Investigation of multiplex minisequencing coupled with denaturing high performance liquid chromatography (dHPLC) as a general genotyping platform for [beta]-thalassemia

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Investigation of multiplex minisequencing coupled with denaturing high performance liquid chromatography (dHPLC) as a general genotyping platform for [beta]-thalassemia


Author: Ngan, Pui-man Pauline
Title: Investigation of multiplex minisequencing coupled with denaturing high performance liquid chromatography (dHPLC) as a general genotyping platform for [beta]-thalassemia
Degree: M.Sc.
Year: 2005
Subject: Hong Kong Polytechnic University -- Dissertations
High performance liquid chromatography
Department: School of Nursing
Pages: xi, 93 leaves : ill. (chiefly col.) ; 30 cm
Language: English
Abstract: The beta (帣) -thalassemia is the most common single gene disorder in the world. The carrier rate is 3.4-6% in Hong Kong. The disease is caused by defective 帣-globin synthesis of adult hemoglobin. The phenotypes range from asymptomatic mild anemia in 3-thalassemia carrier to transfusion-dependent anemia in 帣-thalassemia major. Couples of 帣-thalassemia carriers have 25% risk of giving birth to a child with 帣-thalassemia major. The condition of 帣-thalassemia major is chronic, life-threatening and practically incurable. Experience from prevalent areas shows that the implementation of population screening and prenatal diagnosis can successfully reduce the birth of thalassemia major and subsequently enhance the control of the disease. Conventional laboratory methods have pitfalls as a screening method. The introduction of polymerase chain reaction (PCR) -based technology can provide accurate information of the mutation and definitive diagnosis in genetic counseling and clinical management. As a new genotyping technique, multiplex minisequencing coupled with denaturing high performance liquid chromatography (dHPLC) has the advantage of high throughput, and the economies of cost, time and labor. The platform has potential application in diseases with high allelic heterogeneity. However, extensive experimentation is required in the initial set-up stage in order to obtain simple and unambiguous interpretation. Non-overlapping peaks of various primers and the extension products of multiple mutation sites are crucial to definitive allelic discrimination. The objective of this study was to develop a set of simple criteria for design and empirical optimization of primers and their extension products for multiplex minisequencing coupled with dHPLC analysis. The experience would facilitate the rapid detection of the common mutations in the 帣-globin gene in the local Chinese population. In this study, the 帣-globin gene was amplified by a duplex PCR. The retention ability of multiply-sized primers and their extension products in dHPLC analysis were investigated to facilitate the primer design for multiplex minisequencing reaction. Homopolymeric tails of variable length were tagged to the 5' end of a minisequencing primer. The results demonstrated that prominent retention effect was found in poly-T or poly-A tagged primers but least in poly-C or poly-G tagged primers. Hence, the choice of modification was based on the addition of homopolymer of T or A. A regression equation was derived for preliminary estimation of retention time for different sets of primers in the initial set-up stage. According to the difference in retention time between these primers and their extension products, the separation between two successive primers should be 1-1.5 minutes. In addition, the test platform could be fine-tuned by altering the starting buffer gradient and the rate of gradient change, which might improve the efficiency and assay capacity of the platform. In the second part of the study, based on the experience gained in the first part, a panel of primers was designed for rapid genotyping of six mutations common in local Chinese population. These mutations were four-base deletion at codons 41/42; single base substitution at nucleotide -28, IVS2+654 and codon (CD) 17; insertion of A at codons 71/72 and the Hb E allele (a common 帣-globin variant in Southeast Asia). Samples from patients diagnosed by routine hematological tests to be 帣-thalassemia carriers (n=196) or carrying Hb E variant (n=6) were analyzed using the newly designed primer set on the platform of multiplex minisequencing and dHPLC analysis. The 帣-thalassemia genotypes were successfully determined for 194 samples. Eight samples were found not carrying any of these six mutations. In summary, over 95% 帣-thalassemia carrier samples and all samples carrying Hb E allele Were successfully identified. In conclusion, the experience gained in the study is useful in empirical primer design for multiplex minisequencing. It saves a lot of effort in primer design in the set-up stage without extensive empirical experimentation, but fine-tuning is still needed. Future investigation in the optimization of dHPLC analysis is valuable for fUrther improvement. It facilitates the widespread application of multiplex minisequencing coupled with dHPLC as a high-throughput genotyping platform for large-scale population screening and prenatal diagnosis in at-risk populations.

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