Detection of the Hemoglobin E Mutation Using the Color Complementation Assay: Application to Complex Genotyping

• Published in: Blood Vol. 76; no. 3; pp. 619 - 623
• Main Authors: Embury, Stephen H., Kropp, Gerald L., Stanton, Thomas S., Warren, Tina C., Cornett, Patricia A., Chehab, Farid F.
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01.08.1990; The Americain Society of Hematology
• Subjects: Adult; Biological and medical sciences; DNA - genetics; Electrophoresis - methods; Female; Gene Amplification - genetics; Genetic Counseling; Genotype; Hematology; Hemoglobin E - genetics; Hemoglobins, Abnormal - genetics; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Mutation; Nucleic Acid Amplification Techniques; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Rhodamines; Spectrometry, Fluorescence; Thalassemia - diagnosis; Thalassemia - genetics; Human; Hemoglobinopathy; Genetic counseling; Asia; Fluorescent labelling; Hemopathy; Diagnosis; Mutation; South east asia; Molecular biology; Hemoglobin E
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V76.3.619.619

The color complementation assay (CCA) is a method of allele-specific DNA amplification by which competitive priming and extension of fluorescently labeled oligonucleotide primers determine the color of DNA amplification product. This diagnostic method precludes the need for radioisotopes, electrophoresis, and multiple high-stringency reaction conditions. The multiplicity of mutant globin genes present in Southeast Asians complicates clinical diagnosis and underscores the importance of DNA-based diagnostic methods. We have applied CCA to distinguish βA and βE alleles. Competing 15mer primers were a fluorescein-labeled complement to βA and a rhodamine-labeled complement to βE, identical except for their central nucleotides. A common unlabeled primer was used to amplify DNA product, the color of which was determined by the perfectly complementary primer. Color photography and spectrofluorometry, as well as a method of black-white photography that we developed to distinguish fluorescein- and rhodamine-labeled DNA, were used to record results. We applied CCA to define the complex genotype of a Thai woman with thalassemia intermedia, 96% HbE, and 4% HbF whose possible genotypes included several permutations of α-thalassemia, β-thalassemia, and βE genes. ζ-Globin gene mapping of DNA doubly digested with BglW and Asp 718 showed the α3.7/–SEA genotype, and CCA confirmed homozygous βE/βE. The CCA is useful for diagnosing the compound hemoglobin genotypes of Southeast Asians and could be applied also to prenatal diagnosis in this population.