Co-inheritance of Hb Pak Num Po, a novel alpha1 gene mutation, and alpha0 thalassemia associated with transfusion-dependent Hb H disease.

Hb H disease is generally associated with moderate to severe anemia but rarely requires regular blood transfusion. We recently studied two apparently unrelated patients with transfusion-dependent Hb H disease. Hemoglobin studies demonstrated Hb H and Hb Bart's without other detectable abnormal globin species. Extensive molecular analyses of the alpha globin genes and their regulatory sequence (HS-40) revealed that both patients are compound heterozygotes for alpha0 thalassemia (--(SEA)) and a novel point mutation, a thymidine insertion after codon 131 of the alpha1 gene. The resulting frameshift gives rise to a highly unstable alpha globin chain, which we refer to as "Hb Pak Num Po," containing an additional 34 amino acids. This unusual alpha1 globin variant clearly causes alpha thalassemia, but the unexpectedly severe phenotype suggests that this mutation may have additional effects on red cell physiology. A PCR-based (ARMS) assay was developed for rapid detection of this novel mutation, and this might be useful to study the prevalence of this novel mutation which poses potentially significant clinical consequences in populations of Southeast Asia. Detecting carriers of this mutation using the molecular diagnostic procedures described will provide the means to screen and prevent a potentially severe form of alpha thalassemia in Thailand

Clinical phenotypes and molecular characterization of Hb H-Pakse disease.

BACKGROUND AND OBJECTIVES: Hemoglobin Constant Spring (Hb CS), caused by a termination codon mutation (TAA-->CAA) in the a2 gene, is the most common non-deletional type of a thalassemia in Southeast Asia. This mutation can most easily be detected by loss of an MseI-restriction site (T/TAA) spanning the termination codon. Recently, we sequenced the a globin genes from patients with a thalassemia in whom this MseI site was absent. This revealed, a previously described termination codon mutation (TAA-->TAT) associated with Hb Paksé. This prompted us to re-evaluate the molecular basis of a thalassaemia in other Thai patients with non-deletional types of Hb H disease. DESIGN AND METHODS: DNA samples from 30 patients, previously diagnosed as having Hb H-CS disease, were characterized by direct genomic sequencing and by using a mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Clinical and hematologic data were assessed. RESULTS: Hemoglobin electrophoresis in almost all 30 unrelated patients with non-deletional a thalassemia revealed a slow migrating band resembling Hb CS. Five of these patients were found to have Hb H-Paksé disease and the remainder had Hb H-CS disease. Comparing the hematology in patients with these two genotypes, no significant differences were found except that the proportion of Hb H was higher in patients with Hb H-Paksé disease. INTERPRETATION AND CONCLUSIONS: These results suggest that termination codon mutations may have been previously misidentified in many cases of non-deletional Hb H disease. Findings from six unrelated families described in this study suggest that the proportion of patients with the Hb Paksé mutation might be underestimated and that this mutation could be prevalent in Southeast Asia. Analysis of mismatched-PCR-RFLP, described here, was shown to provide an unequivocal diagnosis and will be applicable in population screening programs