Molecular characterization of hemoglobin F augmentation in Northern Thai subjects with mild HbE/E-Thalassemia

การศึกษาคุณลักษณะระดับโมเลกุลของการกระตุ้นการสร้างฮีโมโกลบิน เอฟในคนไทยภาคเหนือที่เป็นโรคฮีโมโกลบินอี บีตาธาลัสซีเมียชนิด อาการไม่รุนแรง

Name: Lampliamas Tanghan

ThaSH: Hemoglobinopathy -- Thailand, Northern

Classification :.DDC: 612.1111

ThaSH: Thalassemia -- Thailand, Northern

Abstract: Production of hemoglobin is specific to stage of human development. Hemoglobin F (HbF) is the major hemoglobin in fetuses and newborns. Healthy adults have less than 1% of HbF which confines in sub-population of erythrocytes called “F cell (FC)”. HbF levels may be increased in adult life in an inherited condition termed “hereditary persistence of fetal hemoglobin (HPFH)”. Several genetic factors have been shown to be involved in increased -globin gene expression in HPFH. Co-existence of HPFH with -thalassemia reduced degree of imbalanced globin chain synthesis, leading to a mild clinical symptom. This thesis aimed to determine genetic backgrounds of high FC in mild HbE/β-thalassemia of the northern Thai origin and to establish the flow cytometric condition for FC enumeration using both in-house and commercially available monoclonal antibodies. This thesis was successful in optimizing 2 flow cytometric analytic protocols for FC enumeration. The first protocol utilized 4% formaldehyde for membrane fixation, cold acetone : methanol (4:1, v/v) for membrane permeabilization and 50 ng of FITCThal N/B in-house monoclonal antibody (mAb) for detecting intra-erythrocytic HbF (α22). As Thal N/B mAb was specific to -globin chain, falsely high FC may be encountered in those having increased levels of Hbs Bart’s (4) and Portland (22) and further optimization was recommended. In contrast, the second protocol utilized 0.05% glutaradehyde for membrane fixation, 0.1% Triton-X 100 for membrane permeabilization and 20 ng commercially available FITC-3H326 mAb for detecting intraerythrocytic HbF. As the FITC-3H326 mAb was HbF specific, this mAb was utilized in subsequent parts in this thesis. The explore for molecular background of high FC was performed in three northern Thai families having members with mild HbE/-thalassemia phenotypes, compared to four northern families having members with severe HbE/-thalassemia phenotypes. Closed relationship of cis-acting sequent variations including the “XmnIG  : +” site, -globin haplotypes III, “C” allele on HBBP1: rs2071348, PGF2, (TG)13 and ( AT)9N12(AT)10 and  E allele was observed. This particular haplotype was tightly correlated with high FC levels seen mostly in the northern Thai HbE/β-thalassemia families with mild disease severity that were analyzed in this thesis. However, the transsequence elements including A/C of rs766432 in BCL11A gene, C/A of rs9376092 in HMIP gene and G/A of rs140252918 in KLF1 gene showed no consistent relationship with FC levels in these families. It was concluded that the protocols for flow cytometric quantification F cells using the in-house Thal N/B mAb and commercial 3H326 mAb were successfully estabished. It was also concluded that the cis-acting elements including “XmnIG  : +” site, -globin haplotypes III, “C” allele on HBBP1: rs2071348, PGF2, (TG)13 and (AT)9N12(AT)10 that were closely linked to  E allele were most likely to be the molecular background of high HbF/FC productions in these mild HbE/β-thalassemia families. The analyzed trans-acting elements seemed not to be involved in high HbF/FC levels in these families. Further optimization of the flow cytometric analysis of FC using FITC-Thal N/B mAb as well as the explore for associations of other single nucleotide polymorphisms in BCL11A gene, HMIP and KLF1 gene and FC levels were suggested.

Chiang Mai University Library

Address: CHIANG MAI

Email: cmulibref@cmu.ac.th

Name: Suchart Punjaisee

Role: Chairman

Name: Roongsiri Chotpadiwetkul

Role: Member

Name: Pimlak Charoenkwan

Role: Member

Name: Thanusak Tatu

Role: Member

Created: 2015

Modified: 2017-09-21

Issued: 2017-09-21

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: Th/N 612.1111 L238M

eng

DegreeName: Master of Science

Level: ปริญญาโท

Descipline: Medical Technology

Grantor: มหาวิทยาลัยเชียงใหม่

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