Prenatal diagnosis of beta-thalassemia major by high-performance liquid chromatography analysis of hemoglobins in fetal blood samples.
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.
Sanguansermsri
T
T
Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Thailand. tsanguan@mail.med.cmu.ac.th
Thanarattanakorn
P
P
Steger
H F
HF
Tongsong
T
T
Chanprapaph
P
P
Wanpirak
C
C
Siriwatanapa
P
P
Sirichotiyakul
S
S
Flatz
G
G
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D003062:Codon / Q000235:genetics; D018389:Codon, Nonsense; D017218:Cordocentesis; D004252:DNA Mutational Analysis; D005260:Female; D005312:Fetal Blood / Q000737:chemistry*; D005315:Fetal Diseases / Q000097:blood / Q000175:diagnosis*; D005838:Genotype; D005865:Gestational Age; D005914:Globins / Q000235:genetics*; D006455:Hemoglobins, Abnormal / Q000032:analysis*; D006801:Humans; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011446:Prospective Studies; D017384:Sequence Deletion; D013048:Specimen Handling; D013785:Thailand; D017086:beta-Thalassemia / Q000097:blood / Q000175:diagnosis* / Q000196:embryology
D003062:Codon; D018389:Codon, Nonsense; D006455:Hemoglobins, Abnormal; D005914:Globins