Application of third-generation sequencing technology for identifying rare α- and ß-globin gene variants in a Southeast Chinese region.

BACKGROUND: Third-generation sequencing (TGS) based on long-read technology has been gradually used in identifying thalassemia and hemoglobin (Hb) variants. The aim of the present study was to explore genotype varieties of thalassemia and Hb variants in Quanzhou region of Southeast China by TGS. METHODS: Included in this study were 6,174 subjects with thalassemia traits from Quanzhou region of Southeast China. All of them underwent common thalassemia gene testing using the DNA reverse dot-blot hybridization technology. Subjects who were suspected as rare thalassemia carriers were further subjected to TGS to identify rare or novel α- and ß-globin gene variants, and the results were verified by Sanger sequencing and/or gap PCR. RESULTS: Of the 6,174 included subjects, 2,390 (38.71%) were identified as α- and ß-globin gene mutation carriers, including 40 carrying rare or novel α- and ß-thalassemia mutations. The αCD30(-GAG)α and Hb Lepore-Boston-Washington were first reported in Fujian province Southeast China. Moreover, the ßCD15(TGG> TAG), ßIVS-II-761, ß0-Filipino(~ 45 kb deletion), and Hb Lepore-Quanzhou were first identified in the Chinese population. In addition, 35 cases of Hb variants were detected, the rare Hb variants of Hb Jilin and Hb Beijing were first reported in Fujian province of China. Among them, one case with compound αααanti3.7 and Hb G-Honolulu variants was identified in this study. CONCLUSION: Our findings may provide valuable data for enriching the spectrum of thalassemia and highlight the clinical application value of TGS-based α- and ß-globin genetic testing.

Rhabdomyolysis in a Case of Mild Coronavirus Disease 2019 and ß-Thalassemia Minor.

The novel coronavirus disease 2019 (COVID-19) virus primarily causes respiratory disease. Musculoskeletal manifestations are frequent, but rhabdomyolysis is a rare complication of COVID-19. It can be easily missed when there is a lack of high suspicion, especially in mild cases. Serum creatine phosphokinase (CPK) is a simple and affordable test that can screen COVID-19 patients having rhabdomyolysis, especially when they have predominant musculoskeletal symptoms. Early recognition and management of rhabdomyolysis can prevent acute kidney injury (AKI) and its related complications. Here, we present a young male with ß-thalassemia minor who had tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on reverse transcription polymerase chain reaction (RT-PCR). He presented with symptoms suggestive of upper respiratory tract infection and myalgia. He later complained of cola-colored urine. Urine evaluation did not reveal myoglobinuria or hematuria, and there was no evidence of significant hemolysis. Extremely high serum CPK levels and the clinical scenario were suggestive of rhabdomyolysis. He was admitted and adequately hydrated with other symptomatic management. His renal functions and other parameters were monitored. He recovered well with an uneventful course in the hospital.

Hepatic Visualization on DXA Scan: An Ancilliary Finding.

Thalassemia major is a genetic haemoglobinopathy manifesting as severe anaemia, jaundice and hepatosplenomegaly. Due to altered iron metabolism and increased bone resorption it is associated with secondary osteoporosis manifested as decreased bone mineral density (BMD). Dual energy X-ray absorptiometry (DXA) is frequently performed for the diagnosis of secondary osteoporosis. Soft tissues are rarely visualized on DXA unless there is calcification involving those structures like nephro-, cholelithiasis or iatrogenic e.g. surgical clips. Hepatic iron deposition occurs in thalassemia due to repeated blood transfusions which leads to increased density of the liver resulting in visualization of liver on DXA scan. We present an interesting image of hepatic visualization on DXA performed for bone mineral density assessment in a patient with thalassemia major.

Hb SKMC and an unprecedented γδß-thalassemia: first report from Iraq.

BACKGROUND: Thalassemias are genetic disorders of globin chain synthesis. In Iraq, ß-thalassemia is more prevalent than α-thalassemia. This study identifies two unpredicted globin gene mutations, a rare α-globin gene mutation (Hb SKMC) and a novel γδß-thalassemia deletion. METHODS: Over 2 years, the Genetics unit at PAR hospital in Erbil, northern Iraq processed 137 ß-thalassemia and 97 α-thalassemia genetic testing requests. Three symptomatic thalassemia cases with unreported genotypes were identified. Proband-1α and proband-2α had Hb H disease, while proband-1ß had severe transfusion-dependent ß-thalassemia (TDT). Molecular studies included multiplex PCR, reverse hybridization, multiplex ligation-dependent probe amplification (MLPA), and globin gene sequencing. RESULTS: The α-thalassemia probands exhibited moderate microcytic hypochromic anemia with irregular transfusions and splenomegaly. Hb H disease was confirmed by positive Hb H tests and high-performance liquid chromatography (HPLC). Molecular analysis revealed heterozygous -MED deletion in proband-1α and α2Poly-A2 mutation in proband-2α. Sequencing identified the Hb SKMC (HBA1:c.283_300+3dup) mutation in both probands. The ß-thalassemia proband showed anemia and regular transfusions. Molecular studies detected the IVS1.110 G>A mutation and a novel γδß-thalassemia deletion in compound heterozygous form. The maternal sample showed the IVS1.110 G>A mutation, and MLPA confirmed the γδß-thalassemia deletion in the paternal sample. CONCLUSION: These findings highlight the genetic diversity of thalassemias in the region and emphasize the importance of advanced molecular diagnostics in detecting rare mutations.

Expression of microRNA-155 in thalassemic erythropoiesis.

Background: Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in ß-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with ß-thalassemia/HbE. However, the expression and function of miR-155, especially in ß-thalassemia, have not yet been explored. Methods: To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from ß IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. Results: miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in ß-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in ß-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in ß-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. Conclusion: miR-155 regulates IE in ß-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

Evaluation of the immunization efficacy and adverse reactions of hepatitis B vaccination in children with thalassemia minor.

OBJECTIVE: To assess the immunization efficacy and incidence of adverse reactions after hepatitis B vaccination in children with thalassemia based on data from real-world studies. METHODS: A total of 625 children were recruited into this cross-sectional study. Subgroup analyses of different thalassemia types were performed using binary logistic regression, the factors affecting HBsAb levels were identified using multiple linear regression, and the dose-response relationship between the duration of immunization and seroconversion was explored using the restricted cubic spline (RCS) model to further assess the protective duration of the hepatitis B vaccine. RESULTS: HBsAb positivity in enrolled children was 87.3% in the thalassemia group and 81.4% in the control group. Multifactorial analysis revealed that the duration of immunization, age at completion of vaccination, and whether the first dose was delayed were significant factors influencing HBsAb levels in children (P < 0.05). The threshold for HBsAb positivity may be reached when the immunization duration reaches approximately 30 months. A subgroup analysis revealed that the HBsAb positivity rate was lower in children with ß-thalassemia minor compared to those with α-thalassemia minor (P = 0.001, 95% CI: 0.097 ∼ 0.536). Adverse reactions after hepatitis B vaccination were dominated by general reactions, with a statistically significant difference in injection-site redness and swelling between the thalassemia and control groups (P < 0.05). CONCLUSIONS: The immunization response to the hepatitis B vaccine in children with thalassemia minor was comparable to healthy children, with no abnormal adverse effects seen.

An evaluation of exagamglogene autotemcel for the treatment of sickle cell disease and transfusion-dependent beta-thalassaemia.

INTRODUCTION: Sickle cell disease is the most common hereditary hemoglobinopathy followed by beta-thalassemia. Until recently, allogeneic stem cell transplantation was the only curative approach. Based on the Crispr-Cas9-technology enabling targeting specific genes of interest, fetal hemoglobin which is normally shut-off after birth can be switched on and sufficient levels can alleviate symptoms in sickle cell disease and avoid transfusions in beta-thalassemia. Two first-in-human clinical studies in sickle cell disease and beta-thalassemia aiming to increase the level of fetal hemoglobin by using Crispr-Cas9 to modify autologous hematopoietic stem cells in patients aged 12-35 years have proved safety and efficacy and have shown promising clinical outcomes. AREAS COVERED: The paper summarizes the outcome of the results of the two recently published clinical studies and compares them with the other available curative approaches. EXPERT OPINION: Based on the currently available safety and efficacy data of the two published clinical results on gene therapy with Crispr-Cas9 modified autologous stem cells (exagamglogene autotemcel), it can be anticipated that this approach will add significantly to the therapeutic options for patients with sickle cell disease and beta-thalassemia and can be considered for all patients above 12 years of age independent of a suitable allogeneic stem cell donor.

ATG-Thymoglobulin Versus ATG-Fresenius for Conditioning in Thalassemia Patients Who Underwent Allogenic Stem Cell Transplantation from Matched-Sibling Donor: A Tertiary Cancer Care Center Short-Term Experience.

Graft rejection and Graft-versus-host disease (GVHD) are some of the significant factors resulting in morbidity and mortality following allogeneic hematopoietic cell transplantation. Prophylaxis for GVHD using T-cell depleting agents is helpful in reducing the transplant-related mortality and graft rejection. Both tATG and fATG exhibit varied amounts of antibody specificities and perform distinct immunomodulatory effects, regardless of their capacity to deplete T-lymphocytes. We conducted this single-center, retrospective study at our center to compare both formulations. Twenty-six patients were included in the study, 13 in each cohort. The median age at diagnosis of ß-thalassemia was 5 months (range, 3-12 months) in the tATG group and 6 months (range, 3-9 months) in the f-ATG group, respectively. Acute GVHD was observed in 1 (7.7) and 2(15.4) in the tATG and fATG group, respectively. No cases of chronic GVHD were observed in either group. There was no difference in the mixed chimerism observed at 6 months in both groups, tATG (n = 5, 38.5%) and fATG (n = 6, 46.15). There was 1 (7.6) rejection at day +72 observed in the tATG group, whereas no rejection was observed in the fATG group. At a mean follow-up duration of 288 days since transplant, there were no deaths in either of the groups. In conclusion, both ATG preparations showed equivalent effectiveness in preventing rejections and GVHD. However, further larger studies are required to establish the long-term efficacy and safety of both formulations in ASCT.

Characterization of Hemoglobin Malay Phenotypes in Tertiary Hospitals.

Hemoglobin (Hb) Malay is a common ß hemoglobinopathy in Malaysia caused by A > G mutation in codon 19 leading to ß+-thalassemia phenotype. However, screening for Hb Malay is challenging as it is undetectable by routine capillary electrophoresis (CE) or high-performance liquid chromatograpy (HPLC) methods. This study aimed to determine the Hb Malay phenotypes. The study was done on 521 cases with presumed ß thalassemia from UKMMC and Hospital Selayang as well as confirmed Hb Malay cases from Hospital Sultanah Bahiyah, Kedah in over a 5-year period. Hb analysis using CE or HPLC followed by multiplex amplification refractory mutation system polymerase chain reaction and DNA sequencing were performed. Significant differences in mean values of haematological parameters among Hb Malay carriers against ß thalassemia carriers were determined using one-way ANOVA and ROC analysis. A total of 482/521 cases of ß globin mutations were identified. Among these, 54 Hb Malay cases were identified whereby 21 Hb Malay cases were from UKMMC and Hospital Selayang whilst 33 Hb Malay cases were from Hospital Sultanah Bahiyah, Kedah. Fifty-two were Hb Malay carriers whereas two cases were compound heterozygotes. The mean hemoglobin, mean cell volume, mean cell hemoglobin, and HbA of Hb Malay carriers were significantly higher than ß° thalassemia carriers. The HbA2 range of Hb Malay carriers was wider (3.5-5.5%) with median value of 3.9%. A new HbA2 cutoff value ≤4.6% (AUC 0.717, p < 0.001) was proposed. Compound heterozygous Hb Malay/IVS1-5(G > C) showed transfusion-dependent thalassemia phenotype. Hb Malay carriers have different red cell and electrophoretic parameters than classical ß° thalassemia carriers with wider HbA2 range. HbA2 of ≤4.6% should prompt a molecular confirmation for Hb Malay carrier status.

Social and Cultural Influences on Genetic Counseling Acceptability: A Mixed-Methods Study on Beta-Thalassemia Carriers Among Graduate Students in West Bengal, India.

BACKGROUND: Thalassemia is an inherited blood disorder characterized by abnormal production of hemoglobin. The prevalence of thalassemia in India varies depending on the region and population. The study used a pre- and postcounseling cross-sectional design, which involves measuring outcomes before and after the intervention (genetic counseling). OBJECTIVES: Three hundred and eighty-five respondents were screened as thalassemia carriers, between a pool of 2985 participants to depict the quantitative prevalence of thalassemia. Two separate qualitative cross-sectional studies were conducted and compared to validate genetic counseling. The aims of the study are to contribute to the understanding of thalassemia carrier frequency and to improve the education and awareness of college students regarding thalassemia. MATERIALS AND METHODS: Two different questionnaires were used with the same knowledge, attitude, and practice parameters, one before and one after counseling. A two-sample t-test and an analysis of variance (ANOVA) F-test were used to compare the changes in knowledge, attitude, and practice. RESULTS: Using paired samples t-test to compare the pre- and postcounseling outcome showed significant (P < 0.001) elevation in terms of knowledge, attitude, social beliefs, social discomfort, and practice as a thalassemia carrier. Further, ANOVA F-test demonstrates the relationship between demography and the difference in parametric score of the pre- and postcounseling outcome. CONCLUSION: By improving knowledge and attitudes, counseling can help individuals to better understand their condition and the importance of adhering to treatment recommendations. This can lead to improved health outcomes and a better quality of life for affected individuals.

The effect of applying emotional intelligence components on coping strategies in adolescents with beta-thalassemia major: a randomized clinical trial.

BACKGROUND: Thalassemia is one of the most common genetic disorders. Patients with beta-thalassemia major confront serious clinical and psychosocial challenges in their all lives, which require coping strategies. It appears that psychological interventions are necessary to improve their coping skills. The aim of this study was to determine the effect of applying emotional intelligence components on coping strategies in adolescents with beta- thalassemia major. METHODS: This randomized clinical trial study involved 60 teenagers with beta- thalassemia major who were divided equally into intervention and control groups. The experimental group participated in 9 sessions of an emotional intelligence program consisting of 90 min, held both virtually and in person, two sessions per week. We investigated problem-focused and emotion-focused (including positive emotion-focused and negative emotion-focused) coping strategies of both groups of adolescents using the Billings and Moos questionnaire before the intervention, immediately after the intervention, and one month after the intervention. Data were analyzed using SPSS 21. Then, according to the research objectives, independent t-tests, Chi-square, Mann-Whitney, repeated measures Analysis of Variance (ANOVA) and Bonferroni test were used. RESULTS: In experimental group, the mean score of problem-focused (problem-solving, cognitive evaluation) and positive emotion-focused (social support) coping increased from (14.2 ± 2.6) and (5.0 ± 0.5) before the intervention to (29.6 ± 3.1) and (10.9 ± 1.3) one month after the intervention, respectively (P < 0.001). However, the mean score of emotional inhibition and somatic inhibition (negative emotion-focused) decreased from (13.8 ± 1.7) and (6.7 ± 1.5) before the intervention to (8.6 ± 2.0) and (3.8 ± 1.8) one month after the intervention, respectively (P < 0.001). While the mean score of problem-focused and emotion-focused coping strategies before and one month after the intervention remained stable in the control group. CONCLUSIONS: Adolescents with beta-thalassemia suffer from psychosocial disorders and they also cope maladaptive with their illness. Applying emotional intelligence has improved their coping strategies. Caregivers should be encouraged to assess coping skills in teenagers with beta-thalassemia major and use methods such as emotional intelligence to improve them. Therefore, it can help these adolescents to deal effectively with stress and complications of the disease. TRIAL REGISTRATION NUMBER: IRCT20210521051356N1 (17/06/2021).

Cauda equina syndrome with beta thalassemia: a case report.

INTRODUCTION: Cauda equina syndrome (CES) related to beta thalassemia with extramedullary hematopoiesis is a rarely reported and challenging clinical presentation. A thorough literature review revealed only a limited number of documented cases, each demonstrating a variety of treatment modalities with divergent outcomes. CASE PRESENTATION: In this case, a 29-year-old male with beta thalassemia, undergoing frequent blood transfusions, and with a history of splenectomy, presented with 2 days of worsening in his lower back pain, extending to both lower limbs, numbness, and urinary incontinence. Following the ASIA ISNCSCI scoring system for physical assessment, there was a significant decrease in anal tone and perianal sensation, suggesting possible sacral nerve roots involvement, but no other upper or lower extremities sensory or motor deficits were detected. Provided with the patient history of frequent blood transfusion and Thalassemia for which hematology referral was promoted. Spinal MRI revealed extramedullary hematopoiesis, disc protrusion, and cauda equina compression. DISCUSSION: Spine surgery, including decompression and laminectomy, resulted in improved back pain and lower limb symptoms during the one-year follow-up. However, persistent sensory impairment and neurogenic bladder necessitated ongoing urological management. The absence of clear guidelines for the management of such cases underscores the need for further data collection and comprehensive outcome reviews.

Clinical characteristics, laboratory features and genetic profile of hemoglobin E (HBB:c.79 G > A)/ß (nucleotide -28 A > G) (HBB:c.-78 A > G) -thalassemia subjects identified from community- and hospital-recruited cohorts.

Despite several existing laboratory-based studies of hemoglobin (Hb) E (HBB:c.79 G > A)/ ß (nucleotide (NT) -28 A > G) (HBB:c.-78 A > G) -thalassemia, no reports have ever provided clinical severity information as well as dependency of blood transfusion. Previously, a comparative study of community- and hospital-recruited Hb E/ß-thalassemia subjects was conducted in the lower northern Thailand between June 2020 and December 2021. A mobile medical team visited each community hospital on-site, collecting clinical severity parameters, and conducting Hb and DNA analyses. The control included Hb E/ß-thalassemia patients undergoing transfusions. Subgroup study of adult Hb E/ß (NT -28 A > G) -thalassemia subjects was subsequently conducted. Additional pediatric individuals were recruited from prenatal diagnosis databases. Twenty adult and nine pediatric subjects were enrolled; all were classified as having mild disease severity. Twenty-two individuals (75.9 %) were asymptomatic. Six adults (20.7 %) required blood transfusion. The mean Hb level of subjects without transfusion (23 [79.3 %]) was 10.77 ± 1.10 g/dL. Hb analysis revealed a distinct EFA pattern with low Hb F fraction. The positive impact of genetic modifiers could not be statistically demonstrated except rs7482144-XmnI. These findings could provide essential information for parents carrying fetuses with Hb E/ß (NT -28 A > G) -thalassemia.

Novel Insights into Hb Shaare Zedek Associated with ß0-Thalassemia: Molecular Characteristics, Genetic Origin and Diagnostic Approaches.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and ß-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a ß-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with ß0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Loss-of-Function Variants in SUPT5H as Modifying Factors in Beta-Thalassemia.

It is well known that modifiers play a role in ameliorating or exacerbating disease phenotypes in patients and carriers of recessively inherited disorders such as sickle cell disease and thalassemia. Here, we give an overview of the literature concerning a recently described association in carriers of SUPT5H Loss-of-Function variants with a beta-thalassemia-like phenotype including the characteristic elevated levels of HbA2. That SUPT5H acts as modifier in beta-thalassemia carriers became evident from three reported cases in whom combined heterozygosity of SUPT5H and HBB gene variants was observed to resemble a mild beta-thalassemia intermedia phenotype. The different SUPT5H variants and hematologic parameters reported are collected and reviewed to provide insight into the possible effects on hematologic expression, as well as potential disease mechanisms in carriers and patients.

Skin complications during iron chelation therapy for beta-thalassemia: overview and treatment approach.

Thalassemia is an inherited genetic disorder of hemoglobin that affects a large population worldwide, and it is estimated that between 50,000 and 60,000 infants with thalassemia are born each year. The most common treatment for thalassemia is blood transfusion, which leads to iron overload. This in itself is a serious clinical condition, and is commonly managed with iron chelation therapy. However, iron chelators can cause various skin complications, including hyperpigmentation, skin rash, itching, and photosensitivity. These skin side effects can impact patients' quality of life. Therefore, this article provides a comprehensive overview of skin complications caused by iron chelators, along with a proposed comprehensive approach to their management in patients with beta-thalassemia. Key strategies include patient education, regular skin assessment, sun protection measures, symptomatic relief with topical corticosteroids and antihistamines, and consideration of treatment modification if severe complications occur. Collaboration between hematologists and dermatologists, along with psychological support and regular follow-up, is an essential component of this multidisciplinary approach. By implementing these strategies, healthcare providers can optimize skin care for patients with beta-thalassemia treated with iron chelators and improve their quality of life.

Exploring the bone marrow micro environment in thalassemia patients: potential therapeutic alternatives.

Genetic mutations in the ß-globin gene lead to a decrease or removal of the ß-globin chain, causing the build-up of unstable alpha-hemoglobin. This condition is referred to as beta-thalassemia (BT). The present treatment strategies primarily target the correction of defective erythropoiesis, with a particular emphasis on gene therapy and hematopoietic stem cell transplantation. However, the presence of inefficient erythropoiesis in BT bone marrow (BM) is likely to disturb the previously functioning BM microenvironment. This includes accumulation of various macromolecules, damage to hematopoietic function, destruction of bone cell production and damage to osteoblast(OBs), and so on. In addition, the changes of BT BM microenvironment may have a certain correlation with the occurrence of hematological malignancies. Correction of the microenvironment can be achieved through treatments such as iron chelation, antioxidants, hypoglycemia, and biologics. Hence, This review describes damage in the BT BM microenvironment and some potential remedies.

Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in ß-thalassemia and sickle cell disease patients.

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both ß-thalassemia (ß-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.

Noninvasive Prenatal Genetic Screening of Cell-Free Fetal DNA for Early Prediction of ß-Thalassemia Using Fiber Optic Nanogold-Linked Sorbent Assay.

ß-Thalassemia is a prevalent type of severe inherited chronic anemia, primarily identified in developing countries. The identification of single nucleotide polymorphisms (SNPs) plays a vital role in the early diagnosis of genetic diseases. Here, we reported the development of an amplification-free fiber optic nanogold-linked sorbent assay method using a fiber optic particle plasmon resonance (FOPPR) biosensor for rapid and ultrasensitive detection of SNPs. Herein, MutS protein was selected as the biorecognition capture probe and immobilized on the sensing region to capture the target mutant DNA, which was hybridized with a single-base mismatched single-stranded DNA labeled by a gold nanoparticle (AuNP). The AuNP acts as a signaling agent to be detected by the FOPPR biosensor when it is bound on the fiber core surface. The method effectively differentiates mismatched double-stranded DNA by MutS protein from perfectly matched/complementary dsDNA. It exhibits an impressively low detection limit for the detection of SNPs at approximately 10-16 M using low-cost sensor chips and devices. By determination of the ratio of mutant DNA to normal DNA in cell-free genomic DNA from blood samples, this method is promising for diagnosing ß-thalassemia in fetuses without invasive testing techniques.