Immuno-molecular Studies on Sudanese Patients with Hemophilia: Inhibitor prevalence and carrier detection

Abstract

Introduction: Hemophilia is the oldest known hereditary X-linked recessive and an incurable bleeding disorder that affects males whereas females act as carriers with some rare cases among women worldwide. Naturally, women hemophiliacs are rare because it takes two defective X chromosomes in order for the condition to manifest. Approximately 10 in 100,000 males have hemophilia. Persons with hemophilia suffer from frequent bleeds in joints and muscles with severe pain and swelling. Untreated bleeds lead to progressive crippling which is the major cause of disability in hemophiliac patients. The formation of inhibitory Ig G allo-antibodies is the most severe and costly complication of replacement therapy in patients with haemophilia. Many factors predispose to the development of inhibitors: the nature of the molecular defect, level of factor deficiency, ethnic origin, timing and types of factor replacements. The complexity of the immune response to the infused factor becomes more and more obvious. .Antibodies develop as a result of a complex multi-factorial interaction between antigen-presenting cells, T and B-lymphocytes. Genetic susceptibility of cell surface molecules, such as the major histocompatibility complex (MHC; HLA), the T- cell receptor and cytokine receptors, as well as various immunomodulatory molecules and environmental factors have a major impact on inhibitor development. Carrier detection in the hemophilias has received new impetus in the past few years. Early prenatal diagnosis and development of new genetic markers for the clotting factor genes have focused on this area. Until now, carrier diagnosis has relied upon standard pedigree analysis and clotting factors assays. The results obtained using these methods are probabilistic, and the coagulation tests are unavoidably influenced by the effects of random X chromosome inactivation (Lyonization) and the inherent variability of the methods involved. The cloning and characterization of both factor IX and factor VIII genes have revolutionized gene analysis techniques to diagnose the carrier state. This usually involves the detection of restriction fragment length polymorphisms (RFLPs) and their use as linked markers for the detection of defective clotting factor gene. In hemophilia A, the combined use of two intragenic RFLPs markers BclI and Hind III restriction polymorphic sites (closely linked to the genetic defect in Factor VIII) in intron (18) and intron (19) respectively, made carrier detection feasible for approximately 90% of kindred using PCR, RFLPs and gel electrophoresis. Study design and objectives: This was a prospective, longitudinal study with three years duration with regular follow ups that aimed to determine the prevalence of inhibitors to FVIII in a cohort of Sudanese patients with hemophilia and to correlate the timing and frequency of blood and blood products transfusion to the development of inhibitors. The study also aimed to detect carriers in families of haemophilic patients using PCR-based restriction fragment length polymorphisms (PCR-RFLPs) technique. Materials and methods: Following informed consent, patients with suspected bleeding disorders were seen and investigated at the Haemostasis Clinic at the Institute of Endemic Diseases, University of Khartoum. Families of patients with haemophilia A and B were recruited in the study. Demographic data, present and past medical history, family history and clinical examination were recorded in a specially designed case record form (CRF). Ten mls of venous blood were collected in plain, citrate and EDTA containers respectively for HIV, hepatitis B and C serological tests, Complete Blood Count, Coagulation studies (Prothrombin Time, Activated Partial Thromboplastin Time, Thrombin Time, Fibrinogen , and mixing studies and assays for Factor VIII and IX). Inhibitors to factor VIII/IX were tested for using the Bethesda method. DNA was extracted from EDTA blood using Phenol/choloroform/Isoamyl alcohol method. Polymerase Chain Reaction-based RFLPs (PCR-RFLPs) was carried using standard protocols. Indirect analysis (RFLPs) for carrier detection using specific primers and appropriate restriction enzymes (Bcl1 for intron 18 and Hind III for intron19). Intron22/1 inversion was tested for three patients with laboratory FVIII ≥1% and clinical features of severe haemophilia. Results: Two hundred and forty seven families with 694 individuals (Males: Female = 1:2) Patients with haemostatic defects (n= 533, 76.8%) were categorized according to the screening test as hemophilia A (n=342, 64.2%), hemophilia B (n=34, 6.4%) and miscellaneous bleeding disorder rs (n=157; 29.4%). Hemophilia A is most common genetic disease among the tribes of Galleen and Shaigia and less common among Meisairia and Falata. The mean age of haemophilia A patients 15.4 ±12.5 years. The haemoglobin level was significantly reduced in hemophilic patients compared with non-hemophilic individuals (p=0.007). While the platelets counts (PLTs) (p=0.07), white blood cells counts (p=0.05), bleeding time (p=0.05), prothrombin time (p=0.000), thrombin time (p=0.04) and fibrinogen (p=0.25) were comparable to non-diseased family members. The Activated Partial thromboplastin Time (APTT) of haemophilic patients was significantly prolonged (p= 00001). Patients with mild hemophilia (factor levels, range 5-25%) constituted 24%, while those with moderate hemophilia (factor levels 1-5%) constituted 76% of patients. Factor VIII inhibitors could not be detected in the sera of haemophilic patients. One per cent (1%)of hemophilic patients were treated with whole blood, 10%, 29% and 57% were treated with Cryoprecipitate, Fresh frozen plasma(FFP) and factor concentrates respectively. Three per cent (3%) of the patients received no treatment. The treatment of all patients was carried out after diagnosis. Anonymous viral serology screening showed that 0.3% of the patients were reactive to HIVI/II, 1% was reactive for HBsAg but no patient was reactive for HCV. The HIV reactivity was not different from that reported from National Blood Transfusion Services donations in Sudan(NBTS). The HBsAg screening is much lower than that among blood donors in the (NBTS) figures. The study of homo/heterozygosis for carrier/disease status was carried in thirteen families (n= 63 individuals; males =34 & females =29). Twenty one patients were hemophilic, 16 were their sisters. The mothers tested were 13; 8/13 (61.3%) were possible carriers, while the rest were obligate carriers. Sixteen sisters of haemophilic patients were tested, 2 were normal, and 14 were carriers (6/14 were obligate; 8/14 were possible carriers). In all 27/29 females (mothers + sisters of haemophilics) were investigated for homo/heterozygosity of FVIII polymorphic regions. Nineteen (19/27) were heterozygous, while 8/27 were homozygous. Discussion: Hemophilia is an X-Linked recessive inherited bleeding disorder that affects males usually born to unaffected father and an asymptomatic carrier mother. Delayed treatment can lead to marked disabilities. Untreated bleeds lead to progressive crippling which is the major cause of disability in hemophilic patients. Development of neutralizing antibodies to factors VIII and IX is a major complication of haemophilia therapy. All our patients have either mild or moderate disease have levels of FVIII and FIX above 1% with detectable low levels of the FVIIIC Ag, most probably indicating absence of structurally abnormal factor VIII protein. Inhibitors were not detected in the study patients. This could be a fact of life that no severe haemophilic disease situations exist, or that patients die early due to the remoteness of some areas or lack of factor concentrates /blood components. Absence of truly severe haemophilia was confirmed by negative intron 22/1 inversion that was conducted in the three patients with clinically severe disease. Genetic counseling based on DNA diagnostic approaches have assumed an important role in the pathology laboratory. The genetic testing involves carrier analysis; mother of an affected boy can be obligate carrier if she has a haemophiliac father or more than one haemophiliac son. In this study RFLPs was easily applied to detect carriers with females of index cases. The majority of females (mothers & sisters of haemophilic patients) were possible carriers. Carrier detection combined with factor assay can help in identifying dangerous carriers who sometimes present with severe bleeding tendencies and be mistakenly diagnosed as von Will brand disease. Conclusion & Recommendations: Haemophilic patients investigated have mild/moderate disease. No anti-FVIII inhibitor was detected in the sera of our patients. Most of the females of the families with haemophilic patients tested were possible carriers. A network of satellite haemophilia management and carrier detection centers should be established to provide nationwide standard care management to prevent loss of severe haemophilics. A larger and nationwide study should be launched to estimate the true magnitude of the problem. Further molecular studies (sequencing) is recommended to pinpoint the exact molecular defect to calculate exactly the chance of inhibitor development in case haemophilia care improves and patients with severe haemophlia start to live longer.