Jana Makuc

Hemochromatosis gene mutations in the Croatian and Slovenian populations

The study provide the prevalence of the C282Y, H63D and S65C. mutations in two Slavic populations (Croatian and Slovenian) in SE Europe which further support the well documented European NW-too-SE gradient of the C282Y mutation distribution. Our results have an important clinical implication for the detection and management of hemocromatosis in both observed populations.

Polymorphisms XbaI (rs693) and EcoRI (rs1042031) of the ApoB gene are associated with carotid plaques but not with carotid intima-media thickness in patients with diabetes mellitus type 2.

BACKGROUND Apolipoprotein B is a key structural component of all the atherogenic lipoproteins (LDL, VLDL and IDL). Genetic variations of the ApoB gene may affect plasma ApoB and lipid levels, thus influencing atherogenesis. The present study was designed to investigate the association of polymorphisms XbaI (rs693) and EcoRI (rs1042031) of the ApoB gene with plasma ApoB level, lipid levels and the different ultrasound phenotypes of carotid atherosclerosis in patients with diabetes mellitus type 2. PATIENTS AND METHODS 595 patients with diabetes (399 on statin therapy and 196 without) and 200 healthy controls were enrolled in the study. The carotid intima-media thickness (CIMT) and plaque characteristics (presence and structure) were assessed ultrasonographically. Biochemical analyses were performed using standard biochemical methods. Both XbaI (rs693) and EcoRI (rs1042031) genotypes were determined by real-time PCR. RESULTS Genotype distributions and allele frequencies of the XbaI and EcoRI polymorphisms were not statistically significantly different between diabetic patients and controls. No statistically significant difference in lipid parameters, ApoA1, ApoB, hs-CRP and fibrinogen as well as CIMT was observed in diabetic patients regarding XbaI and EcoRI polymorphisms, even after adjustment for statin treatment. The risk of having plaques on carotid arteries was higher in homozygous carriers of the mutant X + allele (OR = 1.74, p = 0.03) and lower in diabetics carrying mutant E- alleles (OR = 0.48, p = 0.02). Neither XbaI nor EcoRI polymorphism was associated with CIMT or presence of unstable plaques in diabetic patients. Plasma ApoB level was not independently associated with any of the ultrasonographic parameters of carotid atherosclerosis. CONCLUSIONS Both XbaI and EcoRI polymorphisms were associated with presence of plaques on carotid arteries but not with CIMT or presence of unstable plaques. Plasma ApoB level was not independently associated with ultrasonographic phenotypes of carotid atherosclerosis in patients with diabetes mellitus.

Pancreatic exocrine insufficiency, diabetes mellitus and serum nutritional markers after acute pancreatitis.

AIM To investigate impairment and clinical significance of exocrine and endocrine pancreatic function in patients after acute pancreatitis (AP). METHODS Patients with AP were invited to participate in the study. Severity of AP was determined by the Atlanta classification and definitions revised in 2012. Pancreatic exocrine insufficiency (PEI) was diagnosed by the concentration of fecal elastase-1. An additional work-up, including laboratory testing of serum nutritional markers for determination of malnutrition, was offered to all patients with low levels of fecal elastase-1 FE. Hemoglobin A1c or oral glucose tolerance tests were also performed in patients without prior diabetes mellitus, and type 3c diabetes mellitus (T3cDM) was diagnosed according to American Diabetes Association criteria. RESULTS One hundred patients were included in the study: 75% (75/100) of patients had one attack of AP and 25% (25/100) had two or more attacks. The most common etiology was alcohol. Mild, moderately severe and severe AP were present in 67, 15 and 18% of patients, respectively. The mean time from attack of AP to inclusion in the study was 2.7 years. PEI was diagnosed in 21% (21/100) of patients and T3cDM in 14% (14/100) of patients. In all patients with PEI, at least one serologic nutritional marker was below the lower limit of normal. T3cDM was more frequently present in patients with severe AP (P = 0.031), but was also present in some patients with mild and moderately severe AP. PEI was present in all degrees of severity of AP. There were no statistically significantly differences according to gender, etiology and number of AP attacks. CONCLUSION As exocrine and endocrine pancreatic insufficiency can develop after AP, routine follow-up of patients is necessary, for which serum nutritional panel measurements can be useful.

Distribution of HFE gene mutations in Slovenian patients with hereditary hemochromatosis

Dear Editor, Hereditary hemochromatosis (HH; HFE-related or type 1 genetic hemochromatosis) is a common autosomal recessive disorder of iron metabolism, its prevalence being two to five per 1,000 in Caucasians, characterized by increased iron absorption and progressive iron storage, resulting in organ damage, especially liver cirrhosis. Because the clinical consequences can be prevented by repeated phlebotomies, early accurate diagnosis is crucial. This can be achieved by genetic testing. By this approach, HH becomes a preventable genetic disorder on secondary level. In the hemochromatosis gene (HFE), two missense mutations were initially identified: C282Y (c.845G>A/p. C282Y) and H63D (c.187C>G/ p.H63D), followed by a third mutation: S65C (c.193A>T/p.S65C) [3, 10]. In European populations, the estimated contribution of C282Y homozygosity to the HH phenotype is up to 96%. The prevalence decreases from northern to southern Europe. The H63D mutation has a frequency of approximately 16% in the European populations, the proportion of the compound C282Y/H63D heterozygotes among HH patients being the highest in southern Europe. The S65C mutation accounts for approximately 8% of HH chromosomes, which are neither C282Y nor H63D [4, 6, 7, 10]. Reports on the prevalence of HFE mutations in HH patients in Slavic populations are scarce. The present study was performed in order to establish the frequencies of the three HFE mutations in Slovenian HH patients. Forty-nine unrelated HH patients of Slovenian origin were ascertained through the Registry at the Gastroenterology Department, University Medical Center Ljubljana, Slovenia (mean age 48 years, age range 22–69 years): there were 39 men and ten women. In all patients included in the study, the clinical diagnosis of HH was established based on the following criteria: increased transferrin saturation (TS): >50% for females and >60% for males; increased serum ferritin: >200 μg/l for females and >400 μg/l for males. Iron serum markers were measured by standard methods. In 47 of the 49 patients, HH diagnosis and staging was confirmed by liver biopsy and iron staining. Hepatic iron index was not calculated. In two patients who refused liver biopsy, liver iron overload was established by magnetic resonance imaging. Exclusion criteria were: alcohol abuse, secondary iron overload and antihepatitis C and B virus positivity. The study was approved by the National Medical Ethics Committee. Informed consent was obtained from all patients. The analysis of the HFE gene mutations was performed using polymerase chain reaction [3, 10]. The mutation analysis of 49 HH patients revealed that 35 (71.4%) were homozygous for C282Y mutation, and two patients (4.1%) were C282Y–H63D compound heterozygotes. Thus, the C282Y mutation was present in 37 of the 49 patients (75.5%). Among the 12 patients without the C282Y mutation, one (2%) was H63D–S65C compound heterozygote, five (10.2%) Ann Hematol (2008) 87:667–669 DOI 10.1007/s00277-008-0463-2

Angiotensinogen (AGT) gene missense polymorphisms (rs699 and rs4762) and diabetic nephropathy in Caucasians with type 2 diabetes mellitus

Gene polymorphisms associated with the renin-angiotensin-aldosterone system (RAAS) have been extensively studied in diabetic nephropathy (DN) patients, due to therapeutic potential of targeting the RAAS and slowing down the disease progression. The aim of our study was to examine the association between angiotensinogen (AGT) gene polymorphisms (rs699 and rs4762) and DN in Caucasians with type 2 diabetes mellitus (T2DM). A total of 651 unrelated Slovenian (Caucasian) T2DM patients were tested for AGT rs699 and rs4762 polymorphisms using a novel fluorescence-based kompetitive allele-specific polymerase chain reaction (KASPar) assay. A study group consisted of 276 T2DM patients with DN, while control group included 375 patients without DN but who have had T2DM for >10 years. For rs699 polymorphism, the frequencies of GG, GA and AA genotypes were 20.6%, 52.2% and 27.2%, respectively in T2DM patients and 23.4%, 48.1% and 28.5%, respectively in controls. The distributions of GG, GA and AA genotypes for rs4762 polymorphism were 73.9%, 23.2% and 2.9%, respectively in T2DM patients and 70.4%, 27.5% and 2.1%, respectively in controls. No significant differences in the allele frequencies were found between T2DM patients and controls for both polymorphisms. AGT rs699 and rs4762 missense polymorphisms are not associated with DN in our subset of Slovenian T2DM patients.

PECAM-1 Leu125Val (rs688) Polymorphism and Diabetic Nephropathy in Caucasians with Type 2 Diabetes Mellitus.

Objectives. Platelet endothelial cell adhesion molecule-1 (PECAM-1) plays a key role in the transendothelial migration of circulating leukocytes during inflammation and in the maintenance of vascular endothelial integrity. We hypothesized that genetic variation in PECAM-1 gene could be associated with diabetic nephropathy (DN) and with the level of soluble PECAM-1 in Caucasians with type 2 diabetes mellitus (T2DM). Design and Methods. We analyzed the rs688 single nucleotide polymorphism of PECAM-1 gene C373G (Leu125Val) at exon 3, which encodes the first extracellular Ig-like domain that mediates the homophilic binding of PECAM-1, in 276 T2DM subjects with documented DN (cases) and 375 T2DM subjects without DN (controls), using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy. Level of plasma soluble PECAM-1 (sPECAM-1) was measured by ELISA in a subpopulation of 120 diabetics with DN. Results. We found no association between the Leu125Val polymorphism and DN in subjects with T2DM. Likewise, the Leu125Val polymorphism was not associated with serum sPECAM-1 levels in a subpopulation of 120 diabetics with DN. Conclusion. The Leu125Val polymorphism of PECAM-1 and the level of sPECAM-1 are not associated with DN in T2DM subjects of Slovenian origin.