The origin of haemoglobin S (HbS) in Palestine

Sickle cell anaemia (SS) is an inherited chronic hemolytic disease due to a single point mutation substituting thymine for adenine at codon 6 of the β–globin gene. The high prevalence of the βˢ mutation in Africa, the Middle East, India and in parts of the Mediterranean has a relation to the selective protective effect of sickle cell hemoglobin (HbS) against malaria. Its spread to other regions of the world may be due to slave trade and/or population migration. The aim of the present study was; 1) To determine the origin of βˢ mutation among Palestinian patients with sickle cell disease. 2) To study the effect of haplotype on the haematological characteristics of sickle cell disease. 3) To further explore the causes of the variability in Hb F levels in sickle cell disease. 4) To explore how frequent and diverse are recombinations in chromosomes carrying the βˢ gene associated with common (or typical) haplotypes. 5) To study the genetic flow of sickle cell disease haplotypes among normal Palestinians (βᴬ chromosomes). 6) To assess the influence of thrombophilic mutations (factor V Leiden (G1691A) and Prothrombin (G20210A)) on the hypergoagability and thrombosis of sickle cell disease. Patients comprised 117 individuals with SCD, of whom 59 were SS (30 males and 29 females) aged 16 ± 9.9 years, 25 were AS (14 males and 11 females) aged 21.2 ± 9.1 years, and 33 were S/β thalassemia (18 males and 15 females) aged 15.1 ± 5.1 years. The control group was 78 apparently healthy individuals (57 males and 21 females) aged 20.6 ± 5.5 years. All patients came from different Palestinian cities and were all followed at the National Center for Blood Diseases “HIPPOCRATES”, Rammallah, Palestine. The β-globin gene cluster haplotypes were determined using the polymerase chain reaction restriction fragment-length polymorphism PCR-RFLP technique. For haplotype determination, 8 regions around and within the β-gene cluster were amplified by polymerase chain reaction. The 8 polymorphic restriction sites studied and the enzyme used in each case were: Hind II 5΄ to the ε gene, Xmn I 5΄ to the ᴳγ gene, Hind III within IVS-II of the ᴳγ and ᴬγ genes, Hind II 3΄ to ψβ gene, RsaI 5΄ to the β gene, AvaII for IVS-II of the β gene and HinfI 3΄ to the β gene. Genotyping of Thrombophilic mutations was carried out by (PCR-RFLP) using Mnl 1 and HindIII for FV-Leiden and prothrombin G20210A, respectively. One hundred seventy six (176) out of a total number of 234 chromosomes were βˢ, thirty-three (33) βThal, while twenty-five (25) had the wild type βᴬ gene. Four different typical haplotypes (Benin, Bantu, Cameroon, and Senegal) and four atypical ones (Bantu A2 (+----+++), Bantu A2c (+----++-), Bantu A2a (+----+-+) and Bantu A6a (--++-+-+)) were found in our patient population. The Benin haplotype was found to be the most prevalent in the study population in its heterozygous and homozygous states. This haplotype accounted for 82.4% of the homozygous form of the disease. The Bantu A2 atypical haplotype was the second most prevalent haplotype among our patients. The Bantu A2 haplotype accounted for 29.5% of the βᴬ haplotypes clarifying its high prevalence in our sample, and that it may be present a haplotype of normal population. Heterogeneity was observed in Hb F production in SS patients, ranging between (1.5-17%), whereas γᴳ ratio was homogeneous among all SS haplotypes with a normal amount of about (41%). Our results are in agreement with previous reports of the Benin haplotype origin in the Mediterranean. Thrombosis may play an important role in the pathophysiology of certain complications of sickle cell disease (SCD). Vascular complications such as ischemic stroke or deep vein thrombosis were diagnosed in seven patients. Age- and sexadjusted logistic regression analysis indicated a significant association between factor V Leiden mutation and sickle cell anemia with an odds ratios (OR) of 0.33 (95% confidence intervals [CI] of 0.12–0.94, P = 0.039) in SS patients. However, increased prevalence of the factor V Leiden in AS individuals and sickle/β-thalassemia patients was not statistically significant compared to controls. The prevalence of prothrombin G20210A mutation was not different between patients and controls, thereby giving no support to an association of single-point mutation with SCD. Although each of these mutations is relatively common in SCD, neither is independently associated with an increased risk of developing thrombophilic complications. This suggested a low impact of inherited hypercoagulability risk factors in the pathogenesis of SCD and/or its complications in Palestine.Η δρεπανοκυτταρική αναιμία (SS) αποτελεί μία κληρονομούμενη χρόνια αιμολυτική νόσο, που οφείλεται σε σημειακή μετάλλαξη αντικατάστασης της θυμίνης από αδενίνη στο κωδίκιο 6 του γονιδίου της β-σφαιρίνης. Ο υψηλός επιπολασμός της μετάλλαξης βˢ στην Αφρική, στη Μέση Ανατολή, στην Ινδία και σε ορισμένες περιοχές της Μεσογείου συσχετίζεται με την επιλεκτική προστατευτική επίδραση της δρεπανοκυτταρικής αιμοσφαιρίνης (HbS) έναντι της ελονοσίας. Η διασπορά της και σε άλλες περιοχές παγκοσμίως μπορεί να οφείλεται στο δουλεμπόριο ή/και στη μετανάστευση πληθυσμιακών ομάδων. Ο σκοπός της παρούσας μελέτης είναι: 1) Ο προσδιορισμός της προέλευσης της μετάλλαξης βˢ σε Παλαιστίνιους ασθενείς με δρεπανοκυτταρική νόσο. 2) Η μελέτη της επίδρασης του απλότυπου στα αιματολογικά χαρακτηριστικά της νόσου. 3) Η περαιτέρω έρευνα των αιτιολογικών παραγόντων στη διαφορά των επιπέδων της εμβρυϊκής Hb (Hb F) στη δρεπανοκυτταρική νόσο. 4) Η διερεύνηση της συχνότητας και της διαφορετικότητας των ανασυνδυασμών στα χρωμοσώματα που φέρουν το γονίδιο βˢ που σχετίζονται με τους συχνούς (τυπικούς) απλότυπους. 5) Η μελέτη της γενετικής ροής των απλοτύπων της δρεπανοκυτταρικής νόσου σε φυσιολογικά άτομα στη Παλαιστίνη (βᴬ χρωμοσώματα). 6) Ο προσδιορισμός της επίδρασης των θρομβοφιλικών μεταλλάξεων (παράγοντας V Leiden (G1691A) και προθρομβίνη (G20210A) στην υπερπηκτικότητα και εμφάνιση θρομβώσεων στη δρεπανοκυτταρική νόσο. Το δείγμα αποτέλεσαν 117 ασθενείς με SCD, εκ των οποίων οι 59 ήταν SS (30 άρρενες και 29 θήλυ) με μέση ηλικία 16 ± 9.9 έτη, οι 25 ήταν AS (14 άρρενες και 11 θήλυ) με μέση ηλικία 21.2 ± 9.1 έτη, και τέλος οι 33 ήταν S/β θαλασσαιμία (18 άρρενες και 15 θήλυ) με μέση ηλικία 15.1 ± 5.1 έτη. Η ομάδα ελέγχου απαρτίζονταν από 78 υγιή άτομα (57 άντρες και 21 γυναίκες) με μέση ηλικία 20.6 ± 5.5 έτη. Να σημειωθεί ότι όλοι οι ασθενείς προέρχονταν από διαφορετικές πόλεις της Παλαιστίνης και παρακολουθούνταν όλοι στο Εθνικό Κέντρο Αιματολογικών Νοσημάτων «Ιπποκράτης» στη πόλη της Ραμάλα της Παλαιστίνης. Οι απλότυποι του γονιδίου της β-σφαιρίνης προσδιορίστηκαν με την μέθοδο της PCR και ανίχνευση πολυμορφισμών μήκους θραύσματος από περιοριστικό ένζυμο (PCR-RFLP), με ειδικά ένζυμα περιορισμού. Για τον προσδιορισμό των απλοτύπων, 8 περιοριστικές θέσεις γύρω και μέσα στη γονιδιακή περιοχή του β-γονιδίου πολλαπλασιάστηκαν με PCR. Οι 8 πολυμορφικές περιοριστικές θέσεις και τα αντίστοιχα περιοριστικά ένζυμα που χρησιμοποιήθηκαν, είναι τα εξής παρακάτω: το ένζυμο Hind II 5΄ για το γονίδιο ε , το Xmn I 5΄ για το ᴳγ γονίδιο, το Hind III για την ενδιάμεση αλληλουχία IVS-II των ᴳγ και ᴬγ γονιδίων, το Hind II 3΄για το ψβ γονίδιο, το RsaI 5΄ για το β γονίδιο, το AvaII για τη περιοχή IVS-II του β γονιδίου, και τέλος το HinfI 3΄ για το β γονίδιο. Ο προσδιορισμός των θρομβοφιλικών μεταλλάξεων πραγματοποιήθηκε με PCR-RFLP χρησιμοποιώντας τα περιοριστικά ένζυμα Mnl 1 και HindIII για τη μετάλλαξη του παράγοντα FV-Leiden και της προθρομβίνης G20210A, αντίστοιχα. Από το σύνολο των 234 χρωμοσωμάτων, τα 176 ήταν βˢ, τα 33 β-θαλασσαιμία, ενώ 25 έφεραν το φυσιολογικό γονίδιο βᴬ. Τέσσερις τυπικοί απλότυποι (Benin, Bantu, Cameroon και Senegal) και τέσσερις άτυποι (Bantu A2 (+----+++), Bantu A2c (+----++-), Bantu A2a (+----+-+) και Bantu A6a (--++-+-+)) ανευρέθηκαν στο δείγμα της μελέτης μας. Ο απλότυπος Benin ήταν ο επικρατέστερος τόσο στην ετερόζυγη όσο και στην ομόζυγη μορφή του. Επιπρόσθετα, ο απλότυπος αυτός βρέθηκε στο 82.4% των ομόζυγων μορφών της νόσου. Ο άτυπος απλότυπος Bantu A2 ήταν ο δεύτερος συχνότερος απλότυπος στο δείγμα των ασθενών μας. Συγκεκριμένα ο απλότυπος Bantu A2 αντιστοιχεί στο 29.5% των βᴬ απλοτύπων, εξηγώντας τον υψηλό επιπολασμό στο δείγμα μας και από την παρουσία του και στο φυσιολογικό πληθυσμό. Η ετερογένεια που παρατηρήθηκε στην παραγωγή της Hb F στους ασθενείς SS, κυμαίνονταν μεταξύ 1.5 και 17%, ενώ ο λόγος γᴳ ήταν ομοιογενής ανάμεσα στους SS ασθενείς, με φυσιολογικές τιμές να κυμαίνονται περίπου στο 41%. Τα αποτελέσματά μας είναι σε συμφωνία με προϋπάρχουσες έρευνες για την προέλευση του απλότυπου Benin στη Μεσόγειο. Η θρόμβωση πιθανόν παίζει πρωτεύοντα ρόλο στην παθοφυσιολογία ορισμένων επιπλοκών της δρεπανοκυτταρικής νόσου (SCD). Οι αγγειακές επιπλοκές, όπως το ισχαιμικό εγκεφαλικό επεισόδιο και η εν τω βάθει φλεβική θρόμβωση διαγνώσθηκαν σε 7 ασθενείς στη μελέτη μας. Η σταθμισμένη για ηλικία και φύλο ανάλυση (λογιστική παλινδρόμιση) έδειξε μία σημαντική συσχέτιση μεταξύ της μετάλλαξης του παράγοντα V Leiden και της δρεπανοκυτταρικής αναιμίας με odds ratios (OR) 0.33 (95% διαστήματα εμπιστοσύνης-confidence intervals [CI] 0.12–0.94, P = 0.039) σε SS ασθενείς. Εντούτοις, η διαφορά του υψηλού επιπολασμού του παράγοντα V Leiden σε AS άτομα και σε sickle/β-θαλασσαιμία ασθενείς δεν ήταν στατιστικώς σημαντική σε σχέση με τους μάρτυρες. Ο επιπολασμός της μετάλλαξης της προθρομβίνης G20210A δεν ήταν σημαντικά διαφορετικός ανάμεσα στους ασθενείς και στους μάρτυρες, απορρίπτοντας την υπόθεση της συσχέτισης της μετάλλαξης αυτής με τη δρεπανοκυτταρική νόσο SCD. Αν και οι μεταλλάξεις αυτές είναι αρκετά συχνές στη SCD, καμία δεν συσχετίστηκε ανεξάρτητα με αυξημένο κίνδυνο θρομβοφιλικών επιπλοκών. Τα αποτελέσματα αυτά προτείνουν τη χαμηλή επίδραση των κληρονομούμενων παραγόντων κινδύνου για υπερπηκτικότητα στην παθογένεια της δρεπανοκυτταρικής νόσου ή/και στις επιπλοκές αυτής, στην Παλαιστίνη

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

Abstract 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 β-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 β-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

Genetic modulators of sickle cell disease

Sickle cell disease is a monogenic hereditary disease characterized by a mutation in the β-globin gene. The molecular nature is substitution of valine for glutamic acid at the sixth amino acid position in the β-globin gene. The clinical heterogeneity of the disease, which is characterized by the presence of hemoglobin S, can be explained in terms of fetal hemoglobin (HbF) levels, ratio of Gγ chains to Aγ chains, linked mutations, βs haplotypes, co-existence of α-thalassemia and environmental factors. Five major haplotypes associated with the βs mutation have been defined: Benin, Bantu, Senegal, Cameroon and Arab/Indian. The inheritance of Senegal and Arab/Indian haplotypes is associated with a milder course; whereas the Bantu, Cameroon, and Benin haplotypes are linked with sever sickle cell disease. Bantu haplotype carries the worst prognosis of all. These DNA polymorphisms also assume great importance as anthropologic and genetic markers. High levels of hemoglobin F protect against several clinical features. α-thalassemia coincides with sickle cell disease, this gene lowers the mean cell Hb concentration, inhibiting polymerization and hence sickling, giving rise to a milder course. The present review discusses the different genetic modulators that ameliorate the clinical picture of sickle cell disease

Prevalence of Sero-Molecular Markers of Hepatitis C and B Viruses among Patients with β-Thalassemia Major in Northern West Bank, Palestine

Background. HCV and HBV present a great challenge in the management of β-thalassemia patients. Objective. The present study aimed to determine the prevalence of both HBV and HCV in multitransfused-dependent β-thalassemia patients in northern West Bank, Palestine, using sero-molecular markers. Methods. Serum sample from 139 multitransfused β-thalassemia patients were tested for HBV and HCV markers including HBsAg, anti-HBc, anti-HBs, HBV-DNA, and anti-HCV and HCV-RNA. Demographic data and selected clinical parameters were collected by means of a questionnaire and from the patients’ medical files. Results and Conclusion. The mean (±SD) age of patients was 18.1 years (±10.6). The overall prevalence of the HCV was 10% (14/139), which is 50 times higher than the normal Palestinian population (0.2%). Of which, 3 were positive for anti-HCV alone, 7 positives for HCV-RNA alone, and 4 positives for both anti-HCV and PCR-RNA. On the other hand, low prevalence of HBV was detected at a level of 0.7% (1/139). Only one patient had HCV-HBV coinfection. Twenty-five patients (19%) were positive for anti-HBc, while 99 (71%) were immune with the anti-HBs level above 10 IU/mL. Anti-HBc was insignificantly high (P=0.07) in HCV-positive cases. In conclusion, the prevalence of HCV among β-thalassemia patients is considered high compared to normal population. Determination of HCV prevalence should be based on the detection of both HCV-RNA and anti-HCV. On the contrary, HBV showed a low prevalence. A follow-up schedule and administration of booster dose of HBV vaccine is strongly recommended for β-thalassemia patients whose anti-HBs level <10 IU/ml

Genetic diversity of the enteroviruses detected from cerebrospinal fluid (CSF) samples of patients with suspected aseptic meningitis in northern West Bank, Palestine in 2017.

BackgroundHuman enterovirus genus showed a wide range of genetic diversity.ObjectivesTo investigate the genetic diversity of the enteroviruses isolated in 2017 in northern West Bank, Palestine.Study design249 CSF samples from aseptic meningitis cases were investigated for HEV using two RT-PCR protocols targeting the 5' NCR and the VP1 region of the HEV genome. The phylogenetic characterization of the sequenced VP1 region of Echovirus18 (E18) and Coxsackievirus B5 (CVB5) isolated in Palestine along with 27 E18 and 27 CVB5 sequences available from the Genbank were described.ResultsE18 and CVB5 account for 50% and 35% of the successfully HEV types, respectively. Phylogenetic tree of E18 and CVB5 showed three main clusters, with all Palestinian isolates uniquely clustering together with those from China and from different countries, respectively. Cluster I of E18, with 13 Palestinian and 6 Chinese isolates, showed the lowest haplotype-to-sequence ratio (0.6:1), haplotype diversity (Hd), nucleotide diversity (π), and number of segregating sites (S) compared to clusters II and III. Furthermore, cluster I showed negative Tajima's D and Fu-Li'sF tests with statistically significant departure from neutrality (PConclusionsThe study divulged close genetic relationship between Palestinian HEV strains as confirmed by population genetics and phylogenetic analyses

Genetic diversity, haplotype analysis, and risk factor assessment of hepatitis A virus isolates from the West Bank, Palestine during the period between 2014 and 2016.

BackgroundHepatitis A virus (HAV) infection is one of the major causes of acute viral hepatitis. HAV genotypes and its genetic diversity is rarely investigated in our region as well as worldwide.AimsThe aims of the present study were to determine the HAV genotypes and its risk factors and to investigate the genetic diversity of the HAV isolates in the West Bank, Palestine.Study designA cohort of 161 clinically and laboratory-confirmed HAV (IgM-positive) cases and 170 apparently healthy controls from all the districts of the West Bank, Palestine during the period of 2014 to 2016 were tested for HAV infection using IgM antibodies, RT-PCR and sequence analysis of the VP3/VP1 junction region of the HAV genome. Phylogenetic analysis, genetic diversity and haplotypes analysis were used to characterize the VP3/VP1 sequences.ResultsAll the 34 sequences of the HAV were found to be of HAV-IB sub-genotype. The phylogenetic analysis showed four main clusters with cluster III exclusively consisting of 18 Palestinian isolates (18/23-78%), but with weak bootstrap values. A high haplotype diversity (Hd) and low nucleotide diversity (π) were observed. Cluster III showed high number of haplotypes (h = 8), but low haplotype (gene) diversity (Hd = 0.69). A total of 28 active haplotypes with some consisting of more than one sequence were observed using haplotype network analysis. The Palestinian haplotypes are characterized by closely related viral haplotypes with one SNV away from each other which ran parallel to cluster III in the phylogenetic tree. A smaller Palestinian haplotype (4 isolates) was three SNVs away from the major haplotype cluster (n = 10) and closer to others haplotypes from Iran, Spain, and South Africa. Young age, low level of parent's education, infrequent hand washing before meals, and drinking of un-treated water were considered the major HAV risk factors in the present study.ConclusionHaplotype network analysis revealed haplotype variation among the HAV Palestinian sequences despite low genetic variation and nucleotide diversity. In addition, this study reconfirmed that age and parent's level of education as HAV risk factors, while hand washing and treating drinking water as protective factors