Abstract
β-Globin gene mutations reduce or terminate the production of beta globin chains, of which approximately 10% are large deletions within the β-globin gene cluster. Because gene deletion leads to loss of heterozygosity at single nucleotide polymorphism (SNP), a novel method for detecting β-globin gene cluster deletions based on SNP heterozygosity analysis was established in this study. The location range of SNPs was selected according to the breakpoint of β-globin gene cluster deletions. SNPs were screened using bioinformatics analysis and population sequencing data. A novel method which enables genotyping of multiplex SNPs based on tetra-primer ARMS-PCR was designed and optimized. Forty clinical samples were tested in parallel by this method and MLPA to verify the performance of this method for detecting β-globin gene cluster deletion. Six informative SNPs were obtained, achieving heterozygote coverage of 93.3% in normal individuals. Genotyping of six SNPs were successfully integrated into two multiplex tetra-primer ARMS-PCR reactions. The sensitivity, specificity, positive predictive value and negative predictive value of the method for detecting β-globin gene cluster deletion were 100%, 96.30%, 92.86%, and 100%, respectively. This is a simple, cost-effective and novel method for detecting β-globin gene cluster deletions, which may be suitable for use in combination with MLPA for thalassemia molecular testing.
Highlights
Β-Globin gene mutations reduce or terminate the production of beta globin chains, of which approximately 10% are large deletions within the β-globin gene cluster
A panel of 6 informative single nucleotide polymorphism (SNP) was obtained as follows: rs7480526, rs713040, rs10742584, rs74234654, rs35755129, and rs11036364, respectively (Table 2). This indicated that 98 out of 105 samples carried at least one heterozygous SNP, revealing a heterozygote coverage of 93.3% in normal individuals that was attributable to the panel of six SNPs
The current study developed a method for detecting β-globin gene cluster deletions based on heterozygosity analyses of SNPs
Summary
Β-Globin gene mutations reduce or terminate the production of beta globin chains, of which approximately 10% are large deletions within the β-globin gene cluster. The sensitivity, specificity, positive predictive value and negative predictive value of the method for detecting β-globin gene cluster deletion were 100%, 96.30%, 92.86%, and 100%, respectively This is a simple, cost-effective and novel method for detecting β-globin gene cluster deletions, which may be suitable for use in combination with MLPA for thalassemia molecular testing. Initial screening for thalassemia via red blood-cell indices and hemoglobin analyses, is followed by genetic testing to further identify the specific mutations underlying β-thalassemia in D NA12. Molecular tests, such as gap-polymerase chain reaction (Gap-PCR), Southern blot analysis, and multiplex ligation dependent probe amplification (MLPA), are commonly used to detect β-globin gene cluster d eletions[13]. In view of the limitations of the current method, a simple, economical and low-tech method is needed to detect the deletion of β-globin gene cluster
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