Association of IL-6 rs1800795, but not TNF-α rs1800629, and IL-1ß rs16944 polymorphisms' genotypes with recovery of ischemic stroke patients following thrombolysis.

OBJECTIVES: Inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 can cause brain injury, slow recovery, and adverse effects (ADEs) in ischemic stroke (IS) patients treated with recombinant tissue plasminogen activator (rtPA). We explored the relationship between selected polymorphisms within TNF-α, IL-1ß and IL-6 genes, and post-IS outcome and ADEs in patients treated with rtPA. METHODS: One hundred and sixty-six patients with IS treated with rtPA were included in this study. The modified Rankin Scale (mRS) was used to assess functional recovery 3 months after IS likewise thrombolytic therapy efficacy. Patients were classified into groups with favorable (0-1) or poor recovery based on their mRS score at the ninetieth day post-IS. During hospitalization, ADEs following rtPA were monitored. TNF-α-308 G/A (rs1800629), IL-1ß-511 G/A (rs16944), and IL-6-174 G/C (rs1800795) polymorphisms were genotyped using Real-Time PCR. SPSS software version 22.0 was used for statistical analyses. RESULTS: Patients with the TNF-α-308 G/A GG genotype had a higher mean NIHSS value at admission (12.75 ± 5.176) than those carrying A-allele (10.56 ± 3.979;p = 0.016). Individuals with the CC genotype of the IL-6-174 G/C polymorphism had significantly lower NIHSS scores (8.79 ± 5.053) than those with G-allele (12.06 ± 6.562) 24 hours after rtPA (p = 0.050). Patients with the GG genotype of the IL-6-174 G/C polymorphism had a significantly poorer outcome (p = 0.024; OR = 2.339; 95%CI 1.121-4.880), while patients who were G-allele carriers of the Il-6-174 G/C polymorphism and had the AA genotype of the IL-1ß-511 G/A polymorphism were statistically significantly more likely to experience hemorrhagic transformation (p = 0.046; OR = 2.7273; 95%CI 1.0414-7.1426). CONCLUSION: GG genotype of the IL-6-174 G/C polymorphism is associated with poor recovery after IS treated with rtPA therapy.

Association of PRDM16 rs12409277 and CtBP2 rs1561589 gene polymorphisms with lipid profile of adolescents.

Introduction: Positive regulatory domain containing 16 (PRDM16) protein represents the key regulator of brown adipose tissue (BAT) development. It induces brown fat phenotype and represses white adipose tissue specific genes through the association with C-terminal binding co-repressor proteins (CtBP1 and CtBP2). In healthy adults presence of BAT has been associated with lower glucose, total cholesterol and low-density lipoprotein (LDL) cholesterol levels. Our aim was to analyze the association of PRDM16 gene (rs12409277) and CtBP2 gene (rs1561589) polymorphisms with body mass index (BMI), fasting glucose level and lipid profile of adolescents. Material and methods: Our study included 295 healthy school children, 145 boys (49.2%) and 150 girls (50.8%), 15 years of age. Genotypes for the selected polymorphisms were detected by the real-time PCR method. Age, gender, height, weight, lipid profile (total cholesterol, high-density lipoprotein (HDL) cholesterol, LDL cholesterol, triglycerides) and fasting glucose levels were recorded. Results: We did not find a statistically significant association of rs12409277 and rs1561589 polymorphisms with BMI, fasting glucose and lipid profile of adolescents. We further analyzed the combined effect of the two SNPs and the statistical analysis showed that carriers of CT genotype of rs12409277 polymorphism and GG genotype of rs1561589 polymorphism had significantly lower total cholesterol (p = 0.001) and LDL cholesterol (p = 0.008) levels compared to all other groups of genotypes. Conclusions: Our study suggests that rs12409277 and rs1561589 polymorphism might have an influence on total and LDL cholesterol levels in adolescents. Larger studies should be performed in order to confirm our results.

Galectin 3 (LGALS3) Gene Polymorphisms Are Associated with Biochemical Parameters and Primary Disease in Patients with End-Stage Renal Disease in Serbian Population.

Galectin 3 plays a significant role in the development of chronic renal failure, particularly end-stage renal disease (ESRD). The aim of our study was to investigate the association between Gal-3 and biochemical parameters and primary disease in ESRD patients, by exploring the polymorphisms LGALS3 rs4644, rs4652, and rs11125. A total of 108 ESRD patients and 38 healthy controls were enrolled in the study. Genotyping of LGALS3 gene rs4644, rs4652, and rs11125 polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). By multivariate logistic regression analysis, we found that LGALS3 rs4644 CC and rs4652 AA genotypes were significantly associated with a higher risk for lower hemoglobin, higher level of parathyroid hormone, and also occurrence of diabetes mellitus and arterial hypertension. The CAA haplotype was significantly more common in patients with diabetes, low hemoglobin level, and normal PTH level. It has been observed as well that the ACT haplotype was more common in patients with low glomerular filtration, low PTH, and normal hemoglobin level. We found that the LGALS3 rs4644 and rs4652 gene polymorphism may be involved in the pathogenesis and appearance of complications in ESRD patients and thus could be considered a new genetic risk factor in this population.

Chromosomal microarray in postnatal diagnosis of congenital anomalies and neurodevelopmental disorders in Serbian patients.

BACKGROUND: Array-based genomic analysis is a gold standard for the detection of copy number variations (CNVs) as an important source of benign as well as pathogenic variations in humans. The introduction of chromosomal microarray (CMA) has led to a significant leap in diagnostics of genetically caused congenital malformations and neurodevelopmental disorders, with an average diagnostic yield of 15%. Here, we present our experience from a single laboratory perspective in four years' postnatal clinical CMA application. METHODS: DNA samples of 430 patients with congenital anomalies and/or neurodevelopmental disorders were analyzed by comparative genome hybridization using oligonucleotide-based microarray platforms. Interpretation of detected CNVs was performed according to current guidelines. The detection rate (DR) of clinically significant findings (pathogenic/likely pathogenic CNVs) was calculated for the whole cohort and isolated or combined phenotypic categories. RESULTS: A total of 140 non-benign CNVs were detected in 113/430 patients (26.5%). In 70 patients at least one CNV was considered clinically significant thus reaching a diagnostic yield of 16.3%. The more complex the phenotype, including developmental delay/intellectual disability (DD/ID) as a prevailing feature, the higher the DR of clinically significant CNVs is obtained. Isolated congenital anomalies had the lowest, while the "dysmorphism plus" category had the highest diagnostic yield. CONCLUSION: In our study, CMA proved to be a very useful method in the diagnosis of genetically caused congenital anomalies and neurodevelopmental disorders. DD/ID and dysmorphism stand out as important phenotypic features that significantly increase the diagnostic yield of the analysis.

Prevalence of five pharmacologically most important CYP2C9 and CYP2C19 allelic variants in the population from the Republic of Srpska in Bosnia and Herzegovina.

The enzymes of the cytochrome P450 superfamily play a critical role in phase I drug metabolism. Among them, CYP2C9 and CYP2C19 are clinically important, as they can mediate severe toxicity, therapy failure, and increased susceptibility to cancer and other diseases caused by chemicals. The aim of this study was to determine the prevalence of pharmacologically most important allelic variants of the CYP2C9 and CYP2C19 genes in the general population of the Republic of Srpska (Bosnia and Herzegovina) and to compare them with other populations. For this purpose we determined the genotype profile and allele frequency of 216 randomly selected healthy volunteers using real-time polymerase chain reaction (RT-PCR). The prevalence of the CYP2C9 *2 and *3 alleles was 13.6 and 7.4 %, respectively. Based on these frequencies, of the 216 participants four (1.86 %) were predicted to be poor metabolisers, 78 (36.11 %) intermediate, and the remaining 134 (62.03 %) normal metabolisers. Based on the prevalence of CYP2C19 *2 and *17 variants - 16.2 and 20.4 %, respectively - nine (4.17 %) were predicted to be poor, 57 (26.39 %) rapid, and nine (4.17 %) ultra-rapid metabolisers. We found no significant differences in allele frequencies in our population and populations from other European countries. These findings suggest that genetically determined phenotypes of CYP2C9 and CYP2C19 should be taken into consideration to minimise individual risk and improve benefits of drug therapy in the Republic of Srpska.

TT genotype of the MMP-9-1562C/T polymorphism may be a risk factor for thrombolytic therapy-induced hemorrhagic complications after acute ischemic stroke.

INTRODUCTION: Levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) influence recombinant tissue plasminogen activator (rtPA) therapy response in patients with acute ischemic stroke (AIS). Serum levels of MMPs and TIMPs along with the expression of genes coding these proteins are related to the recovery and appearance of adverse effects (AE) after AIS. Consequently, it is important to explore whether polymorphisms in regulatory sequences of MMPs and TIMPs are associated with rtPA response in AIS patients. OBJECTIVES: To determine whether selected polymorphic variants within MMP-2, MMP-9, and TIMP-2 genes may influence rtPA therapy response with regard to outcomes in patients with AIS and the occurrence of AE. METHODS: Our study included 166 patients suffering AIS, treated with rtPA. Patients' recovery was estimated using the Modified Rankin Scale (mRS) 3 months after the AIS occurred. Favorable outcome was defined with scores 0-1 and poor outcome with scores 2-6. Genotyping was performed using real-time PCR (rs243866, rs243865, rs243864, rs2277698, and rs8179090) and PCR-RFLP (rs2285053, rs3918242) methods. Additionally, rtPA AE were followed during the hospitalization. RESULTS: There was no significant association between genotypes and alleles of selected polymorphisms and rtPA therapy response measured through the decrease of the mRS score in patients with AIS. Intracranial hemorrhage, as well as parenchymal hematoma type 2, was significantly more frequent in patients with TT genotype of the MMP-9-1562C/T polymorphism (p = 0.047, p = 0.011, respectively). Patients with intracranial hemorrhages after rtPA were significantly more likely to have the TT genotype of TIMP-2-303C/T polymorphism and the TT genotype of MMP-9-1562C/T polymorphism (p < 0.001). CONCLUSION: TT genotype of the MMP-9-1562C/T polymorphism may be a risk factor for rtPA-induced hemorrhagic complications after AIS.

Premutations in the FMR1 gene in Serbian patients with undetermined tremor, ataxia and parkinsonism.

Introduction: Although one of the most common monogenic late-onset neurodegenerative disorders, fragile-X-associated tremor/ataxia syndrome (FXTAS) is still underdiagnosed. The aim of the present study was to estimate the frequency of premutation carriers in patients with unexplained degenerative ataxias, action tremor or parkinsonism, and action tremor with or without associated cognitive impairment.Methods: The study comprised 100 consecutive patients with the disease onset >49 years who had any form of unexplained action tremor, cerebellar ataxia, followed by parkinsonism with or without incipient dementia, and in whom the FMR1 repeats size was determined.Results: Premutation in the FMR1 was identified in two patients (2%): the first, male patient had 83 CGG repeats and the second, female patient had 32 and 58 CGG repeats.Discussion/Conclusion: FXTAS was relatively rare among older patients with unexplained ataxia and action tremor, with or without parkinsonism and/or cognitive impairment. Tremor and ataxia were major clinical features in our two patients, although parkinsonism, autonomic dysfunction and psychiatric problems might be an important part of the spectrum. Probable FXTAS should be considered in the differential diagnosis of patients with unexplained action tremor and ataxia, and undetermined parkinsonism, especially when there was a positive family history for involuntary movement disorders in other family members and/or autism spectrum disorders in younger cousins.

Analysis of association of ADORA2A and ADORA3 polymorphisms genotypes/haplotypes with efficacy and toxicity of methotrexate in patients with Rheumatoid arthritis.

Adenosine receptors ADORA2A and ADORA3 are part of the adenosine-mediated antiinflammatory pathway and are overexpressed in patients with Rheumatoid arthritis (RA). Methotrexate (MTX) antiinflammatory effects are partially mediated via increased release of adenosine into extracellular space. Polymorphisms in ADORA2A and ADORA3 genes may have an impact on the efficacy and toxicity of MTX in RA patients. The study included 127 RA patients. Treatment efficacy was estimated using the changes in Disease activity score (DAS28) after 6 months of MTX monotherapy, according to EULAR response criteria. Patients with good and moderate response were classified as "responders", and with a poor response as "nonresponders". Adverse effects were collected during the follow-up period. Genotyping for polymorphisms within ADORA2A gene (rs2298383, rs2236624, rs5751876, rs17004921) and ADORA3 gene (rs2298191, rs1544223, rs3393) was performed using the KASPar assays. Among patients 112 (88.19%) were responders (18.8% good, 81.2% moderate). We observed no association between analyzed genotypes or alleles and MTX response by EULAR criteria but carriers of ADORA2A rs17004921 T allele (CT + TT) had a higher DAS28 decrease after 6 months of treatment than patients with CC genotype (p = 0.013). Adverse effects were reported in 31 patients (24.41%). Bone erosions were present in 82 (64.6%) patients. Haplotype block was observed among all 3 analyzed polymorphisms within ADORA3 gene and TAA haplotype was associated with bone erosions (29% vs 15.6%, p = 0.023) and hepatotoxicity (51.3% vs 21.6%, p = 0.013). According to our study, ADORA3 TAA haplotype may be associated with bone erosions and hepatotoxicity in RA patients treated with MTX.

Genetic variant rs16944 in IL1B gene is a risk factor for early-onset sepsis susceptibility and outcome in preterm infants.

OBJECTIVE: Interleukin-1-B (IL1B) is a proinflammatory cytokine that plays an important role in sepsis. The aim of this study was to evaluate the relationships between IL1B-511G/A polymorphism and susceptibility and outcome of early-onset sepsis (EOS) in preterm infants. METHODS: DNA was extracted from the buccal swabs of 471 (285 with EOS and 186 control) preterm infants. Genotypes of rs16944 polymorphism were determined with real-time PCR method. RESULTS: We found statistically significant higher frequency of IL1B-511AA genotype in EOS group than in control group (p = 0.012). Also, IL1B-511AA genotype is statistically significantly more frequent in patients with lethal EOS outcome (p = 0.011). CONCLUSION: Genotype IL1B-511AA was associated with susceptibility to EOS and it is a significant predictor of lethal outcome in preterm infants with EOS.

Methotrexate pharmacogenetics in the treatment of rheumatoid arthritis.

For many decades, methotrexate (MXT) has remained the drug of choice in the treatment of rheumatoid arthritis (RA). Unfortunately, a considerable number of patients do not achieve an appropriate therapeutic response. Pharmacogenetics studies do not give usable results regarding differences in MTX response among RA patients. The mechanism of MTX action in RA is not completely understood. We present and discuss data regarding the molecular basis of folate and adenosine pathways, the most obvious MTX targets, to explain possible causes of therapy failure. The molecular basis of the disease could also have an impact on therapy outcomes and in this review we explore this. Finally, we make a short review of available pharmacogenetics study results.

High-quality draft genome sequence of Pseudomonas aeruginosa san ai, an environmental isolate resistant to heavy metals.

The strain Pseudomonas aeruginosa san ai, isolated from an extreme environment (industrial mineral cutting oil, pH 10), is able to survive and persist in the presence of a variety of pollutants such as heavy metals and organic chemicals. The genome of P. aeruginosa san ai is 6.98 Mbp long with a GC content of 66.08% and 6485 protein encoding genes. A large number of genes associated with proteins, responsible for microbial resistance to heavy metal ions and involved in catabolism of toxic aromatic organic compounds were identified. P. aeruginosa san ai is a highly cadmium-resistant strain. Proteome analysis of biomass after cadmium exposal confirmed a high tolerance to sublethal concentrations of cadmium (100 mg/L), based on: extracellular biosorption, bioaccumulation, biofilm formation, controlled siderophore production and a pronounced metalloprotein synthesis. Proteins responsible for survival in osmostress conditions during exposure to elevated concentrations of cadmium (200 mg/L) demonstrate a strong genetic potential of P. aeruginosa san ai for survival and adaptation. Sequencing of P. aeruginosa san ai genome provides valuable insights into the evolution and adaptation of this microbe to environmental extremes at the whole-genome level, as well as how to optimally use the strain in bioremediation of chemically polluted sites.

Analysis of the Association Between Polymorphisms within PAI-1 and ACE genes and Ischemic Stroke Outcome After rt-PA Therapy.

PURPOSE: Treatment of Ischemic stroke (IS) in acute phase is based on the use of thrombolytic rt-PA therapy. We aimed to determine whether different alleles and genotypes of I/D ACE gene and 4G/5G PAI-1 gene polymorphisms may influence outcome of rt-PA therapy in patients with IS and the occurrence of haemorrhagic transformation (HT). METHODS: Our study included 94 consecutive patients with IS treated with rt-PA. Modified Rankin Scale (mRS) at 3rd month after IS was used to determine the stroke outcome, with scores 0-1 defining the favourable outcome, and scores 2-6 defining poor outcome. Genotypisation of the ACE-1 I/D polymorphism was performed by polymerase chain reaction and of the PAI-1 4G/5G polymorphism by polymerase chain reaction - restriction fragment length analysis. RESULTS: Regarding PAI-I 4G/5G polymorphism, 44 patients (46.8%) were heterozygotes, and the number of 4G/4G and 5G/5G homozygotes was the same - 25 each (26.6%). Number of heterozygotes for the ACE I/D polymorphism was 54 (57.4%), 9 patients (9.6%) had II, and 31 (33%) DD genotypes. A favourable outcome was recorded in 26 (28.0%) and the poor outcome in 67 (72.0%) patients. Favourable and poor outcome groups did not differ significantly in PAI-1 4G/5G and ACE I/D polymorphisms genotype or allele frequencies. There was a statistically significant difference in the occurrence of HT between patients with ACE II and patients with ACE ID or DD genotypes (p=0.035). CONCLUSION: Results of our study suggest that stroke patients with ACE II genotype, treated with rt-PA, may be at risk of HT.

Association between Tumor Necrosis Factor-α Promoter -308 G/A Polymorphism and Early Onset Sepsis in Preterm Infants.

Early-onset neonatal sepsis (EOS) is diagnosed during the first 7 days of neonatal life and is the major cause of morbidity and mortality among preterm infants. Genetic predisposition may have an impact on EOS susceptibility and outcome. The aim of our study was to explore the association between TNF-α -308 G/A or IL-6 -174 G/C gene polymorphism and the susceptibility and outcome of EOS in preterm infants. The study included 471 preterm infants: 282 with EOS (151 with culture proven sepsis and 131 with clinical sepsis) and 189 without infection (control group). TNF-α -308 G/A and IL-6 -174 G/C were genotyped using Real-time RCR method. We observed significantly higher frequency of A allele of TNF-α -308 G/A polymorphism in blood culture proven EOS (p = 0.017) or clinical EOS (p = 0.025) compared with the control group. Logistic regression confirmed significant association between TNF-α -308 GA+AA genotypes and development of culture proven EOS (B = -0.718, p = 0.013) or clinical EOS (B = -0.602, p = 0.027). No significant differences in IL6 -174G/C alleles or genotypes distribution have been observed between culture proven EOS group, clinical EOS group and the control group. An association between TNF-α -308 G/A or IL-6 -174 G/C genotypes and EOS lethal outcome was not observed (p = 0.652 and p = 0.384, respectively). According to our analysis of large cohort of preterm infants with clearly defined EOS groups, the TNF-α -308 A allele may be a risk factor for the EOS occurrence.

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.

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.

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.

Nonsense mutations in FAM161A cause RP28-associated recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a degenerative disease of the retina leading to progressive loss of vision and, in many instances, to legal blindness at the end stage. The RP28 locus was assigned in 1999 to the short arm of chromosome 2 by homozygosity mapping in a large Indian family segregating autosomal-recessive RP (arRP). Following a combined approach of chromatin immunoprecipitation and parallel sequencing of genomic DNA, we identified a gene, FAM161A, which was shown to carry a homozygous nonsense mutation (p.Arg229X) in patients from the original RP28 pedigree. Another homozygous FAM161A stop mutation (p.Arg437X) was detected in three subjects from a cohort of 118 apparently unrelated German RP patients. Age at disease onset in these patients was in the second to third decade, with severe visual handicap in the fifth decade and legal blindness in the sixth to seventh decades. FAM161A is a phylogenetically conserved gene, expressed in the retina at relatively high levels and encoding a putative 76 kDa protein of unknown function. In the mouse retina, Fam161a mRNA is developmentally regulated and controlled by the transcription factor Crx, as demonstrated by chromatin immunoprecipitation and organotypic reporter assays on explanted retinas. Fam161a protein localizes to photoreceptor cells during development, and in adult animals it is present in the inner segment as well as the outer plexiform layer of the retina, the synaptic interface between photoreceptors and their efferent neurons. Taken together, our data indicate that null mutations in FAM161A are responsible for the RP28-associated arRP.