Josef Vcelak

Is a Pro12Ala Polymorphism of the PPARγ2 Gene Related to Obesity and Type 2 Diabetes Mellitus in the Czech Population

Abstract: The peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear hormone receptor subfamily of transcription factors. PPARγ2 plays a key role in regulation of adipocyte differentiation and energy homeostasis. Recent studies provide evidence that the Pro12Ala polymorphism is linked to obesity and type 2 diabetes mellitus, but the results are controversial and depend on ethnicity. The aim of this study was to determine allele frequencies and to study the influence of the polymorphism on biochemical and anthropometric parameters in a Czech healthy adult population, in type 2 diabetics, and in a group of obese women. Results: The frequency of the Pro12Ala PPARγ2 gene polymorphism in Czech probands is similar to other central European populations. Frequency of the Pro12Ala substitution tends to be higher in obese women and diabetics compared with controls. The fasting insulin levels in the 12Ala carriers were significantly lower within the group of diabetics even after adjustment for age, BMI, and the length of diabetes duration. In obese women, higher WHR was found in subjects with the 12Ala allele. Conclusions: This study indicates that the substitution Pro12Ala is not associated with a decreased obesity or diabetes risk in the Czech population. However, the present data show that fasting insulin concentrations are lower in diabetics with the 12Ala allele than in those without it. This finding provides evidence that the polymorphism may influence glucose homeostasis.

Determinants of circulating adiponectin in women with polycystic ovary syndrome.

Background and Aim: Adiponectin is regarded as a possible link between adiposity and insulin resistance. Ghrelin and leptin are considered as signals of energy status. We evaluated the relationships between these peptides, androgens and insulin sensitivity in women affected by polycystic ovary syndrome. Methods: Thirty-six women with PCOS were examined with euglycemic hyperinsulinemic clamp (to determine M/I, index of insulin sensitivity). Leptin, ghrelin, adiponectin, androgens, and SHBG were determined. Statistics was done using correlation analysis and backward stepwise multiple regression. Results: The positive correlation of adiponectin with testosterone remains significant even after adjustment for BMI (p = 0.01), M/I (p = 0.009) and for both M/I and BMI (p = 0.02). In multiple regression with testosterone, M/I, leptin and ghrelin as independent variables, the model including testosterone (p = 0.03) and ghrelin (p = 0.002) explained 49% of the variability (p < 0.0012) of adiponectin. Conclusions: Both adiponectin and ghrelin can be involved in the pathophysiology of PCOS but their relation must be delineated further.

Role of D327N sex hormone-binding globulin gene polymorphism in the pathogenesis of polycystic ovary syndrome.

SHBG (sex hormone-binding globulin) is a transport protein specific for dihydrotestosterone, testosterone and estradiol. The missense mutation in exon 8 (GAC-->AAC) causing the amino acid exchange Asp-->Asn in codon 327 (D327N) correlates according to the published data with increased SHBG levels. We studied possible association of this polymorphism with polycystic ovary syndrome (PCOS) and anthropometric and biochemical parameters in 248 PCOS patients and 109 healthy control women. The D327N polymorphism (wild-type and variant allele) was detected using PCR-RFLP method (restriction enzyme Bbs-I). For statistical evaluation chi(2) test, Mann-Whitney test, ANCOVA, ANOVA (NCSS 2004, Statgraphics Plus v.5.1, USA) were used. There was no significant difference in genotype distribution between PCOS and controls (chi(2)=1.03, p=0.59). Moreover, we did not find an association of the variant allele with plasma SHBG level, steroid hormones, or screened parameters of lipid and glucose metabolism. In conclusion, the D327N polymorphism of the SHBG gene does not influence susceptibility to PCOS.

MTNR1B Genetic Variability Is Associated with Gestational Diabetes in Czech Women.

The gene MTNR1B encodes a receptor for melatonin. Melatonin receptors are expressed in human β-cells, which implies that genetic variants might affect glucose tolerance. Meta-analysis confirmed that the rs10830963 shows the most robust association. The aim of the study was to assess the rs10830963 in Czech GDM patients and controls and to study relations between the SNP and biochemical as well as anthropometric characteristics. Our cohort consisted of 880 women; 458 were diagnosed with GDM, and 422 were normoglycemic controls without history of GDM. Despite similar BMI, the GDM group showed higher WHR, waist circumference, abdominal circumference, and total body fat content. The risk allele G was more frequent in the GDM group (38.3 versus 29.4% in controls, OR 1.49 CI95% [1.22; 1.82]; P OR = 0.0001). In spite of higher frequency, the G allele in the GDM group was not associated with any markers of glucose metabolism. In contrast, controls showed significant association of the allele G with FPG and with postchallenge glycemia during the oGTT. Frequency analysis indicates that rs10830963 is involved in gestational diabetes in Czech women. However, the association of the SNP with glucose metabolism, which is obvious in controls, is covert in women who have experienced GDM.

Is a Mutation of the β3‐Adrenergic Receptor Gene Related to Non‐Insulin‐dependent Diabetes Mellitus and Juvenile Hypertension in the Czech Population?a

B. B E N D L O V A , ~ ~ I. M A Z U R A , ~ J. VCELAK,~ J. PERU SIC OVA,^ D. PALYZOVA,~ I . KLIMES,~ AND E. SEBOKOVA~ Institute of Endocrinology Prague, Czech Republic General Teaching Hospital First Medical Faculty Clinic for Children and Adolescents Third Medical Faculty Charles University Prague, Czech Republic flnstitute of Experimental Endocrinology Slovak Academy of Sciences Bratislava, Slovak Republic

Polymorphisms in selected DNA repair genes and cell cycle regulating genes involved in the risk of papillary thyroid carcinoma.

BACKGROUND Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. In addition to causal somatic mutations in the BRAF gene and RET/PTC rearrangements, the contribution of single nucleotide polymorphisms (SNPs) in low-penetrance genes in the development of PTC has been proposed. METHODS Four SNPs in the XRCC1 (Arg399Gln, Arg280His, Arg194Trp and T-77C) and one SNP from each of three other genes participating in DNA repair pathways and/or cell cycle regulation (ATM Asp1853Asn, TP53 Arg72Pro, CDKN1B Val109Gly) were selected. The allelic and genotypic distributions of these variants as well as haplotypes of the XRCC1 were examined in 583 individuals comprising well-characterized cohorts of 209 PTC patients and 374 healthy volunteers. Correlations of polymorphism with clinical-pathological data and mutation status were performed. RESULTS XRCC1 T-77C polymorphism affects the genetic susceptibility for PTC development in men, the specific combination of XRCC1 haplotypes correlates with RET/PTC incidence, CDKN1B Val109Gly significantly influences the risk of developing PTC regardless of gender and in PTC cases, selected genotypes of TP53 Arg72Pro and ATM Asp1853Asn were significantly associated with monitored tumour characteristics. CONCLUSION It seems that SNPs in studied regulating genes contribute to the development of PTC and modify the tumour behaviour or characteristics.

RET Variants and Haplotype Analysis in a Cohort of Czech Patients with Hirschsprung Disease

Hirschsprung disease (HSCR) is a congenital aganglionosis of myenteric and submucosal plexuses in variable length of the intestine. This study investigated the influence and a possible modifying function of RET proto-oncogenes single nucleotide polymorphisms (SNPs) and haplotypes in the development and phenotype of the disease in Czech patients. Genotyping of 14 SNPs was performed using TaqMan Genotyping Assays and direct sequencing. The frequencies of SNPs and generated haplotypes were statistically evaluated using chi-square test and the association with the risk of HSCR was estimated by odds ratio. SNP analysis revealed significant differences in frequencies of 11 polymorphic RET variants between 162 HSCR patients and 205 unaffected controls. Particularly variant alleles of rs1864410, rs2435357, rs2506004 (intron 1), rs1800858 (exon 2), rs1800861 (exon 13), and rs2565200 (intron 19) were strongly associated with increased risk of HSCR (p<0.00000) and were over-represented in males vs. females. Conversely, variant alleles of rs1800860, rs1799939 and rs1800863 (exons 7, 11, 15) had a protective role. The haploblock comprising variants in intron 1 and exon 2 was constructed. It represented a high risk of HSCR, however, the influence of other variants was also found after pruning from effect of this haploblock. Clustering patients according to genotype status in haploblock revealed a strong co-segregation with several SNPs and pointed out the differences between long and short form of HSCR. This study involved a large number of SNPs along the entire RET proto-oncogene with demonstration of their risk/protective role also in haplotype and diplotype analysis in the Czech population. The influence of some variant alleles on the aggressiveness of the disease and their role in gender manifestation differences was found. These data contribute to worldwide knowledge of the genetics of HSCR.

A common variant near BDNF is associated with dietary calcium intake in adolescents

Specific targets for most obesity candidate genes discovered by genomewide association studies remain unknown. Such genes are often highly expressed in the hypothalamus, indicating their role in energy homeostasis. We aimed to evaluate the associations of selected gene variants with adiposity and dietary traits. Anthropometric parameters, fat mass, dietary intake (total energy, fat, protein, carbohydrate, fiber, and calcium) and 10 gene variants (in/near TMEM18, SH2B1, KCTD15, PCSK1, BDNF, SEC16B, MC4R and FTO) were analyzed in 1953 Czech individuals aged 10.0 to 18.0 years (1035 nonoverweight and 918 overweight: body mass index [BMI] ≥90th percentile). Obesity risk alleles of TMEM18 rs7561317, SEC16B rs10913469, and FTO rs9939609 were related to increased body weight and BMI (P < .005). The FTO variant also showed a significant positive association with waist circumference and fat mass (P < .001). Overweight adolescents had a lower total energy intake (P < .001) but a higher percentage of fat (P = .009) and protein intake (P < .001) than the nonoverweight subjects. There was also a lower calcium intake in the overweight group (P < .001). An association with at least one component of dietary intake was found in 3 of 10 studied gene variants. The MC4R rs17782313 was associated negatively with protein (P = .012) and positively associated with fiber (P = .032) intakes. The obesity risk alleles of BDNF rs925946 and FTO rs9939609 were related to a lower calcium intake (P = .001 and .037). The effects of FTO and MC4R variants, however, disappeared after corrections for multiple testing. Our results suggest that the common BDNF variant may influence dietary calcium intake independent of BMI.

A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.

Reconstruction of elbow flexion in arthrogryposis multiplex congenita type I: results of transfer of pectoralis major muscle with follow-up at skeletal maturity.

Background: The purpose of this study was to analyze the results of a pectoralis major transfer to restore active elbow flexion in patients with extension elbow contracture in arthrogryposis. The hypotheses were: (1) this transfer ensures permanent useful elbow flexion; and (2) flexion elbow deformity will not progress during growth and after its cessation. Methods: Unipolar transfer of the 3 distal parts of the pectoralis major muscle was used in 9 extremities of 5 patients (age range, 5 to 9 y; average age, 6.3 y) and the results were prospectively followed in the period of 13 to 16 years. Posterior elbow release was necessary in 5 extremities to achieve passive flexion of 90 degrees before the transfer. The subjective evaluation of daily living activities and data on the physical examination of the range of movement of the elbow, muscle strength, and electrical activity of the transferred muscle were assessed. Two specimens from transferred muscles were histologically examined. Results: All extremities achieved the active elbow flexion. Significant improvement of function for daily living activities was achieved in 5 extremities (55.5%). It includes the following results: 1 very good with flexion of 90 degrees and a deficit of extension of 35 degrees; 2 good with flexion of 92 and 100 degrees and a lack of extension of 42 and 45 degrees; and 2 satisfactory with a limited arc of motion between 20 and 45 degrees. Four extremities remained unsatisfactory with the arc of motion of 5 to 15 degrees. Significant elbow flexion contracture of 70 to 80 degrees developed in 4 extremities. Extremities with a necessity of posterior elbow release achieved a limited range of movement or significant elbow flexion contracture. Electromyography corresponded to a partial denervation of the transferred muscle followed by reinervation. Histologic examinations showed partial atrophy with signs of ongoing regeneration. Conclusions: The hypotheses of the study were not confirmed, because this muscle transfer restores useful elbow flexion without flexion deformity if the passive flexion at children’s age exceeds 90 degrees without a necessity of posterior release. In these cases, bilateral pectoralis to biceps transfer is recommended. Level of Evidence: Level II.