controls. Genomic DNA was amplified by PCR method for I/D and A1166C polymorphism. Results: Frequencies for II, ID, DD ACE genotypes in patients were 16,67%, 59, 52%, 23,81% respectively and I/D allele frequencies were 0,46/0,54, not significantly different from our healthy population. Frequencies for AT1 genotypes in patients were 39,0%, 43,9%, 17,1% for AA, AC, CC, respectively. Significant difference between patients and controls was found for genotype distribution for A1166C polymorphism (p<0,05). Allele C ¹¹⁶⁶was slightly but not significantly frequent in patients than in controls (0,39 vs. 0,27). Conclusion: Further study with larger number of patients with micro and macrovascular complications could reveal possible association of ACE and AT1 genes polymorphism with progression of these complications.

B. I. Freedman¹, F. I. N. D. Research Group²;
¹Wake Forest University School of Medicine, Winston-Salem, NC, ²Case Western Reserve University, Cleveland, OH.

FIND is a multi-center consortium acquiring DNA from sets of families or individuals with well-characterized diabetic nephropathy and performing a genome scan to identify chromosomal regions linked with diabetic nephropathy. FIND includes families of African-, European-, Native- and Hispanic-American ethnicity. Affected sibling pair, discordant sibling pair, affected relative pair, and discordant relative pair linkage analyses will be performed in the "family" arm of the study. Mapping by Admixture Linkage Disequilibrium analyses will be performed in the "MALD" arm. Markers at candidate genes or chromosomal regions containing putative renal failure susceptibility genes identified in previous genome scans, or syntenic to regions of renal failure susceptibility in model organisms, will be examined. Approximately 10,000 individuals will be recruited in FIND. Participants provide medical histories and urine, plasma, and serum samples for determination of glycosylated hemoglobin, serum and urine creatinine concentration, and urinary albumin:creatinine ratio (UAC). Lymphocytes from eligible participants are immortalized and a repository kept for serum, plasma and urine samples. The family study is recruiting multiplex diabetic families identified by a proband with overt nephropathy or end-stage renal disease (ESRD) attributed to diabetes mellitus. Family eligibility is determined by at least one additional diabetic sibling with either nephropathy (UAC > 0.03 mg/mg) or without nephropathy (UAC < 0.03 mg/mg and normal serum creatinine concentration) after ten years' diabetes duration. FIND will contain a large collection of multiplex diabetic families enriched for the presence of nephropathy. These resources should provide significant power to detect genes contributing to diabetic kidney disease.

S. S. Rich, L. A. Lange, L. E. Wagenknecht, D. M. Herrington, C. D. Langefeld, B. I. Freedman, D. W. Bowden;
Wake Forest University School of Medicine, Winston-Salem, NC.

High blood pressure is a recognized cardiovascular risk factor. Although emphasis in treatment has focused on lowering diastolic blood pressure (DBP) and pulse pressure (PP), PP may be a better predictor of events. The role of genetic factors in blood pressure, including systolic (SBP) and DBP, is well known. Fewer data are available on the extent of genetic control of PP. We hypothesize that in families with multiple members having diabetes, blood pressure phenotypes are influenced by both genetic and environmental factors. To determine the extent of the familial aggregation of SBP, DBP and PP, we studied 245 individuals with type 2 diabetes from 122 families. Other measured factors included duration of diabetes, ethnicity, body mass index (BMI), cigarette smoking, and self-reported medical history. Heritability estimates were obtained using genetic variance components (SOLAR). The sample was 89% Caucasian, 59% female and had a mean±SD for age and duration of diabetes of 60.6±10.4 and 11.2±7.9 years, respectively. Adjusting for age, gender and ethnicity, heritability (h²±SE) for SBP, DBP and PP was estimated to be 0.21±0.18 (P=0.13), 0.35+0.16 (P=0.01), and 0.38+0.17 (P=0.01). No changes in residual heritability were noted after further adjusting for BMI, duration of diabetes or current smoking status. Adjustment for diagnosis of hypertension only slightly altered the residual heritability estimates (0.26 for SBP, 0.33 for DBP, 0.37 for PP). These data provide empirical evidence that blood pressure phenotypes, including pulse pressure, have a significant genetic component in families with multiple members with type 2 diabetes.

T. Nikopensius¹, P. Peterson²^,3, A. Kurg¹, K. J. E. Krohn²^,3, M. Saha⁴, J. Ilonen⁵, M. Knip⁴^,6, A. Metspalu¹;
¹Institute of Molecular and Cell Biology, University of Tartu, Tartu, ESTONIA, ²Institute of Medical Technology, University of Tampere, Tampere, FINLAND, ³Department of Pathology, Tampere University Hospital, Tampere, FINLAND, ⁴Department of Pediatrics, Tampere University Hospital, Tampere, FINLAND, ⁵Department of Virology, University of Turku, Turku, FINLAND, ⁶Hospital for Children and Adolescents, University of Helsinki, Helsinki, FINLAND.

Type 1 diabetes (IDDM) is an autoimmune multifactorial disease characterized by destruction of the pancreatic islet cells, with the major susceptibility gene in HLA region on chromosome 6p21. IDDM often occurs in autoimmune polyendocrinopathy syndromes (APS). In APS1, a rare autosomal recessive autoimmune disease with Mendelian inheritance, the patients have IDDM independently of the HLA association. Nevertheless, IDDM in APS1 patients strongly resembles HLA-associated IDDM with high titers of GAD65 autoantibodies and specific lymphocytic destruction of pancreatic ß-cells. The gene responsible for APS1 - AIRE (autoimmune regulator) - has been mapped to chromosome 21q22.3 and characterised as a transcriptional activator. IDDM and APS1 are enriched in Finnish population. We designed a DNA chip with AIRE sequence polymorphisms and mutations searching for allelic variants associated with IDDM. For analysis we used arrayed primer extension (APEX) technology, a resequencing method based on two-dimensional array of oligonucleotides. We report the allelic and genotype frequencies for 11 SNPs in 123 patients with IDDM and 135 controls from the Finnish population.
The genotype frequency of two SNP-s, 1197T>C and 1411C>T was increased among patients, compared to controls. We suggest that 1411C>T polymorphism, resulting in amino acid change R471C, may be associated with IDDM autoimmunity in some of the Finnish patients. We also show that the most common APS1 mutations, R257X and 13-bp deletion in exon 8, as well as the third Finnish mutation K83E, do not contribute to a haplotype carrying susceptibility to IDDM.