Genetic heterogeneity of glucose-6-phosphate dehydrogenase deficiency revealed by single-strand conformation and sequence analysis

• Published in: American journal of human genetics 1993-03, Vol.52 (3), p.527-536
• Main Authors: CALABRO, V, MASON, P. J, FILOSA, S, CIVITELLI, D, CITTADELLA, R, TAGARELLI, A, MARTINI, G, BRANCATI, C, LUZZATTO, L
• Format: Article
• Language: English
• Subjects: Anemias. Hemoglobinopathies; Base Sequence; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; BIOLOGICAL MARKERS; Child; deficiency; DETECTION; DEVELOPED COUNTRIES; DISEASES; Diseases of red blood cells; DNA - genetics; DNA SEQUENCING; DNA, Single-Stranded - genetics; ENZYMES; EUROPE; Exons; GENE MUTATIONS; genetic variance; Genetic Variation; glucose-6-phosphate dehydrogenase; Glucosephosphate Dehydrogenase - genetics; Glucosephosphate Dehydrogenase Deficiency - genetics; Hematologic and hematopoietic diseases; heterogeneity; Humans; ITALY; Kinetics; Male; man; Medical sciences; METABOLIC DISEASES; Molecular Sequence Data; MUTATIONS; Nucleic Acid Conformation; Oligodeoxyribonucleotides; ORGANIC COMPOUNDS; OXIDOREDUCTASES; Polymerase Chain Reaction - methods; PROTEINS; Restriction Mapping; single-strand conformation polymorphism; STRUCTURAL CHEMICAL ANALYSIS 550400 > Genetics
• ISSN: 0002-9297; 1537-6605

We have carried out a systematic study of the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency on a sample of 53 male subjects from Calabria, in southern Italy. Our sequential approach consisted of the following steps: (1) Partial biochemical characterization was used to pinpoint candidate known variants. The identity of these was then verified by restriction-enzyme or allele-specific oligonucleotide hybridization analysis of the appropriate PCR-amplified fragment. (2) On samples for which there was no obvious candidate mutation, we proceeded to amplify the entire coding region in eight fragments, followed by single-strand conformation polymorphism (SSCP) analysis of each fragment. (3) The next step was M13 phage cloning and sequencing of those individual fragments that were found to be abnormal by SSCP. Through this approach we have identified the molecular lesion in 51 of the 53 samples. In these we found a total of nine different G6PD-deficient variants, five of which (G6PD Mediterranean, G6PD A-, G6PD Coimbra, G6PD Seattle, and G6PD Montalbano) were already known, whereas four are new (G6PD Cassano, G6PD Cosenza, G6PD Sibari, and G6PD Maewo). G6PD Mediterranean is the commonest variant, followed by G6PD Seattle. At least seven of the variants are present, at polymorphic frequencies, in the Calabria region, and some have a nonrandom distribution within the region. This study shows that the genetic heterogeneity of G6PD deficiency in Calabria, when analyzed at the DNA level, is even greater than had been anticipated from biochemical characterization. The sequential approach that we have followed is fast and efficient and could be applied to other populations.