CARDIOVASCULAR RISK ASSESSMENT IN THE ADULT (AGED 40-79 YEARS) ROMANIAN POPULATION.

Atherosclerotic Cardiovascular Diseases are the leading cause of death worldwide. AIM: To estimate the prevalence of cardiovascular risk (CVR) categories in the adult population (aged 40-79 years) of Romania. DESIGN: The present study was part of the epidemiological, cross-sectional PREDATORR study (PREvalence of DiAbeTes mellitus, prediabetes, overweight, Obesity, dyslipidemia, hyperuricemia and chronic kidney disease in Romania). SUBJECTS AND METHODS: Exclusion criteria: age <40/or>79 years old and diagnosis of ischemic vascular disease. The CVR was evaluated using charts developed by the World Health Organization/ International Society of Hypertension (WHO/ISH) available for Europe B (epidemiological sub-region where Romania was included). The CVR was divided into 5 categories: <10%, 10-20%, 20-30%, 30-40%, > 40%. RESULTS: A total of 1631 subjects (57.0±10.7 years, 45.1% males) were included in the present study.The age and sex-adjusted prevalence of CVR >40% was 2.9% (95%CI 2.8-3.1%), CVR 30-40% was 1.85% (95%CI 1.8-1.9%), CVR 20-30% was 5.8% (95%CI 5.6-6.0%) and 13.0% (95%CI 12.8-13.3%) of the adult Romanian population has a 10-20% CVR, these CVR categories being more frequent in male and older age. Diabetes, overweight/obesity and smoking were associated with high CVR categories. CONCLUSION: Romania is one of the countries with high CVR, requiring CVD prevention measures.

A COMMENTARY ON CLASSIFICATION OF DIABETES: LATENT AUTOIMMUNE DIABETES IN ADULTS (LADA) OR INTERMEDIARY DIABETES MELLITUS (IDM)?

Diabetes Mellitus is a huge syndrome which can be detected from the first day of life until the last year of life of a centenarian. In the current classification of diabetes among the so-called "idiopathic phenotypes", apart Type 1 Diabetes (T1D) and Type 2 Diabetes (T2D) has been included provisionally term "Latent Autoimmune Diabetes in Adults" (LADA). This has unclear characterization regarding the age at onset, the presence of anti-ß-cell antibodies and the level of insulin secretory function, in conformity with C-peptide levels. According to several recent publications, there are no specific biochemical or genetic markers for Latent Autoimmune Diabetes in Adults (LADA), but only a gradual transition from T1D to T2D. In addition, the word "latent" in the construction of "LADA" term is inaccurate because in this phenotype nothing is latent: both the autoimmunity and diabetes are present and are even parts of the diagnosis. So that, the best term should be what in reality this sub-phenotype is: an Intermediary Diabetes Mellitus (IDM). Some recent genetic data strongly support this designation.

PATIENTS TREATED WITH INSULIN AND SULPHONYLUREA ARE AT INCREASED MORTALITY RISK AS COMPARED WITH THOSE TREATED WITH INSULIN PLUS METFORMIN.

AIMS: To investigate the effect of sulphonylurea (SU) treatment on all-cause and cardiovascular mortality as compared with metformin (MET), when used in combination with insulin (INS) in type 2 diabetes. METHODS: All type 2 diabetes patients aged ≥40 years were included at their first prescription of INS+MET or INS+SU, during 2001-2008. They were considered at risk until death or December 31st, 2011. Mortality rates were calculated per 1000 person-years. Crude and adjusted rate ratios (RR) were calculated using time dependent analysis with INS+MET as reference. RESULTS: There were 7122 patients (60.8% women) included in the analysis, with a mean age at baseline of 62.0±9.9 years. During the 11 years of study, patients on INS+MET contributed 13620 person-years and 330 deaths (mortality rate 24, CI95% 22-27), while those on INS+SU contributed 8720 person-years and 393 deaths (mortality rate 45, CI95% 41-50). Adjusted all-cause mortality RR were: SU 1.6 (CI95% 1.21-2.11, p<0.001), glimepiride 1.18 (CI95% 0.73-1.91, p=0.51), gliclazide 1.78 (CI95% 1.07-2.95, p=0.024), glibenclamide 1.66 (CI95% 0.71-3.88, p=0.23), glipizide 1.24 (CI95% 0.68-2.27, p=0.49), and gliquidonum 2.32 (CI95% 1.54-3.50, p=0.001). CONCLUSIONS: When combined with insulin as dual therapy, patients treated with SU were at increased mortality risk as compared with insulin + MET.

Prevalence of overweight/obesity, abdominal obesity and metabolic syndrome and atypical cardiometabolic phenotypes in the adult Romanian population: PREDATORR study.

PURPOSE: The objectives were to assess the prevalence of overweight/obesity, abdominal obesity and metabolic syndrome (MetS), and to evaluate the characteristics of the metabolically unhealthy lean (MUHL) and metabolically healthy overweight/obese (MHO) phenotypes in a Romanian population-based sample from the PREDATORR study. METHODS: PREDATORR was an epidemiological study with a stratified, cross-sectional, cluster random sampling design. Participants were classified into four cardiometabolic phenotypes based on the BMI, the cut-off value being 25 kg/m(2), and the presence of MetS (defined according to the Harmonization definition 2009): MUHL, MHO, metabolically healthy lean (MHL) and metabolically unhealthy overweight/obese (MUHO). RESULTS: Overall, 2681 subjects aged 20-79 years were included in the analysis. The overall age and sex-adjusted prevalence of obesity was 31.90 %, overweight was 34.7 %, abdominal obesity was 73.90 % and MetS was 38.50 %. The age- and sex-adjusted prevalence of MHO phenotype was 31.60 %, while MUHL phenotype prevalence was 3.90 %. MUHL and MHO participants had a cardiometabolic profile, kidney function and CVD risk intermediary between MHL and MUHO. MUHL had higher odds of being associated with CVD risk (OR 5.8; p < 0.001), abdominal obesity, prediabetes, diabetes, hypertriglyceridemia and hypo-HDL cholesterolemia than MHL, while MHO phenotype was associated with hypo-HDL cholesterolemia (OR 3.1; p = 0.002), prediabetes (OR 2.9; p < 0.001) and abdominal obesity. CONCLUSIONS: PREDATORR study showed a high prevalence of obesity/overweight, abdominal obesity and MetS in the adult Romanian population, and their association with kidney function and several cardiometabolic factors.

The dynamics of life expectancy over the last six decades in elderly people with diabetes.

AIM: To investigate the historical changes in survival with diabetes in elderly people with diabetes. RESEARCH DESIGN AND METHODS: We analyzed 6504 deaths (44.5% males) registered in a large urban population, aged ≥65 years, and deceased between 1943 and 2009. We split the analysis into three time periods according to year of death: 1943-1965, 1966-1988 and 1989-2009. The parallel changes in the corresponding general population were available. RESULTS: The mean age at diabetes onset was 70.8 ± 4.7 years, with mean disease duration at death 7.5 ± 5 years, and mean age at death 78.3 ± 5.9 years. The mean survival loss due to diabetes (expected minus observed survival) was 4.5 ± 5.1 years (4.9 ± 5.1 years for females versus 4.1 ± 5.2 years for males, p<0.001). The mean disease duration at death was 6.4 ± 5.7 years in the period 1943-1965, followed by a significant (p=0.019) rise to 7 ± 5 years in 1966-1988, and 8.3 ± 4.9 years (p<0.001) in 1989-2009. There was a significant increase in coronary heart disease and stroke, and a significant decrease in infections and end-stage renal disease as causes of death. CONCLUSIONS: We found a significant increase in age at onset and survival with diabetes leading to a significant increase in age at death. Females had a higher survival loss due to diabetes compared with males.

Combined liver and islet transplantation using steroid-free immunosuppression.

Due to a vicious circle in which HCV favors insulin resistance and, alternatively, insulin resistance facilitates the persistence of HCV, HCV patients have often diabetes associated with liver cirrhosis. We present the case of combined liver and pancreatic islets transplantation performed in a patient with HCV liver cirrhosis associated with insulin-dependent diabetes. This is also the first case of islet allotransplantation in Romania. A 40-year-old male diagnosed with liver cirrhosis due to HCV infection and insulin dependent diabetes underwent combined liver and islet transplantation. Our therapeutic design was based on data provided by both the use of Edmonton immunosuppressive steroid-free protocol in islets cell transplantation and the findings of international studies on the effects of this protocol in liver transplantation for patients with HCV infection. Good metabolic control of the diabetes was obtained. The absence of anti beta cell autoimmunity could explain also the good tolerance for the transplanted islets, proved by the rapid and durable decrease of the insulin need, from 64 U/day to 20 U/day at one month post-transplantation, dose that was maintained for 16 months when the patient died due to recurrent HCV hepatitis. Islet transplantation can be associated to liver transplantation in order to improve the associated diabetes in cirrhotic patients.

Strong association of insulin gene INS-VNTR polymorphisms with type 1 diabetes in the Romanian population.

Type 1 diabetes (T1DM) is a common, chronic disease with autoimmune pathogeny, conditioned by genetic factors. Class II HLA DR and DQ and insulin gene polymorphisms encode for most of the T1DM genetic susceptibility. We have previously shown that class I alleles of the insulin gene INS-VNTR locus are strongly associated with T1DM in the Romanian population. The aim of our study was to confirm the contribution of INS-VNTR to T1DM genetic susceptibility in Romania. For this we typed the insulin gene -23HphI A/T polymorphism (an accurate marker for the INS-VNTR alleles) on 219 Romanian T1DM families using Taqman. Allele transmission to diabetics and unaffected siblings was assessed using the Transmission Disequilibrium Test (TDT). We found a significantly increased transmission of -23HphI A allele to diabetics (78.31% transmission, pTDT = 2.4 e-07) which confirms our previous findings. Combined with the data from the first 204 Romanian T1DM families, the transmission of -23HphI A allele to diabetics is almost 80% (79.78%, pTDT = 2.8 e-15). This percentage indicates the same level of predisposition as for the most diabetogenic HLA's. In conclusion, our results indicate an exceptionally strong association of the class I INS-VNTR alleles with T1DM for the Romanian population.

Continuous glucose monitoring: physiologic and pathophysiologic significance.

Diabetes mellitus is a complex disorder of the energy metabolism. In the present paper, we have tried to illustrate the changes in the regulation of blood glucose levels encountered in the two main types of diabetes: Type 2 (T2DM) and Type 1 (T1DM) diabetes mellitus, compared with healthy, non-diabetic subjects. For this we used the MiniMed CGMS (Continuous Glucose Monitoring System) which allows the continuous in vivo blood glucose measurement over a 3-day period. The study group comprised 19 diabetic patients (14 T1DM and 5 T2DM cases) and 4 non-diabetic controls. The recording in normal subjects showed a glycemic variation between 46 and 118 mg/dl, suggesting the existence of a strong and efficient glycemic control mechanism. In T2DM patients, both on diet only or on oral antidiabetic treatment, the oscillation of blood glucose levels was significantly higher compared to that recorded in non-diabetic subjects. In T1DM patients with stable metabolic control blood glucose fluctuations were comparable with those recorded in long-term type 2 diabetic patients but the "mean" values of blood glucose over 72 hours were lower. The CGMS is a valuable tool in the detection of unrecognized hypoglycemic episodes and hyperglycemic postprandial peaks and allows the patient and the health care team to adjust the treatment regimen in order to improve glycemic control. From our point of view, the CGMS could offer valuable information for the knowledge of glycemic regulation in normal people and for the diabetogenic mechanisms in prediabetic IGT and IFG patients.

NIDDM: new once-daily intervention for type 2 diabetes mellitus: Diaprel MR.

UNLABELLED: Pharmacological treatment of hyperglycemia should address to both abnormalities in T2DM treatment, that is reduction of insulin resistance and restoration of normal insulin secretion. Gliclazide is a sulfonylurea compound oral hypoglycemic drug that has a unique feature of restoring the first-phase insulin secretion, which is lost in T2DM being one of the early features of disease. MATERIAL AND METHOD: Laboratoires Servier conducted in Romania a an open non randomized surveillance on the efficacy and safety of Diaprel MR in type 2 diabetic patients. 199 patients in 14 treatment centers were enrolled. Eligibility criteria were as it follows: men and women with diabetes, previously on diet alone and not treated with other OAD, over 35 years old with FPG (mg/dl) at enrollment between 126 and 180. The clinical trial lasted for 16 weeks. During this period the doctors examined the patients 6 times. First visits were at a 2 weeks interval and the last two visits at a 4 weeks interval. At each visit the doctor renewed the prescription for the subsequent period according to the following protocol: the starting dose was 30 mg Diaprel MR/day, if the FPG (mg/dl) was over 140 (at the next visit) the dosage was increased with 30 mg Diaprel MR/day, if the FPG (mg/dl) was under 140 the dosage remained the same as the previous dosage. The maximum dosage was 120 mg Diaprel MR/day. The following parameters were measured on first and last (seventh) visit: blood pressure (systolic and diastolic), heart rate (bpm), body mass index-BMI (kg/m2), fasting plasma glucose FPG (mg/dl and mmol/l), glycated hemoglobin HbA1c (%) and Hb-Hct (mg/dl-%), creatinine (mg/dl), SGPT (UI/I), cholesterol (mg/dl) and triglycerides (mg/dl). Blood pressure (systolic and diastolic), heart rate, BMI and FPG were measured from the second to sixth visit also. On each visit there was registered other data such as: associated illnesses, concomitant medication and adverse events. RESULTS: Primary end points. The average values of end points HbA1c (%) and PFG (mg/dl) registered a significant decrease during the 16 weeks of medication, from the enrollment moment (S0) to the last week (S16). The decrease was significant on the total sample of the main analysis group but also on subsamples of age, gender and BMI. HbA1c (%) average values decreased in the main analysis group (S16 compared to S0): with 22% on the total sample (from 7.7 to 6.0); p < 0.05. FPG (mg/dl) average values decreased in the main analysis group (S16 compared to S0): with 21% on the total sample (from 159 to 126); p < 0.05. Secondary end points. There were no significant changes registered in the average level of cholesterol and triglycerides, BMI, diastolic blood pressure, heart rate, creatinine, SGPT. A significant decline of the average systolic blood pressure was registered. CONCLUSION: Diaprel MR can be used safely in diabetic patients newly diagnosed, uncontrolled on diet or other oral antidiabetic drugs, overweight, safely in those with cardio-vascular disease, or in patients with creatinine clearance 50-80 ml/min.

An increasing trend in the incidence of type 1 diabetes mellitus in children aged 0-14 years in Romania--ten years (1988-1997) EURODIAB study experience.

AIM: To assess the incidence of type 1 diabetes mellitus (DM1) in Romanian children aged 0-14 years using EURODIAB Study methodology. METHODS: Data were collected for a 10-year interval (1988-1997) for the whole country, using the capture-recapture method. RESULTS: We registered 1,418 newly diagnosed patients. The mean total incidence rate was 3.051/100,000/year. There was a wide geographic variation (6.71-fold) between the highest and the lowest incidence rates in different districts. We noticed a progressive increase of age-specific incidence rates from 1.43/100,000/year for the 0-4 year age subgroup to 4.37/100,000/year (10-14 years). The annual mean incidence rate rose from 1.91/100,000/year (1988) to 3.94/100,000/year (1996). CONCLUSIONS: Romania is one of the European countries with the lowest incidence rates (3.051/100,000/year) for DM1 in children. There was an evident increasing trend of DM1 incidence in children between 1988 and 1997, with an annual increase of 7.57%.

Type 1 diabetes genetic susceptibility encoded by HLA DQB1 genes in Romania.

Most cases of type 1 diabetes (T1DM) are due to an immune-mediated destruction of the pancreatic beta cells, a process that is conditioned by multiple genes and environmental factors. The main susceptibility genes are represented by the class II HLA-DRB1 and DQB1 alleles. The aim of our study was to reconfirm the contribution of HLA-DQB1 polymorphisms to T1DM genetic susceptibility for the Romanian population. For this, 219 Romanian T1DM families were genotyped at high resolution for HLA DQB1 using the PCR-SSOP method (Polymerase Chain Reaction - Sequence Specific Oligonucleotide Probes). Allele transmission to diabetics and unaffected siblings was studied using the Transmission Disequilibrium Test (TDT). We found an increased transmission of DQB1*02 (77.94% transmission, p(TDT) = 7.18 x 10(-11)) and DQB1*0302 (80.95% transmission, p(TDT) = 2.25 x 10(-10)) alleles to diabetics, indicating the diabetogenic effect of these alleles. Conversely, DQB1*0301, DQB1*0603, DQB1*0602, DQB1*0601 and DQB1*05 alleles are protective, being significantly less transmitted to diabetics. In conclusion, our results confirmed the strong effect of HLA-DQB1 alleles on diabetes risk in Romania, with some characteristics which can contribute to the low incidence of T1DM in this country.

Cost-effective analysis of candidate genes using htSNPs: a staged approach.

We have previously shown that the selection of haplotype tag single nucleotide polymorphisms (htSNPs) and their statistical analysis in a multi-locus transmission/disequilibrium test (TDT) results in a more cost-effective genotyping strategy in disease association studies of genes by minimising redundancy due to linkage disequilibrium between SNPs. Further savings can be achieved by the use of a two-stage genotyping strategy. This approach is illustrated here in conjunction with the multi-locus TDT in determining whether common alleles of the immune regulatory genes RANK and its ligand TRANCE (RANKL) are associated with type 1 diabetes (T1D). A saving of approximately 75% of potential genotyping reactions could be made with minimal loss of power. There was little evidence from our analysis for association between the TRANCE and RANK genes and T1D in the populations tested.

Testing the possible negative association of type 1 diabetes and atopic disease by analysis of the interleukin 4 receptor gene.

Variations in the interleukin 4 receptor A (IL4RA) gene have been reported to be associated with atopy, asthma, and allergy, which may occur less frequently in subjects with type 1 diabetes (T1D). Since atopy shows a humoral immune reactivity pattern, and T1D results from a cellular (T lymphocyte) response, we hypothesised that alleles predisposing to atopy could be protective for T1D and transmitted less often than the expected 50% from heterozygous parents to offspring with T1D. We genotyped seven exonic single nucleotide polymorphisms (SNPs) and the -3223 C>T SNP in the putative promoter region of IL4RA in up to 3475 T1D families, including 1244 Finnish T1D families. Only the -3223 C>T SNP showed evidence of negative association (P=0.014). There was some evidence for an interaction between -3233 C>T and the T1D locus IDDM2 in the insulin gene region (P=0.001 in the combined and P=0.02 in the Finnish data set). We, therefore, cannot rule out a genetic effect of IL4RA in T1D, but it is not a major one.

The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population.

Several studies suggested that part of the genetic susceptibility for Type 1 diabetes (T1DM) is encoded by some polymorphisms of CTLA-4 gene (2q33) and of Vitamin D Receptor gene (VDR; 12q12-14). Our aim was to assess their contribution to T1DM genetic susceptibility in the Romanian population. We typed CTLA-4 49 A/G and VDR FokI (F/f), ApaI (A/a) and TaqI (T/t) polymorphisms by Sequence Specific Primer PCR (SSP-PCR) in 204 Romanian diabetic families (756 individuals: 212 T1DM probands and 544 unaffected parents and siblings). We studied alleles transmission using the Transmission Disequilibrium Test (TDT). We found an increased transmission of CTLA-4 49G allele to diabetics (54.8%, p=0.11). The transmission of F (56.1%, p=0.063), a (55.7%, p=0.061) and T (51.8%, p=0.37) alleles of VDR gene to diabetics was increased but did not reach statistical significance. In conclusion we found the same increased transmission of CTLA-4 49 G allele to diabetics as previously reported. VDR FoqI F allele seems to be predisposing while TaqI T allele seems to be protective.

Low frequency of HLA DRB1*03 - DQB1*02 and DQB1*0302 haplotypes in Romania is consistent with the country's low incidence of Type I diabetes.

AIMS/HYPOTHESIS: Our study aimed to determine the association of HLA class II HLA-DQB1 alleles with Type I (insulin-dependent) diabetes mellitus and the frequencies of these alleles in the Romanian population, which has one of the lowest incidences of Type I diabetes in children aged 0-14 years in Europe at 3-4 cases per 100,000 person-years. METHODS: We used the sequence specific primer-polymerase chain reaction (PCR-SSP) technique to type HLA-DQB1 alleles, the HLA-DRB1alleles DRB1*03 and one single nucleotide polymorphism (SNP) in the insulin gene (INS). We studied 204 Type I diabetic Romanian families, 196 of which were simplex with 70.3 % of subjects diagnosed under 14 years of age. Data was analysed using a modified version of the Transmission Disequilibrium Test, the Transmission Disequilibrium Test itself, and the affected family-based control method. RESULTS: We found, as expected, the strong positive DQB1*02-DRB1*03 and DQB1*0302, and negative DQB1*0602, HLA class II allele associations with Type I diabetes in these Romanian families. However, using the affected family-based control method, we found relatively low population frequencies of DQB1*02-DRB1*03 and DQB1*0302 alleles in Romania (15.8%) compared with Sardinia (31.3%), a high incidence European region (35 cases per 100,000 person-years in children aged 0-14years). The INS locus had a strong effect in this data set with 80.5 % transmission of the susceptible INS allele from parents to affected siblings (relative risk = 4.1). CONCLUSION/INTERPRETATION: Part of the explanation for the low incidence of Type I diabetes in Romania could be the lower frequency of the DRB1*03 DQB1*02 and DQBI*0302 susceptibility haplotypes in this country.

Evaluation of fine mapping strategies for a multifactorial disease locus: systematic linkage and association analysis of IDDM1 in the HLA region on chromosome 6p21.

The positional cloning of multifactorial disease genes is a major challenge in human genetics. We have therefore empirically tested the utility of the available polymorphic microsatellite map to locate the already identified type 1 diabetes locus IDDM1 (sibling risk/population prevalence ratio lambda(s)= 2.7) within a 14 Mb region of chromosome 6p21 linked to disease. In a two-stage approach to fine mapping, linkage was evaluated in 385 affected sib-pair families using 13 evenly spaced polymorphic microsatellite markers. The whole 14 Mb showed strong linkage. Then, each marker was analysed for evidence of allelic association, revealing evidence of disease association at one marker located within the 95% confidence interval of 1.7 cM obtained by linkage. Analysis of an additional 12 markers flanking this marker revealed a highly specific region of 570 kb associated with disease ( P = 7.5 x 10(-35)), which included the HLA class II genes, known to be the primary determinants of IDDM1. The peak of association was as close as 85 kb centromeric of the disease-predisposing class II gene HLA-DQB1. We investigated the importance of the underlying inter-marker linkage disequilibrium, marker informativity and recombination for fine mapping and demonstrate that the majority of disease association in the region can be explained by linkage disequilibrium with the class II susceptibility genes. Recombination within the major histocompatibility complex was rare and nearly absent in the class III region. We demonstrate that fine mapping of a multifactorial disease gene is possible with high accuracy even in a region with extraordinary linkage disequilibrium across distances of several Mb. The results will be applicable to association studies of disease loci with lambda(s)values <2.7 except that much larger data sets will be required.