Identification of the Hb Lepore phenotype by HPLC.

BACKGROUND AND OBJECTIVE: Hb Lepore is a structurally abnormal hemoglobin in which the abnormal globin chain is a hybrid or fused globin chain (db). Three different Lepore hemoglobins have been identified, differing from each other in the point at which the db fusion occurs; Hb Lepore Hollandia (d22/b50), Hb Lepore Baltimore (d59/b86) and Hb Lepore Boston (d87/b116). In Spain only Hb Lepore Boston and Hb Lepore Baltimore have been identified. Hb Lepore is easily detected by electrophoretic and chromatographic studies, whereas the type of Hb Lepore is distinguished by chromatography of tryptic peptides of abnormal db chain. In this work, we show an easier chromatography technique for identifying the Hb Lepore phenotype. DESIGN AND METHODS: Thirteen different unrelated families (23 patients) from different parts of Spain were studied. The existence of Hb Lepore was diagnosed by standard methodology and quantified by ionic exchange HPLC. The globin chains were studied by reversed phase HPLC, which showed us the phenotype of Hb Lepore; this phenotype was corroborated by a gold standard test using molecular biology techniques. The statistical analysis was designed to determine the behavior of the quantitative (hematologic) variables using the independent variable (Hb Lepore Baltimore or Hb Lepore Boston) categorized by Student's t-test for independent groups. The distribution of the variable was established using theoretical models and the variance homogeneity hypothesis was tested. The validity of the hematologic data was estimated by creating a receiver operating characteristic (ROC) curve. RESULTS: In the study of globin chains by reversed phase HPLC, in 14 patients (8 families) three peaks were eluted; they corresponded to a, b and db globin chains. In these cases when DNA was studied by PCR followed by digestion with the restriction enzyme Pvu II, the phenotype of Hb Lepore was identified as being the Boston variant, whereas in the rest of patients (9 in total), the peak identified with hybrid chain globin (db) was not present and the molecular study showed that these patients were heterozygotes for Hb Lepore Baltimore. INTERPRETATION AND CONCLUSIONS: The study of globin chains by reversed phase HPLC is sufficient to know the phenotype of Hb Lepore and thus tedious techniques such as chromatography of tryptic digestion product of db abnormal chains are not essential, a particularly important consideration in those laboratories that do not have the possibility of carrying out molecular biology studies. Neverteheless, we should continue to use a gold standard molecular biology test in cases of prenatal diagnosis because this technique is the most exact and reproducible that we have.

Red blood cell phenotypes in alpha-thalassemias in the Spanish population.

BACKGROUND AND OBJECTIVE: alpha-thalassemia is very common on all thalassemic geographical regions. The present work aimed at analyzing the relationship between the degree of microcytosis and hematological parameters and the type of alpha-thalassemic mutation. DESIGN AND METHODS: Five hundred and thirty-six subjects with 4 kinds of alpha-thalassemia were examined using established techniques that determined all hematological parameters, and globin synthesis and molecular biological techniques to study the DNA of globin genes by Southern blotting. RESULTS: Adult carriers of alpha (+)-thalassemia (-alpha/alpha alpha) present very few hematological alterations. In a statistical comparison with normal individuals (alpha alpha/alpha alpha), significant differences were found between the hemocytometric data and the MCV and MCH of heterozygous alpha + thalassemia and the heterozygous alpha zero or homozygous alpha + genotype. Hb H disease was detected in 15 patients, presenting a severe degree of anemia, a significant increase in RDW and globin chain synthesis with an alpha/beta ratio of 0.5 +/- 0.1. INTERPRETATION AND CONCLUSIONS: These data provide reference values for geographical areas where alpha + thalassemia is common. These hematocytometric data, together with hemoglobin analysis, could be useful as a future reference data for new patients diagnosed with alpha-thalassemia.

[Molecular analysis of glucose-6-dehydrogenase deficiency in Spain]. / Análisis molecular del déficit de glucosa-6-fosfato deshidrogenasa (G6PD) en España.

PURPOSE: G6PD deficiency is the most frequent enzymopathy-producing genetic polymorphism in humans. Up to now, over 400 putative variants of G6PD have been distinguished on the basis of biochemical characterization of the deficient enzyme. Analysis of the G6PD gene has made possible a precise classification of the G6PD molecular variants by identification of about 80 different point mutations causing much of the phenotypic heterogeneity. In the Spanish population, the analysis of G6PD has led to the identification of 15 different point mutations that underlay the phenotypic heterogeneity of G6PD previously reported by biochemical analysis. The purpose of the study has been to identify the genetic mutation responsible of the G6PD deficiency and to improve the knowledge of its genetic homogeneity. PATIENTS AND METHODS: From 50 Spanish males with G6PD deficiency 34 came from out consultation and 16 from the Spanish Study Group on Red Cell Pathology (GEHBTA-Eritropatología) The methods employed included screening of prevalent mutations by ER-PCR, SSCP-PCR, genetic segmentation and biochemical characterization of the deficient enzyme. RESULTS: In 31 cases the mutations were characteristic of the four most frequent polymorphic variants found in Spain (G6PD A-376G/202A, G6PD Mediterranean 563T G6PD Union 1360T and G6PD Seattle 344C). Since these mutations either create or abolish a specific site recognized by a restriction endonuclease (RE), they can be rapidly detected by RE digestion of a PCR-amplified product (PCR-RE). In patients where none of these mutations were present (17 cases), the G6PD gene was subjected to PCR single-strand conformation polymorphism (PCR-SSCP) analysis combined with direct PCR-sequencing. By using this procedure, 9 new mutations have been identified, five of them have been also found in other geographical areas and were associated with favism (G6PD A-376G/968C, G6PD Santamaria 376G/542T, G6PD Aures 143C and G6PD Chatham 1003A) or chronic haemolytic anaemia (G6PD Tomah 1153C). The other four mutations are unique and not reported so far: Three of them are associated with favism (G6PD Málaga 542T, G6PD Murcia 209G and G6PD Valladolid 406T) and one with chronic haemolytic anaemia (G6PD Madrid 1155G). The remaining eight cases are under study. CONCLUSION: The present study confirms the marked genetic heterogeneity of G6PD deficiency in Spain and demonstrate that the PCR-RE analysis is an easy tool for rapid diagnosis of the molecular defect in subjects with the common forms of G6PD deficiency. Furthermore the fact that G6PD A-376G/202A is the most common variant within Spanish population and the finding of G6PD Aures 43C and G6PD Santamaría 76G/542T, who are polymorphic in Algeria is consistent with a significant gene flow from Africa to Europe through Spain.