Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis
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Margarita Petropoulou
Abstract
Background: Screening for “non-deletion” α-chain haemoglobin variants resulting from point mutations or short deletions/insertions has attracted an increased interest during recent years, especially in areas where α-thalassaemia is prevalent. We describe a method utilising high resolution melting analysis for detecting the 13 most common “non-deletion” α-thalassaemia mutations in populations around the Mediterranean and Middle East.
Methods: The method comprises: (1) amplification of a 1087 bp fragment for each of the duplicated α-globin genes (HBA1 and HBA2) flanking all 13 mutations using a common forward primer and different reverse primers specific for HBA1 and HBA2, respectively; (2) nested amplification of three fragments in HBA2 flanking 10 mutations and two fragments in HBA1 flanking 5 mutations; (3) High resolution melting analysis of the amplicons using a LightScanner Instrument and LC Green.
Results: All 13 “non-deletion” α-chain haemoglobin variants were successfully detected by high resolution melting analysis. All heterozygote samples and eight out of 10 available homozygotes were clearly differentiated from each other and from wild type in the same amplicon. Although not all homozygote samples were distinguishable from wild type samples, this should not present a problem in a clinical setting since all DNA results should be evaluated alongside the haematological and (if relevant) clinical findings in each case.
Conclusions: The 13 “non-deletion” α-chain haemoglobin variants were successfully genotyped by high resolution melting analysis using LightScanner instrument and LCGreen Plus saturating dye. High resolution melting analysis is an accurate mutation scanning tool, advantageous as a closed-tube method, involving no post-PCR manipulations and requiring only around 5 min post-PCR analysis.
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©2015 by De Gruyter
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- Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients
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- A functional variant in the γ-glutamyltransferase (GGT)1 gene is associated with airflow obstruction in smokers
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Articles in the same Issue
- Frontmatter
- Editorial
- Biomarkers of inflammatory bowel disease: ready for prime time?
- Review
- Discriminant indices for distinguishing thalassemia and iron deficiency in patients with microcytic anemia: a meta-analysis
- Mini Reviews
- Cell-free DNA for diagnosing myocardial infarction: not ready for prime time
- The Laboratory Medicine and the care of patients infected by the Ebola virus. Experience in a reference hospital of Madrid, Spain
- Opinion Papers
- Theranos phenomenon − part 2
- Considerations in parathyroid hormone testing
- Quantity quotient reporting. Comparison of various models
- Genetics and Molecular Diagnostics
- Assessing quality and functionality of DNA isolated from FFPE tissues through external quality assessment in tissue banks
- Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience
- Non-invasive fetal ABO genotyping in maternal plasma using real-time PCR
- Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis
- General Clinical Chemistry and Laboratory Medicine
- European views on patients directly obtaining their laboratory test results
- Diagnostic performances of clinical laboratory tests using Triton X-100 to reduce the biohazard associated with routine testing of Ebola virus-infected patients
- Thrombin generation, D-dimer and protein S in uncomplicated pregnancy
- Multi-analyte analysis of non-vitamin K antagonist oral anticoagulants in human plasma using tandem mass spectrometry
- Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing
- Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease
- Reference Values and Biological Variations
- Total folate and 5-methyltetrahydrofolate in the cerebrospinal fluid of children: correlation and reference values
- Effect of age and gender on reference intervals of red blood cell distribution width (RDW) and mean red cell volume (MCV)
- Infectious Diseases
- Inosine triphosphate pyrophosphohydrolase activity: more accurate predictor for ribavirin-induced anemia in hepatitis C infected patients than ITPA genotype
- The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites
- Letter to the Editors
- Misleading high-sensitivity troponin algorithm for NSTEMI in the ESC guidelines
- Adjustment of serum potassium for age and platelet count. A simple step forward towards personalized medicine
- The impact of Tween 20 on repeatability of amyloid β and tau measurements in cerebrospinal fluid
- Effect of storage time and temperature on the generation of reactive oxygen species in peripheral blood leukocytes
- Iohexol interference in the α2-globulin fraction of the serum protein capillary electrophoresis
- A functional variant in the γ-glutamyltransferase (GGT)1 gene is associated with airflow obstruction in smokers
- Comparison of measured venous carbon dioxide and calculated arterial bicarbonates according to the PaCO2 and PaO2 cut-off values of obesity hypoventilation syndrome
- Creatinine, Jaffe, and glucose: another inconvenient truth
- Analytical evaluation of a new liquid immunoturbidimetric assay for the determination of ferritin in serum
- Analytical assessment of the novel homocysteine liquid enzymatic assay on Beckman Coulter AU5800
- Immunoglobulin IgA, IgD, IgG, IgM and IgG subclass reference values in adults
