Detection of sequence variability of the collagen type II alpha 1 3 ' variable number of tandem repeat

The variable number of tandem repeat (VNTR) 3' of the collagen type II (COL 2A1) gene has been shown to be highly variable with a complex molecular str ucture. In a previous pilot experiment we observed discordance between meth ods to genotype this informative marker. To further investigate the extent and molecular nature of this discordance, we genotyped a random sample of 2 07 Caucasian individuals with two genotyping methods and sequenced new alle les. We compared single-strand (SS) analysis, which is based on detection o f size differences between the different alleles, and heteroduplex analysis (HA), which is sensitive to both size and sequence differences. Overall, 2 6% of discordance between the two methods was detected. Approximately two t hirds of this discordance was caused by subdivision of SS-alleles 13R1 and 14R2 into HA-alleles 4A + 4B and 3B + 3C, respectively. Sequence analysis o f the COL2A1-VNTR alleles 4B and 3C showed that these alleles differed in s equence, but not in size, from already described SS-alleles, which explains why they escape detection by SS. The 4B allele is a frequent allele in the population (14%) and is, therefore, important to distinguish in associatio n studies. We conclude that HA is a reliable method when the described opti mized electrophoretic conditions are used; HA is a sensitive genotyping met hod to document allelic diversity at this locus, which can distinguish more alleles compared to the SS method.