A multiplex quantitative fluorescent PCR test for prenatal diagnosis of Hb Barts hydrops fetalis.

tion, which removes both the 1 and 2 genes from 1 allele, leaving only 2 functional genes. This finding explains the comparatively high expression level (40%) of the variant chain. In an alignment of 449 globin sequences from different species, 91 is absolutely conserved as leucine, and this extraordinary level of conservation suggests an important functional role. X-ray structural analyses show that the alkyl side chain heads internally toward the heme plate, where it comes to within 2.4 Å of the Fe -bound proximal histidine ( 87) and within 3.6 Å of the heme (Fig. 1B). Its main chain carbonyl group is located on the surface and forms an H-bond with the guanidine group of Arg40 in the adjacent 2 subunit. On oxygenation, movements of the heme Fe are transmitted through 87His and 91Leu onto the adjacent 2 subunit to regulate cooperatively and produce the characteristic sigmoidal O2 binding curve (3 ). As this model correctly predicts, mutation of 40 Arg also impacts O2 binding; its mutation to Ser in Hb Austin alters both affinity and cooperativity (4 ). There are 2 predictable consequences of the novel 91Leu3Phe mutation, altered O2 affinity and decreased molecular stability. We were unable to measure O2 affinity, but we were able to demonstrate decreased molecular stability. A small amount of precipitate was collected after extending a standard isopropanol stability test to 40-min incubation. The precipitate was then dissolved in 1% formic acid and analyzed directly by mass spectrometry (Fig. 1A). This showed selective precipitation of the variant Hb, which was enriched from 40% in lysate to 56% in the precipitate. Only one mutation has been previously reported at this position, 91Leu3Pro. In this case the less conservative mutation was associated with mild anemia and a more marked instability (5 ). This case highlights the importance of investigating abnormal Hbs at biochemical, hematological, and genetic levels to understand the pathological implications of the mutation and the risks it poses to carriers.