Danuta Januszkiewicz

Multiple sclerosis-associated virus-related pol sequences found both in multiple sclerosis and healthy donors are more frequently expressed in multiple sclerosis patients

In the etiopathogenesis of multiple sclerosis (MS), both genetic and environmental factors play an important role. Among environmental factors, viral infections are most likely connected with the etiology of MS. There are many evidence suggesting possible involvement of retroviruses in the development of autoimmune diseases including MS. Multiple sclerosis-associated retrovirus (MSRV) seems to be the important candidate for viral etiology of MS. The aim of the study was to analyze MSRV pol sequences in patients with MS. As control, groups of myasthenia gravis, Parkinson’s disease, and migraine patients, and healthy individuals have been studied. The MSRV pol sequences have been analyzed in RNA isolated from the serum and in DNA and RNA of peripheral blood lymphocytes from untreated MS patients and control groups. The MSRVpol sequences have been detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR technique, using specific oligonucleotide primers. In the serum RNA (cDNA), MSRV pol sequences have been identified in 31/32 MS patients. MSRV pol sequences were detected in serum cDNA of 9/17 myasthenia gravis patients, 7/16 Parkinson’s disease patients, 10/21 migraine patients, and 13/27 healthy individuals. MSRV pol sequences were observed also in RNA from lymphocytes of all MS patients, 12/17 myasthenia gravis patients, 9/16 Parkinson’s disease patients, 14/21 migraine patients, and 18/27 healthy donors. In the DNA from peripheral blood lymphocytes of all studied patients and healthy individuals, MSRV pol sequences have been found. The observed pattern of fiber-fluorescence in situ hybridization (FISH) signals suggests the presence of multiple copies of MSRV pol sequences, most likely tandemly dispersed in the genome. It can be concluded that MSRV pol sequences are endogenous, widespread in lymphocytes DNA, and transcribed into RNA of MS patients as well as of other studied patients and healthy individuals. However, more frequent expression of MSRV sequences detected in lymphocytes RNA (cDNA), as well as their presence in higher frequency in the serum of MS patients, may suggest the involvement of MSRV in the etiopathogenesis on MS.

Heterozygous carriers of the I171V mutation of the NBS1 gene have a significantly increased risk of solid malignant tumours

Homozygous mutation 657del5 within the NBS1 gene is responsible for the majority of Nijmegen breakage syndrome (NBS) cases. NBS patients are characterised by increased susceptibility to malignancies mainly of lymphoid origin. Recently it has been postulated that heterozygous carriers of 657del5 NBS1 mutation are at higher risk of cancer development. The aim of the study was to analyse the frequency of I171V mutation in NBS1 gene in 270 women with breast cancer, 176 patients with larynx cancer, 81 with second primary tumours of head and neck, 131 with colorectal carcinoma and 600 healthy individuals. I171V mutation was present in 17 cancer patients compared with only one in healthy individuals. This constitutes 2.58% in studied patients with malignancies and 0.17% in the control group (P=0.0002; relative risk 1.827; odds ratio 15.886; 95% confidence interval 2.107-119.8). Since DNA was isolated from non malignant cells, all mutations found in cancer patients appeared to be of germinal origin. It can be concluded that NBS1 allele I171V may be a general susceptibility gene in solid tumours.

Activity and expression of human telomerase in normal and malignant cells in gastric and colon cancer patients.

Background The reactivation of telomerase is believed to play an important role in immortalization and carcinogenesis. Objective To investigate the expression of three components of the telomerase complex (hTR, hTERT and TP1), along with telomerase activity in malignant and normal cells. Methods Cells were isolated from gastric and colon cancer, and from normal mucosa from the stomach and colon of participating patients. Expression of hTERT, hTR and TP1 has been studied by the reverse transcriptase polymerase chain reaction (PCR) technique. The telomerase repeat amplification protocol and PCR enzyme-linked immunosorbent assay were used for analysis of telomerase activity. Results All telomerase components were consistently expressed in colon and gastric cancer cells. Neoplastic RNA produced consistently very strong amplification signals either for hTR, hTERT or TP1. The expression of hTR was observed in RNA isolated from all normal mucosa samples and from peripheral blood lymphocytes. The expression of TP1 and hTERT has been found in the majority of normal cells; however, the amplification signals produced were usually much weaker than in malignant cells. The limiting dilution experiments indicated that the cancer cells have at least 100-fold higher telomerase activity and at least 25-fold higher TP1 and hTERT expression in comparison to normal cells. Conclusions It can be concluded that all the cancer cells tested have higher telomerase expression and activity than normal cells. Therefore, telomerase can be a good cancer marker, provided that quantitative analysis is carried out.

Is the NBN gene mutation I171V a potential risk factor for malignant solid tumors in children

NBN gene is considered as one of the low-to-moderate cancer susceptibility gene. At least 4 germline NBN mutations have been found in several malignancies in adults. In our studies, we observed the high incidence of germline mutation I171V of NBN gene in breast, colorectal, larynx cancer, and in multiple primary tumors. In this study, we would like to answer the question whether I171V germline mutation of NBN gene may constitute risk factor for solid tumors in children. The frequency of this mutation has been analyzed in patients with neuroblastoma (n=66), Wilms tumor (n=54), medulloblastoma (n=57), and rhabdomyosarcoma (n=82) hospitalized in Pediatric Oncology, Hematology and Bone Marrow Transplantation Department in the years between 1987 and 2010. About 2947 anonymous blood samples collected on Guthrie cards drawn from the newborn screening program of the Wielkopolska region have been used as controls. All the patients and controls came from the same geographical region. I171V mutation of the NBN gene has been observed in 5 controls. Among children with solid tumors only in 1 child with medulloblastoma I171V variant has been found. In conclusion, I171V germline mutation in contrary to adults cannot be considered as a risk factor for children malignancies. However, owing to low number of patients with solid tumors the possibility of a Type II error may exist.

No evidence for association of the polymorphisms in NLRP1 gene with type 1 diabetes in Poland

Type 1 diabetes (T1D) is a common autoimmune disorder caused by T-cell mediated destruction of the insulin-producing pancreatic beta cells. Its pathogenesis is a complex process, influenced by both genetic and environmental factors. To date, the class II HLA genes, the insulin gene promoter, the CTLA4 locus and PTPN22 have been identified as significant immune-response loci [1–5]. Recently, the NLRP1 has been linked to vitiligo and associated autoimmunity [6]. The NLRP1geneencodes leucine-rich repeatprotein1,a memberofa group of cytoplasmic pattern recognition receptors, which recognize microbial lipopolysaccharide, thereby stimulating innate immunity [7]. We hypothesized that polymorphisms in the NLRP1 gene might affect susceptibility to T1D. The six variants (rs6502867, rs12150220, rs2670660, rs878329, rs8182352, rs4790797) of the NLRP1 region were genotyped by PCR–RFLP and PCR–SSCP in a case-control study comprising 221 T1D patients and 254 healthy controls, issued from the Polish population of Caucasian origin. The mean age ( SD) at disease onset was 8.3 ( 4.3) years. The diagnosis of T1D was based on World Health Organization criteria and all patients required insulin therapy. The patients were recruited from the inpatients and outpatients endocrine clinics at the Poznan University of Medical Sciences. This research was approved by the local ethics committee and informed consent was obtained from study participants. All analysed single nucleotide polymorphisms (SNPs) were in Hardy–Weinberg equilibrium in both patients and control cohorts (P > 0.1). The frequencies of alleles and genotypes of the six SNPs in the NLRP1 region did not present significant differences between patients with T1D and controls (Table 1). Linkage disequilibrium measurement (LD; Levontin’s D0 and r coefficient) for the studied SNPs showed strong LD between rs12150220 and rs2670660, rs12150220 and rs8182352, rs2670660 and rs8182352, rs878329 and rs8182352 (D0 0.82, r 0.62). In contrast, the LD between rs6502867 and the five other SNPs was absent (D0 < 0.2, r = 0). The construction of haplotypes based on rs12150220, rs2670660, rs878329, rs8182352 and rs4790797 polymorphisms did not reveal significant differences between the T1D group and healthy controls (P > 0.4, data not shown). However, the haplotype carrying the risk alleles of rs12150220, rs878329 and rs8182352 observed in 5 of 221 (2.5%) patients with T1D, was not found in control group. The present study demonstrated lack of association of the rs6502867, rs12150220, rs2670660, rs878329, rs8182352 and rs4790797 polymorphisms in the NLRP1 region with T1D in the Polish population. Similar analysis conducted among the Norwegian T1D patients revealed a significant association of the major allele of the coding variant rs12150220 with disease [8]. The L155H variant in the NLRP1 gene have also been reported to confer increased risk for vitiligo [6] and autoimmune Addison’s disease [8,9] in Caucasian populations. Diabetic patients and healthy controls enrolled in our study were ethnically and geographically matched, therefore the observed lack of association could not result from population stratification. However, relatively modest sample size in the current study may limit its power to detect variants with minor effect. Nonetheless, our results are in agreement with the data of the genome-wide association studies, which have not indicated the NLRP1 locus as associated with T1D [10]. In conclusion, polymorphisms in NLRP1 gene do not confer risk for type 1 diabetes in the Polish population.

Juvenile xanthogranuloma with clonal proliferation in the bone marrow.

The triple association between juvenile xanthogranuloma (JXG), juvenile myelomonocytic leukemia and neurofibromatosis was described in literature in about 20 cases. In this paper, the case of an 11-month-old infant boy with a disseminated JXG with unusual cytogenetic representation in the bone marrow was reported. Neurofibromatosis and juvenile myelomonocytic leukemia were excluded, just the same as other leukemias. Bone marrow and peripheral blood cytogenetic analysis revealed a karyotype with many rearrangements 46,XY,-6,der(12)t(6;12)(p21;p13),del(7)(p13p22),+9 once described in the literature as a B-acute lymphoblastic leukemia case. On the contrary, in our patient immunologic testing demonstrated a high activity of T lymphocytes, however, inflammation was excluded. To the best of our knowledge this is the first described case of systemic JXG with determined karyotype representing unusual chromosomal aberrations.

Limited junctional diversity of Vδ5-Jδ1 rearrangement in multiple sclerosis patients

T-cell receptor (TCR) δ gene repertoire, as assessed by Vδ-Jδ rearrangements, has been analyzed in nine multiple sclerosis (MS) cases and in 30 healthy individuals by seminested PCR technique. Among the Vδ-Jδ junctional diversities studied, the most striking result has been observed in Vδ5-Jδ1 rearrangement. The detection of repeated Vδ5-Jδ1 nucleotide sequences in all analyzed clones from seven out of nine patients studied proved the monoclonal nature of γ δ T-cells with Vδ5-Jδ1 rearrangement. The clonal nature of this rearrangement proved by PAGE and sequencing analysis may suggest an antigen-driven expansion of γδT cells and argues for a significant role of γδ T-cells with Vδ5-Jδ1 rearrangement in MS pathogenesis. However, it cannot be excluded that clonal expansion of these lymphocytes may represent secondary change to central nervous system damage.

Restricted T cell receptor delta chain genes repertoire in peripheral blood of multiple sclerosis patients

Multiple sclerosis (MS) is a chronic inflammatory disease characterized by focal demyelination of central nervous system (CNS). Susceptibility to MS is thought to be affected by multiple genes including HLA and T cell receptor (TCR) genes. In view of the recent evidence, that in addition to α/β T lymphocytes also γ/δ T cells may have autoreactive potential, TCR delta repertoire in peripheral blood of MS patients has been studied. TCR delta repertoire, as assessed by Vδ‐Jδ rearrangements, has been analysed in 13 MS cases and in 30 healthy individuals by seminested PCR technique. Oligonucleotide primers specific for six Vδ regions and for Jδ1 gene were used for amplification of Vδ‐Jδ junctional region responsible for the diversity of γ/δ TCR. In the majority of MS patients PAGE analysis of Vδ1‐Jδ1, Vδ3‐Jδ1 and Vδ5‐Jδ1 rearrangements showed single‐band or two‐band pattern. The most striking result has been observed in Vδ5‐Jδ1 rearrangement, where in nine cases studied single band and in four patients two bands have been found. In all but one MS cases multi‐band pattern of Vδ2‐Jδ1 rearrangement was obtained. None of the 13 MS patients showed single‐band rearrangement pattern of Vδ4‐Jδ1 and Vδ6‐Jδl. Contrary to the MS group almost all healthy individuals produced smear‐like or multi‐band pattern of Vδ1‐Vδ5 to Jδ1 rearrangements. On the basis of the banding pattern produced by Vδ‐Jδ rearrangement in MS, it can be suggested that T lymphocytes had undergone clonal expansion in vivo, most likely due to stimulation by antigen related to CNS. In particular a very consistent single‐band pattern of Vδ5‐Jδ1 rearrangement observed in almost all MS patients studied, argues very strongly for a significant role of γ/δ T cells with Vδ5 rearrangement in the pathogenesis of MS. However, it cannot be excluded that the observed patterns of TCR δ gene rearrangement in MS patients may represent secondary changes to CNS damage.

Analiza aberracji chromosomowych we wczesnej diagnostyce wznowy ALL i przełomu blastycznego CML – opis dwóch przypadków

Streszczenie Cytogenetyka klasyczna obejmująca określenie kariotypu pacjenta z uzyciem klasycznej techniki prązkowania GTG, a takze zastosowanie fluoroscencyjnej hybrydyzacji in situ (FISH) oraz diagnostyka molekularna pozwalają na wykrycie klonalnych zaburzen u ponad 70% pacjentow z bialaczką w czasie jej rozpoznania. Wiekszośc z tych aberracji jest wysoce swoista, dlatego tez identyfikacja zmian chromosomowych w komorkach nowotworowych jest istotnym czynnikiem nie tylko diagnostycznym, ale rowniez prognostycznym. Przedstawiono 5-letniego chlopca z pro-B ALL, u ktorego po 2 latach od remisji badanie kariotypu i analiza FISH wykazaly obecnośc klonu komorek o kariotypie 47,XY,add(6)(p21.3),+11 wskazującym na wznowe bialaczki. Ocena cytomorfologiczna i immunofenotypowa wykazaly obecnośc blastow dopiero 3 miesiące po stwierdzeniu cytogenetycznej wznowy ALL. Drugi przypadek to 14-letni chlopiec z CML w fazie przewleklej, u ktorego rok po przeszczepieniu komorek macierzystych szpiku od dawcy niespokrewnionego wykazano, przy braku wznowy klinicznej i hematologicznej, wznowe (kariotyp zlozony 46,XY,inv(2)(p11;q13),t(3;21)(q26;q22),t(9;22)(q34;q11.2) z dwoma genami fuzyjnymi: BCR/ABL charakterytycznym dla CML oraz EVI1/AML1 wystepującym w fazie przelomu blastycznego).