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Screening for α(0)-thalassemia is usually associated with a high false-positive rate, leading to an unnecessary PCR workload for accurate diagnosis. We have developed a modified Hb H inclusion test for use as a secondary screening. This test was performed on young red blood cell enriched fractions using dextran sedimentation. The study was performed in 100 subjects positive on initial screening. Confirmatory tests included Hb analysis and a multiplex PCR assay to identify α(0)-thalassemia deletions. A modified Hb H inclusion test was positive in 31 cases, 30 of whom were α(0)-thalassemia carriers (97%). The remaining case (3.0%) was homozygous for α(+)-thalassemia. The remaining 69 cases with a negative Hb H inclusion test included normal subjects, α(+)-thalassemia carriers and β-thalassemia carriers. Two of them (2/69, 3.0%) were found to be double heterozygotes for β(0)-thalassemia and α(0)-thalassemia. The overall sensitivity and specificity of the modified Hb H inclusion test for screening of α(0)-thalassemia were 94.0 and 99.0%, respectively. Therefore, we recommend the use of this test in combination with Hb analysis to exclude cases with αβ-thalassemia. This should lead to a significant reduction in the number of cases referred for PCR analysis of α(0)-thalassemia by about 50.0%., (© 2013 S. Karger AG, Basel.)

The clinical characteristics of 357 patients with hemoglobin H (HbH) disease from the Guangxi province of Southern China were studied. One hundred and ninety-one (53.3%) patients were diagnosed with HbH-Constant Spring, 19 were diagnosed with HbH Westmead. Ten patients were shown to have coinherited HbH-Constant Spring/QS with a β-thalassemia mutation. Coinheritance of the β-thalassemia gene does not alleviate anemia (8.2 ± 2.3 vs. 7.6 ± 1.7 g/dl, p = 0.276), or influence age at diagnosis (20.2 ± 19.6 vs. 12.9 ± 11.0 years, p = 0.276). Ferritin levels were significantly higher in the group of patients with the nondeletional form of the disease (475 ± 719 vs. 249 ± 264 ng/ml, p = 0.005)., (Copyright © 2010 S. Karger AG, Basel.)

We utilized the PCR method to amplify the alpha-thalassemia-1 breakpoint area of the Southeast Asia type and several regions of the alpha-globin gene cluster to diagnose rightward deletion (-alpha 3.7), leftward deletion (-alpha 4.2) or nondeletion forms of the Hb H disease. For the nondeletion form, a natural restriction site of MseI was used to detect the Hb Constant Spring (Hb CS) or other termination codon mutations. Another naturally occurring restriction site of MspI was used to detect the Hb Quong-Sze. For the deletion form of the Hb H disease, the differences among nonhomologous I, II and III of the rightward or leftward deletion were used to distinguish the mutations. For further characterization of the subtype of -alpha 3.7 deletion, the same primers for detecting termination codon mutations were used to amplify part of the alpha-globin gene, then the PCR product was digested by the restriction enzyme ApaI. In the 57 cases which were studied, 19 were deletion forms while 38 were nondeletion forms. In the deletion form cases, 13 were rightward deletion (-alpha 3.7) and the other 6 were leftward deletion (-alpha 4.2). However, all of the nondeletion form cases were alpha-thalassemia-1 with Hb CS. After the subtyping of -alpha 3.7 deletion, 11 out of 13 were type I deletions and the other 2 were type II deletions.

In this study we have defined the molecular basis and correlated the clinical phenotype with the alpha-globin genotype in a large series of patients of Sardinian descent with HbH disease. The most prevalent molecular defect was the deletion of 3 alpha-globin structural genes most commonly the (--/-alpha 3.7) genotype (83.6%) and rarely the (--/-alpha 4.2) genotype (1.4%), followed in decreasing order of incidence by the combination of deletion alpha zero-thalassemia and initiation codon mutation of the alpha 2-gene (--/alpha NcoI alpha = 9.8%), deletion alpha zero-thalassemia and pentanucleotide deletion of IVS-I of the alpha 2-globin gene, (--/alpha HphI alpha = 3.3%) deletion alpha zero-thalassemia and initiation codon mutation of the alpha 1-gene (--/alpha alpha NcoI = 1.3%), a homozygous state for initiation codon mutation of the alpha 2-gene (alpha Nco alpha/alpha NcoI alpha = 0.7%). Patients with the (--/alpha thal alpha) genotypes showed severer clinical and hematological features as compared to those with the (--/-alpha) or those with the (--/alpha alpha thal) genotypes. The single patient with the (alpha Nco alpha/alpha Nco alpha) genotype had a clinical phenotype intermediate between HbH disease and the alpha-thalassemia carrier status. This heterogeneity depends on the fact that the alpha 2-globin gene produces 2-3 times alpha-globin chains than the alpha 1-gene and the single remaining alpha 1-like globin gene in the -alpha 3.7 chromosome has a compensatory increase in the alpha-globin chain output. alpha-Globin gene mapping of HbH disease patients may be useful for predicting the clinical outcome and to improve genetic counseling.