Deletional Α-thalassemia Research Articles

Routine Screening of (--SEA) α-Thalassemia Deletion by an Enzyme-Linked Immunosorbent Assay for Embryonic ζ-Globin Chains

We evaluated an enzyme-linked immunosorbent assay (ELISA) for embryonic ζ-globin chains as a routine screening test for (--^SEA) α-thalassemia deletion (SEA deletion). A total of 174 consecutive patient samples with a request for Hb analysis were recruited. The ELISA method was evaluated against a polymerase chain reaction (PCR)-based technique that was taken as the standard. Among 56 simple carriers of SEA deletion diagnosed by PCR and 112 subjects without the SEA deletion, the sensitivity and specificity of the ELISA method was 89.3–96.4 and 98.2–100%, respectively, depending on the cutoff value for optical density that was adopted. The ELISA method was able to detect both subjects with SEA deletion and concurrent β-thalassemia trait in this series, but only 1 out of 4 patients (25%) with Hb H disease. We speculate that incomplete lysis of hypochromic microcytic red cells together with the low red cell count in Hb H disease might account for the false-negative results. We showed that the ELISA method for embryonic ζ-chains was a sensitive method of screening for SEA deletion carriers at our locality, and should be easily adopted in a routine diagnostic laboratory. The method was rapid and also amendable to automation. In areas with a high prevalence of α-thalassemia, improved detection of SEA deletion carriers would ultimately facilitate the identification of pregnancies at risk of hydrops fetalis and its prevention through prenatal diagnosis.

“Reconstituted” α-Thalassemia Genomic Samples as Positive Controls for the Molecular Diagnostic Laboratory

We and others recently described strategies for multiplex-PCR analysis of deletional determinants of α-thalassemia (1)(2)(3), culminating in the development of a single-tube assay for simultaneous screening of seven common deletions (4). Since then, several molecular diagnostic laboratories wanting to set up the test have requested DNA samples carrying these deletions for use as validation and positive controls. These requests have led to a critical shortage of our limited stocks of genomic samples, especially those with the rarer deletions, something that has caused our inability to fulfill all requests. The ideal solution to limited genomic DNA is to establish immortal lymphoblastoid cell lines from peripheral-blood leukocytes of patients by Epstein–Barr virus transformation (5)(6). To do so, however, requires that patients be contacted again to provide renewed consent and a fresh aliquot of blood for transformation, something that may be inconvenient or impractical for third-party referral laboratories to implement. We have devised an alternative strategy for creating a renewable resource of positive control samples of known α-thalassemia genotype, derived from existing patient DNA samples. Briefly, genomic DNA of known α-thalassemia genotype was initially used as a template to amplify each deletion junction fragment individually by PCR. The relevant primer pairs and thermo-cycling conditions were as described previously (4). Junction fragments of seven α-thalassemia deletions were PCR-amplified separately from patient DNA, gel-purified with the GFXTM reagent set (Amersham Pharmacia Biotech), and ligated to a pBluescript (Stratagene) T-vector (Fig. 1A⇓ ). Each 5-μL ligation reaction contained 15 ng of T-vector and an amount of …

PCR-based diagnosis of the Filipino (--(FIL)) and Thai (--(THAI)) alpha-thalassemia-1 deletions.

In southeast Asia, the carrier frequency of two-gene alpha-thalassemia deletions is quite high, ranging from 4% to 14% depending on the population. The most common alpha-thalassemia-1 deletion is the so-called southeast Asian deletion (--(SEA)). In addition, a significant proportion of cases involve two other deletions, the Filipino (--(FIL)) and Thai (--(THAI)) deletions. In this report, we identify the deletion breakpoints for the (--(FIL)) and (--(THAI)) deletions, and describe PCR-based protocols for rapid and reliable DNA diagnosis of these deletions.

Frequency of deletional alpha-thalassemia genotypes in a predominantly Asian-American population.

alpha-Thalassemia is prevalent among Asian-Americans. Most cases involve deletions of one or more alpha-globin genes. Parents with two-gene cis deletions can have offspring with hemoglobin Bart's hydrops fetalis or hemoglobin H disease. The diversity of deletions poses special challenges to laboratories that offer DNA-based testing for alpha-thalassemia. The purpose of this study was to determine both the frequency of alpha-thalassemia genotypes in a predominantly Asian-American population (n = 78) and the mutation detection rate of a comprehensive Southern blot method. The ethnic composition of the study population was similar to the major Asian-American groups that reside in the United States as a whole. Three mutations (the Southeast Asian type, --SEA; -alpha3.7; and the Filipino type, --FIL) accounted for all mutant alleles present in the Asian-American patients tested for reproductive reasons. The relative frequencies of these three mutations were 62%, 27%, and 11%, respectively. The mutation detection rate was 100%. Laboratories that perform DNA-based alpha-thalassemia testing in populations similar to the one residing in Washington State should use a testing system that allows for the unequivocal identification of the haplotypes detected in the study population, namely --SEA, -alpha3.7, and --FIL.

The Unusual Pathobiology of Hemoglobin Constant Spring Red Blood Cells

Hemoglobin Constant Spring (HbCS) is the most common nondeletional α-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other α-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV “normalizing” effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional α-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and α-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional α-thalassemic variants and found that in addition to oxidized β-globin chains, oxidized αcs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized αcs- and β-globin chains. The membrane alterations induced by αcs chains are more akin to those induced by βA-globin chains than those induced by the αA-globin chains that accumulate in the β-thalassemias. Thus, each globin chain, αcs, αA, βA, appears to produce its own form of membrane perturbation.

The Unusual Pathobiology of Hemoglobin Constant Spring Red Blood Cells

AbstractHemoglobin Constant Spring (HbCS) is the most common nondeletional α-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other α-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV “normalizing” effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional α-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and α-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional α-thalassemic variants and found that in addition to oxidized β-globin chains, oxidized αcs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized αcs- and β-globin chains. The membrane alterations induced by αcs chains are more akin to those induced by βA-globin chains than those induced by the αA-globin chains that accumulate in the β-thalassemias. Thus, each globin chain, αcs, αA, βA, appears to produce its own form of membrane perturbation.

Hb Sun Prae or α213o(H13)Ala → Proβ2A New Unstable Variant Occurring In Low Quantities

A severe hemolytic anemia with microcytosis and hypochromia was present in a young adopted Indian patient. Reversed phase high performance liquid chromatographic methodology and heat stability tests detected an unstable alpha chain which was present in 3 to 5% of the total hemoglobin. A larger quantity of the alpha X chain was obtained by preparative reversed phase high performance liquid chromatography. Structural analyses identified an Ala----Pro replacement at position 130 of the alpha chain. The instability of the variant, named Hb Sun Prairie, is comparable to that of Hb Bibba [alpha 136 (H19)Leu----Pro]. Gene mapping failed to detect an alpha-thalassemia deletion (alpha alpha/alpha alpha), while dot-blot analysis of amplified DNA with synthetic probes localized a G----C mutation in codon 130 (resulting in the Ala----Pro mutation) of the alpha 2-globin genes of both chromosomes. These results suggest a homozygosity for the G----C mutation and the condition alpha 2(G----C)alpha 1/alpha 2(G----C)alpha 1 adequately explains the rather severe clinical status of this child, including the marked microcytosis and hypochromia. Unfortunately, family studies to exclude the presence of a large deletion involving all zeta- and alpha-globin genes were not possible.

Frequency and molecular types of deletional α-thalassemia in Egypt

The frequency of deletional alpha-thalassemia in the Egyptian population was estimated at 0.08 by DNA analysis of a newborn random sample. No alpha 0 determinants were found. The most frequent alpha+ determinant was the -alpha 3.7 type I in association with the medium allele at inter-zeta HVR. The -alpha 4.2 and alpha alpha alpha anti 3.7 arrangements were found at very low frequencies.

Alpha-Thalassemia Is Related to Prolonged Survival in Sickle Cell Anemia

AbstractWe have determined the frequency of deletional α-thalassemia in black populations in the USA and Africa that harbor sickle cell anemia. In normals, the frequency of the chromosome bearing a deletion of one of the two normal α gene loci, designated (−α). ranged from 0.12 to 0.16, and in sickle trait subjects, the frequency ranged from 0.18 to 0.20. By contrast, in sickle cell anemia subjects, the frequency was significantly greater and ranged from 0.22 to 0.33. Analysis demonstrated that the greater frequency in the last group was primarily a result of an increased number of subjects with α-thalassemia trait (also called homozygous α-thalassemia-2). In addition, the frequency of the (−α) chromosome was found to increase progressively with age, supporting the hypothesis that α-thalassemia is favorable to the survival of subjects with sickle cell anemia. Thus, individuals who inherit α-thalassemia and sickle cell anemia may represent a subgroup of patients with a longer life expectancy.