Prenatal diagnosis of beta-thalassemia major by high-performance liquid chromatography analysis of hemoglobins in fetal blood samples

Hemoglobin. 2001 Feb;25(1):19-27. doi: 10.1081/hem-100103066.

Abstract

In Thailand and adjacent countries, most of the beta-thalassemia genes are beta(0)-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 beta-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 beta-globin gene mutations were characterized by beta-globin gene sequencing. The 4 bp deletion at codons 41/42 (-TTCT) was the most frequent of the 40 beta-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 beta(+)-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 beta(0)-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 beta-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.89 +/- 1.47% (range 2.9-7.4%) and normal beta-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for beta(0)-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 beta-thalassemia major.

Publication types

  • Evaluation Study

MeSH terms

  • Adult
  • Chromatography, High Pressure Liquid*
  • Codon / genetics
  • Codon, Nonsense
  • Cordocentesis
  • DNA Mutational Analysis
  • Female
  • Fetal Blood / chemistry*
  • Fetal Diseases / blood
  • Fetal Diseases / diagnosis*
  • Genotype
  • Gestational Age
  • Globins / genetics*
  • Hemoglobins, Abnormal / analysis*
  • Humans
  • Polymerase Chain Reaction
  • Pregnancy
  • Prenatal Diagnosis / methods*
  • Promoter Regions, Genetic / genetics
  • Prospective Studies
  • Sequence Deletion
  • Specimen Handling
  • Thailand
  • beta-Thalassemia / blood
  • beta-Thalassemia / diagnosis*
  • beta-Thalassemia / embryology

Substances

  • Codon
  • Codon, Nonsense
  • Hemoglobins, Abnormal
  • Globins