Top-down proteomics and direct surface sampling of neonatal dried blood spots: diagnosis of unknown hemoglobin variants.

We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as ß-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-ß, ß, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and ß thalassemia) were found in the area ratio of α/pre-ß+ß applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/ß and α/pre-ß+ß area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating ß thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/ß for ß thalassemia carriers and 0.626 of α/pre-ß+ß for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work.

Prevalence and molecular characterization of abnormal hemoglobin in eastern Guangdong of southern China.

Abnormal hemoglobins (Hbs) are the most commonly inherited disorders in humans. Their frequency and types change considerably with geographic location and ethnic group. To investigate the molecular epidemiological characterization of abnormal Hbs in eastern Guangdong of southern China, a total of 11,450 'healthy' subjects were subjected to hemoglobin electrophoresis screening. Samples of EDTA-K(2) blood with abnormal Hbs were analyzed by CELL-DYN1700 blood analyzer; thalassemia genotypes and Hb E variant were identified by gap-PCR and/or reverse dot blot (RDB). The genotypes of Hb variants were detected by PCR and sequencing. The incidence of abnormal Hbs was 0.358%(41/11,450) in Chaozhou, including 12.2% (5/41) Hb J, 4.9% (2/41) Hb K, 9.7% (4/41) Hb Q, 31.7% (13/41) Hb G/D and 41.5% (17/41) Hb E. Eight types of Hb variants were found, including 3 cases of Hb J-Bangkok, 2 cases of Hb J-Wenchang-Wuming, 2 cases of Hb New York, 4 cases of Hb Q-Thailand, 5 cases of Hb G-Waimanalo, 4 cases of Hb Ottawa, 4 cases of Hb G-Chinese and 17 cases of Hb E. In comparison with other areas of Guangdong, Chaozhou had a different pattern of abnormal Hbs with a high prevalence of Hb G/D. This study describes the prevalence and molecular characterization of abnormal Hbs in eastern Guangdong.

Hemoglobin variants in North Africa.

The populations of Morocco, Algeria, and Tunisia are composed of different ethnic groups including Arabs, Berbers, Sub-Saharan Africans, Europeans, and Turks. Between 1981 and 1991, we studied more than 3,000 individuals from these North African countries. One-hundred and eighty-one carried one (or more) unusual hemoglobin variant(s) other than Hb S and Hb C which are the most frequent variants in these countries. Each of these 181 individuals was heterozygous for at least one of the 49 abnormal alpha or beta alleles identified by electrophoretic and/or structural studies, and some homozygotes were detected. A few mutants are common in North Africa: Hb O-Arab, Hb D-Punjab and Hb G-Philadelphia. Other mutants encountered in European or African populations are found in relatively few North African families. The observed polymorphisms in the populations of North Africa probably result largely from their complex ethnic origins.

The unstable haemoglobins.

The unstable haemoglobin haemolytic anaemias result from the presence in the red cell of a structurally abnormal haemoglobin variant. There are many mutations producing unstable haemoglobins; most are single amino acid replacements that affect a few key areas of the haemoglobin structure. A wide range of haemoglobin instability is evident from in vitro studies, extending from mutants with a subclinical degree of instability to those associated with severe haemolytic disease. The characteristic feature of variants associated with haemolysis is a markedly decreased stability which is readily detectable by simple screening tests. The in vivo consequence is the precipitation of the unstable haemoglobin to give Heinz bodies which are associated with the red cell membrane and lead to premature cell destruction. The unstable haemoglobins have a greater tendency to spontaneously oxidise to methaemoglobin with subsequent formation of haemichromes and precipitation. This process is significantly accelerated by external factors such as exposure to oxidative substances and increased temperature; thus haemolytic crises are frequently associated with infections in otherwise asymptomatic carriers of unstable haemoglobins. The clinical expression of the unstable haemoglobin mutation may also be modified by proteolysis of the unstable globin chain in the bone marrow. This proteolytic mechanism can predominate in the case of extremely unstable globin chains to produce primarily a thalassaemic phenotype with little if any circulating unstable haemoglobin or evidence of haemolysis.

Molecular epidemiology of the thalassaemias (including haemoglobin E).

The thalassaemias are the most common genetic disorders of man, and over the last decade the molecular epidemiology of these defects has been studied in detail. After briefly reviewing the great diversity of mutations giving rise to these conditions, four global regions are discussed in more detail. The thalassaemias, of which haemoglobin E is one, are most frequent in Asia, where recent work has defined the molecular basis of the beta thalassaemias and the frequencies of the various types of alpha + and alpha 0 thalassaemia. Oceanic populations have a range of globin gene variants remarkably different to those of south-east Asia. Most is known about the nature and frequencies of thalassaemia mutations in Mediterranean countries, where prenatal diagnosis programmes have been very successful in reducing the frequency of new cases of thalassaemia major. alpha + Thalassaemia is the most common haemoglobinopathy in sub-Saharan Africa, and molecular studies of American Blacks with beta thalassaemia have elucidated the probable molecular basis of the mild form of this disorder in Africans. Although each geographical region has its own group of common beta thalassaemia mutations, with little overlap, most of these appear to have had a single origin. The question of single or multiple origins for HbE in south-east Asia is unresolved. Recombination events producing alpha + thalassaemia deletions are frequent, whereas alpha 0 thalassaemia is produced by a variety of large deletions, each of which has had a single origin. The evidence favouring natural selection by P. falciparum malaria as the primary cause of high frequencies of the thalassaemias throughout the tropics and subtropics is reviewed. While the mechanism of protection remains unclear, epidemiological evidence supporting the hypothesis is strong, but more information is required from case-control studies on the amount of protection provided by the various thalassaemia genotypes.