Danuta Januszkiewicz-Lewandowska

A coding variant in NLRP1 is associated with autoimmune Addison's disease.

Autoimmune Addisons disease (AAD) is a complex disorder with several susceptibility loci. Variations in the NLRP1 (previously, NALP1) gene have recently been reported to confer risk for vitiligo and associated autoimmune conditions. We hypothesized that polymorphisms in this gene may affect susceptibility to AAD. The aim of this study was to analyze the associations of six NLRP1 single-nucleotide polymorphisms (SNPs) with AAD within a Polish cohort. The study comprised 101 AAD patients and 254 healthy control individuals. Genotyping was performed by polymerase chain reaction followed by restriction fragment length polymorphism and single strand conformation polymorphism methods. The minor allele of the coding SNP rs12150220 appeared significantly more frequently in AAD compared with healthy individuals (OR = 1.5, 95% CI, 1.08-2.08, p = 0.015). The distribution of genotypes also demonstrated significant differences. The frequency of high-risk genotype AA of rs12150220 SNP was significantly increased among AAD subjects versus controls (p = 0.006 and p = 0.036, respectively; significant after Bonferroni correction), yielding an OR of 2.96 (95% CI, 1.34-6.55). Likewise, the heterozygous genotype TA was observed more frequently in the patient group [OR = 3.09 (95% CI, 1.53-6.24), p = 0.001 and p = 0.006 after Bonferroni correction]. In conclusion, this study confirms an association between the coding polymorphism in NLRP1 and AAD.

PTPN22, PDCD1 and CYP27B1 polymorphisms and susceptibility to type 1 diabetes in Polish patients

Type 1 diabetes (T1DM) is a common autoimmune disease with a complex genetic background. This study was aimed to investigate the association of PTPN22 G(‐1123)C and C1858T, PDCD1 G7146A and CYP27B1 C(‐1260)A polymorphisms with T1DM among Polish subjects. The PTPN22 gene encodes lymphoid tyrosine phosphatase, a potent negative regulator of T cell activation. PDCD1 gene gives rise to an inhibitory cell‐surface receptor, expressed on activated lymphocytes. CYP27B1 encodes 1‐alpha hydroxylase, responsible for conversion of the vitamin D3 precursor into its active form, involved in the immune function. Polymorphic variants of these genes have previously been associated with various autoimmune disorders. The four polymorphisms were genotyped by PCR‐restriction fragment assays in a case–control study comprising 215 T1DM patients and 236 healthy controls. The PTPN22 T1858 allele appeared significantly increased in T1DM compared to the control group (P = 0.004), yielding an OR of 1.73 (95% CI 1.19–2.51). The difference in distribution of C1858T genotypes also demonstrated statistical significance (P = 0.015). The frequencies of PTPN22 G(‐1123)C alleles and genotypes did not differ between T1DM cases and controls, although the haplotype comprising both mutant PTPN22 alleles, C(‐1123) and T1858, was significantly more frequent in affected individuals (P = 0.003). G(‐1123)C and C1858T were in linkage disequilibrium (D′ = 0.98; r2 = 0.61 in T1DM and D′ = 0.97; r2 = 0.41 in controls). No significant differences in the allele and genotype frequencies of PDCD1 and CYP27B1 polymorphisms were found between patients and controls. This study confirms the association of PTPN22 C1858T polymorphism with T1DM, whereas the effects of PTPN22 G(‐1123)C, PDCD1 G7146A and CYP27B1 C(‐1260)A seem unlikely, at least in the Polish population.

Polymorphisms in microRNA target sites modulate risk of lymphoblastic and myeloid leukemias and affect microRNA binding

BackgroundMicroRNA dysregulation is a common event in leukemia. Polymorphisms in microRNA-binding sites (miRSNPs) in target genes may alter the strength of microRNA interaction with target transcripts thereby affecting protein levels. In this study we aimed at identifying miRSNPs associated with leukemia risk and assessing impact of these miRSNPs on miRNA binding to target transcripts.MethodsWe analyzed with specialized algorithms the 3′ untranslated regions of 137 leukemia-associated genes and identified 111 putative miRSNPs, of which 10 were chosen for further investigation. We genotyped patients with acute myeloid leukemia (AML, n = 87), chronic myeloid leukemia (CML, n = 140), childhood acute lymphoblastic leukemia (ALL, n = 101) and healthy controls (n = 471). Association between SNPs and leukemia risk was calculated by estimating odds ratios in the multivariate logistic regression analysis. For miRSNPs that were associated with leukemia risk we performed luciferase reporter assays to examine whether they influence miRNA binding.ResultsHere we show that variant alleles of TLX1 _rs2742038 and ETV6 _rs1573613 were associated with increased risk of childhood ALL (OR (95% CI) = 3.97 (1.43-11.02) and 1.9 (1.16-3.11), respectively), while PML _rs9479 was associated with decreased ALL risk (OR = 0.55 (0.36-0.86). In adult myeloid leukemias we found significant associations between the variant allele of PML _rs9479 and decreased AML risk (OR = 0.61 (0.38-0.97), and between variant alleles of IRF8 _ rs10514611 and ARHGAP26 _rs187729 and increased CML risk (OR = 2.4 (1.12-5.15) and 1.63 (1.07-2.47), respectively). Moreover, we observed a significant trend for an increasing ALL and CML risk with the growing number of risk genotypes with OR = 13.91 (4.38-44.11) for carriers of ≥3 risk genotypes in ALL and OR = 4.9 (1.27-18.85) for carriers of 2 risk genotypes in CML. Luciferase reporter assays revealed that the C allele of ARHGAP26 _rs187729 creates an illegitimate binding site for miR-18a-3p, while the A allele of PML _rs9479 enhances binding of miR-510-5p and the C allele of ETV6 _rs1573613 weakens binding of miR-34c-5p and miR-449b-5p.ConclusionsOur study implicates that microRNA-binding site polymorphisms modulate leukemia risk by interfering with the miRNA-mediated regulation. Our findings underscore the significance of variability in 3′ untranslated regions in leukemia.

Functional variants of gene encoding folate metabolizing enzyme and methotrexate-related toxicity in children with acute lymphoblastic leukemia

Methotrexate (MTX) is commonly used agent in therapy of malignancies, including acute lymphoblastic leukemia (ALL). Based on the literature data it is known that MTX elimination and toxicity can be affected by polymorphisms in genes encoding enzymes involved in MTX metabolism. The aim of our study was to investigate the influence of C677T and A1298C polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene on MTX-induced toxicity during treatment of children with ALL. We also tried to answer the question whether simultaneous occurrence of these two polymorphisms has a clinical significance. MTHFR polymorphisms were assessed in 47 pediatric ALL patients, treated according to intensive chemotherapy for childhood ALL, ALL IC BFM 2009. Prolonged MTX elimination and higher incidence of toxicity were observed for patients with 677T-1298A haplotype. On the other hand, occurrence of 677C-1298A haplotype had protective effect on MTX clearance and toxicity, that was not observed in carriers of 677C-1298C haplotype. In patients with coexistence of studied variants 677CT/1298AC heterozygotes as well as in 677TT/1298AA homozygotes more frequently toxicity incidents were noted. The obtained results suggest that occurrence of 677T allele and coexistence of 677T and 1298C alleles may be associated with lower MTX clearance and elevated risk of adverse effects during MTX-treatment of pediatric ALL patients.

Utility of quantitative EBV DNA measurements in cerebrospinal fluid for diagnosis and monitoring of treatment of central nervous system EBV-associated post-transplant lymphoproliferative disorder after allogenic hematopoietic stem cell transplantation.

BACKGROUND Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorder (PTLD) after hematopoietic stem cell transplantation (HSCT) is a rare but life- threatening condition. Despite routine EBV DNA-emia monitoring in blood, central nervous system involvement of the disease is still associated with diagnostic difficulties and poor outcome. CASE REPORT We describe a 17-year-old female patient with severe aplastic anemia who developed central nervous system EBV-associated PTLD after matched unrelated allo-HSCT. EBV DNA concentration in cerebrospinal fluid was initially tested due to central nervous system-related symptoms. The sample was found to be highly EBV DNA-positive. Using quantitative real-time polymerase chain reaction (PCR) measurements, therapy efficiency was monitored until complete remission of the EBV-associated PTLD. CONCLUSIONS The case report illustrates the high utility of quantitative real-time PCR EBV measurements in cerebrospinal fluid for rapid diagnosis and therapy monitoring of central nervous system EBV-associated PTLDs after allo-HSCT.

Analysis of TLR2, TLR4, and TLR9 single nucleotide polymorphisms in children with bacterial meningitis and their healthy family members

BACKGROUND The aim was to analyse TLR2 rs5743708, TLR2 rs4696480, TLR4 rs4986790, TLR9 rs5743836, and TLR9 rs352140 single nucleotide polymorphisms (SNPs) in children with pneumococcal and meningococcal meningitis and their family members. METHODS The study group consisted of 39 children with bacterial meningitis (25 with meningococcal meningitis and 14 with pneumococcal meningitis) and 49 family members. Laboratory test results and the course of the diseases were analyzed. Genomic DNA was extracted from 1.2ml of peripheral blood in order to analyze the five SNPs. RESULTS Patients with pneumococcal and meningococcal meningitis showed a similar male/female ratio, mean age, and duration of symptoms. There were no statistically significant differences in biochemical markers between the two groups. All patients possessed at least one polymorphic variant of the analyzed SNPs. The most common SNP was TLR9 rs352140, detected in 89.7% of patients. No significant differences in SNP frequency were found between patients, family members, and the general population. CONCLUSIONS The allele frequencies in the population studied are in accordance with the literature data. The study did not find an association between the analyzed SNPs and susceptibility to bacterial meningitis. The role of SNPs in genes coding toll-like receptors and the interactions between them in controlling inflammation in the central nervous system needs further evaluation.

FKBP5 polymorphism is associated with insulin resistance in children and adolescents with obesity

OBJECTIVE Since metabolic syndrome shares several clinical features with hypercortisolism, it was hypothesised that genes altering individual glucocorticoid (GC) sensitivity might be implicated in pathogenesis of obesity and its adverse outcomes. FKBP5 gene encodes a chaperon protein in the GC receptor (GR) complex, which modulates steroid action upon target genes. Its functional variant, rs1360780, may enhance FKBP5 gene transcription, affect GR signalling and thereby influence the hypothalamo-pituitary-adrenal axis. We investigated the association of rs1360780 with obesity and metabolic characteristics in 250 obese children and adolescents (mean age 12.3±3.6years, BMI ≥95th percentile). METHODS Anthropometric measurements, body composition, biochemical and hormonal results were analysed. Genotyping of rs1360780 was compared with 568 lean controls. RESULTS Impaired fasting glucose was present in 8.8%, glucose intolerance in 10.4%, diabetes in 2.8% and dyslipidemia in 28.8% obese individuals. Hypertension was diagnosed in 34 out of 143 patients. No difference was found in FKBP5 polymorphism distribution between subjects with obesity and controls (p>0.05). Stratification by rs1360780 revealed no differences in body mass and composition. However, carriers of the minor allele displayed enhanced insulin resistance (p=0.009) and elevated serum triglyceride (p=0.006), whereas cholesterol, HbA1c, and oral glucose challenge results were similar for all genotypes. Morning ACTH and cortisol did not differ but evening cortisol was higher in minor allele carriers (p=0.039), although this association was lost in logistic regression analysis. CONCLUSION This study does not support the association of FKBP5 with obesity but demonstrates plausible implication of its variant in susceptibility to obesity-related insulin resistance and hypertriglyceridemia.

Interleukin-2 and subunit alpha of its soluble receptor in autoimmune Addison's disease – An association study and expression analysis

Abstract Autoimmune Addisons disease (AAD) results from T cell-mediated destruction of the adrenal cortex, commonly accompanied by autoantibodies to 21-hydroxylase (21OH). In order to gain insight into the obscure aetiology of this disease, we investigated the roles of the IL2 and IL2RA genes, encoding interleukin-2 and subunit alpha of its receptor (IL2Ra), respectively. The association of AAD with IL2 and IL2RA polymorphisms (rs6822844, rs2069762, rs3136534, rs11594656, rs3118470 and rs2104286) was tested in 223 patients and 672 healthy controls. Functional studies consisted of gene expression analysis in cultured PBMCs exposed to 21OH and evaluation of serum interleukin by ELISA assays. The frequency of the minor C allele of rs3136534 was significantly decreased in AAD subjects compared to controls (OR 0.71; 95%CI 0.561–0.887; p = 0.003). Only AAD cells responded to 21OH with an elevated IL2 and IL2RA mRNA synthesis (p = 0.004 and p = 0.009 versus controls, respectively), paralleled by increased supernatant levels of both cytokines (p = 0.031 and p = 0.001 versus controls). IL2 mRNA level in 21OH-stimulated AAD PBMCs correlated negatively with age (p = 0.036) and positively with serum antibodies to 21OH (p = 0.006). Carriers of the rs2104286 AA genotype demonstrated higher IL2RA mRNA (p = 0.022) and soluble IL2Ra secretion (p = 0.029) upon 21OH stimulation. Serum interleukin-2 in AAD subjects was significantly higher compared to controls (4.61 ± 4.3 versus 1.71 ± 3.2 pg/mL, p < 0.001), whereas sIL2Ra levels remained similar in both groups (p = 0.885). In conclusion, the study reveals an association between AAD and IL2 locus. It confirms specific 21OH-directed reactivity of the peripheral AAD lymphocytes, which display increased synthesis of interleukin-2 and sIL2Ra.

Genes and their single nucleotide polymorphism involved in innate immune response in central nervous system in bacterial meningitis: review of literature data

BackgroundThere are many studies analysing the effect of SNPs in genes coding proteins which are involved in innate immune response on susceptibility to invasive bacterial disease. Many of them gave inconclusive results. Regarding the complexity of immune response and cooperation between particular elements, number of SNPs may have a cumulative effect on the susceptibility to bacterial meningitis.FindingsIn most studies cooccurrence of several SNPs was not analysed. These studies were performed on small groups of patients and usually only few SNPs were checked simultaneously. Additionally, comparison of the results across the studies is hard to conduct. We hypothesise that the number of variants of genes involved in innate immune response plays a role in susceptibility to bacterial meningitis. However, the role of toll-like receptors and other part of innate immune response in the eradication of bacteria, and initiation of the inflammatory response in CNS need further studies.ConclusionLarge multicentre studies assessing multiple SNPs in patients with microbiologically proven pneumococcal or meningococcal meningitis are needed to find real genetic risk factors for developing bacterial meningitis. This is necessary to design more effective treatment and prevention strategies for severe infections.

No Overt Clinical Immunodeficiency Despite Immune Biological Abnormalities in Patients With Constitutional Mismatch Repair Deficiency

Immunoglobulin class-switch recombination (CSR) and somatic hypermutations (SHMs) are prerequisites for antibody and immunoglobulin receptor maturation and adaptive immune diversity. The mismatch repair (MMR) machinery, consisting of homologs of MutSα, MutLα, and MutSβ (MSH2/MSH6, MLH1/PMS2, and MSH2/MSH3, respectively) and other proteins, is involved in CSR, primarily acting as a backup for nonhomologous end-joining repair of activation-induced cytidine deaminase-induced DNA mismatches and, furthermore, in addition to error-prone polymerases, in the repair of SHM-induced DNA breaks. A varying degree of antibody formation defect, from IgA or selective IgG subclass deficiency to common variable immunodeficiency and hyper-IgM syndrome, has been detected in a small number of patients with constitutional mismatch repair deficiency (CMMRD) due to biallelic loss-of-function mutations in one of the MMR genes (PMS2, MSH6, MLH1, or MSH2). To elucidate the clinical relevance of a presumed primary immunodeficiency (PID) in CMMRD, we systematically collected clinical history and laboratory data of a cohort of 15 consecutive, unrelated patients (10 not previously reported) with homozygous/compound heterozygous mutations in PMS2 (n = 8), MSH6 (n = 5), and MLH1 (n = 2), most of whom manifested with typical malignancies during childhood. Detailed descriptions of their genotypes, phenotypes, and family histories are provided. Importantly, none of the patients showed any clinical warning signs of PID (infections, immune dysregulation, inflammation, failure to thrive, etc.). Furthermore, we could not detect uniform or specific patterns of laboratory abnormalities. The concentration of IgM was increased in 3 out of 12, reduced in 3 out of 12, and normal in 6 out of 12 patients, while concentrations of IgG and IgG subclasses, except IgG4, and of IgA, and specific antibody formation were normal in most. Class-switched B memory cells were reduced in 5 out of 12 patients, and in 9 out of 12 also the CD38hiIgM− plasmablasts were reduced. Furthermore, results of next generation sequencing-based analyses of antigen-selected B-cell receptor rearrangements showed a significantly reduced frequency of SHM and an increased number of rearranged immunoglobulin heavy chain (IGH) transcripts that use IGHG3, IGHG1, and IGHA1 subclasses. T cell subsets and receptor repertoires were unaffected. Together, neither clinical nor routine immunological laboratory parameters were consistently suggestive of PID in these CMMRD patients, but previously shown abnormalities in SHM and rearranged heavy chain transcripts were confirmed.