Molecular genetic confirmatory testing from newborn screening samples for the common African‐American, Asian Indian, Southeast Asian, and Chinese β‐thalassemia mutations

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Abstract
β‐Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high‐performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with β‐thalassemia. Our purpose was to develop single‐tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for β‐thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic‐specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for β‐thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African‐American descent. Specimens from African‐American neonates were tested for two mutations (−88 and −29); Asian Indians for five mutations (IVSI‐1, IVSI‐5, codons (Cd) 41/42, Cd 8/9, and 619‐bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, −28, IVSII‐654, Cd 71/72, IVSI‐5, and IVSI‐1). We identified each of these β‐thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with β‐thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic‐specific β‐thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation. Am. J. Hematol. 78:249–255, 2005.