Association of inherited thrombophilia mutations and their combinations among palestinian women with unexplained recurrent miscarriage.

BACKGROUND: Inherited thrombophilia (IT) has a complex pathophysiology and is associated with recurrent miscarriage (RM) by causing placental insufficiency and inhibiting fetal development. However, thrombophilia screening in unexplained RM cases is still questionable. This study aimed to investigate the association between the common eight IT mutations and their combinations among Palestinian women with unexplained RM. METHODS: This is an unmatched case-control study with 200 women (100 unexplained RM cases, 100 controls). Eight common IT mutations namely Factor V Leiden (FVL), prothrombin gene (FII) G202120A, Methylenetetrahydrofolate Reductase (MTHFR) gene (C677T and A1298C), B-fibrinogen gene - 455G > A, FV HR2 A4070G, Plasminogen activator inhibitor 1 (PAI1) 5G/4G and Factor XIIIA (FXIIIA) V34L; were analyzed. The first five mutations were analyzed by Restriction Fragment Length Polymorphism PCR and the other three mutations were analyzed using Amplification Refractory Mutation System PCR. RESULTS: The prevalence of the eight IT mutations among the control group was in the order PAI1 5G/4G (69%), MTHFR C677T (53%) and A1298C (47%), BFG - 455G > A (35%), FVL and FV HR2 (each 18%), FXIIIA V34L (16%) and FII G20210A (3%). Patients had a higher percentage of MTHFR A1298C (heterozygotes and mutant homozygote) compared to controls (p = 0.016). Frequencies of mutant alleles MTHFR A1298C (p < 0.001) and FXIIIA V34L (p = 0.009) were higher among patients compared to controls. No significant differences were observed for all other mutations or mutant alleles. Most patients (75%) and controls (75%) have 2-4 mutant alleles out of 8 mutant alleles studied, while 1% of patients and 2% of controls have zero mutant alleles. None of the combinations of the most often studied mutations (FVL, FII G20210A, MTHFR C1677T, and MTHFR A1298C) showed a significant difference between patients and controls. CONCLUSIONS: There was a significant association between unexplained RM and the mutant alleles of MTHFR A1298C and FXIIIA V34L. No significant association was observed between unexplained RM and the combination of both mutant alleles for the mutations studied. This study is the first Palestinian report that evaluates eight inherited thrombophilia mutations and their alleles' combinations in unexplained RM cases.

Evaluation of platelet parameters, coagulation markers, antiphospholipid syndrome, and thyroid function in palestinian women with recurrent pregnancy loss.

BACKGROUND: Multiple etiologies contribute to recurrent pregnancy loss (RPL) including immunological, endocrine, anatomical, genetic and infection but more than 50% of cases remain unexplained. Evidences of thrombotic and inflammatory processes were observed at maternal-fetal interface and considered pathological findings in most RPL cases including unexplained cases. This study aimed to evaluate the association between RPL and several risk factors: platelet parameters, coagulation factors, antiphospholipid syndrome, and thyroid function. METHODS: This is an unmatched case-control study that included 100 RPL and 100 control women. Anthropometric and health data were collected and a gynecologist examined participants to assure fitting the inclusion criteria. Platelet parameters [including Mean Platelet Mass (MPM), Concentration (MPC) and Volume (MPV)] and ratios (MPV/Platelet, MPC/Platelet, MPM/Platelet, Platelet/Mononuclear cells), coagulation markers [Protein C (PC), Protein S (PS), Antithrombin III, D-dimer], antiphospholipid antibodies [Anti-phospholipid (APA), Anti-cardiolipin (ACA) and anti-B2-glycoprotein 1], Lupus anticoagulant, Antinuclear antibodies, and thyroid function (Thyroid stimulating hormone and anti-thyroid peroxidase) were measured. RESULTS: Mean ages of cases and controls at marriage were 22.5 years for both, and their current ages were 29.4 and 33.0, respectively. 92% of cases and 99% of controls aged blow 30 years at marriage. 75% of cases have 3-4 miscarriages and 9% have ≥ 7 miscarriages. Our results indicated significantly lower male/female age ratio (p = .019), PC (p = .036) and PS (p = .025) in cases compared to controls. Plasma D-dimer (p = .020) and antiphospholipid antibodies [ACA (IgM and IgG), APA (IgM)] were significantly higher in cases compared to controls. No significant differences were observed between cases and controls concerning APA (IgG), anti-B2-glycoprotein 1 (IgM and IgG), Lupus anticoagulant, Antinuclear antibodies, platelet parameters, thyroid markers, family history of miscarriage, consanguineous marriage, and other health data. CONCLUSIONS: This is the first study that investigated the association between platelet, coagulation, antiphospholipid, autoimmune and thyroid parameters, and RPL in Palestinian women. Significant associations between male/female age ratio, PC, PS, D-dimer, ACA (IgM, IgG), APA (IgM) and RPL were observed. These markers could be used in evaluating RPL. These findings confirm the heterogeneous nature of RPL and emphasize the need for further studies to find out risk factors for RPL.

Plasma Lipids and Lipoproteins in Sickle Cell Disease Patients in the Northern West Bank, Palestine.

BACKGROUND: Lipid metabolism may be altered in red cell genetic disorders. The erythrocyte and plasma lipids are defected which may increase the risk of cardiovascular disease. In the present study, we hypothesized a possible link between severity of anemia and altered lipid profile in SCD. METHODS: A total of 151 SCD patients, including 62 patients with sickle cell anemia (SS), 54 patients with sickle ß-thalassemia (ST), and 35 individuals with sickle cell trait (AS), were studied. The control group consisted of 160 healthy individuals. Total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) were enzymatically measured. RESULTS: Total cholesterol and LDL-C were significantly lower (P value < 0.001) in SS and ST patients compared to AS individuals and AA controls. However, LDL-C was significantly lower in AS individuals (both males and female) compared to AA controls (P value < 0.001). The HDL-C in SS and ST patients (both males and females) was significantly lower than that in AS individuals (P value < 0.001). In addition, the HDL-C was significantly higher in SS and ST males and AS (males and females) compared to AA controls (P value < 0.001). The HDL-C was also significantly higher in SS males (P value < 0.001) and females (P value < 0.05) compared to ST patients. The HDL-C was significantly higher in AS individuals (P value < 0.001) compared to AA controls. The triglycerides in SS males was significantly lower than that in ST patients (P value < 0.001), but there was no significant difference when compared to AS individuals and AA controls. In contrast, triglycerides in SS females were significantly lower than those in ST (P value < 0.05), AS (P value < 0.001), and AA controls (P value < 0.001). In males of ST patients, triglycerides were significantly higher than those observed in AS males and AA males (P value < 0.001). In contrast, females of ST patients have a significantly lower triglycerides compared to AS and AA females (P value < 0.001). CONCLUSIONS: In SCD, the plasma is affected in some way, especially the plasma cholesterol that was investigated in this study. Further prospective studies should examine the contribution of an altered lipid profile to the severity and clinical complications in SCD patients.

Molecular characterization of ß-thalassemia intermedia in the West Bank, Palestine.

BACKGROUND: We aimed to investigate the molecular basis of ß-Thalassemia intermedia (TI) in the West Bank region and its management practices. METHODS: This was a case series multi-center study and included 51 cases of TI. DNA sequencing was used to analyze ß-globin gene mutations. Common α-globin gene mutations were screened by Gap-PCR (-α3.7, -α4.2, --MED, αααanti3.7) or DNA sequencing (α2-IVS II 5 nt del). XmnI -158 C > T polymorphisms of Gγ-globin gene was determined by RFLP-PCR. RESULTS: Seven ß-globin gene mutations were observed, namely IVS-I -6 C > T, IVS-I-110 G > A, IVS-II-1 G > A, IVS-I-1 G > A, Codon 37 Trp > Stop, beta - 101 and IVS-II-848 C > A. Ten genotypes were observed. Homozygosity for IVS-I-6 accounted for the majority of TI cases with a frequency of 74.5%. The second common ß-globin gene genotype was homozygote IVS-I-110 G > A (5.8%) and homozygote IVS-II-1 G > A (5.8%). The remaining seven genotypes were each detected in about 2% of patients. α-Thalassemia mutations were seen in five patients (9.8%), and included (-α3.7, αααanti3.7 and α2-IVSII-5 nt del). XmnI polymorphism was observed in four patients (7.8%), three homozygotes and one heterozygote. CONCLUSIONS: Homozygosity for the mild ß-globin gene IVS-I-6 allele was the major contributing factor for the TI phenotype among the study subjects. The role of XmnI SNP and α-thalassemia mutations in ameliorating the TI phenotype was observed in few patients for each factor. The beta - 101 C > T mutation was diagnosed in one patient in homozygote state for the first time in Palestine.

An unusual report of anti-N antibody presenting as ABO discrepancy in an old female patient in Palestine.

The MNS is a highly complex immune blood group system which is almost equal to Rh in size and complexity. Anti-N antibody is naturally occurring in general, cold reactive IgM or IgG saline agglutinin and relatively rare when compared with anti-M. The immune type anti-N is seldom encountered. Anti-N antibody is not clinically significant unless it reacts at 37°C. Clinically significant anti-N antibody is reactive at 37°C or in the anti-human globulin phase, which may cause delayed hemolytic transfusion reactions or hemolytic disease of newborn. Here, we report a rare case presented as blood group discrepancy of a naturally occurring anti-N that reacts at 37°C.

Expression of the GBGT1 Gene and the Forssman Antigen in Red Blood Cells in a Palestinian Population.

BACKGROUND: The Forssman antigen (FORS1 Ag) is expressed on human red blood cells (RBCs). We investigated its presence on RBCs from Palestinian subjects and Swedish subjects by serological testing and by sequencing part of exon 7 of the GBGT1 gene, which encodes Forssman synthase. MATERIALS AND METHODS: Blood samples from Palestinian subjects (n = 211 adults and n = 73 newborns) and from Swedish subjects (n = 47 adults) were analyzed in the study. RBCs from the Palestinian samples were typed for the FORS1 Ag using a monoclonal anti-Forssman antibody. The GBGT1 gene was genotyped by DNA sequencing (all adult samples) or by using amplification refractory mutation system PCR (newborn samples). RESULTS: All of the studied samples were negative for the FORS1 Ag by serologic typing. DNA sequencing of the 3' end of exon 7 of the GBGT1 gene, which includes Arg296, showed that all samples had the wild-type Arg296 sequence, which is associated with an inactive form of Forssman synthase. We detected four single nucleotide polymorphisms in the adult samples; two were silent (p.Tyr232=, p. Gly290=), and two were missense (p. Arg243Cys, p. Arg243His). The allele frequencies ranged from 0.2 to 3.6%. The p. Arg243Cys SNP was a novel SNP that was detected in one Palestinian sample. CONCLUSION: Our results confirmed the allelic diversity of GBGT1 and identified a novel nucleotide polymorphism in this gene, p. Arg243Cys. Our results also confirmed that the FORS blood group system is a low-frequency system.

Frequency of Red Blood Cell Alloimmunization in Patients with Sickle Cell Disease in Palestine.

BACKGROUND: Transfusion of red blood cells (RBC) is an essential therapeutic tool in sickle cell disease (SCD). Repeated RBC transfusions can cause alloimmunization which causes difficulty in cross-matching and finding compatible blood for transfusions. This study aimed to investigate the frequency of RBC alloimmunization and related risk factors among Palestinian SCD patients. MATERIALS AND METHODS: A multicenter cross-sectional study on 116 previously transfused SCD patients from three centers in West Bank, Palestine. Demographic, medical data and history of transfusion were recorded. Blood samples were collected from transfused consenting SCD patients. Gel card method was used for antibody screening and identification. In all patients, autocontrol and direct antiglobulin (DAT) test were performed using polyspecific (anti-IgG + C3d) anti-human globulin (AHG) gel cards for the detection of autoantibodies. RESULTS: Of the SCD patients, 62 (53.4%) patients were HbSS and 54 (46.6%) patients were sickle ß-thalassemia (S/ß-thal). There were 53 (45.7%) females and 63 (54.3%) males. Mean age was 18.8 years (range 3-53 years). The frequency of RBC alloimmunization among SCD patients was 7.76%, with anti-K showing the highest frequency (33.3%) followed by anti-E (22.2%), anti-D (11.1%), anti-C (11.1%), and anti-c (11.1%). All reported IgG alloantibodies were directed against antigens in the Rh (66.7%) and Kell (33.3%) systems. Older ages of patients, increased number of blood units transfused, and splenectomy were the commonest risk factors for alloimmunization in our study. CONCLUSIONS: RBC alloimmunization rate among Palestinian SCD patients is low compared to neighboring countries and countries all over the world but still warrants more attention. Phenotyping of donors/recipients' RBC for Rh antigens and K1 (partial phenotype matching) before their first transfusion may reduce the incidence of alloimmunization.

Factor V Leiden G1691A and prothrombin G20210A mutations among Palestinian patients with sickle cell disease.

BACKGROUND: Vascular thrombosis is an important pathophysiological aspect of sickle cell disease (SCD). This study aimed to investigate the prevalence and clinical impact of factor V Leiden G1691A (FVL) and prothrombin G20210A mutations among Palestinian sickle cell disease (SCD) patients. METHODS: A total of 117 SCD patients, including 59 patients with sickle cell anemia (SS), 33 patients with sickle ß-thalassemia and 25 individuals with sickle cell trait (AS) were studied. The control group consisted of 118 healthy individuals. FVL and prothrombin G20210A mutations were determined by RFLP PCR. RESULTS: Analysis of the clinical history of SCD patients revealed that seven patients have had vascular complications such as ischemic stroke or deep vein thrombosis. In SCD patients, the inheritance of the FVL mutation showed a significantly higher incidence of pain in joints, chest and abdomen as well as regular dependence on blood transfusion compared to SCD with the wild type. Age- and sex-adjusted logistic regression analysis revealed a significant association between FVL and sickle cell anemia with an odds ratio (OR) of 5.6 (95% confidence intervals [CI] of 1.91-39.4, P = 0.039) in SS patients. However, increased prevalence of the FVL in AS subjects and sickle ß-thalassemia patients was not statistically significant compared to controls (OR 3.97, 95% CI 0.51-28.6, P = 0.17 and OR 3.59, 95% CI 0.35-41.6, P = 0.26, respectively). The distribution of prothrombin G20210A mutation among SCD patients compared to controls was not significantly different, thus our findings do not support an association of this mutation with SCD. CONCLUSIONS: FVL was more prevalent among SS patients compared to controls and it was associated with higher incidence of disease complications among SCD patients.

Prevalence of antibodies to a new histo-blood system: the FORS system.

BACKGROUND: In 1987, three unrelated English families were reported with a putative blood subgroup called Apae. Swedish researchers later found evidence leading to abolishment of the Apae subgroup and establishment instead of the FORS blood group system (System 31 - ISBT, 2012). It is important to know the prevalence of antibodies in order to make the best decisions in transfusion medicine. Cells expressing the Forssman saccharide, such as sheep erythrocytes, are needed to detect the anti-Forssman antibody. The aim of this study was to define the prevalence of human anti-Forssman antibody. MATERIALS AND METHODS: Plasma samples from 800 individuals were studied. Sheep erythrocytes or Forssman "kodecytes" were mixed with the plasma samples using the tube technique. Plasma from an Apae individual was used as a negative control and monoclonal anti-Forssman antibody (M1/22.25.8HL cell line supernatant) was used as the positive control. RESULTS: Of the 800 individuals tested, one was negative for the presence of anti-Forssman antibody. We compared the anti-Forssman antibody reaction pattern between genders and found that males have weaker reactions than females, both at room temperature (p=0.026) and at 37 °C (p=0.043). We also investigated the reaction pattern of anti-Forssman antibody in relation to ABO and Rh blood group types without finding any significant differences. DISCUSSION: Sheep erythrocytes are suitable for searching for human anti-Forssman antibody. The quantity of anti-Forssman antibodies in plasma is higher in females than in males. In the population (n=800) studied here, we found one individual lacking the anti-Forssman antibody. These results contribute to the data already published, confirming that FORS is a rare blood group.

Prevalence of Anemia and Iron Deficiency among Palestinian Pregnant Women and Its Association with Pregnancy Outcome.

BACKGROUND: Anemia is a public health problem especially among pregnant women. This study aimed to investigate the prevalence of anemia and iron deficiency among pregnant women and its association with pregnancy outcome in Hebron Governorate in southern Palestine. METHODS: This is a cross-sectional study that included 300 pregnant women in their first trimester and 163 babies. Maternal anthropometric and socioeconomic and newborns' data were collected. Complete blood count for study subjects and maternal serum ferritin were measured. RESULTS: The prevalence of iron deficiency anemia among pregnant women was 25.7% and 52% of them had depleted iron stores. When pregnant women were grouped into three hemoglobin (Hb) tertile groups, a significant difference was observed between maternal Hb and newborns' birth weight (P= 0.009), height (P= 0.022), head circumference (P= 0.017), and gestational age (P= 0.012). There was a significant association between maternal serum ferritin and frequency of low birth weight (P= 0.001) and frequency of preterm delivery (P= 0.003). No significant association was observed between maternal anthropometric measures or the socioeconomic status and pregnancy outcomes. CONCLUSION: Iron deficiency is a moderate public health problem among the study subjects. Maternal Hb and serum ferritin significantly affect pregnancy outcomes.

Regulation of human factor IX expression using doxycycline-inducible gene expression system.

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000). Thus, it should be possible to maintain transgene expression within the therapeutic range. Therefore, we inserted elements of the tetracycline (Tet)-dependent Tet-On gene regulatory system into replication deficient adenovectors. The new system consists of two adenovectors: a response vector expressing hFIX (Ad5.TRE.hFIX), and a regulator vector expressing a second generation reverse tetracycline transactivator controlled by a CMV- (Ad5.CMV.rtTA) or human alpha1-antitrypsin-promoter (Ad5.hAAT.rtTA). Expression studies in four human cell lines showed high expression of hFIX from Ad5.TRE.hFIX in all cell lines in combination with Ad5.CMV.rtTA regulator vector, but only high specific expression in HepG2-cells in combination with Ad5.hAAT.rtTA regulator vector. Additionally, up- and down-regulation of hFIX expression could be demonstrated in vitro with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination and modulating doxycycline concentrations. When SCID-mice were infected with the Ad5.TRE.hFIX/Ad5.CMV.rtTA combination, up- and down-regulation of hFIX expression was achieved by oral doses of doxycycline for a period of at least two months. Replacement of the Ad5.CMV.rtTA vector by the Ad5.hAAT.rtTA vector showed minimal expression of hFIX in vivo. Although hFIX expression showed a slow and gradual decrease over time in vivo with the Ad5.CMV.rtTA vector, it remained within the therapeutic range. To date, regulation of hFIX has not been described in this way.