Association of C35T polymorphism in dihydrofolate reductase gene with toxicity of methotrexate in rheumatoid arthritis patients.

BACKGROUND: Methotrexate (MTX), a folate analogue, is the most commonly used disease-modifying drug in the treatment of rheumatoid arthritis (RA). However, high interindividual differences in drug response are present among RA patients. RESEARCH DESIGN AND METHODS: In a group of 234 RA patients treated with MTX, we investigated whether rs1650697 polymorphism in DHFR gene may have an impact on MTX efficacy and/or adverse drug effects (ADEs). Relative DAS28 values (rDAS28) were used for the estimation of MTX therapy and all ADEs were recorded. Patients were genotyped for selected polymorphism by real-time PCR method. RESULTS: According to the European League Against Rheumatism criteria after 6 months of MTX therapy, 196 patients (83.8%) were classified as responders (25 (10.7%) were good and 171 (73.1%) were moderate) and 38 patients (16.2%) as nonresponders. ADEs were observed in 55 patients (23.5%). CONCLUSIONS: Our results showed that the presence of T allele might be protective against MTX hepatotoxicity measured by transaminase levels (p = 0.05). Furthermore, among patients who also received low-dose corticosteroids, we have found a lower rDAS value in patients with CC genotype (p = 0.039).

Association of 63/91 length polymorphism in the DHFR gene major promoter with toxicity of methotrexate in patients with rheumatoid arthritis.

AIM: Our aim was to explore the influence of 9-bp insertion/deletion and variable number of 9 bp elements (63/91) length polymorphism in noncoding interfering RNA and major promoter of DHFR gene on methotrexate (MTX) efficacy and toxicity in patients with rheumatoid arthritis (RA). PATIENTS & METHODS: Response to the MTX therapy and adverse effects were estimated in 243 RA patients genotyped for the selected polymorphism. RESULTS: The presence of allele 1 of analyzed polymorphism had significant protective effect against MTX toxicity (odds ratio: 0.37 [95% CI: 0.19-0.70]; p = 0.002). Results remained significant in multiple logistic regression analysis with the inclusion of disease and treatment features in the model (p = 0.03). CONCLUSION: Polymorphism 63/91 in DHFR gene promoter can modulate the onset of MTX-related adverse effects in RA patients.

Evaluation of cytokine genetic polymorphisms in adult patients with common variable immunodeficiency: A single-center study.

Common variable immunodeficiency (CVID) is a heterogeneous disease characterized by impaired B-cell differentiation and maturation accompanied with the defective antibody production. Several investigators addressed the possibility that disturbed cytokine production of TNF, IL-6, IFN-γ and IL-10, among a variety of others, may be implicated in CVID. The aim of this study was to test the hypothesis that genetic polymorphisms involving TNF (-308G/A), IFNG (+874 T/A), IL10 (-1082G/A, -819T/C and -592A/C), and IL6 (-174G/C) cytokine genes might contribute to susceptibility to CVID. Thirty five patients with CVID and 250 healthy controls were genotyped for indicated single nucleotide polymorphisms (SNP) in TNF, IL6, IFNG and IL10 using Taqman-based assays. CVID patients had significantly higher frequency of TNF A allele and AA genotype than in healthy subjects (p=0.006; OR=2.27; 95%CI=1.24-4.17 and p=0.038, OR=15.64; 95%CI=1.38-177.20, respectively). In addition, the frequency of GG genotype was significantly higher in healthy controls than in patient group (p=0.019, OR=0.43, 95%CI=0.21-0.89). Genetic analysis of IL6 SNP showed that allele G confers increased risk for CVID (p=0.037, OR=1.78, 95% CI=1.03-3.08) while IFNG allele T was associated with splenomegaly in CVID (p=0.032; OR=2.86; 95% CI=1.08-7.56). We observed no association between genotypes, alleles and haplotypes of IL-10 gene and CVID or its clinical complications. In conclusion, our results indicated association between CVID and cytokine gene polymorphisms -308G/A TNF and -174G/C IL6. In addition, we demonstrated that splenomegaly, one of the most common complications in this disease, is associated with +874T/A IFNG polymorphism. These findings add further support to the notion that cytokines may play significant role in pathogenesis of this primary antibody deficiency. However, further investigation that would involve a larger study group of CVID patients is warranted to confirm our findings.

Polymorphisms of the eNOS gene are associated with disease activity in rheumatoid arthritis.

Nitric oxide (NO) is a mediator in autoimmune responses and thus involved in the pathogenesis of a variety of rheumatic diseases. Genetic factors that influence the expression of the enzyme endothelial nitric oxide synthase (eNOS) that catalyzes NO synthesis are important for the control of NO level and consequently its activity. We have analyzed three functionally relevant polymorphisms of eNOS gene: T-786C, G894T and VNTR (4a/b), to investigate whether they are predisposing factors in pathogenesis of RA in Serbian population and to evaluate their role in clinical manifestations of RA. We performed genotyping of 196 patients with RA and the control group of 132 healthy individuals from Serbian population, using PCR and polymerase chain reaction-restriction fragment length polymorphism methods. Disease activity was prospectively assessed using number of tender joints, number of swollen joints and 28-joints disease activity score (DAS28). There were no differences between the patients and control groups in the genotypes and alleles frequencies of the three analyzed SNPs. Our results showed statistically significant differences in all three analyzed parameters of disease severity between 786TT/786CT and 786CC genotypes and between 894GG/894GT and 894TT genotypes. In the case of 4a/b polymorphism, carriers of minor allele had significantly lower DAS28 values. In conclusion, our results do not support the implication of analyzed eNOS gene polymorphisms in susceptibility to RA but associate them with the disease activity and give assumption that minor alleles are indicators of better clinical course.

Subtelomeric screening in Serbian children with dysmorphic features and unexplained developmental delay/intellectual disabilities.

Developmental delay and intellectual disabilities (DD/ID) are significant health problems affecting 3% of the human population. Submicroscopic chromosomal rearrangements involving subtelomeric regions are often considered to be the cause of unexplained DD/ID. Screening of subtelomeric regions was performed in 80 unrelated patients with DD/ID and normal GTG-banded chromosomes using the MLPA method with two kits (SALSA P070-B1 and P036-E1). The MLPA screening revealed subtelomeric chromosome aberrations in four cases (5%). The aberrations detected were: 1p deletion, 1p deletion combined with 12q duplication, 4p deletion, and 9p deletion combined with 15q duplication. The deletions detected were classified as causative for the patients' observed phenotypes. This study confirms the high frequency of subtelomeric rearrangements in unexplained DD/ID and reinforces the argument for routine subtelomeric screening in order to get a correct diagnosis, establish genotype-phenotype correlations and offer accurate genetic counseling.

Could spindle cell lung carcinoma be considered and treated as sarcoma, according to its clinical course, morphology, immunophenotype and genetic finding?

The actual nature of spindle cell carcinoma has been debated extensively because of its rarity. It carries a poor prognosis, even when early-stage disease is diagnosed and resected. In view of the rarity and the significance of the histological diagnosis, we report a patient with rapidly progressing spindle cell lung carcinoma with soft tissue metastasis. Diagnosis was confirmed by immunohistochemistry finding. Analysis of the TP53 gene mutations by polymerase chain reaction and DNA sequencing revealed insertion of single thymine resulting in frameshift mutation in the exon 8. Prognosis of spindle cell lung carcinoma might be determined by the sarcoma component of the tumor and, based on that, we wonder if this type of lung carcinoma could be followed-up and treated by strategies for soft tissue sarcomas, because of its rapid, sarcomatous type of growth, beside the properly lung carcinoma oncological treatment.

Association of the TYMS 3G/3G genotype with poor response and GGH 354GG genotype with the bone marrow toxicity of the methotrexate in RA patients.

PURPOSE: Gamma-glutamyl hydrolase (GGH), cyclin D1 (CCND1) and thymidylate synthase (TS) genes encode enzymes that are involved in methotrexate (MTX) action. In a group of 184 RA patients treated with MTX, we have investigated whether selected polymorphisms in these genes modulate MTX efficacy and/or have impact on adverse drug effects (ADEs). METHODS: The efficacy of the MTX therapy has been estimated using the disease activity score in 28 joints (DAS28-ESR) based on EULAR criteria and relative DAS28 values (rDAS28). All adverse drug events were recorded. Patients were genotyped for selected polymorphisms of the GGH (-354 G > T and 452 C > T), CCND1 (870 A > G) and TYMS (variable number of tandem repeats, VNTR, and G to C substitution of triple repeat, 3R allele) gene. Association studies have been performed between obtained genotypes and the efficacy and toxicity of MTX. RESULTS: According to the EULAR response criteria, 146 RA patients (79.3 %) were classified as responders (good/moderate response) and 38 (20.7 %) as non-responders (poor response). Higher frequency of the TYMS 3 G/3 G genotype has been found among non-responders as compared to individuals with remaining genotypes (p = 0.02). ADEs were recorded in 53 patients. Among those patients eight experienced bone marrow toxicity, all of them carried GGH -354GG genotype (p = 0.003). No other significant association were observed. CONCLUSION: The 3 G/3 G genotype of the TYMS gene may indicate predisposition of poor response to MTX and GG genotype of GGH -354 T > G polymorphism may have high predictive value for myelosuppression in RA patients.

Association of dihydrofolate reductase (DHFR) -317AA genotype with poor response to methotrexate in patients with rheumatoid arthritis.

OBJECTIVES: Identifying genetic predictors of methotrexate (MTX) treatment response in patients with rheumatoid arthritis (RA) may have great importance for optimising drug doses required for clinical benefit without toxicity. In a group of 125 RA patients treated with MTX we investigated whether selected polymorphisms in genes relevant for MTX action (aminoimidazole-4-carboxiamide ribonucleotide transformylase, ATIC, and dihydrofolate reductase, DHFR) modulate disease activity and/or have impact on therapy side effects. METHODS: The efficacy of treatment was estimated both by the disease activity score in 28 joints (DAS28), based on EULAR criteria, and relative DAS28 (rDAS28) score. Adverse drug events (ADEs) were also recorded. RA patients were genotyped using the PCR-RFLP method, followed by an association study between ATIC -129T>G, DHFR -216T>C and DHFR -317A>G polymorphisms and the efficacy and toxicity of MTX. RESULTS: According to the EULAR response criteria, 96 RA patients (76.8%) were classified as responders (good/moderate response) and 29 (23.2%) as non-responders (poor response). rDAS28 values ranged from -0.01 to 0.80 (mean value 0.31±0.19). Among 125 patients enrolled in this study 39 experienced at least one side effect. The DHFR -317AA genotype was associated with the less favourable response (reduction in rDAS28 score, p=0.05). None of the analysed polymorphisms was associated with MTX toxicity. CONCLUSIONS: RA patients with DHFR-317AA genotype had less favourable response to MTX. Further studies in larger patient populations are necessary to confirm the relationship between the analysed polymorphisms and MTX treatment response.

Aberrant promoter methylation of CDH13 and MGMT genes is associated with clinicopathologic characteristics of primary non-small-cell lung carcinoma.

UNLABELLED: Non­small-cell lung carcinoma (NSCLC) (n = 65) were analyzed for promoter methylation of RASSF1A, CDH13, MGMT, ESR1, and DAPK genes in matching lung tumors, normal lung tissue, and blood samples. Aberrant methylation in CDH13 and MGMT was associated with clinicopathologic features of NSCLC. Hypermethylation detected in primary tumors was not observed in corresponding blood samples, which rendered this an unsuitable blood-based test for NSCLC detection. INTRODUCTION: Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue and whether DNA methylation changes can be detected in paired blood samples. MATERIAL AND METHODS: Sixty-five patients were enrolled in a surgical case series of non-small-cell lung carcinoma at a single institution. By using bisulfite pyrosequencing, CpG methylation was quantified at 5 genes (RASSF1A, CDH13, MGMT, ESR1, and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases. RESULTS: The analyses of methylation in tumors compared with normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, whereas ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the 3 aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, which indicated a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly, we probed whether tumor methylation was associated with clinical and demographic characteristics. Histology and sex were associated with methylation at the CDH13 gene, whereas, stage was associated with methylation at MGMT. CONCLUSION: Our results showed higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared with normal lung. The lack of reflection of these methylation changes in blood samples from patients with non-small-cell lung carcinoma indicates their poor suitability for a screening test.

Synchronous primary lung cancers: a multidisciplinary approach in diagnosis.

UNLABELLED: BACKGROUND. For patients with two or more primary cancers a correct diagnosis is critically important because prognosis and treatment vary considerably between multiple primary cancers and metastatic disease. CASE REPORT: Two bilateral synchronous primary lung malignancies of different histological types were diagnosed and immunohistochemically confirmed in a 60-year-old woman. In biopsy specimens of the right lung pure squamous cell carcinoma was detected (stage IIIa). The tumor expressed AE1/AE3, cytokeratin 5 and 34ßE12. In biopsy specimens of the left lung small cell carcinoma was detected (stage IIIa). The small cells expressed synaptophysin, chromogranin A and CD56. DNA was extracted from paraffin-embedded tissue of both tumors. Exons 5-9 of the TP53 gene were examined for genetic mutations by polymerase chain reaction and DNA sequencing analysis. Direct sequencing of DNA isolated from the small cell carcinoma revealed a TGC to TTC mutation at codon 404 of TP53 exon 5. In DNA isolated from the squamous cell carcinoma no TP53 mutation was found. The tumors' different response to chemotherapy also suggested that they belonged to different histological types. The patient lived 24 months after the diagnosis, which is more typical for stage III than for stage IV lung carcinoma. CONCLUSION: Discrimination of synchronous primary lung cancers from intrapulmonary metastases based only on clinical findings can be very difficult. Multidisciplinary diagnostic evaluation is therefore very helpful in cases like this, because a correct diagnosis will determine the best treatment for the patient and consequently a better prognosis.

Large cell lung carcinoma with unusual imaging feature, immunophenotype and genetic finding.

We present a case of large cell lung carcinoma in sixty-one year old male with typical lung cancer symptoms but unusual radiological presentation and immunophenotype. Tumor morphological finding related to its radiological finding was suggestive for large cell lymphoma or carcinoma, but its immunophenotype made confusion for pathological diagnosis. No p53 mutations were detected in genetic investigation. Multidisciplinary diagnostic approach to some tumors is useful for their final diagnosis.

Association between the methylenetetrahydrofolate reductase polymorphisms and risk of acute lymphoblastic leukemia in Serbian children.

SUMMARY: Methylenetetrahydrofolate reductase (MTHFR) regulates the metabolism of folate and methionine, essential components of DNA synthesis and methylation. Polymorphisms in the MTHFR gene have been associated with susceptibility to some types of cancer. We investigated a possible association of MTHFR polymorphisms (677C>T and 1298A>C) and increased risk for acute lymphoblastic leukemia in 78 affected children. The frequencies of both MTHFR 677 genotypes and alleles were significantly different between patients and controls. A significant association between CT/TT individuals and reduced risk of acute lymphoblastic leukemia was found. The odds ratios were 0.53 (95% confidence interval, 032-0.89) and 0.30 (95% confidence interval, 0.12-0.81). Polymorphism 1298 did not show statistical difference between patients and controls.

Degree of genetic homozygosity and distribution of AB0 blood types among patients with spina bifida occulta and spina bifida aperta.

INTRODUCTION: Assuming that spina bifida (SB) is a genetically controlled disease, the aim of our study was to evaluate the degree of genetic homozygosity and the distribution of AB0 blood types among patients with SB occulta and SB aperta by the homozygously recessive characteristics (HRC) test. MATERIAL AND METHODS: Our study included an analysis of the presence, distribution and individual combination of 15 selected genetically controlled morpho-physiological traits in a sample of 100 patients with SB (SB occulta N = 50 and SB aperta N = 50) and a control group of individuals (N = 100). RESULTS: We found a statistically significant difference between the mean values for genetic homozygosity (SB 4.5 ±0.3; control 3.0 ±0.2, p < 0.001) and also differences in the presence of certain individual combinations of such traits. In 12 (80.0%) of the 15 observed characteristics, recessive homozygosity was expressed to a greater degree among the group of SB patients, while for 9 (60.0%) of the traits this level of difference was statistically significant (Σ(χ) (2) = 266.3, p < 0.001). There was no difference in average homozygosity of such genetic markers between groups of SB occulta and SB aperta patients, but the type of individual variation in the two studied groups significantly differed. In the group of patients with SB the frequency of 0 blood group was significantly increased while B blood group was significantly decreased. CONCLUSIONS: Our results clearly show that there is a populational genetic difference in the degree of genetic homozygosity and variability between the group of patients with SB and individuals without clinical manifestations, indicating a possible genetic component in the aetiopathogenesis of spina bifida.

Single institute study of FLT3 mutation in acute myeloid leukemia with near tetraploidy in Serbia.

Patients with de novo acute myeloid leukemia (AML) and near-tetraploid or completely tetraploid karyotype at presentation are rare. We present four patients with near-tetraploidy/tetraploidy in a cohort of 426 consecutive AML patients (0.98%) in respect to their cytogenetic findings, immunophenotype pattern, response to chemotherapy, course of disease and molecular analyses including tyrosine kinase receptor FLT3 gene, NRAS gene, and tumour suppressor gene, p53. We have found FLT3/ITD mutation only in one patient among the four with near-tetraploidy. The main finding is that these patients had a variable clinical course, with two having a long period of remission (36 and 12 months) and two died, not having achieved remission.

[Y chromosome microdeletions in infertile male candidates for microfertilization].

INTRODUCTION: Y chromosome microdeletions are the second most frequent genetic cause of male infertility after Klinefelter's syndrome. OBJECTIVE: The aim of the study was to determine the frequency ofY chromosome microdeletions in a group of infertile men with an idiophatic cause of infertility, candidates for microfertilization (Intra-cytoplasmic Sperm Injection--ICSI) in Serbia and to correlate genotype-phenotype in patients with Y chromosome microdeletions. METHOD: One hundred and sixty patients with low sperm count (less than 5 x 10(6) spermatozoa/ml) were enrolled in the study. Forty patients were excluded from the study: ten because they were diagnosed with cytogenetic abnormality and thirty patients were diagnosed with other known causes of infertility. The control group consisted of 150 men who fathered at least one child in the last two years. Genomic DNA was extracted from peripheral blood samples and two multiplex polymerase chain reactions (PCR) analyses were performed using specific primers to confirm the presence or absence of Y chromosome microdeletions. RESULTS: Microdeletions were detected in 12 of 120 (10%) cases, while no deletions were detected in the control group. Of total number of 12 deletions, nine were detected in AZFc region (75%), one in AZFa (8%), and two in AZFbc (17%). CONCLUSION: Testing for Y chromosome microdeletions should be considered as an important element in diagnosis and genetic counselling of infertile couples in Serbia. Decisions regarding the assisted reproduction should be made based on the detailed clinical, endocrinological and cytogenetic examinations, spermogram, presence or absence and type of AZF microdeletions and CFTR gene mutations.

New RB1 oncogenic mutations and intronic polymorphisms in Serbian retinoblastoma patients: genetic counseling implications.

The purpose of this work was to identify germ line RB1 mutations in 16 Serbian retinoblastoma patients for genetic counselling. Mutation analysis was carried out by PCR directed sequencing of the 27 exons. Loss of heterozygosity for two RB1 intragenic markers was also analyzed in 14 tumour samples. Five new RB1 oncogenic mutations (g.2078 del C, g.77047_48 del GC, g.78117_8 del TT, g.160797 del T, and g.64439+2 T>C) and two recurrences (R445X and Q383X) have been found in this study. In addition, four intronic variants were observed germ line in some unilateral patients. Two of these variants (g.44668-15T/G, and g.166204-8T/A) are discussed as potential oncogenic mutation candidates. The results show the relevance of studies aimed to investigate the role of intronic variants in exon splicing regulation. Such studies will help to disclose hidden retinoblastoma susceptibilities, important for accurate genetic counselling.

Lack of TP53 and FMS gene mutations in children with myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) are rare disorders in children. Molecular mechanisms underlying MDS in children are not yet completely understood. Considering the role of FMS and TP53 gene mutations in adult MDS patients, we analyzed mutations of these genes in a cohort of 35 children with MDS. Single-strand conformation polymorphism polymerase chain reaction analysis performed on FMS codon 969 and TP53 exons 5-9 showed no mutations in the analyzed sequences. Our results suggest that molecular mechanisms of MDS evolution in children are different from those in adults.

Low frequency of NRAS and KRAS2 gene mutations in childhood myelodysplastic syndromes.

In children, myelodysplastic syndromes (MDS) represent less then 10% of all hematological malignancies; consequently, molecular genetic studies dealing with this group of patients are scarce. We have analyzed 35 archival bone marrow samples of children with MDS for the presence of mutations in the first and second exons of the NRAS and KRAS2 genes. Mutations were detected with single-strand conformation polymorphism analysis in three patients. One patient harbored a mutation in the second exon of NRAS and two patients in the second exon of KRAS2. Sequencing was performed in two samples and novel mutations were found in both. One patient had a missense mutation in codon 45 of NRAS; the other had a silent mutation in codon 53 and a missense mutation in codon 55 of KRAS2.