Therapeutic gene editing strategies using CRISPR-Cas9 for theβ-hemoglobinopathies

With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

Retinal Damage of Hemoglobinopathies in Adults: About 181 Cases in Campus-Teaching Hospital of Lomé

Aims: To study the frequency of sickle cell retinopathy and its severity. Material and Methods: Retrospective study over a period of 3 years (July 2013 thru June 2016) of cases of adults over 18 years old sent by the Hematology Department of the Campus-Teaching Hospital of Lomé in the context of a sickle-cell disease check-up. The parameters studied were age, sex, genotype, examination of the fundus by the Goldmann lens. The classi-fication after the fundus was in non-proliferative retinopathy and in 5 proliferative stages according to Goldberg’s classification. Results: Hundred and eighty-one patients were included with an average age of 29.39 ± 9.84. The sex ratio was 0.72 in favor of female. The SC genotype was 50.3%;SS 37.6%;CC 11% and SF 1.1%. Sickle cell retinopathy was found in 22.7% of patients, among which 14.4% were proliferative and 8.3% non-proliferative. Seventy-three percent of patients with proliferative retinopathy were SC genotype, 23% SS and 3% CC genotype. Among SC patients, 20.9% had proliferative retinopathy;8.8% SS;and 5% CC. Fifty percent of patients with proliferative retinopathy had Goldberg’s stage ≥ 3. Conclusion: We found 22.7% of sickle cell retinopathy which proliferative form was strongly represented by the SC genotype.

Comparative Study of Neurosurgical Complications of Thalassemia and Sickle Cell Disease

Introduction: Sickle cell disease and thalassemia are the most frequent hemoglobinopathies. During their evolution, they present certain complications, among which are two neurosurgical emergencies, namely spontaneous cranial epidural hematoma and non traumatic radiculo-medullary compression, with some particularities for each. Method: In order to highlight these particularities, we compared the characteristics of these two complications, from a number of publications reported between 2000 and 2021. Results: Sickle cell disease was complicated by spontaneous cranial epidural hematoma. Forty-two cases were reported, the mean age was 14.7 years (2 - 21 years) and the sex ratio was 6.4. The clinical presentation combined, in a non-traumatic context, signs of intracranial hypertension with those of neurological focalization. Neuroimaging showed epidural-type collection, often frontal and parietal in location. The incriminating mechanisms were ischemia, hemorrhage and extra medullary hematopoiesis. The treatment was surgical. Non traumatic radiculo-medullary was the complication of thalassemia. Of the 77 cases reported, the mean age was 27.5 years (9 - 66 years) and the sex ratio was 4.1. The lesions were epidural with a clear thoracic predominance and resulted from extra marrow hematopoiesis. Treatment included: hypertransfusion, radiotherapy, hydroxyurea and surgery. Vital and functional prognosis were globally satisfactory when the management was rapid. Conclusion: Cranial and spinal epidural lesions, respective complications of sickle cell disease and thalassemia, result from similar mechanisms. Their prognosis depends on the rapidity of management. .

Homozygous Hemoglobinosis CC: A Series of 3 Cases and a Review of the Literature

Hemoglobinosis C occurs mainly in Africa and America with a high frequency in West Africa. In Senegal, homozygous hemoglobinopathy CC constitutes a very rare profile of which only 3 cases are followed in the clinical hematology department of Dakar. The 1st case is a 49-year-old female patient, with notion of 1st degree consanguinity, and a long history of abdominal pain who presented a poorly tolerated anemic syndrome and splenomegaly. The biological assessment showed moderate anemia (7.6 g/dL) with microcytic hypochromia and a CC profile (HbC = 99.2%;HbA2 = 0.8%) on hemoglobin electrophoresis. The second case was a 22-year-old female patient with a notion of 2nd degree consanguinity who presented a Chauffard triad. The haemogram showed mild anaemia (11 g/dL), microcytic and hypochromic. Hemoglobin electrophoresis confirmed a CC profile (HbC = 95.3%;HbA2 = 4.7%). The third patient was 27 years old, with a history of diffuse abdominal pain and 2nd degree consanguinity. The haemogram and haemoglobin electrophoresis confirmed the CC profile (HbC = 94.6%;HbA2 = 5.4%). The negativity of the Emmel test in front of this presentation suggestive of sickle cell disease means that this type of hemoglobinopathy is diagnosed late in our regions. We therefore recommend the systematic performance of hemoglobin electrophoresis in the presence of any chronic hemolytic anemia.

Perforated duodenal ulcer secondary to deferasirox use in a child successfully managed with laparoscopic drainage:A case report

BACKGROUND A perforated gastroduodenal ulcer is rarely observed in children.Certain medications have been reported to cause ulcerations.Deferasirox,an iron chelating agent,has been previously reported to be associated with the development of gastroduodenal ulcers.CASE SUMMARY We report a case of a 3-year-old boy who was diagnosed with beta thalassemia major and treated with deferasirox.He presented to the emergency department with an acute abdomen.A perforated duodenal ulcer was suspected after X-ray imaging and laparoscopic exploration.It was successfully managed with laparoscopic washout and drainage.CONCLUSION Due to the rarity and severity of this case,it is a reminder that prevention and early recognition of gastrointestinal complications in patients receiving deferasirox are crucial.Minimally invasive laparoscopic surgery is both safe and feasible to treat perforated duodenal ulcers in selected patients.

GENE DIAGNOSIS OF HEMOGLOBINOPATHY IN CHINESE BY AMPLIFIED DNA

This paper describes the application of DNA amplification in vitro with the polymerase chain reaction on the prenatal diagnosis of thalassemia syndromes and on detection of HbS and HbD Punjab genes. DNA polymerase chain reaction was performed on lysed amniotic fluid cells or chorionic villus samples without prior DNA extraction and on DNA sampling from dried blood spots on filter paper blotters, α-thalassemia was prenatally diagnosed by direct analysis of amplified fetal target sequences on gel electrophoresis; and β-thalassemia was predicted by Hgi AI RFLP linkage analysis with amplified β-globin DNA. HbS and HbD Punjab genes were identified by Mst Ⅱor Eco RI mapping of the amplified β-globin DNA directly on the electrophoretic gels. The analysis of the amplified DNA does not require radioactive DNA probes and Southern hybridization. The total procedure can be completed within five hours.

Preimplantation HLA typing: Practical tool for stem cell transplantation treatment of congenital disorders

It is well known that to achieve an acceptable engraftment and survival in stem cell therapy, an human leukocyte antigens(HLA) identical stem cell transplant is strongly required. However, the availability of the HLA matched donors even among family members is extremely limited, so preimplantation HLA typing provides an attractive practical tool of stem cell therapy for children requiring HLA matched stem cell transplantation. The present experience of preimplantation genetic diagnosis(PGD) for HLA typing of over one thousand cases shows that PGD provides the at-risk couples with the option to establish an unaffected pregnancy, which may benefit the affected member of the family with hemoglobinopathies, immunodeficiencies and other congenital or acquired bone marrow failures. Despite ethical issues involved in preimplantation HLA typing, the data presented below show an extremely high attractiveness of this option for the couples with affected children requiring HLA compatible stem cell transplantation.

Cation-exchange high-performance liquid chromatography for variant hemoglobins and HbF/A2:What must hematopathologists know about methodology?

Cation-exchange high-performance liquid chromatography(CE-HPLC) is a widely used laboratory test to detect variant hemoglobins as well as quantify hemoglobins F and A2 for the diagnosis of thalassemia syndromes. It's versatility, speed, reproducibility and convenience have made CE-HPLC the method of choice to initially screen for hemoglobin disorders. Despite its popularity, several methodological aspects of the technology remain obscure to pathologists and this may have consequences in specific situations. This paper discusses the basic principles of the technique, the initial quality control steps and the interpretation of various controls and variables that are available on the instrument output. Subsequent sections are devoted to methodological considerations that arise during reporting of cases. For instance, common problems of misidentified peaks, totals crossing 100%, causes of total area being above or below acceptable limits and the importance of pre-integration region peaks are dealt with. Ultimately, CE-HPLC remains an investigation, the reporting of which combines in-depth knowledge of the biological basics with more than a working knowledge of the technological aspects of the technique.

Association of Thalassemia and Trait, Sickle Cell and Trait and Haemoglobin C with Iron-Deficiency Anemia in Colombians of African Descent

The conditions of poverty and violence in Colombia have increased the displacement of people of African descent. The research group concerned with the health of these communities has performed research to find the association of alterations in the hemoglobin molecule with the presence of iron deficiency anemia. This research was performed with the support of the epidemiological public health laboratory. The objective of the study is the association of sickle cell disease and sickle cell trait, and the presence of hemoglobin C and its trait, with iron deficiency anemia in Afro-Colombians between 18 and 50 years of age, by investigation of hemograms, peripheral blood study, serum ferritin, soluble receptor for transferrin （sTfR） and index of sTfR, and hemoglobin electrophoresis. This research is a descriptive, quantitative, transverse, structured, non-experimental and correlational study, with a total of 56 samples （10 men, 46 women）. The results showed that ferritin in men had normal values, compared to 32% （15/46） of women who had low levels of ferritin. Men＇s sTfR levels were normal, while in women 2% （1/46） had iron deficiency in stage I, 41% （19/46） iron deficiency in stage If, and 9% （4/46） possibly without anemia iron deficiency. In hemoglobin electrophoresis, 84% （46/56） of the population presents hemoglobin A, 4% （2/56） increased hemoglobin A2 and fetal hemoglobin related to a possible thalassemia trait, 11% （6/56） had hemoglobin AS related to sickle cell trait, and 2% （1/56） show hemoglobin AC, related to hemoglobin AC trait. This project supports investigative and social projects, and also contributes to the improvement of the quality of life and dissemination of good health practices in these communities.

Age and genotype dependent erythropoietin protection in COVID-19

Erythropoietin(EPO)is the main mediator of erythropoiesis and an important tissue protective hormone that appears to mediate an ancestral neuroprotective innate immune response mechanism at an early age.When the young brain is threatened-prematurity,neonatal hyperbilirubinemia,malaria-EPO is hypersecreted disproportionately to any concurrent anemic stimuli.Under eons of severe malarial selection pressure,neuroprotective EPO augmenting genetic determinants such as the various hemoglobinopathies,and the angiotensin converting enzyme(ACE)I/D polymorphism,have been positively selected.When malarial and other cerebral threats abate and the young child survives to adulthood,EPO subsides.Sustained high ACE and angiotensin II(Ang II)levels through the ACE D allele in adulthood may then become detrimental as witnessed by epidemiological studies.The ubiquitous renin angiotensin system(RAS)influences theα-klotho/fibroblast growth factor 23(FGF23)circuitry,and both are interconnected with EPO.Here we propose that at a young age,EPO augmenting genetic determinants through ACE D allele elevated Ang II levels in some or HbE/beta thalassemia in others would increase EPO levels and shield against coronavirus disease 2019,akin to protection from malaria and dengue fever.Human evolution may use ACE2 as a“bait”for severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)to gain cellular entry in order to trigger an ACE/ACE2 imbalance and stimulate EPO hypersecretion using tissue RAS,uncoupled from hemoglobin levels.In subjects without EPO augmenting genetic determinants at any age,ACE2 binding and internalization upon SARS-CoV-2 entry would trigger an ACE/ACE2 imbalance,and Ang II oversecretion leading to protective EPO stimulation.In children,low nasal ACE2 Levels would beneficially augment this imbalance,especially for those without protective genetic determinants.On the other hand,in predisposed adults with the ACE D allele,ACE/ACE2 imbalance,may lead to uncontrolled RAS overactivity and an Ang II induced proinflammatory state and immune dysregulation,with interleukin 6(IL-6),plasminogen activator inhibitor,and FGF23 elevations.IL-6 induced EPO suppression,aggravated through co-morbidities such as hypertension,diabetes,obesity,and RAS pharmacological interventions may potentially lead to acute respiratory distress syndrome,cytokine storm and/or autoimmunity.HbE/beta thalassemia carriers would enjoy protection at any age as their EPO stimulation is uncoupled from the RAS system.The timely use of rhEPO,EPO analogs,acetylsalicylic acid,bioactive lipids,or FGF23 antagonists in genetically predisposed individuals may counteract those detrimental effects.

Hemoglobinopathy and Systemic Lupus: A Rare Association

The authors report a case of systemic lupus associated with a composite heterozygosis SC with thalassemic component in a 19-year-old patient hospitalized for anemia and polyarthralgia who has a staturoponderal delay, macular erythematous lesions in butterfly wings on the face and ears, photosensitivity, puffy face, alopecia, pubic and axillary hair loss, scalp dermatophytosis, painful swelling of the interphalangeal joints, wrists and knees. The hemoglobin electrophoresis showed a compound heterozygote SC associated with β thalassemia. Antinuclear antibodies were positive with an anti-Sm positive antibody. Conclusion: The diagnosis of both diseases can be difficult when symptoms are concomitant and look alike.

Genetic Polymorphisms of HBS1L-MYB (rs4895441 and rs9376090) in Egyptian Patients with Hemoglobinopathy

Objective: Study the HBS1L-MYB (rs4895441 and rs9376090) genetic polymorphisms in Egyptian patients with β-thalassemia major and sickle cell disease and its relation to Hb F and severity of the disease. Background: Hb F is a predominant modulator for the severity of β-thalassemia major & sickle cell disease. Genetic polymorphism in the intergenic region (HBS1L-MYB) between GTP-binding elongation factor HBS1L and myeloblastosis oncogene MYB on chromosome 6q is associated with high fetal hemoglobin levels. Subjects and Methods: 150 subjects were included in this study. For all studied groups: Complete blood picture and serum ferritin were evaluated. For patients, hemoglobin variants were separated by High-performance liquid chromatography. Genotyping of HBS1L-MYB (rs4895441 & rs9376090) was evaluated by real-time polymerase chain reaction technique using TaqMan probe. Results: AG, CT genotypes, and G, C alleles of HBS1L-MYB (rs4895441 & rs9376090) were significantly high in sickle cell patients [OR (3.400);95% C.I (1.482 - 7.799)], (p = 0.003) & [OR (4.522);95% C.I (1.854 - 11.029)], (p = 0.001) respectively. Also, a significant association was detected between polymorphisms and disease severity. However, in β-thalassemia major, no significant association was detected. Conclusion: In sickle cell disease patients, Genetic polymorphisms in HBS1L-MYB (rs9376090 & rs4895441) affect the level of Hb F which could improve the prognosis of these patients.

SC Hemoglobinopathy(HbSC) with Osteoarticular Complications:Case Report

Background: Among hemoglobinopathies, the most prevalent in our population are hemoglobins S and C, which are capable of producing disease when homozygous. In cases of double heterozygotes with sickle hemoglobin C (SC), the disease is less expressive in its clinical condition and rarer. Case report: Patient has a previous hospitalization with pain in the joints in knee and hip and several febrile peaks. Upon physical examination, the patient had difficulty in walking, without edema, pedal and tibial posterior pulses present, with no signs of compartment syndrome. Complementary exams revealed anemia, leukocytosis and lymphopenia. The hemoglobin electrophoresis showed the SC Hemoglobinopathy. The treatment with antibiotic therapy according to the protocol (Oxacillin and Ceftriaxone) was restarted and submitted to joint drainage in affected limb. Conclusion: Osteomyelitis and septic arthritis in patients in the pediatric age group should be considered as serious infections that deserve hospitalization and more expressive treatment.

Recent advances in lentiviral vectors for gene therapy

Lentiviral vectors(LVs), derived from human immunodeficiency virus, are powerful tools for modifying the genes of eukaryotic cells such as hematopoietic stem cells and neural cells. With the extensive and in-depth studies on this gene therapy vehicle over the past two decades, LVs have been widely used in both research and clinical trials. For instance, third-generation and selfinactive LVs have been used to introduce a gene with therapeutic potential into the host genome and achieve targeted delivery into specific tissue. When LVs are employed in leukemia, the transduced T cells recognize and kill the tumor B cells;in β-thalassemia, the transduced CD34^(+) cells express normal β-globin;in adenosine deaminase-deficient severe combined immunodeficiency, the autologous CD34^(+) cells express adenosine deaminase and realize immune reconstitution. Overall, LVs can perform significant roles in the treatment of primary immunodeficiency diseases, hemoglobinopathies, B cell leukemia, and neurodegenerative diseases. In this review, we discuss the recent developments and therapeutic applications of LVs. The safe and efficient LVs show great promise as a tool for human gene therapy.