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Sequence variations of the alpha-globin genes: scanning of high CG content genes with DHPLC and DG-DGGE.
Lacerra, Giuseppina; Fiorito, Mirella; Musollino, Gennaro; Di Noce, Francesca; Esposito, Maria; Nigro, Vincenzo; Gaudiano, Carlo; Carestia, Clementina.
Afiliação
  • Lacerra G; Istituto di Genetica e Biofisica Adriano Buzzati Traverso, Consiglio Nazionale delle Ricerche, Napoli, Italy.
Hum Mutat ; 24(4): 338-49, 2004 Oct.
Article em En | MEDLINE | ID: mdl-15365991
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.
Assuntos
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Base de dados: MEDLINE Assunto principal: Globinas / Análise Mutacional de DNA / Talassemia alfa Tipo de estudo: Evaluation_studies / Observational_studies / Prognostic_studies / Risk_factors_studies Limite: Humans Idioma: En Ano de publicação: 2004 Tipo de documento: Article
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Base de dados: MEDLINE Assunto principal: Globinas / Análise Mutacional de DNA / Talassemia alfa Tipo de estudo: Evaluation_studies / Observational_studies / Prognostic_studies / Risk_factors_studies Limite: Humans Idioma: En Ano de publicação: 2004 Tipo de documento: Article