Impact of the methylenetetrahydrofolate reductase [MTHFR] C677T gene polymorphism on clinical outcomes of HLA-matched sibling allogeneic hematopoietic stem cell transplantation

Background: allogeneic hematopoietic stem cell transplantation [HSCT] is widely used to treat various hematological malignant and non-malignant diseases. The occurrence of complications following HSCTas graft versus host disease[GVHD], hepatic veno-occlusive disease [VOD], oral mucositis [OM], drug induced hepatic and renal adverse events- is highly variable and dependent on a multitude of host, donor, and treatment factors. Identifying important genetic variables will allow for better prediction of HSCTrelated outcomes, and in the process of identifiing these susceptibilities, that could help to develop targeted interventions

Objectives: to evaluate impact of the C677T polymorphism of 5,10-methylenetetrahydrofolate reductase [MTNFR] on the clinical outcomes of patients treated using human leukocyte antigen-matched sibling stem cell transplantation as acute gruff versus host disease [GVHD],oral mucositis ,drug induced hepatic and renal toxicity, transplant related mortality[TRM] and overall survival[OS]

Patients and Methods: we examined the association of a single nucleotide polymorphism [SNP] at position 677 in the MTHFR gene of patients with outcomes of allogeneic HSCT. MTHFR genotyping was performed by po2ymerase chain reaction-restriction fiagment length polymorphism [PCR-RFLP]

Results: 46 Patients with complete clinical records were recruited. Median age at the time of HSCT was 22 years [range 3-42 years]; 32 patients [69.6%] above >/=18 years, and the median follow-up period of survivors was 21 months. 212efrequencies of the MTHFR C677T genotypes in patients were 43.5% [20 patients] for 677CC, 50% [23 patients] for 677CX and 6.5% [3 patients] for 677TT; the allelic frequency of the 677T was 31.5%. Recipient MTHFR677 in CT or TT versus CC showed non-statistically significant higher incidence of acute GVHD [7/26] 26.9% versus [2/20] 10%; p=0.15, hepatic toxicity [11/26] 42.3% versus [5/20] 25%, p= 0.22 and TRM [5/26] 19.2% versus [2/20] 10%; p=0.45. Recipients with variant allele MTHFR 677T were associated with lower non statistically signijicant overall survival; p=0.281. Conclusion: Genofyping for WHFR C677T before HSCT could have clinical significance, not statistically proven in our study, in prediction of patients at high risk of developing poor outcomes. Larger studies with homogeneous HSCT cohort are needed to identifi such potential phar]nacogenetic markers with suflciently strong evidence to be used in clinical practice

Lack of association of CTLA-4 + 49 A/G polymorphism with predisposition to type 1 diabetes in a cohort of Egyptian families

Type 1 diabetes is one of the most common chronic childhood illnesses. Interplay between genetic susceptibility and environmental factors is thought to provide the fundamental element for the disease. Apart from the Major Histocompatibility locus which is the main contributor to risk susceptibility, more than 40 loci are recognized. One among these is the CTLA-4, however data from the literature are controversial. The aim of our study was to investigate the role of CTLA4 49 A/G as a risk susceptibility factor for the development of type 1 diabetes in a cohort of Egyptian families. This is a case control study including 88 Egyptian families with one or more index cases [< 18 years]. The control group comprised 369 healthy unrelated subjects with no family history of diabetes or autoimmune disease. Using PCR-RFLP methodology, CTLA4 49 A/G was analyzed in 738 samples representing 88 families [88 patients, 125 siblings and 156 parents] and 369 control. The age of onset was 6 days-12.5 years with a mean of 5.3 +/- 3.6 and a median of 5 years. The mode of presentation was classic symptoms in 51 and diabetic ketoacidosis in 37 cases. Twenty-two cases had a history of viral infection or exanthematous disease and four had associated autoimmune diseases. No significant differences were encountered between the different groups with regard to CTLA4 +49 A/G genotype or allele frequencies. Neither was there a relation between the various genotypes and age of onset or the mode of presentation. CTLA4 49 A/G polymorphism was not recognized as a risk susceptibility factor in our cohort. This may be attributed to the low co-incidence of autoimmune diseases. Up to our best knowledge, this is the first study involving families. We recommend that all studies performed on risk susceptibility to type 1 diabetes should include proper investigation for other autoimmune diseases to exclude their confounding effect on data analysis

HLA-DQA and DQB alleles and predisposition to insulin dependent diabetes mellitus

Type I insulin-dependent diabetes mellitus [IDDM] is an autoimmune disease. Onset of the disease is attributed to interplay between genetic and environmental risk factors. It is strongly associated with the presence of arginine in position 52 of DQ alpha [alpha] chain and the absence of aspartic acid in position 57 of the DQ beta [alpha] chain. In this study we assessed the relative contribution of DQ alpha and DQ alpha chains to susceptibility to type I diabetes among the Egyptian patients. We identified those genetically at risk of development among their siblings in order to detect early development of autoantibodies allowing early application of preventive programs. Genomic DNA of forty Egyptian type I IDDM patients, 13 non diabetic siblings and 22 non diabetic controls were amplified using polymerase chain reactionamplification refractory mutation system [ARMS] and genotyped for HLA-DQA and DQB alleles. A significant high frequency of homozygous genotype for DQB1 non- Asp allele was detected in patients 50%, p=0.01, odd ratio [OR] =10 at 95% confidence interval [CI] =2.1-48.6 with susceptible results to the disease. The frequency of diabetogenic heterodimer Arg/non-Asp was significantly high in patients [82%, p=0.044, OR= 3.26, at 95% CI= 1.005-10.6]. On the other hand, a significant lower frequency of homozygous genotype for DQB1 Asp allele was detected in patients 12.5%, p=0.065; it was associated with protection from the disease. In conclusion, in Egyptian patients susceptibility and protection from type I diabetes is mainly associated with the DQ alpha chain. Siblings have potential risk to the disease. Non affected siblings should be targeted in a larger study for counselling. At risk individuals should be subjected to regular monitoring for the early development of autoantibodies which start years before the overt diabetes.

Synergistic effect of methyltetrahydrofolate reductase [MTHFR] C677T and a1298C polymorphism as risk modifiers of pediatric acute lymphoblastic leukemia

ALL is the most common pediatric cancer. The causes of the majority of pediatric acute leukemia are unknown and are likely to involve an interaction between genetic and environmental factors. Therefore, unfavourable gene-environmental interactions might be involved in the genesis of ALL. The aim of this work was to evaluate, in a case-control study, whether the common polymorphisms in 5, 10-methylenetetrahydro-folate reductase [MTHFR] namely [C677T and A1298C] and methionine synthase [MS] [A2756G] genes may play a role in altering susceptibility to pediatric ALL as individual genes and in combination. DNA of 88 ALL patients [age <18 years] and 311 healthy control subjects was analyzed for the polymorphisms of MTHFR and MS genes using PCR-RFLP method. The frequencies of the wild types of MTHFR 677CC, MTHFR 1298AA and MS 2756AA, the homozygous genotypes of MTHFR 677TT, MTHFR 1298CC and MS 2756GG and heterozygous genotypes of MTHFR 677CT and MS 2756AG showed no statistically significant differences between patients and controls. The frequency of the MTHFR 1298AC heterozygous genotype was 25% among patients compared to 45.0% among controls; the difference was found to be statistically significant [p value =0.001, O.R=0.382 and 95% C.1=0.222-0.658]. The frequency of the MTHFR 1298AC heterozygous genotype plus 1298CC homozygous genotype was 34% among patients compared to 54.3% among controls and the difference was statistically significant [p value=0.001]. A synergistic effect of 677CT and 298AC [CTAC] was observed, [p value=0.002] with 3.65 fold protection [OR 0.273 and 95% C.1=0.155-0.9] compared to 2.6 folds for MTHFR 1298AC alone. This protective effect of CTAC polymorphism was abolished when combined with MS 2756AA or AG. The present study provided further evidence for the protective role of MTHFR 1298AC mutant alleles in acute lymphoblastic leukemia in children [2.6 fold protection]. This suggests that folate and methionine metabolism play an important role in the pathogenesis of pediatric ALL. In contrast to the main bulk of literature, we did not find any protective role of either MTHFR C677T or MS A2756G polymorphisms. This may reflect the ethnic variation in both the polymorphism frequencies, variation in plasma level of folate in addition to the possible role of gene-environment interaction mainly dietary availability of folate. The synergistic effect of MTHFR 1298AC and 677CT and its abolishment by MS 2756AA or AG further emphasizes that the interaction of genes, rather than the polymorphism in any single one, determines risk susceptibility to disease

Enhanced apoptosis of peripheral blood mononuclear cells from chronic hcv patients is associated with reduced caspase 3 activity

Background: Infection with hepatitis C virus [HCV] is a major cause of chronic liver disease throughout the world. Down-regulation of the immune response plays a major role in HCV persistence. Recent investigation suggest that apoptosis of peripheral blood mononuclear cells [PBMCs] contributes to such down-regulation

Objective: The current study investigates apoptotic changes in PBMCs and their relation to caspase-3 and -8 activities in patients with chronic HCV infection

Methods: Apoptosis were investigated by measuring annexin-V binding using flowcytometry and DNA fragmentation using agarose gel electrophoresis, and caspases-3 and -8 specific activities were also measured in 43 chronic HCV patients and 10 normal control subjects

Results: A significantly higher percent of annexin-V positive PBMCs was found in chronic HCV patients than controls [p<0.0001]. DNA fragmentation was detected in PBMCs from 20/43 patients [46.5%] but not from controls. There was no statistically significant difference between HCV-PCR positive and negative patients as regards the degree of PBMCs apoptosis. Caspase-3 activity was significantly lower in patients than controls [p=0.001], was significantly lower in HCV-PCR positive than controls [p=0.001] and was significantly higher in patients with PBMCs DNA fragmentation [p=0.005]. On the other hand, caspase-8 activity was comparable in both patients and control groups. However, patients with PBMCs DNA fragmentation showed statistically significant higher activity than those without [p=0.023]. There was a statistically significant direct correlation between caspase-3 and caspase-8 activities in the patients groups [r=0.56,p<0.0001]

Conclusion: Chronic HCV infection is associated with PBMCs apoptosis irrespective of the presence of viremia. However, this apoptosis is independent on activation of either caspase-3 or caspase-8

Immunologic changes in HD patients under the effect of chemotherapy

The immune status was tested in 51 HD patients at diagnosis and after MOPP chemotherapy and 10 normal controls. Absolute lymphocyte and absolute T cells were slightly lower in pretreated patients compared to controls. Further reduction was observed after chemotherapy that was significantly different from the control values [P values 0.001 and 0.02 respectively]. The T4/T8 ratio of controls was higher than pre and post treated patients being 1.8 +/- 0.28 for patients after therapy. The difference between all groups was highly statistically significant. The absolute level of HNK1 + cells was reduced at diagnosis 263 +/- 245 compared to control 322 +/- 281 but the difference was statistically insignificant. The NHK + cells was elevated after treatment with statistically significant difference compared to pretreated patients [P 0.03] but its level did not reach the normal value. The Leu 11 [CD 11a] positive cells were markedly reduced at diagnosis than normal controls but were elevated after treatment even exceeding the normal values. The immunoglobulins [IgM, IgG and IgA] were reduced at diagnosis than normal controls and even further reduction after treatment, except for elevated IgG levels in patients at diagnosis which may be due to chronic infections. The impaired cell mediated immunity in HD patients tends to be improved by chemotherapy

Immunological phenotypic pattern of acute lymphoblastic leukemia in Egypt

Immunological characterization of acute lymphoblastic leukemia [ALL] in the Egyptian population was performed on 186 cases. An immunoperoxidase technique using monoclonal antibodies [Mo Abs] was used. Mo Mbs originally used included T11 [CD2], T4/Leu 3a [CD4], T8/Leu 2a [CD8] as T cell markers, J5 [CD10], B1 [CD20] anti-kappa [K] and anti-lambda [lambda] light chains as markers of the B cell line and anti class II [Ia/HLADR]. Subsequently after 62 cases had been phenotyped, Leu 9 [CD7] and B4 [CD19] were added to the antibody panel. Leu M1 and T200 were used to exclude myeloid and nonhemopoietic malignancies, respectively. Cases that remained unclassifiable were further tested with a wider panel of T cell markers including T6 [CDI], XII and G 144 [CD2] T3/Leu4 [CD3], TI, Leu1, A50 and 173D9 [CD5] and 121 [CD7]. The relative incidences of CALL and BALL were calculated from the total number of cases and were found to be 39.2% and 3.2%, respectively, while those of TALL and null ALL were calculated from the subset of cases tested by the complete panel of Mo Abs and were found to be 50% and 4.8%, respectively. A 2-5 years age peak of CALL was evident in the series, while TALL was more common between 4 and 12 years of age. Male predominance in TALL was not evident. This study shows that the phenotypic pattern of ALL in Egypt is different from that in the Western countries. There is a lower incidence of CALL and a higher incidence of TALL and male predominance in TALL was not observed

Phenotypic analysis of T-cell leukemia in Egypt

Leukemic cells from 46 T acute lymphoblastic leukemia [ALL] cases and 2 T chronic lymphatic leukemia [CLL], one T prolymphocytic leukemia [PLL] and one T lymphocytic lymphoma in leukemic phase [DLL] were studied with a wide panel of T cell markers. The panel included monoclonal antibodies [Mo Abs] belonging to CD1 [OKT6], CD2 [T11, X11, G144], CD3 [OKT3/Leu4], CD4[OKT4/Leu3a], CD5 [Leu1, T1, A50, L73D9], CD7 [Leu9, I21], CD8 [OKT8/Leu2a]. Other Mo Abs used included OKT9, OKT10, and HNK1. Immunoperoxidase technique using the Avidin-Biotin complex [ABC] method was used for staining. The 46 ALL cases expressed 46 different phenotypes reflecting an extreme degree of heterogeneity. Twelve L1 cases studied showed equal distribution among the 3 intrathymic stages with 4 cases in each group, while 33 L2 cases showed 9 in stage I, 19 in stage II and 5 cases in stage III. The most sensitive CDs in diagnosing T ALL both in children and adult respectively CD5 [100%, and 92.9%], CD7 [92.3% and 81.3%] and CD2 [80.8% and 82.4%]. The most sensitive Mo Ab were Leu1 [81.8%] followed by I21 [71.1%], T11 [70.2%] and Leu9 [68.9%]. The most sensitive combination of 2 Mo Abs was Leu1, I21 [100%] followed by Leu1, T11 [97.4%]. Five of the 46 ALL cases reacted with HNK1 denoting an origin from the natural killer [NK] subset. The PLL cases expressed the helper phenotype. The 2 CLL cases and the DLL case expressed the suppressor phenotype; one of the CLL cases reacted with HNK1. The findings encountered in ALL cases as regards the degree of heterogeneity, distribution according to intrathymic stage of origin in children and adults, sensitivity of various CDs and Mo Abs or prevalence of HNK1 + cases are different from those reported in the western countries; a finding that may reflect a different biological nature of the T ALL