title: Sequence variations of the alpha-globin genes: scanning of high CG content genes with DHPLC and DG-DGGE.
abstract: The alpha-globin chains are encoded by two duplicated genes (HBA2 and HBA1, 5'-3') showing overall sequence homology >96% and average CG content >60%. alpha-Thalassemia, the most prevalent worldwide autosomal recessive disorder, is a hereditary anemia caused by sequence variations of these genes in about 25% of carriers. We evaluated the overall sensitivity and suitability of DHPLC and DG-DGGE in scanning both the alpha-globin genes by carrying out a retrospective analysis of 19 variant alleles in 29 genotypes. The HBA2 alleles c.1A>G, c.79G>A, and c.281T>G, and the HBA1 allele c.475C>A were new. Three pathogenic sequence variations were associated in cis with nonpathogenic variations in all families studied; they were the HBA2 variation c.2T>C associated with c.-24C>G, and the HBA2 variations c.391G>C and c.427T>C, both associated with c.565G>A. We set up original experimental conditions for DHPLC and DG-DGGE and analyzed 10 normal subjects, 46 heterozygotes, seven homozygotes, seven compound heterozygotes, and six compound heterozygotes for a hybrid gene. Both the methodologies gave reproducible results and no false-positive was detected. DHPLC showed 100% sensitivity and DG-DGGE nearly 90%. About 100% of the sequence from the cap site to the polyA addition site could be scanned by DHPLC, about 87% by DG-DGGE. It is noteworthy that the three most common pathogenic sequence variations (HBA2 alleles c.2T>C, c.95+2_95+6del, and c.523A>G) were unambiguously detected by both the methodologies. Genotype diagnosis must be confirmed with PCR sequencing of single amplicons or with an allele-specific method. This study can be helpful for scanning genes with high CG content and offers a model suitable for duplicated genes with high homology.
authors.affiliation: Istituto di Genetica e Biofisica Adriano Buzzati Traverso, Consiglio Nazionale delle Ricerche, Napoli, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Lacerra
authors.forename: Mirella
authors.initials: M
authors.lastname: Fiorito
authors.forename: Gennaro
authors.initials: G
authors.lastname: Musollino
authors.forename: Francesca
authors.initials: F
authors.lastname: Di Noce
authors.forename: Maria
authors.initials: M
authors.lastname: Esposito
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Nigro
authors.forename: Carlo
authors.initials: C
authors.lastname: Gaudiano
authors.forename: Clementina
authors.initials: C
authors.lastname: Carestia
mesh_terms: D000483:Alleles; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D018740:Genetic Heterogeneity; D014644:Genetic Variation; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D015203:Reproducibility of Results; D012189:Retrospective Studies; D017085:alpha-Thalassemia / Q000235:genetics*
chemical_list: D005914:Globins