Detection of Clinically Silent Alpha-Globin Gene Mutations in Chinese Using High Resolution Melting Analysis

This dissertation, "Detection of Clinically Silent Alpha-globin Gene Mutations in Chinese Using High Resolution Melting Analysis" by Sophia KW, Ho, 何廣慧, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: α-thalassemia is an inherited globin gene disorder commonly found among the Chinese population. It is composed of both non-deletional and deletional α-globin gene mutations. Classical α-thalassemia presents with red cell microcytosis but silent cases with a normal mean corpuscular volume (MCV) are also seen. Routine laboratory testing methods for large-scale detection of silent α-thalassemia mutations are onerous and time-consuming. Furthermore, methods such as denaturing high performance liquid chromatography (HPLC) or denaturing gradient gel electrophoresis (DGGE) for scanning of point mutations are costly and they require post-PCR separation. High resolution melting (HRM) analysis is an economical, sensitive, and fast method for large scale point mutation scanning. Contamination is significantly reduced with HRM because the process is performed in a closed-tube environment and does not require post-PCR manipulation. We used HRM and multiplex gap-PCR analysis to determine the prevalence of silent α-thalassemia carriers in Hong Kong. Of the 223 hematologically normal blood samples scanned by Roche LightCycler 480(R), HRM did not show any sample with a non-deletional α-globin gene mutation of clinical significance. α-multiplex gap-PCR analysis revealed 36 samples (16.1%) with single α-globin gene deletions. The detection of single α-globin gene deletions in samples with a MCV greater than 80 fL indicates that the previously reported prevalence of α-thalassemia mutations in our Chinese population based on MCV screening is under-estimated. The data also suggest that non-deletional α-thalassemia mutations presenting with a normal MCV are very rare, and they most likely present with microcytosis. The fact that most silent α-thalassemia mutations are due to large deletions supports the use of traditional molecular techniques such as gap-PCR for their detection. HRM can be used as an adjunct tool for large-scale population screening of non-deletional mutations. This study provides more accurate data on the prevalence of silent α-thalassemia carriers in the Hong Kong Chinese population. The information will facilitate genetic counseling and risk assessment in families carrying α-thalassemia mutations. DOI: 10.5353/th_b5318929 Subjects: Thalassemia - Diagnosis