Hemoglobin Constant Spring (Hb CS) Missed by HPLC in an Hb E Trait Pregnancy Resulting in Hb H-CS Disease in a Thai Girl: Utility of Capillary Electrophoresis.

Hemoglobin Constant Spring [Hb CS; α142, Term→Gln (TAA>CAA IN α2)] is often missed by routine laboratory testing, especially in subjects with co-inheritance of ß-thalassemia or ß-variants. We reported the case of a 1-year-old female with Hb H-CS disease who was born from a father with heterozygous of α-thalassemia-1 Southeast Asian type deletion and a mother with the combination of Hb CS and Hb E [ß26 (B8) Glu→Lys, GAG>AAG] trait. A very tiny peak of Hb CS of the mother was easily ignored on the high performance liquid chromatography chromatogram while it was clearly seen on the capillary electrophoresis (CE) electrophoregram. Therefore, the CE is useful in screening for heterozygous Hb CS in a person with Hb E trait. This is of potential benefit for prevention of new cases of Hb H-CS disease.

Detection of α-thalassemia-1 Southeast Asian and Thai type deletions and ß-thalassemia 3.5-kb deletion by single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting analysis.

BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and ß-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous ß-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, ß-thalassemia 3.5-kb gene deletion, and the wild-type ß-globin gene had specific peak heights at mean melting temperature (T(m)) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and ß-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.