Hemoglobinopathies, G6PD deficiency, and hereditary elliptocytosis in Bahrain

The native population of Bahrain has a high prevalence of hemoglobinopathies and G6PD deficiency, probably as a result of past malarial endemism. We used the Biorad-Variant hemoglobin testing system for primary screening of hemoglobinopathies in 20,000 individuals. Hemoglobin abnormalities were detected in 7,206 (36.3%) cases.     

Treating hemoglobinopathies using gene-correction approaches: promises and challenges

Hemoglobinopathies are genetic disorders caused by aberrant hemoglobin expression or structure changes, resulting in severe mortality and health disparities worldwide. Sickle cell disease (SCD) and β-thalassemia, the most common forms of hemoglobinopathies, are typically treated using transfusions and pharmacological agents. Allogeneic hematopoietic stem cell transplantation is the only curative therapy, but has limited clinical applicability. Although gene therapy approaches have been proposed based on the insertion and forced expression of wild-type or anti-sickling β-globin variants, safety concerns may impede their clinical application. A novel curative approach is nuclease-based gene correction, which involves the application of precision genome-editing tools to correct the disease-causing mutation. This review describes the development and potential application of gene therapy and precision genome-editing approaches for treating SCD and β-thalassemia. The opportunities and challenges in advancing a curative therapy for hemoglobinopathies are also discussed.     

In silico analysis of single nucleotide polymorphism (SNPs) in human β-globin gene

Single amino acid substitutions in the globin chain are the most common forms of genetic variations that produce hemoglobinopathies--the most widespread inherited disorders worldwide. Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB) gene mutations, such as that producing sickle cell hemoglobin (HbS), HbC, HbD and HbE. Several of these mutations are deleterious and result in moderate to severe hemolytic anemia, with associated complications, requiring lifelong care and management. Even though many hemoglobinopathies result from single amino acid changes producing similar structural abnormalities, there are functional differences in the generated variants. Using in silico methods, we examined the genetic variations that can alter the expression and function of the HBB gene. Using a sequence homology-based Sorting Intolerant from Tolerant (SIFT) server we have searched for the SNPs, which showed that 200 (80%) non-synonymous polymorphism were found to be deleterious. The structure-based method via PolyPhen server indicated that 135 (40%) non-synonymous polymorphism may modify protein function and structure. The Pupa Suite software showed that the SNPs will have a phenotypic consequence on the structure and function of the altered protein. Structure analysis was performed on the key mutations that occur in the native protein coded by the HBB gene that causes hemoglobinopathies such as: HbC (E→K), HbD (E→Q), HbE (E→K) and HbS (E→V). Atomic Non-Local Environment Assessment (ANOLEA), Yet Another Scientific Artificial Reality Application (YASARA), CHARMM-GUI webserver for macromolecular dynamics and mechanics, and Normal Mode Analysis, Deformation and Refinement (NOMAD-Ref) of Gromacs server were used to perform molecular dynamics simulations and energy minimization calculations on β-Chain residue of the HBB gene before and after mutation. Furthermore, in the native and altered protein models, amino acid residues were determined and secondary structures were observed for solvent accessibility to confirm the protein stability. The functional study in this investigation may be a good model for additional future studies.     

Taking the good out of the bad: lentiviral-based gene therapy of the hemoglobinopathies

Sickle cell disease and beta-thalassemia are excellent candidates for gene therapy since transfer of a single gene into hematopoietic stem cells should theoretically elicit a therapeutic response. Initial attempts at gene therapy of these hemoglobinopathies have proved unsuccessful due to limitations of available gene transfer vectors. With the extensive research on human immunodeficiency virus-1 due to the acquired immune deficiency syndrome pandemic, researchers have realized that this lentivirus, engineered to be devoid of any pathogenic elements, can be an effective gene transfer vector. This review discusses the gene therapy strategy for the hemoglobinopathies and outlines why lentiviral-derived vectors are particularly suited for this type of application, keeping past failures at gene therapy of these hemoglobinopathies in mind. Development, improvement, and methods for preparation of lentiviral-derived vectors are examined. Recently published results of successful gene therapy treatment of beta-thalassemic and sickle cell diseased mice using lentiviral-derived vectors are described. Finally, criticisms and future directions of lentiviral-based biotechnology are considered.     

Ocular manifestations of sickle hemoglobinopathies

Sickle cell disease is a hereditary disorder that is characterized by the production of structurally abnormal hemoglobin molecules. Clinical manifestations depend upon the amount and types of abnormal hemoglobin present. This paper reviews the genetic and molecular basis of sickle hemoglobinopathies and thalassemias including sickle cell anemia, SC disease, sickle cell-Beta Thalassemia and sickle trait. The systemic and ocular manifestations of these diseases are presented. Treatment regimens pertaining to hyphema, proliferative retinopathy, vitreous hemorrhage and retinal detachment are also discussed.     

Incidence of Sickle Cell Disease and Other Hemoglobin Variants in 10,095 Lebanese Neonates

Hemoglobinopathies are highly prevalent diseases and impose a public health burden. Early diagnosis and treatment can ameliorate the course of these diseases and improve survival. Despite purported high incidence of hemoglobinopathies in Lebanon, there are no nationwide screening programs. In this study, newborn screening utilizing high pressure liquid chromatography was executed in all public hospitals across Lebanon between 2010 and 2013. All newborns with an abnormal hemoglobin (Hb) were offered genetic counseling and all those with disease were enrolled in comprehensive hemoglobinopathy clinics. Among newborns, 2.1% were found to have an abnormal Hb variant with sickle Hb being the most common while 0.1% were found to have sickle cell disease (SCD). The majority of those with SCD had non-Lebanese origins. The most common causes of hospitalizations in infants with SCD were acute splenic sequestration and pain crises. No bacteremia or other life threatening infections were noted. At a median follow up 14 months (follow up range 7 to 34 months), all children with disease are alive and compliant with treatment. Systematic screening for SCD and other Hb variants was shown to be feasible, cost effective, and of accurate predictive value. This program was also clinically effective because it led to the identification of babies with disease and to providing them with free early multidisciplinary care. Conclusively, a newborn screening program should be implemented across Lebanon to detect hemoglobinopathies and initiate early therapeutic and preventive strategies and genetic counseling.     

Disorders of the synthesis of human fetal hemoglobin

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (alpha(2)(G)gamma(2) and alpha(2)(A)gamma(2)) that differ only at position 136 reflecting the products of two nonallelic gamma-globin genes. At the time of birth, HbF accounts for approximately 70% of the total Hb. The (G)gamma:(A)gamma globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a gamma- to beta-globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to (G)gamma and (A)gamma chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as gamma-thalassemias, duplications, triplications, and even sextuplications of the gamma-globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the beta-globin gene cluster, such as beta-thalassemia, sickle cell disease, deltabeta-thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of gamma-globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching.     

Epistasis and the genetics of human diseases

Epistasis or modifier genes, that is, gene-gene interactions of non-allelic partners, play a major role in susceptibility to common human diseases. This old genetic concept has experienced a major renaissance recently. Interestingly, epistatic genes can make the disease less severe, or make it more severe. Hence, most diseases are of different intensities in different individuals and in different ethnicities. This phenomenon affects sickle-cell anemia carriers and other hemoglobinopathies, systemic lupus erythematosus, cystic fibrosis, complex autoimmune diseases, venous thromboembolism, and many others. It is likely, and fortunate, than 20 years form now, patients entering a medical facility will be subjected to a genomic scanning, including pathogenic genes as well as epistatic genes.     

Future prospects for treatment of hemoglobinopathies.

Strategies for the treatment of sickle cell anemia and beta-thalassemia are founded on the knowledge that these disorders result from structural or functional defects in an adult gene for which an intact fetal counterpart exists. During the past decade, several pharmacologic agents have been investigated for their potential to ameliorate sickle cell anemia and beta-thalassemia by increasing the synthesis of fetal hemoglobin in adults. Progress in understanding globin gene regulation is now being combined with advances in retrovirus-mediated gene transfer, and the once-distant goal of providing gene therapy for hemoglobinopathies is rapidly approaching reality.     

Curative therapy for hemoglobinopathies: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee review comparing outcomes,accessibility and cost of ex vivo stem cell gene therapy versus allogeneic hematopoietic stem cell transplantation

Thalassemia and sickle cell disease (SCD) are the most common monogenic diseases in the world and represent a growing global health burden. Management is limited by a paucity of disease-modifying therapies; however, allogeneic hematopoietic stem cell transplantation (HSCT) and autologous HSCT after genetic modification offer patients a curative option. Allogeneic HSCT is limited by donor selection, morbidity and mortality from transplant conditioning, graft-versus-host disease and graft rejection, whereas significant concerns regarding long-term safety, efficacy and cost limit the broad applicability of gene therapy. Here the authors review current outcomes in allogeneic and autologous HSCT for transfusion-dependent thalassemia and SCD and provide our perspective on issues surrounding accessibility and costs as barriers to offering curative therapy to patients with hereditary hemoglobinopathies.     

Elephant behaviour and conservation: social relationships, the effects of poaching, and genetic tools for management

Genetic tools are increasingly valuable for understanding the behaviour, evolution, and conservation of social species. In African elephants, for instance, genetic data provide basic information on the population genetic causes and consequences of social behaviour, and how human activities alter elephants' social and genetic structures. As such, African elephants provide a useful case study to understand the relationships between social behaviour and population genetic structure in a conservation framework. Here, we review three areas where genetic methods have made important contributions to elephant behavioural ecology and conservation: (1) understanding kin-based relationships in females and the effects of poaching on the adaptive value of elephant relationships, (2) understanding patterns of paternity in elephants and how poaching can alter these patterns, and (3) conservation genetic tools to census elusive populations, track ivory, and understand the behavioural ecology of crop-raiding. By comparing studies from populations that have experienced a range of poaching intensities, we find that human activities have a large effect on elephant behaviour and genetic structure. Poaching disrupts kin-based association patterns, decreases the quality of elephant social relationships, and increases male reproductive skew, with important consequences for population health and the maintenance of genetic diversity. In addition, we find that genetic tools to census populations or gather forensic information are almost always more accurate than non-genetic alternatives. These results contribute to a growing understanding of poaching on animal behaviour, and how genetic tools can be used to understand and conserve social species.     

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells.     

Human and mouse hemoglobin association with the transgenic mouse erythrocyte membrane

Transgenic mouse models of hemoglobinopathies unravel pathophysiological mechanisms; yet the validity of the red blood cell (RBC) model of human hemoglobin (hHb) enveloped by a mouse (m) membrane has been questioned. Isoelectric focusing of hHb and mHb from transgenic mRBC shows a greater association of mHb to the mouse membrane compared to normal hHbA, supporting a species-specific Hb-mRBC membrane interaction. Enhanced hmutant Hb (HbE, HbS and HbC)-mRBC membrane affinities correlates with enhanced membrane lipid peroxidation and parallel those reported in hRBC, lending support to transgenic mRBC as models of hemoglobinopathies. Species-specific Hb-membrane interaction may be overridden by Hb charge and conformational alterations.     

Chromosomes: the missing link — young people's understanding of mitosis,meiosis, and fertilisation

This paper considers school students' understanding of the processes of cell division and fertilisation towards the end of their compulsory science education. The difficulties which students have in understanding the purposes and products of these processes are discussed, and the origins of some of these problems are identified. In particular, it notes the widespread lack of understanding of the physical link between chromosomes and genetic material, and the relationship between the behaviour of chromosomes at cell division and the continuity of genetic information - both within and between organisms. Key words: Cell division, Chromosomes, Genetic information, Students' understandings.     

Regulation of iron absorption in hemoglobinopathies

Beta-thalassemia and sickle cell anemia (SCD) represent the most common hemoglobinopathies caused, respectively, by deficient production or alteration of the beta chain of hemoglobin (Hb). Patients affected by the most severe form of thalassemia suffer from profound anemia that requires chronic blood transfusions and chelation therapies to prevent iron overload. However, patients affected by beta-thalassemia intermedia, a milder form of the disease that does not require chronic blood transfusions, eventually also show elevated body iron content due to increased gastrointestinal iron absorption. Even SCD patients might require blood transfusions and iron chelation to prevent deleterious and painful vaso-occlusive crises and complications due to iron overload. Although definitive cures are presently available, such as bone marrow transplantation (BMT), or are in development, such as correction of the disease through hematopoietic stem cell beta-globin gene transfer, they are potentially hazardous procedures or too experimental to provide consistently safe and predictive clinical outcomes. Therefore, studies that aim to better understand the pathophysiology of the hemoglobinopathies might provide further insight and new drugs to dramatically improve the understanding and current treatment of these diseases. This review will describe how recent discoveries on iron metabolism and erythropoiesis could lead to new therapeutic strategies and better clinical care of these diseases, thereby yielding a much better quality of life for the patients.     

The promise of epigenetics in personalized medicine

Numerous preclinical and clinical trials, with older as well as some newer drugs, have demonstrated the targeting of aberrant epigenetic marks to be a viable means of preventing and treating certain human disorders, including myelodysplastic and leukemic syndromes and various hemoglobinopathies. These findings are encouraging, and although the risks associated with such therapy are largely unknown, precise maps of epigenetic marks are becoming increasingly available through advancements in sequencing protocols that combine chromatin immunoprecipitation and gene expression analyses. Indeed, progress in understanding gene regulation at promoter regions and chromatin organization in health and disease has been substantial. New insights into the proteins that are targeted by therapeutic agents that alter epigenetic programs may provide important inroads into personalized medicine.     

Application of diagnostic methods and molecular diagnosis of hemoglobin disorders in Khuzestan province of Iran

BACKGROUND:

The hemoglobinopathies refer to a diverse group of inherited disorders characterized by a reduced synthesis of one or more globin chains (thalassemias) or the synthesis of structurally abnormal hemoglobin (Hb). The thalassemias often coexist with a variety of structural Hb variants giving rise to complex genotypes and an extremely wide spectrum of clinical and hematological phenotypes. Hematological and biochemical investigations and family studies provide essential clues to the different interactions and are fundamental to DNA diagnostics of the Hb disorders. Although DNA diagnostics have made a major impact on our understanding and detection of the hemoglobinopathies, DNA mutation testing should never be considered a shortcut or the test of first choice in the workup of a hemoglobinopathy.

MATERIALS AND METHODS:

A careful three-tier approach involving: (1) Full blood count (2) Special hematological tests, followed by (3) DNA mutation analysis, provides the most effective way in which to detect primary gene mutations as well as gene-gene interactions that can influence the overall phenotype. With the exception of a few rare deletions and rearrangements, the molecular lesions causing hemoglobinopathies are all identifiable by PCR-based techniques. Furthermore, each at-risk ethnic group has its own combination of common Hb variants and thalassemia mutations. In Iran, there are many different forms of α and β thalassemia. Increasingly, different Hb variants are being detected and their effects per se or in combination with the thalassemias, provide additional diagnostic challenges.

RESULTS:

We did step-by-step diagnosis workup in 800 patients with hemoglobinopathies who referred to Research center of Thalassemia and Hemoglobinopathies in Shafa Hospital of Ahwaz Joundishapour University of medical sciences, respectively. We detected 173 patients as iron deficiency anemia (IDA) and 627 individuals as thalassemic patients by use of different indices. We have successfully detected 75% (472/627) of the β-thalassemia mutations by using amplification refractory mutation system (ARMS) technique and 19% (130/627) of the β-thalassemia mutations by using Gap-PCR technique and 6% (25/627) as Hb variants by Hb electrophoresis technique. We did prenatal diagnosis (PND) for 176 couples which had background of thalassemia in first pregnancy. Result of PND diagnosis in the first trimester was 35% (62/176) affected fetus with β-thalassemia major and sickle cell disease that led to termination of the pregnancy.

CONCLUSION:

Almost all hemoglobinopathies can be detected with the current PCR-based assays with the exception of a few rare deletions. However, the molecular diagnostic service is still under development to try and meet the demands of the population it serves. In the short term, the current generation of instruments such as the capillary electrophoresis systems, has greatly simplified DNA sequence analysis.     

Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes

Hb Hasharon has an electrophoretic mobility similar to that of Hb S in cellulose acetate and a mobility between Hb S and C at acid pH. In high-performance liquid chromatography, Hb Hasharon shows a distinct chromatographic profile and retention time. The origin of this variant is a mutation in codon 47 (GAC --> CAC) of the alpha2-globin gene, resulting in the replacement of asparagine by histidine during the translation process. Ten blood samples from individuals suspected of being Hb Hasharon carriers were analyzed. In addition to classic laboratory tests and high-performance liquid chromatography, molecular analysis by polymerase chain reaction with restriction fragment length polymorphism designed in the laboratory was performed to confirm this mutation. The study of these cases showed that a combination of classical and molecular methodologies is necessary in the diagnosis of hemoglobinopathies for a correct hemoglobin mutant identification. The accurate identification of hemoglobin variants is essential for genetic counseling and choice of therapy.     

Birth defects in India: Hidden truth,need for urgent attention

Birth defects (structural, functional and metabolic disorder present from birth, may be diagnosed later) rising up as an important cause of infant mortality even in developing countries where infant mortality has been reduced to much extent. Seventy percent of birth defects are preventable through the application of various cost effective community genetic services.Indian people are living in the midst of risk factors for birth defects, e.g., universality of marriage, high fertility, large number of unplanned pregnancies, poor coverage of antenatal care, poor maternal nutritional status, high consanguineous marriages rate, and high carrier rate for hemoglobinopathies. India being the second most populous country with a large number infant born annually with birth defects should focus its attention on strategies for control of birth defects. Many population based strategies such as iodization, double fortification of salt, flour fortification with multivitamins, folic acid supplementation, periconceptional care, carrier screening and prenatal screening are some of proven strategies for control of birth defects. Strategies such as iodization of salt in spite of being initiated for a long time in the past do have a very little impact on its consumption (only 50% were using iodized salt). Community genetic services for control of birth defects can be easily flourished and integrated with primary health care in India because of its well established infrastructure and personnel in the field of maternal and child health care. As there is wide variation for infant mortality rate (IMR) in different states in India, so there is a need of deferential approach to implement community genetic services in states those had already achieved national goal of IMR. On the other hand, states those have not achieved the national goal on IMR priority should be given to management of other causes of infant mortality.     

Toward a population genetic framework of developmental evolution: the costs,limits, and consequences of phenotypic plasticity

Adaptive phenotypic plasticity allows organisms to cope with environmental variability, and yet, despite its adaptive significance, phenotypic plasticity is neither ubiquitous nor infinite. In this review, we merge developmental and population genetic perspectives to explore costs and limits on the evolution of plasticity. Specifically, we focus on the role of modularity in developmental genetic networks as a mechanism underlying phenotypic plasticity, and apply to it lessons learned from population genetic theory on the interplay between relaxed selection and mutation accumulation. We argue that the environmental specificity of gene expression and the associated reduction in pleiotropic constraints drive a fundamental tradeoff between the range of plasticity that can be accommodated and mutation accumulation in alternative developmental networks. This tradeoff has broad implications for understanding the origin and maintenance of plasticity and may contribute to a better understanding of the role of plasticity in the origin, diversification, and loss of phenotypic diversity.