New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease­Serbian experience.

UNLABELLED: Perthes disease is one of the most common forms of pediatric femoral head osteonecrosis with an unknown etiology. Coagulation factors were the first genetic factors suspected to have a role in the pathogenesis of this disease, but studies showed inconsistent results. It is described that inflammation is present during early stages of Perthes disease, but its genetic aspect has not been studied extensively. Little is known regarding the status of apoptotic factors during the repair process that leads to the occurrence of hip deformity in patients. Therefore, the aim of this study was to analyze major mediators involved in coagulation, inflammation, and apoptotic processes as possible causative factors of Perthes disease. The study cohort consisted of 37 patients. Gene variants of TNF-α, FV, FII, and MTHFR genes were determined by PCR-RFLP, while IL-3 and PAI-1 were genotyped by direct sequencing. The expression level of Bax, Bcl-2, Bcl2L12, Fas and FasL was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique. Our results showed a significantly increased level of expression of pro-apoptotic factor Bax along with significantly higher Bax/Bcl-2 ratio in the patient group. CONCLUSION: The results presented indicate that apoptosis could be one of the factors contributing to the lack of balanced bone remodeling process in Perthes patients.

Association of gene variants in TLR4 and IL-6 genes with Perthes disease.

INTRODUCTION: Perthes disease is idiopathic avascular osteonecrosis of the hip in children, with unknown etiology. Inflammation is present during development of Perthes disease and it is known that this process influences bone remodeling. OBJECTIVE: Since genetic studies related to inflammation have not been performed in Perthes disease so far, the aim of this study was to analyze the association of frequencies of genetic variants of immune response genes, toll-like receptor 4 (TLR4) and interleukin-6 (IL-6), with this disease. METHODS: The study cohort consisted of 37 patients with Perthes disease and 50 healthy controls. Polymorphisms of well described inflammatory mediators: TLR4 (Asp299Gly, Thr39911e) and 11-6 (G-174C, G-597A) were determined by polymerase chain reaction restriction fragment length polymorphism method. Results IL-6 G-174C and G-597A polymorphisms were in complete linkage disequilibrium. A statistically significant increase of heterozygote subjects for IL-6 G-174C/G-597A was found in controls in comparison to Perthes patient group (p = 0.047, OR = 2.49, 95% CI = 1.00-6.21). Also, the patient group for IL-6 G-174C/G-597A polymorphisms was not in Hardy-Weinberg equilibrium. No statistically significant differences were found between patient and control groups for TLR4 analyzed polymorphisms. A stratified analysis by the age at disease onset also did not reveal any significant difference for all analyzed polymorphisms. Conclusion Our study revealed that heterozygote subjects for the IL-6 G-174C/G-597A polymorphisms were significantly overrepresented in the control group than in the Perthes patient group. Consequently, we concluded that children who are heterozygous for these polymorphisms have a lower chance of developing Perthes disease than carriers of both homozygote genotypes.

The influence of novel transcriptional regulatory element in intron 14 on the expression of Janus kinase 2 gene in myeloproliferative neoplasms.

The expression of Janus kinase 2 (JAK2) gene is altered in myeloproliferative neoplasms (MPN) and the regulation of transcription could be a mechanism that modulates JAK2 gene expression. We analyzed the transcriptional potential of single-nucleotide polymorphism (SNP) rs12343867 T > C in JAK2 intron 14, tagging 46/1 haplotype, and its influence on JAK2 gene expression. Functional analysis of JAK2 intron 14 was performed using the pBLCAT5 reporter system in K562 cells. Identification of the proteins binding to the intron 14 regulatory element was accomplished by electrophoretic mobility shift assay (EMSA) and supershift assays. Quantification of the expression of JAK2 gene in a cohort of 51 MPN patients and 12 healthy controls was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Functional analysis revealed that the intronic DNA element harboring SNP rs12343867 T > C acts as a transcriptional repressor in vitro. The repressor activity was significantly attenuated by the presence of nucleotide C. Supershift analysis showed the enrolment of transcriptional factor Meis1 in this process. RQ-PCR experiments showed increased JAK2 expression in patients with the JAK2V617F mutation, with a significant difference between essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) patients. SNP rs12343867 showed no statistically significant influence on the expression of JAK2 gene in MNP patients.

-174G/C interleukin-6 gene promoter polymorphism predicts therapeutic response to etanercept in rheumatoid arthritis.

To examine whether -174G/C interleukin-6 (IL-6) gene polymorphism, previously reported to correlate with IL-6 level, influences response to etanercept therapy in patients with rheumatoid arthritis. Seventy-seven patients with active RA were studied, at baseline and 6- and 12-month follow-up after etanercept therapy. Treatment response was estimated according to the European League Against Rheumatism response criteria. RA patients were genotyped for -174G/C IL-6 gene polymorphism by the PCR-RFLP method, and influence of genotype at this polymorphism to clinical response to etanercept was assessed. After 12 months of treatment, the percentage of responders (patients who had DAS28 improvement >1.2) was significantly increased in patients carrying the IL-6 -174G/G genotype (95.7 %) compared with those with the G/C (75.6 %) or CC (44.4 %) genotype (p = 0.006 by Chi-square test). No significant difference in the mean values of DAS28 improvement was observed between groups with different genotype. RA patients with an IL-6 -174GG genotype respond to etanercept better than patients with GC or CC genotype. This finding, if confirmed in future studies, suggests that the -174G/C IL-6 polymorphism may be a genetic marker of responsiveness to tumor necrosis factor-alpha (TNF-α) blockers in RA.

Mutational status and gene repertoire of IGHV-IGHD-IGHJ rearrangements in Serbian patients with chronic lymphocytic leukemia.

UNLABELLED: The mutational status and configuration of immunoglobulin heavy variable (IGHV) gene rearrangements was analyzed in 85 Serbian patients with chronic lymphocytic leukemia (CLL). We found that 55.3% of cases belonged to mutated and 44.7% to unmutated CLL, progressive disease predominating in the unmutated subset. IGHV gene use resembled that obtained for Mediterranean countries, except for underrepresentation of the IGHV4 subgroup in our cohort. BACKGROUND: Chronic lymphocytic leukemia (CLL) results from the clonal expansion of mature B lymphocytes and is characterized by extreme clinical heterogeneity. One of the most reliable prognostic markers in chronic lymphocytic leukemia (CLL) is the mutational status of immunoglobulin heavy variable (IGHV) genes, which defines 2 subsets, mutated CLL (M-CLL) and unmutated CLL (U-CLL), with different clinical courses. Biased IGHV gene use between M-CLL and U-CLL clones, as well as population differences in the IGHV gene repertoire have been reported. PATIENTS AND METHODS: In this study, mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 85 Serbian patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. RESULTS: We found that 55.3% of cases belonged to M-CLL and 44.7% belonged to U-CLL, with progressive disease predominating in the unmutated subset. Most frequently expressed was the IGHV3 subgroup (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%), and IGHV6 (1.1%). The distribution of IGHD subgroups was as follows: IGHD3, 39.1%; IGHD2, 21.8%; IGHD6, 12.6%; IGHD1, 10.3%; IGHD4, 8%; IGHD5, 6.9%; and IGHD7, 1.1%. The most frequent IGHJ gene was IGHJ4 (48.9%), followed by IGHJ6 (28.4%), IGHJ3 (11.4%), and IGHJ5 (11.4%). In 15.3% of cases, heavy complementarity-determining region 3 (VH CDR3) amino acid sequences could be assigned to previously defined stereotyped clusters. CONCLUSIONS: Our study showed a strong correlation between IGHV gene mutational status and clinical course of CLL. IGHV gene use was comparable to that obtained for Mediterranean countries, with the exception of the IGHV4 subgroup, which was underrepresented in our cohort.