HBB Gene Mutations and Their Pathological Impacts on HbE/ß-Thalassaemia in Kuala Terengganu, Malaysia.

BACKGROUND: ß-thalassaemia is a disorder caused by mutations in the ß-globin gene, leading to defective production of haemoglobins (Hb) and red blood cells (RBCs). It is characterised by anaemia, ineffective erythropoiesis, and iron overload. Patients with severe ß-thalassaemia require lifelong blood transfusions. Haemoglobin E beta-thalassaemia (HbE/ß-thalassaemia) is a severe form of ß-thalassaemia in Asian countries. More than 200 alleles have been recognised in the ß-globin region. Different geographical regions show different frequencies of allelic characteristics. In this study, the spectrum of ß-thalassaemia (ß-thal) alleles and their correlation with iron overload, in HbE/ß-thalassaemia patients, ß-thalassaemia trait, and HbE trait were studied. METHODS: Blood samples (n = 260) were collected from 65 ß-thalassaemia patients, 65 parents (fathers and/or mothers) and 130 healthy control individuals. Haematological analyses, iron profiles, and serum hepcidin levels were examined for all participants. DNA was extracted from patients' and their parents' blood samples, then subjected to PCR amplification. Multiplex amplification refractory mutation system PCR (MARMS-PCR) was conducted for eighteen primers to detect the mutations. RESULTS: There was severe anaemia present in HbE/ß-thalassaemia patients compared to their parents and healthy controls. The ferritin and iron levels were significantly increased in patients compared to their parents and healthy controls (p = 0.001). Two common mutations were detected among the patient group and three mutations were detected among their parents, in addition to seven novel mutations in HbE/ß-thalassaemia patients (explained in results). CONCLUSION: Some mutations were associated with severe anaemia in ß-thalassaemia patients. The detection of mutations is a prognostic marker, and could enhance the appropriate management protocols and improve the haematological and biochemical statuses of ß-thalassaemia patients.

Thymoquinone Inhibits JAK/STAT and PI3K/Akt/ mTOR Signaling Pathways in MV4-11 and K562 Myeloid Leukemia Cells.

Constitutive activation of Janus tyrosine kinase-signal transducer and activator of transcription (JAK/STAT) and Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathways plays a crucial role in the development of acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Thymoquinone (TQ), one of the main constituents of Nigella sativa, has shown anti-cancer activities in several cancers. However, the inhibitory effect mechanism of TQ on leukemia has not been fully understood. Therefore, this study aimed to investigate the effect of TQ on JAK/STAT and PI3K/Akt/mTOR pathways in MV4-11 AML cells and K562 CML cells. FLT3-ITD positive MV4-11 cells and BCR-ABL positive K562 cells were treated with TQ. Cytotoxicity assay was assessed using WSTs-8 kit. The expression of the target genes was evaluated using RT-qPCR. The phosphorylation status and the levels of proteins involved in JAK/STAT and PI3K/Akt/mTOR pathways were investigated using Jess western analysis. TQ induced a dose and time dependent inhibition of K562 cells proliferation. TQ significantly downregulated PI3K, Akt, and mTOR and upregulated PTEN expression with a significant inhibition of JAK/STAT and PI3K/Akt/mTOR signaling. In conclusion, TQ reduces the expression of PI3K, Akt, and mTOR genes and enhances the expression of PTEN gene at the mRNA and protein levels. TQ also inhibits JAK/STAT and PI3K/Akt/mTOR pathways, and consequently inhibits proliferation of myeloid leukemia cells, suggesting that TQ has potential anti-leukemic effects on both AML and CML cells.

Activation of STAT and SMAD Signaling Induces Hepcidin Re-Expression as a Therapeutic Target for ß-Thalassemia Patients.

Iron homeostasis is regulated by hepcidin, a hepatic hormone that controls dietary iron absorption and plasma iron concentration. Hepcidin binds to the only known iron export protein, ferroportin (FPN), which regulates its expression. The major factors that implicate hepcidin regulation include iron stores, hypoxia, inflammation, and erythropoiesis. When erythropoietic activity is suppressed, hepcidin expression is hampered, leading to deficiency, thus causing an iron overload in iron-loading anemia, such as ß-thalassemia. Iron overload is the principal cause of mortality and morbidity in ß-thalassemia patients with or without blood transfusion dependence. In the case of thalassemia major, the primary cause of iron overload is blood transfusion. In contrast, iron overload is attributed to hepcidin deficiency and hyperabsorption of dietary iron in non-transfusion thalassemia. Beta-thalassemia patients showed marked hepcidin suppression, anemia, iron overload, and ineffective erythropoiesis (IE). Recent molecular research has prompted the discovery of new diagnostic markers and therapeutic targets for several diseases, including ß-thalassemia. In this review, signal transducers and activators of transcription (STAT) and SMAD (structurally similar to the small mothers against decapentaplegic in Drosophila) pathways and their effects on hepcidin expression have been discussed as a therapeutic target for ß-thalassemia patients. Therefore, re-expression of hepcidin could be a therapeutic target in the management of thalassemia patients. Data from 65 relevant published experimental articles on hepcidin and ß-thalassemia between January 2016 and May 2021 were retrieved by using PubMed and Google Scholar search engines. Published articles in any language other than English, review articles, books, or book chapters were excluded.

Thymoquinone Inhibits Growth of Acute Myeloid Leukemia Cells through Reversal SHP-1 and SOCS-3 Hypermethylation: In Vitro and In Silico Evaluation.

Epigenetic silencing of tumor suppressor genes (TSGs) plays an essential role in cancer pathogenesis, including acute myeloid leukemia (AML). All of SHP-1, SOCS-1, and SOCS-3 are TSGs that negatively regulate JAK/STAT signaling. Enhanced re-expression of TSGs through de-methylation represents a therapeutic target in several cancers. Thymoquinone (TQ) is a major component of Nigella sativa seeds with anticancer effects against several cancers. However, the effects of TQ on DNA methylation are not entirely understood. This study aimed to evaluate the ability of TQ to re-express SHP-1, SOCS-1, and SOCS-3 in MV4-11 AML cells through de-methylation. Cytotoxicity, apoptosis, and cell cycle assays were performed using WSTs-8 kit, Annexin V-FITC/PI apoptosis detection kit, and fluorometric-red cell cycle assay kit, respectively. The methylation of SHP-1, SOCS-1, and SOCS-3 was evaluated by pyrosequencing analysis. The expression of SHP-1, SOCS-1, SOCS-3, JAK2, STAT3, STAT5A, STAT5B, FLT3-ITD, DNMT1, DNMT3A, DNMT3B, TET2, and WT1 was assessed by RT-qPCR. The molecular docking of TQ to JAK2, STAT3, and STAT5 was evaluated. The results revealed that TQ significantly inhibited the growth of MV4-11 cells and induced apoptosis in a dose- and time-dependent manner. Interestingly, the results showed that TQ binds the active pocket of JAK2, STAT3, and STAT5 to inhibit their enzymatic activity and significantly enhances the re-expression of SHP-1 and SOCS-3 through de-methylation. In conclusion, TQ curbs MV4-11 cells by inhibiting the enzymatic activity of JAK/STAT signaling through hypomethylation and re-expression of JAK/STAT negative regulators and could be a promising therapeutic candidate for AML patients.

Reduced hepcidin expression enhances iron overload in patients with HbE/ß-thalassemia: Α comparative cross-sectional study.

Iron homeostasis is regulated by hepcidin (HEPC) that controls the dietary iron absorption and iron recycling. HEPC deficiency contributes to iron overload in ß-thalassemia patients. The present study aimed to investigate the correlation between HEPC concentration and serum iron status among hemoglobin E (HbE)/ß-thalassemia patients and their parents (HbE trait and ß-thalassemia trait) compared with healthy controls. This study is a comparative cross-sectional study in which iron profile and HEPC level were examined in 65 HbE/ß-thalassemia patients (pretransfusion) and 65 parents at the Hospital Sultanah Nur Zahirah and in 130 students as healthy controls from Univesiti Sultan Zainal Abidin, Terengganu, Malaysia. Furthermore, six samples from each group (HbE/ß-thalassemia patients, parents and healthy controls) were randomly selected for gene expression analysis of HEPC and ferroportin1 (FPN1) using reverse transcription quantitative PCR. The results demonstrated that serum HEPC level were significantly decreased in HbE/ß-thalassemia patients and their parents (P<0.001) compared with healthy controls. In addition, the gene expression analysis showed a dramatically downregulated HEPC in HbE/ß-thalassemia patients and their parents (P=0.001) compared with healthy controls. However, there was a marked upregulation of FPN1 in HbE/ß-thalassemia patients and their parents (P=0.001) compared with healthy controls. Iron profiling results revealed a significantly increased serum ferritin in HbE/ß-thalassemia patients and their parents compared with healthy controls (P<0.001). In summary, the present study demonstrated that HEPC expression level and serum level were significantly decreased in HbE/ß-thalassemia patients and their parents, which was combined with a marked increased FPN1 expression level and serum ferritin level compared with healthy volunteers. These findings supported the hypothesis that downregulated HEPC could lose its function as a negative regulator of FPN1, resulting in iron overload in HbE/ß-thalassemia patients. Subsequently, assessing HEPC and FPN1 gene expression may be a useful tool to determine the risk of iron toxicity in patients with HbE/ß-thalassemia and their parents, and could therefore be considered as a therapeutic target in the management of iron burden in these patients.

Evidence of new intragenic HBB haplotypes model for the prediction of beta-thalassemia in the Malaysian population.

This study sought to determine the potential role of HBB haplotypes to predict beta-thalassemia in the Malaysian population. A total of 543 archived samples were selected for this study. Five tagging SNPs in the beta-globin gene (HBB; NG_000007.3) were analyzed for SNP-based and haplotype association using SHEsis online software. Single-SNP-based association analysis showed three SNPs have a statistically significant association with beta-thalassemia. When Bonferroni correction was applied, four SNPs were found statistically significant with beta-thalassemia; IVS2-74T>G (padj = 0.047), IVS2-16G>C (padj = 0.017), IVS2-666C>T (padj = 0.017) and 3'UTR + 314G>A (padj = 0.002). However, 3'UTR + 233G>C did not yield a significant association with padj value = 0.076. Further investigation using combined five SNPs for haplotype association analysis revealed three susceptible haplotypes with significant p values of which, haplotypes 1-2-2-1-1 (p = 6.49 × 10-7, OR = 10.371 [3.345-32.148]), 1-2-1-1-1 (p = 0.009, OR = 1.423 [1.095-1.850] and 1-1-1-1-1 (p = 1.39 × 10-4, OR = 10.221 [2.345-44.555]). Three haplotypes showed protective effect with significant p value of which, 2-2-1-1-1 (p = 0.006, OR = 0.668 [0.500-0.893]), 1-1-2-2-1 (p = 0.013, OR = 0.357 [0.153-0.830]) and 1-1-2-1-1 (p = 0.033, OR = 0.745 [0.567-0.977]). This study has identified the potential use of intragenic polymorphic markers in the HBB gene, which were significantly associated with beta-thalassemia. Combining these five SNPs defined a new haplotype model for beta-thalassemia and further evaluation for predicting severity in beta-thalassemia.

Thymoquinone, as a Novel Therapeutic Candidate of Cancers.

To date, natural products are widely used as pharmaceutical agents for many human diseases and cancers. One of the most popular natural products that have been studied for anticancer properties is thymoquinone (TQ). As a bioactive compound of Nigella sativa, TQ has shown anticancer activities through the inhibition of cell proliferation, migration, and invasion. The anticancer efficacy of TQ is being investigated in several human cancers such as pancreatic cancer, breast cancer, colon cancer, hepatic cancer, cervical cancer, and leukemia. Even though TQ induces apoptosis by regulating the expression of pro- apoptotic and anti-apoptotic genes in many cancers, the TQ effect mechanism on such cancers is not yet fully understood. Therefore, the present review has highlighted the TQ effect mechanisms on several signaling pathways and expression of tumor suppressor genes (TSG). Data from relevant published experimental articles on TQ from 2015 to June 2020 were selected by using Google Scholar and PubMed search engines. The present study investigated the effectiveness of TQ alone or in combination with other anticancer therapeutic agents, such as tyrosine kinase inhibitors on cancers, as a future anticancer therapy nominee by using nanotechnology.

Sequence polymorphism and haplogroup data of the hypervariable regions on mtDNA in Semoq Beri population.

Orang Asli is the aboriginal people in Peninsular Malaysia who have been recognized as indigenous to the country and still practicing traditional lifestyle. The molecular interest on the Orang Asli started when the earliest prehistoric migration occurred approximately 200 kya and entering Peninsular Malaysia 50 kya in stages. A total of three groups of Orang Asli present in Peninsular Malaysia, namely, Negrito also known as Semang, Senoi and Proto Malays. Through records, there is no research has been conducted on mtDNA variations in the Semoq Beri population, one of the tribes in Senoi group. In this report, variations of mtDNA were analysed in the population in Hulu Terengganu as an initial effort to establish the genetic characterisation and elucidating the history of Orang Asli expansion in Peninsular Malaysia. An array of mtDNA parameters was estimated and the observed polymorphisms with their respective haplogroups in comparison to rCRS were inferred respectively. The DNA sequences are registered in the NCBI with accession numbers KY853670-KY853753.

The PTPN22 R263Q polymorphism is a risk factor for rheumatoid arthritis in Caucasian case-control samples.

OBJECTIVE: Recently, a functional PTPN22 variant (R263Q; rs33996649) was found to be associated with systemic lupus erythematosus (SLE). This study was undertaken to analyze the influence of this polymorphism on the risk of rheumatoid arthritis (RA). METHODS: RA patients (n = 5,579) were recruited from outpatient clinics from 6 different countries (Spain, New Zealand, the UK, Norway, The Netherlands, and Germany). Healthy controls (n = 5,392) were recruited from the same areas. There was 100% power to detect an effect equivalent to that observed in SLE. Samples were genotyped for the PTPN22 R263Q (rs33996649) and PTPN22 R620W (rs2476601) polymorphisms using a TaqMan 5'-allele discrimination assay. The effect of the R263Q variant was analyzed in isolation and in combination with the effect of R620W, using Unphased and Stata 10 software. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined. RESULTS: The minor allele A of PTPN22 R263Q was significantly associated with a lower risk of RA in the pooled analysis of the 6 populations (P = 0.016, Mantel-Haenszel pooled OR 0.80 [95% CI 0.67-0.96]), independent of the effect of the R620W polymorphism. Both polymorphisms had an additive effect. The more RA risk alleles carried (R263Q G allele, R620W T allele), the higher the RA risk (for 2 versus 1 risk allele P = 0.014, OR 1.28 [95% CI 1.05-1.55], for 3 versus 1 risk allele P = 6.67 × 10(-11) , OR 2.01 [1.63-2.48], and for 4 versus 1 risk allele P = 6.50 × 10(-11) , OR 3.55 [2.42-5.20]). CONCLUSION: Our findings indicate that the minor allele of the PTPN22 R263Q polymorphism is associated with a lower risk of RA. This association is independent of the well-established association between PTPN22 R620W and RA. Both polymorphisms have an additive effect on the risk of RA.