Direct cord blood LAMP colorimetric phenol red assay for detecting α0-thalassemia (SEA deletion); the validation and post-natal screening in Thailand.

Post-natal or newborn screening for thalassemia and hemoglobinopathies is useful for genetic counseling and managing thalassemia in children. We characterized thalassemia genotypes in newborns from the eastern part of Thailand. The results demonstrated a high heterogeneity of thalassemia and hemoglobinopathies with seventeen genotypes. We focused on α0- thalassemia (Southeast Asian [SEA] deletion) in this study. We developed and validated the loop-mediated isothermal amplification (LAMP) colorimetric assay for detecting α0- thalassemia (SEA deletion) using simple direct cord blood sampling compared to genomic DNA. A total of 160 cord blood samples were evaluated with the LAMP assay. The sensitivity and specificity of the LAMP colorimetric assay for α0-thalassemia (SEA deletion) using direct cord blood showed 100% (6/6 x 100) and 98.05% (151/154 x 100) whereas, genomic DNA showed 100% (6/6 x 100) and 100% (154/154 x 100), respectively. Moreover, we demonstrated other simple screening tools for α0-thalassemia with %Hb Bart's, MCV, and MCH values and found that these parameters were not diagnostic in our samples. The direct cord blood with colorimetric LAMP assay is simple, rapid, and does not require a post-LAMP step compared to conventional PCR. These techniques could be applied in post-natal or large population screening for α0-thalassemia (SEA deletion). Finally, this could support early prevention of complications, early management, genetic counseling for α-thalassemia disease in children, or a long-term prevention and control program of severe thalassemia in Thailand.

Molecular Spectrum of ß-Thalassemia Mutations in Central to Eastern Thailand.

The aim of this study was to determine the molecular spectrum of ß-thalassemia (ß-thal) mutations in eastern Thailand. We identified ß-thal mutations using allele specific-polymerase chain reaction (ASPCR) and direct DNA sequencing. We found 18 different ß-thal mutations in a total of 191 unrelated subjects. Six common ß-thal mutations comprised 86.91% of all the mutations, including codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) (35.60%), codon 17 (A>T) (HBB: c.52A>T) (18.85%), -28 (A>G) (HBB: c.-78A>G) (15.71%), IVS-II-654 (C>T) (HBB: c.316-197C>T) (6.28%), IVS-I-1 (G>T) (HBB: c.92+1G>T) (5.76%) and codon 19 (A>G) (HBB:(c.59A>G) (4.71%). In addition, a novel 60 kb deletion in two unrelated cases was characterized and initially suspected to originate from eastern Thailand. Moreover, we demonstrated the molecular spectrum of recent ß-thal mutations in Thailand, and data from this study were compared with five reference laboratory centers in Thailand. This study is the first to identify the comprehensive molecular spectrum of ß-thal mutations in eastern Thailand, information that may be essential for screening, genetic counseling and prenatal diagnosis (PND) in this region.

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.

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.

Acute monoblastic leukemia with t(10;11)(p12;q23) presenting with pulmonary involvement: a case report and literature review.

A forty-three-year-old Thai man presented with acute fever and dyspnea for one week with bilateral patchy infiltration, pancytopenia with monoblast. Bone marrow study was consistent with acute monoblastic leukemia. Lung lesions rapidly progressed to acute respiratory failure, which required intubation. Bronchoscopy with bronchoalveolar lavage revealed monotonous monoblast infiltration. Induction chemotherapy with 7 + 3 regimen was administered to halt the progression of leukemic pulmonary infiltration. Although there was clinical improvement, the chest radiograph developed crescent formation in the right upper lung field. Invasive pulmonary aspergillosis was suspected and successfully treated with antifungal agent. After peripheral blood recovery, bone marrow evaluation was performed and complete remission was established. HLA matching was sent to prepare for hematopoietic stem cell transplantation (HSCT). The literature review showed that the appropriate treatment for the patients with t(10;11)(p12;q23) was HSCT, but there was no data concerning correlation of t(10;11)(p12;q23) and pulmonary infiltration. This may be due to the low incidence of leukemic infiltration of acute leukemia patients, which is 0.48% and 3.06% in acute myeloid leukemia and acute monoblastic leukemia, respectively.