Accurate Prenatal Diagnosis of Hb Bart’s Hydrops Fetalis in Daily Practice with a Double-Check PCR System

Abstract

Hemoglobin (Hb) Bart’s hydrops fetalis is a fatal condition associated with homozygous α⁰-thalassemia. Prenatal diagnosis of the disease is usually done by gap-PCR; however, misdiagnosis can occur with allelic dropout. Diagnosis using more than one method is preferred. We describe a double-check PCR assay for accurate prenatal diagnosis. The study was conducted on 64 fetuses at risk of homozygous α⁰-thalassemia encountered at our routine thalassemia diagnosis laboratory. Chorionic villus sample (CVS), amniotic fluid or fetal blood specimens were obtained from pregnant women at risk and analyzed by two PCR methods. In the first method, the SEA α⁰-thalassemia deletion of parents and fetuses were determined by gap-PCR routinely run in our laboratory. In another method, two specific fragments located 5′ to the ζ₂ gene (XbaI fragment) and the α₂-globin gene (RsaI fragment) together with the gap-PCR fragment were multiply co-amplified to determine the presence or absence of normal and α⁰-thalassemia alleles. The molecular diagnosis of α⁰-thalassemia was possible in all 64 fetuses using the two PCR approaches. The final diagnoses included 13 normal, 29 unaffected heterozygote and 22 homozygote α⁰-thalassemia fetuses.The two PCR assays disclosed no discordant result in the diagnosis of the Hb Bart’s hydrops fetalis caused by α⁰-thalassemia.The combined PCR assay for gap-PCR, ζ₂ XbaI and α₂ RsaI fragments, described here, is simple, accurate and applicable in the prenatal diagnosis of Hb Bart’s hydrops fetalis in a routine setting.

Anemia, Iron Deficiency and Thalassemia among Adolescents in Northeast Thailand: Results from Two Independent Surveys

“…We found prevalence as high as 62.2 and 58.8% for thalassemia in the 2 surveys, the data confirming the high prevalence of thalassemia and hemoglobinopathies in northeast Thailand. As shown in table 3, all forms of common thalassemia and hemoglobinopathies in Thailand, including α⁰-thalassemia, α + -thalassemia, Hb Constant Spring, Hb Paksé, β-thalassemia and Hb E, were detected in as many as 20 different genotypes in our subjects, the data indicating the heterogeneity of thalassemia and hemoglobinopathies in this area [4,5,12,13,14]. We have shown previously that, as compared to the thalassemia and hemoglobinopathies, the contribution of ID to pregnancy-associated anemia in this region is low [5].…”

“…Identification of α⁰-thalassemia (SEA and THAI deletions), α + -thalassemia (3.7-and 4.2-kb deletions), Hb Constant Spring and Hb Paksé were routinely performed in our laboratory using PCR methods described elsewhere [12,13,14,15]. The β E and β-thalassemia mutations were identified using allele-specific PCR assays [16,17].…”

Hydrops fetalis – has there been a change in diagnostic spectrum and mortality?

“…Occurrence also varies between countries. While in the United States the rate for HF has been estimated to be *1 for 600-4,000 pregnancies, [10,11] in Thailand HF secondary to homozygous athalassemia or hemoglobin Bart alone accounts for HF with an expected frequency of 1 to 500-1,500 pregnancies [12][13][14][15][16][17].…”

Prenatal Diagnosis of Hemoglobin Bart’s Hydrops Fetalis with Gap-PCR System

“…As such, this multiplex assay is just as prone to allele dropout misdiagnosis as a simple 3-primer gap-PCR assay that detects the deletion and non-deletion alleles. Indeed, the considerably larger size of the fragment that indicates a non-deletion allele (820 bp) compared to the deletion junction fragment (195 bp) may increase the propensity for allele dropout of the non-deletion allele I read with interest the recent article by Karnpean et al [1] , which described a double-check PCR assay for accurate prenatal diagnosis of hemoglobin (Hb) Bart's hydrops fetalis. In their method, 2 specific fragments located 5 to the 2 gene (XbaI fragment) and the ␣ 2 -globin gene (RsaI fragment) together with the gap-PCR fragment were multiply co-amplified to determine the presence or absence of normal and ␣ 0 -thalassemia alleles.…”