Objectives: Certain point mutations in the alpha globin gene may lead to unstable hemoglobins such as Hb Constant-Spring and Hb Quong-Sze. These alpha chain variants, when inherited with deletional α-thalassemia mutations cause more severe forms of HbH disease. To evaluate the utility of the Luminex xMAP platform for rapid screening of non-deletional α-thalassemia mutations, we developed a multiplexed genotyping assay to detect six mutations in the α2-globin gene. that are clinically significant or, when coinherited with α-thalassemia-1, result in HbH disease.

Methods: A panel of positive controls with known heterozygosity for Hb Constant-Spring, Hb Quong-Sze, Hb J-Toronto, Hb Hasharon, Hb G-Philadelphia and Hb G-Norfolk, was obtained from anonymized whole blood or newborn dried blood spots. The α2 gene was amplified using nested PCR and single base chain extension (SBCE) was carried out using the PCR products, capture-tagged primers, thermosequenase, and one of four biotinylated ddNTPs. Biotinylated amplicons were hybridized to fluorescent microspheres containing allele-specific capture probes and analyzed by flow cytometry on the Luminex100.

Results: Median flourescent intensity (MFI) values uniformly discriminated between wild type and mutant alleles in three independent experiments. Mean background-subtracted MFIs ranged from 255 to 1000 for all positive alleles and genotypes were correctly identified in all of the samples tested.

Conclusions: As HbH disease is becoming more prevalent in the U.S., early detection of this disorder using accurate, affordable diagnostic methods will permit timely delivery of health care and prevention of severe clinical manifestations of disease. As the severity of many of these disorders has not been well-characterized, an understanding the phenotypic expression of these hemoglobin disorders is essential to providing adequate counseling and treatment options.

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