Hematological Profile of Hb Adana Among High School Students in Northeast Peninsular Malaysia.

Background Hb Adana is a non-deletional alpha (α)-thalassaemia variant resulting from mutations in α1- or α2-globin codon 59 (αCD59), leading to the production of unstable α-globin. Clinical manifestations can vary from silent carrier status to dependence on blood transfusions, hepatosplenomegaly, skeletal deformities, and spinal cord compression. Despite the significance of Hb Adana inheritance, studying this variant poses challenges due to the scarcity of molecular tests and the potential for routine diagnoses to be overlooked. This study aims to investigate the prevalence of Hb Adana among local high school students and assess the hematological parameters and hemoglobin analysis of Hb Adana in Malaysia. Methodology This retrospective study analyzed 13,721 blood samples collected from high school students participating in Malaysia's National Thalassaemia Screening Program at Hospital Raja Perempuan Zainab II (HRPZ II). Deletional α-thalassaemia was detected using multiplex gap-polymerase chain reaction (PCR), while common non-deletional α-thalassaemia was identified using multiplex amplification refractory mutation system (ARMS) PCR. Data were extracted from the HRPZ II database for analysis. Results Among the participants, 2327 individuals were found to have either common deletional (n=1037, 44.6%) or non-deletional (n=1290, 55.4%) α-thalassaemia. Hb Constant Spring was the most prevalent non-deletional α-thalassaemia, accounting for 53.03% of cases. Thirty-one participants (1.33%) exhibited αCD59α/αα, and one (0.04%) had αCD59α/-α3.7. Among the 32 subjects with Hb Adana, 87.5% were Malay, and 12.5% were Orang Asli. Additionally, seven cases of HbE/Hb Adana co-inheritance were identified. Hemoglobin levels in heterozygous Hb Adana individuals ranged from mild anemia to normal, between 95 g/L and 153 g/L. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were approximately 73 fL and 23 pg, respectively. Conclusion This study delineates the distribution of α-thalassaemia mutation patterns among high school students in Kelantan, Northeast Peninsular Malaysia. Our findings indicate that Hb Adana is rare in our region and co-inheritance with an α-gene deletion results in α+-thalassaemia and with HbE, α0-thalassaemia. All heterozygous Hb Adana individuals exhibited low MCVs and MCHs.

The Role of Epigenetics in the Development and Progression of Multiple Myeloma.

Multiple myeloma (MM) is an exceptionally complicated and heterogeneous disease that is caused by the abnormal proliferation of malignant monoclonal plasma cells initiated in the bone marrow. In disease progression, a multistep process including differentiation, proliferation, and invasion is involved. Despite great improvement in treatment outcomes in recent years due to the substantial discovery of novel therapeutic drugs, MM is still regarded as an incurable disease. Patients with MM are afflicted by confronting remission periods accompanied by relapse or progression outcomes, which inevitably progress to the refractory stage. In this regard, MM may need new medications or modifications in therapeutic strategies to overcome resistance. A variety of genetic abnormalities (e.g., point mutations, translocations, and deletions) and epigenetic changes (e.g., DNA methylation, histone modification, and non-coding RNA) contribute to the pathogenesis and development of MM. Here, we review the significant roles of epigenetic mechanisms in the development and progression of MM. We also highlight epigenetic pathways as potential novel treatment avenues for MM, including their interplay, use of epigenetic inhibitors, and major involvement in immuno-oncology.

Genetic Manipulation Strategies for ß-Thalassemia: A Review.

Thalassemias are monogenic hematologic diseases that are classified as α- or ß-thalassemia according to its quantitative abnormalities of adult α- or ß-globin chains. ß-thalassemia has widely spread throughout the world especially in Mediterranean countries, the Middle East, Central Asia, India, Southern China, and the Far East as well as countries along the north coast of Africa and in South America. The one and the only cure for ß-thalassemia is allogenic hematopoietic stem cell transplantations (HSCT). Nevertheless, the difficulty to find matched donors has hindered the availability of this therapeutic option. Therefore, this present review explored the alternatives for ß-thalassemia treatment such as RNA manipulation therapy, splice-switching, genome editing and generation of corrected induced pluripotent stem cells (iPSCs). Manipulation of ß-globin RNA is mediated by antisense oligonucleotides (ASOs) or splice-switching oligonucleotides (SSOs), which redirect pre-mRNA splicing to significantly restore correct ß-globin pre-mRNA splicing and gene product in cultured erythropoietic cells. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) are designer proteins that can alter the genome precisely by creating specific DNA double-strand breaks. The treatment of ß-thalassemia patient-derived iPSCs with TALENs have been found to correct the ß-globin gene mutations, implying that TALENs could be used as a therapy option for ß-thalassemia. Additionally, CRISPR technologies using Cas9 have been used to fix mutations in the ß-globin gene in cultured cells as well as induction of hereditary persistence of fetal hemoglobin (HPFH), and α-globin gene deletions have proposed a possible therapeutic option for ß-thalassemia. Overall, the accumulated research evidence demonstrated the potential of ASOs-mediated aberrant splicing correction of ß-thalassemia mutations and the advancements of genome therapy approaches using ZFNs, TALENs, and CRISPR/Cas9 that provided insights in finding the permanent cure of ß-thalassemia.

Epigenetic Insights and Potential Modifiers as Therapeutic Targets in ß-Thalassemia.

Thalassemia, an inherited quantitative globin disorder, consists of two types, α- and ß-thalassemia. ß-thalassemia is a heterogeneous disease that can be asymptomatic, mild, or even severe. Considerable research has focused on investigating its underlying etiology. These studies found that DNA hypomethylation in the ß-globin gene cluster is significantly related to fetal hemoglobin (HbF) elevation. Histone modification reactivates γ-globin gene expression in adults and increases ß-globin expression. Down-regulation of γ-globin suppressor genes, i.e., BCL11A, KLF1, HBG-XMN1, HBS1L-MYB, and SOX6, elevates the HbF level. ß-thalassemia severity is predictable through FLT1, ARG2, NOS2A, and MAP3K5 gene expression. NOS2A and MAP3K5 may predict the ß-thalassemia patient's response to hydroxyurea, a HbF-inducing drug. The transcription factors NRF2 and BACH1 work with antioxidant enzymes, i.e., PRDX1, PRDX2, TRX1, and SOD1, to protect erythrocytes from oxidative damage, thus increasing their lifespan. A single ß-thalassemia-causing mutation can result in different phenotypes, and these are predictable by IGSF4 and LARP2 methylation as well as long non-coding RNA expression levels. Finally, the coinheritance of ß-thalassemia with α-thalassemia ameliorates the ß-thalassemia clinical presentation. In conclusion, the management of ß-thalassemia is currently limited to genetic and epigenetic approaches, and numerous factors should be further explored in the future.

Conformation sensitive gel electrophoresis for the detection of calreticulin mutations in BCR-ABL1-negative myeloproliferative neoplasms.

INTRODUCTION: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients. METHODS: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing. RESULTS: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2. CONCLUSION: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.