Length and sequence variation in the apolipoprotein B intron 20 Alu repeat

We have developed a single-stranded conformation polymorphism (SSCP) protocol for typing both sequence and length variations in an Alu element located in intron 20 of the human apolipoprotein B (apo B) gene. Using the polymerase chain reaction (PCR), we simultaneously amplified and isotopically labeled the apo B intron 20 Alu. The Alu tail, which is composed of two arrays of variable numbers of tandem repeats, (TTTX)_y (X = A or G) and (T)_z, was separated from the rest of the PCR product by restriction enzyme digestion with PstI. Length variation in the Alu tail (IN20-REP) was thus separated from sequence variation in the Alu body (IN20-SEQ), rendering the SSCP patterns both easier to interpret and more informative. In a sample of 242 unrelated individuals from Nancy, France, we observed 11 SSCP alleles at the IN20-SEQ locus that differed only in sequence. At the IN20-REP locus, we observed 7 alleles that differed in both sequence and length. All alleles at both loci were subcloned and sequenced. One additional allele that did not undergo a detectable mobility shift in SSCP gels was uncovered at each locus during sequencing of the SSCP alleles. The additional IN20-SEQ allele was typed by restriction enzyme digestion. Although the number of IN20-SEQ and IN20-REP alleles was large, most were uncommon; the three most common alleles at each locus represented more than 94% of those sampled. We also typed the children of the 242 unrelated French individuals, enabling verification of the Mendelian segregation of the two loci and construction of haplotypes. Twenty-three out of a possible 84 haplotypes were observed with a heterozygosity of 0.813. As expected given their close proximity, these two loci are in significant linkage disequilibrium. Using maximum parsimony, we were able to unambiguously place 11 of the 12 IN20-SEQ alleles on a phylogenetic network. We conclude that Alu length and sequence polymorphisms are a source of extensive and widely dispersed variation for a variety of genetic applications.