Sequence variations of the α‐globin genes: Scanning of high CG content genes with DHPLC and DG‐DGGE

The a-globin chains are encoded by two duplicated genes (HBA2 and HBA1, 5 0 –3 0 ) showing overall sequence homology 496% and average CG content 460%. a-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 a-globin genes by carrying out a retrospective analysis of 19 variant alleles in 29 genotypes. The HBA2 alleles c.1A4G, c.79G4A, and c.281T4G, and the HBA1 allele c.475C4A were new. Three pathogenic sequence variations were associated in cis with nonpathogenic variations in all families studied; they were the HBA2 variation c.2T4C associated with c.–24C4G, and the HBA2 variations c.391G4C and c.427T4C, both associated with c.565G4A. 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.2T4C, c.95+2_95+6del, and c.523A4G) 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. Hum Mutat 24:338–349, 2004. r 2004 Wiley-Liss, Inc.