PRENATAL DIAGNOSIS OF β-THALASSEMIA MAJOR BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF HEMOGLOBINS IN FETAL BLOOD SAMPLES

Abstract

In Thailand and adjacent countries, most of the β-thalassemia genes are β⁰-thalassemia mutations that prevent the production of Hb A. We propose the quantitation of the Hb A fraction in fetal blood in the mid-trimester of pregnancy by automated high performance liquid chromatography as a reasonable prenatal diagnostic method to be applied in areas with limited laboratory facilities. Forty pregnant women at risk of delivering a child with β-thalassemia major were identified using an erythrocyte osmotic fragility test and quantitation of Hb A₂. Cordocentesis was performed at the gestational age of 18–22 weeks and fetal blood was analyzed for hemoglobin fractions by automated high performance liquid chromatography. The β-globin gene mutations were characterized by β-globin gene sequencing. The 4 bp deletion at codons 41/42 (−TTCT) was the most frequent of the 40 β-thalassemia mutations observed (20/40 = 50%), followed by the splice site mutation IVS-I-1 (G → T) (7/40 = 17.5%), the nonsense mutation at codon 17 (A → T) (7/40 = 17.5%), the nonsense mutation at codon 35 (C → A) (3/40 = 7.5%), and the β⁺ -thalassemia promoter mutation at −28 (A → G) (3/40 = 7.5%). High performance liquid chromatography revealed nine fetuses which had only Hb F and no Hb A. All were homozygotes or compound heterozygotes for β⁰-thalassemia mutations. In the remaining 31 fetuses, a Hb A peak was present in the chromatograms. One fetus with 0.5% Hb A was a compound heterozygote for the −28 (A → G) and codons 41/42 (−TTCT) mutations. In the remaining 30 fetuses, the Hb A values ranged between 0.8 and 7.4%. Twenty of these, with a Hb A concentration of 1.82 ± 0.49% (range 0.8–2.8%), were β-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.89 ± 1.47% (range 2.9–7.4%) and normal β-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for β⁰-thalassemia mutations and the presence of measurable amounts of Hb A in heterozygotes and normal homozygotes, permits the diagnosis of fetuses expected to develop postnatal β-thalassemia major.