Coinheritance of Hb A2-Melbourne (HBD: c.130G>A) and Hb E (HBB: c.79G>A) in Laos and Simultaneous High Resolution Melt Detection of Hb A2-Melbourne and Hb A2-Lampang (HBD: c.142G>A) in a Single Tube

Abstract

We report the molecular and hematological identifications of a Hb A₂ variant [coinheritance of Hb A₂-Melbourne (HBD: c.130G>A) and Hb E (HBB: c.79G>A)] found for the first time in the Lao People’s Democratic Republic (PDR). The subject was a 29-year-old pregnant Laotian woman who was a foreign worker in Thailand and was diagnosed with thalassemia and hemoglobinopathies. Capillary electrophoresis (CE) demonstrated 1.6% of Hb A₂, with a minor unknown peak at the initial Z1 zone (1.7%). Identification of abnormal hemoglobin (Hb) using direct DNA sequencing showed a genetic defect causing a δ-globin gene missense mutation at codon 43 (GAG>AAG) causing a glutamic acid to lysine substitution corresponding to Hb A₂-Melbourne. The origin of Hb A₂-Melbourne in Lao PDR may be similar to a case found in Thailand with the [+ –?– –?– + +] haplotype. We developed a method that could clearly detect Hb A₂-Melbourne and Hb A₂-Lampang (HBD: c.142G>A) mutations in a single tube using high resolution melt (HRM) analysis. The HRM analysis is a more effective method for rapid detection than conventional polymerase chain reaction (PCR), as there is no need for a post-PCR step, and no exposure to ethidium bromide. This new method would be a useful addition for the first investigation of a suspected Hb A₂ variant in the routine molecular setting.     

Clinical and hematological relevance of JAK2 V617F and CALR mutations in BCR-ABL-negative ET patients

Background: Classical BCR-ABL1-negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis frequently harbor JAK2, MPL, and CALR somatic mutations.

Methods: AS-PCR for JAK2 V617F, pyrosequencing for MPL W515L/K, and PCR-fragment analysis for CALR exon 9 mutations were established to analyze genomic DNA isolated from peripheral blood samples of 58 newly diagnosed ET patients in Thailand.

Results: JAK2 V617F was detected in 41 patients (71%) and CALR exon 9 mutation was positive in eight patients (14%), whereas no mutation of MPL W515L/K was observed in this study. Patients with CALR mutation were older (p?=?0.023) and exhibited lower number of platelet count (p?=?0.041) than patients without CALR mutation. Two previously known CALR mutation types were identified in this study (six patients with CALR-type 1 and two patients with CALR-type 2). Additionally, no co-existence of JAK2 V617F and CALR mutations was identified in this work.

Conclusion: We reported the frequency of JAK2 V617F, MPL W515L/K, and CALR mutations in Thai patients with ET. Clinical and hematological phenotypes of patients were associated with JAK2 and CALR mutation statuses. The combination of laboratory testing for the detection of JAK2, CALR, and MPL mutations is necessary to improve the diagnosis and classification of BCR-ABL1-negative MPN.     

The Frequency of SF3B1 Mutations in Thai Patients with Myelodysplastic Syndrome

Genetic mutations in genes encoding critical component of RNA splicing machinery including SF3B1 are frequentlyidentified and recognized as the pathogenesis in the development of myelodysplatic syndrome (MDS). In this study,PCR sequencings specific for SF3B1 exon 13, 14, 15, and 16 were performed to analyse genomic DNA isolated frombone marrow samples of 72 newly diagnosed MDS patients. We found that 10 of 72 (14%) patients harbor SF3B1missense mutations including E622D (1/72), R625C/G (2/72), H662Q (1/72), K666T (1/72), K700E (4/72) and G740E(1/72), respectively. Mutations were predominantly located on exon 14 and 15 of SF3B1 coding sequence. Interestingly,patients with SF3B1 mutations exhibited higher platelet counts (195×109/L VS. 140×109/L, p-value = 0.025) as well aslower hemoglobin levels (81 g/L VS. 92 g/L, p-value = 0.009) and associated with ring sideroblast phenotype (p-value< 0.001) when compared with patients without the SF3B1 mutation. In summary, we reported the frequency of SF3B1mutations in Thai patients with different subtypes of MDS. SF3B1 mutations were predominantly occurred in MDS-RSand considered as favourable prognosis value. This study further highlighted the clinical important of SF3B1 mutationsanalysis for the classification of MDS.