Alena Buretić-Tomljanović

Sex-specific differences of craniofacial traits in Croatia: The impact of environment in a small geographic area

Background: Craniometric variation in humans reflects different genetic and environmental influences. Long-term climatic adaptation is less likely to show an impact on size and shape variation in a small local area than at the global level. Aim: The aim of this work was to assess the contribution of the particular environmental factors to body height and craniofacial variability in a small geographic area of Croatia. Subjects and methods: A total of 632 subjects, aged 18–21, participated in the survey. Body height, head length, head breadth, head height, head circumference, cephalic index, morphological face height, face breadth, and facial index were analysed regarding geographic, climatic and dietary conditions in different regions of the country, and correlated with the specific climatic variables (cumulative multiyear sunshine duration, cumulative multiyear average precipitation, multiyear average air temperatures) and calcium concentrations in drinking water. Significant differences between groups classified according to geographic, climatic or dietary affiliation, and the impact of the environmental predictors on the variation in the investigated traits were assessed using multiple forward stepwise regression analyses. Results: Higher body height measures in both sexes were significantly correlated with Mediterranean diet type. Mediterranean diet type also contributed to higher head length and head circumference measures in females. Cephalic index values correlated to geographic regions in both sexes, showing an increase from southern to eastern Croatia. In the same direction, head length significantly decreased in males and head breadth increased in females. Mediterranean climate was associated with higher and narrower faces in females. The analysis of the particular climatic variables did not reveal a significant influence on body height in either sex. Concurrently, climatic features influenced all craniofacial traits in females and only head length and facial index in males. Mediterranean climate, characterized by higher average sunshine duration, higher average precipitation and higher average air temperatures, was associated with longer, higher and narrower skulls, higher head circumference, lower cephalic index, and higher and narrower faces (lower facial index). Calcium concentrations in drinking water did not correlate significantly with any dependent variable. Conclusion: A significant effect of environmental factors on body height and craniofacial variability was found in Croatian young adult population. This effect was more pronounced in females, revealing sex-specific craniofacial differentiation. However, the impact of environment was low and may explain only 1.0–7.32% variation of the investigated traits.

Chromosome studies in patients with defective reproductive success.

PROBLEM: The objective of this study was to evaluate the contribution of chromosomal anomalies to decreased fertility in humans.
 METHOD OF STUDY: In order to investigate the aetiology of infertility in our population and to assess the karyotype in a group of infertile couples and individuals with fertility problems, 782 persons (259 couples, 158 male and 106 female) with different clinical diagnoses of sterility and infertility were analysed cytogenetically.
 RESULTS: The overall frequency of major chromosomal aberration was 13.1% (103/783), which suggests that fertility or sterility problems in this population are due to chromosomal aberrations. Couples experiencing repeated spontaneous abortions, having malformed children or having sterility problems had chromosomal abnormalities in 18.0% (47/259 couples) of the population studied, and constituted chromosomal disorders occured in couples seeking IVF and ICSI with prevalence of 22.2% (8/38 couples), especially minor mosaicism of sex chromosomes in the female partners. The prevalence of chromosome abnormalities in infertile men was 17.7% (28/158), and in subfertile females, it was 26.4% (28/106).
 CONCLUSIONS: These results could indicate an increased tendency to miotic sex chromosome non‐disjuction in humans.

BanI polymorphism of cytosolic phospholipase A2 gene is associated with age at onset in male patients with schizophrenia and schizoaffective disorder

The enzymes phospholipases A2 are believed to be involved in the pathology of schizophrenia. We investigated allelic and genotype frequencies of PLA2G4A BanI polymorphism and the rs4375 in PLA2G6A in Croatian schizophrenic patients (n=81) and controls (n=182), using PCR/RFLP. Genotype and allelic frequencies of both loci, alone or in combination did not show significant difference (chi2-test). Allele-wise and genotype-wise meta-analyses of BanI polymorphism in case-control and family-based studies also revealed no significant association with schizophrenia. Multiple logistic regression analyses revealed statistically significant association between several items from PANSS general psychopathology scale and BanI polymorphism in PLA2G4A. BanI polymorphism further showed a significant impact on mean age of the onset of disease in males (betaA1=0.351, P=0.021; Spearmans rA1=0.391, P=0.010) indicating lower mean age at admission in homozygous A2A2 males.

Quantitative Analysis of Constitutive Heterochromatin in Couples with Fetal Wastage

PROBLEM: Heteromorphism of constitutive heterochromatin is a stable evolutionary feature that is thought to cause no phenotypic alterations. Nevertheless, the role of constitutive heterochromatin is still unknown. The instability of constitutive heterochromatin was generally restricted to T‐lymphocytes and was associated with variable immunodeficiency. The heterochromatin regions of chromosomes 1, 9, 16, and Y have been postulated to play a role in the immune response and during early embryo development.

The impact of PLA2G4A and PTGS2 gene polymorphisms, and red blood cell PUFAs deficit on niacin skin flush response in schizophrenia patients

We investigated the etiology of the attenuated niacin skin flush response in schizophrenia patients. Skin response to topical niacin of 0.1M, 0.01 M, 0.001 M, and 0.0001 M concentrations was rated using method of volumetric niacin response (VNR) and correlated to two functional A/G polymorphisms in genes: phospholipase A2 group IVA (BanI of the PLA2G4A), and rs689466 of the prostaglandin synthase-2 (PTGS2). We further tested the possible correlation between niacin response and fatty acid (FA) content of red blood cells (RBCs). We detected statistically significant but weak impact of both polymorphisms on niacin flush response in schizophrenia patients. The dosage of the G alleles of both polymorphisms was associated with higher VNR values, although each polymorphic variant accounted for only 1% of the overall flush response variability. Regarding FA content, both n-3 and n-6 polyunsaturated FAs (PUFAs) were significantly reduced in the patient group, but an association with niacin sensitivity was not detected.

Functional inference of methylenetetrahydrofolate reductase gene polymorphisms on enzyme stability as a potential risk factor for Down syndrome in Croatia.

Understanding the biochemical structure and function of the methylenetetrahydrofolate reductase gene (MTHFR) provides new evidence in elucidating the risk of having a child with Down syndrome (DS) in association with two common MTHFR polymorphisms, C677T and A1298C. The aim of this study was to evaluate the risk for DS according to the presence of MTHFR C677T and A1298C polymorphisms as well as the stability of the enzyme configuration. This study included mothers from Croatia with a liveborn DS child (n = 102) or DS pregnancy (n = 9) and mothers with a healthy child (n = 141). MTHFR C677T and A1298C polymorphisms were assessed by PCR-RFLP. Allele/genotype frequencies differences were determined using χ2 test. Odds ratio and the 95% confidence intervals were calculated to evaluate the effects of different alleles/genotypes. No statistically significant differences were found between the frequencies of allele/genotype or genotype combinations of the MTHFR C677T and A1298C polymorphisms in the case and the control groups. Additionally, the observed frequencies of the stable (677CC/1298AA, 677CC/1298AC, 677CC/1298CC) and unstable (677CT/1298AA, 677CT/1298AC, 677TT/1298AA) enzyme configurations were not significantly different. We found no evidence to support the possibility that MTHFR polymorphisms and the stability of the enzyme configurations were associated with risk of having a child with DS in Croatian population.

The impact of hemochromatosis mutations and transferrin genotype on gonadotropin serum levels in infertile men

OBJECTIVE To address the possibility that HFE mutations and TF gene polymorphism cause dysfunction of spermatogenesis and/or the hypothalamic-pituitary-gonadal axis via contribution to long-term iron overload in the testes and brain. DESIGN Case-control and association study. SETTING Clinic of obstetrics and gynecology and university-based research laboratory. PATIENT(S) 127 infertile men (including 97 with idiopathic infertility) and 188 controls of proven fertility. INTERVENTION(S) Polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). MAIN OUTCOME MEASURE(S) HFE mutations and transferrin allelic polymorphism, and testosterone, prolactin, and gonadotropin serum levels. RESULT(S) The frequencies of the analyzed alleles and genotypes showed no statistically significant difference between infertile men and controls. Sperm count and progressive sperm motility did not correlate with HFE or TF genotype, or their combination. After excluding patients with clinical hypogonadism or varicocele from further analysis, a statistically significant correlation between serum follicle-stimulating hormone and luteinizing hormone levels and the combined HFE H63D/TFC2 genotype was found in 97 men with idiopathic infertility. CONCLUSION(S) The combined HFE H63D/TF-C2 genotype contributed to 4.1% and 10.6% of follicle-stimulating hormone and luteinizing hormone variation, respectively, in infertile men, raising mean hormonal values above the normal physiologic range. Therefore, HFE and TF genes together may influence the hypothalamic-pituitary-gonadal axis, functioning at the pituitary or testes level.

Angiotensin-converting enzyme gene insertion/deletion polymorphism is not associated with schizophrenia in a Croatian population.

Because angiotensin converting enzyme (ACE) plays an important role in dopamine system functioning in the brain and the insertion/deletion (I/D) polymorphism of a 287 nucleotide fragment of the ACE gene correlates with enzyme activity, several studies have investigated the role of ACE I/D polymorphism in psychiatric diseases (Segman et al., 2002 ; Crescenti et al., 2009). Two recent studies yielded contradictory results: the D allele was identified as a protective factor in a Spanish population, while a protective effect toward schizophrenia and bipolar disorder was attributed to the I allele in a Turkish population (Crescenti et al., 2009 ; Kucukali et al., 2010). We tested whether schizophrenia risk was associated with ACE I/D polymorphism in a Croatian population, and examined its possible impact on schizophrenia symptom severity. Our study group consisted of 211 Croatian patients (115 male, 96 female) who met DSM-IV criteria for schizophrenia (n = 187) and schizoaffective disorder (n = 24), and 270 healthy blood donors (135 male, 135 female). All participants gave informed consent for the analysis. The study was approved by the Ethics Committee of the School of Medicine, University of Rijeka, Croatia. Genotyping was performed by polymerase chain reaction (PCR) as previously described (Rigat et al., 1990). To exclude mistyping of the heterozygotes as DD homozygotes, all DD genotype samples were confirmed with insertion-specific PCR (Shanmugan et al., 1993). The significance of differences in genotype and allele frequencies between patients and controls was determined using the χ2 test. Potential correlation between Positive and Negative Symptom Scale (PANSS) scores and I/D polymorphism was tested using linear regression analysis (stepwise selection), adjusted for age at PANSS assessment and sex, in patients with schizophrenia. PANSS evaluation was performed during the last hospitalization due to a psychotic episode. P-values < 0.01 were considered statistically significant. Allelic and genotypic frequencies of I/D polymorphisms were consistent with Hardy-Weinberg equilibrium and were not significantly different between groups. Allele frequencies were 230 (D) and 192 (I) in the patient group, and 285 (D) and 255 (I) in the control group. Genotype frequencies were 0.299 (DD = 63), 0.493 (ID = 104) and 0.208 (II = 44) in the patient group, and 0.274 (DD = 74), 0.507 (ID = 137) and 0.219 (II = 59) in the control group. Therefore, our data did not support results from Spanish or Turkish samples. However, after adjusting for sex and age at PANSS assessment we observed a significant correlation between ACE genotype and psychopathology evaluated by PANSS. Increased negative and total PANSS scores were significantly correlated with the number of D alleles (b = 0.26, F = 7.803 ; P = 0.006 and b = 0.29, F = 7.557 ; P = 0.002, respectively). Increased symptom severity of the general psychopathology scale in males with schizophrenia could also be attributed to the number of D alleles (b = 0.37, F = 9.910 ; P = 0.008). This is the first study reporting significant correlation between clinical expression of illness and I/D polymorphic variants. I/D polymorphism may affect symptom severity by modulating ACE expression/activity. Further studies in other populations/ethnic groups may help clarify the relationship between ACE activity and schizophrenia.

Peptidomics as a tool for characterizing bioactive milk peptides

Food peptidomics is a sub-field of proteomics that focuses on the composition, interactions, and properties of bioactive peptides present in different food matrices. The milk peptidome is considered a valuable source of a number of biologically active peptides. Increasing use of peptidomic techniques-including the application of high-resolution techniques, such as mass spectrometry-has led to enhancements of our knowledge regarding the health benefits of dairy products, as well as improved monitoring for food control and food safety. Chromatographic techniques, both at the analytical and preparative scale, are used also in the identification of novel peptides, including those synthesized and those obtained through fermentation processes. The present review focuses on peptidomic approaches to the investigation of bioactive milk peptides, including bioinformatics, chemometric tools, and proteomic/peptidomic methods.

The impact of ACE gene I/D polymorphism on plasma glucose and lipid concentrations in schizophrenia patients.

A functional 287 nucleotide fragment insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene, by affecting ACE levels, may influence various pleiotropic effects of ACE. The I/D polymorphism has been extensively associated with the development of the metabolic syndrome and its separate components such as diabetes mellitus and dyslipidemia in the general population (Mittal et al., 2011). Since the ACE enzyme plays an important role in the functioning of the dopamine system in the brain, the I/D polymorphism has also been investigated in schizophrenia, yet with conflicting results (Crescenti et al., 2009; Nadalin et al., 2012; Hui et al., 2014; Zhang et al., 2014). Schizophrenia patients have a significantly higher risk of developing diabetes, dyslipidemia and obesity and it has been determined that treatment with atypical antipsychotic medications particularly contributes to abnormalities in the glucose and lipid metabolism in those patients (Kapur and Remington, 2001). In our recent study we found no association between the I/D polymorphism and schizophrenia risk in the Croatian population, but we revealed a significant polymorphisms impact on the clinical expression of the illness (Nadalin et al., 2012). We undertook the current study to determine whether and to what extent plasma glucose and lipid concentrations in schizophrenia patients may be influenced by ACE-I/D polymorphism. Our study group consisted of 211 chronically ill schizophrenia patients (115 males, 96 females) from the Department of Psychiatry of the Clinical Medical Centre in Rijeka, Croatia (N1⁄4110) and the Psychiatric Hospital in Rab, Croatia (N1⁄4101), aged 43.1710.8 years, all Croatian citizens. Diagnoses were assessed according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria using the structural clinical interview. The investigation was carried out in accordance with the latest version of the Declaration of Helsinki. The study was approved by the Ethics Committee of the School of Medicine, University of Rijeka, Croatia. All patients gave informed consent to the analysis. Allele/genotype frequencies were determined by polymerase chain reaction/restriction fragment length polymorphism in the Laboratory for Molecular Genetics (Department of Biology and Medical Genetics, School of Medicine, Rijeka). Biochemical measurements (determination of fasting plasma glucose, total cholesterol, HDL cholesterol, LDL cholesterol and triglyceride levels) were carried out in the Department of Clinical Laboratory Diagnostics of the Clinical Medical Centre Rijeka. The difference between means of plasma glucose and lipid levels according to the ACE-I/D polymorphism was revealed by the one-way ANOVA analysis of variance (ANOVA). The association between glucose and lipid levels and ACE-I/D allele/genotype variants was tested using multiple stepwise regression analysis, adjusted for the patients age. Considering the possible interaction of the biochemical measurements with the patients gender, we performed separate analyses among males and females. Statistical analyses were conducted using Statistica for Windows, version 9 (StatSoft, Inc., Tulsa, OK, USA). The P values less than 0.05 (Po0.05) were considered statistically significant. The frequencies of the genotypes DD, ID and II were 0.299 (N1⁄463), 0.493 (N1⁄4104) and 0.208 (N1⁄444), and the frequencies of the D and I alleles were 0.545 (N1⁄4230) and 0.455 (N1⁄4192), respectively. We did not find significant gender differences neither in the frequencies of the genotypes DD, ID and II (males vs. females: 0.278, 0.470 and 0.252 vs. 0.323, 0.521 and 0.156) nor in the frequencies of the D and I alleles (males vs. females: 0.513 and 0.487 vs. 0.583 and 0.417) (P40.05). The multiple stepwise regression analysis revealed a significant association between the ACE genotype andmean plasma glucose levels in females (β1⁄4 0.60, F1⁄48.40, P1⁄40.005). Females heterozygous for the ACE genotype (ID; N1⁄450) had significantly higher glucose levels when compared to those DD homozygous (N1⁄431) and II homozygous (N1⁄415) (6.171.2 vs. 5.370.7 and 5.470.9; one-way ANOVA test: F1⁄43.84, Po0.026). The ACE genotype accounted for approximately 9% of plasma glucose levels variability (R change1⁄40.09). Furthermore, the ACE-ID polymorphism affected plasma triglyceride levels in males, although its impact only approached statistical significance (multiple stepwise regression analysis: β1⁄40.31, F1⁄43.92, P1⁄40.055). Males carrying the D allele in the ACE genotype (DD homozygous or ID heterozygous; N1⁄486) trended toward higher triglyceride levels than those II homozygous (N1⁄429) (2.371.4 vs. 1.570.8; one-way ANOVA test: F1⁄43.92, P1⁄40.055). This is the first report investigating the possible influence of ACE-I/ D polymorphism on plasma glucose and lipid levels in schizophrenia. Our results suggest gender dependent effect of the ACE-I/D polymorphism on glucose and lipid metabolism. Thus, males carrying the D allele in their ACE genotype (DD homozygous or ID heterozygous) trended toward an elevated risk for developing dyslipidemia. Furthermore, female patients heterozygous for the ACE genotype (ID) had significantly greater risk for diabetes mellitus than those DD homozygous and II homozygous. The strength of the observed association (P1⁄40.005) between glucose levels in female patients and ACE genotype is strong, and, to our opinion, the ACE genotype satisfactorily described plasma glucose levels (multiple stepwise regression: R change1⁄40.09). Intriguingly, this is also the first study reporting highest plasma glucose concentrations in ACE-ID heterozygous individuals while investigating the association between ACE-I/D polymorphism and glucose levels. Previous studies revealed highest plasma glucose levels in the ACE homozygous (DD) genotype as well as in the ACE homozygous (II) genotype, in patients with the metabolic syndrome (Alvarez-Aguilar et al., 2007; Mittal et al., 2011). Moreover, Zingone et al. (1994) determined the highest glucose levels in the ACE homozygous (DD) genotype among a random sample of male subjects from the general population. Thus, our results indicate that the mechanism by which the ACE may maintain glucose homoeostasis possibly differs in schizophrenia compared to