The G → T mutation at base 1 of intron 1 at the binding site of the Sp1 transcription factor of the collagen type Iα1 gene (COLIA1, GenBank accession no. AF017178) is a putative marker for low bone mineral density and osteoporotic fractures. A new method for the detection of this mutation is presented, based on the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), which utilizes two separate and simultaneous PCRs to detect the normal and mutated alleles. The forward primer (positions 1307-1336 of the gene) is common to both amplifications. Two reverse primers (positions 1566-1546) are used, differing in the 3′ base (3′-C for the normal S allele and 3′-A for the mutated s allele). The former amplification uses the reverse primer specific for the S allele; the latter uses the reverse primer with the 3′-base complementary to the mutated base of the s allele. In the SS condition, amplification occurs only in the former reaction and in the ss condition only in the latter. Both reactions give a product in the Ss condition. Direct DNA sequencing of a COLIA1 region containing the G → T polymorphism demonstrates the validity of this ARMS-PCR method. The new method is more reliable than a previously published detection method, which utilizes a mismatched reverse primer, introducing a restriction site in the T-substituted (s) allele. However, the restriction enzyme is costly, its digestion time long, and incomplete digestion can lead to an underestimation of the frequency of ss homozygosity. The latter can result in incorrect conclusions about a linkage between osteoporosis and the COLIA1 polymorphism. In a survey of the COLIA1 polymorphism in 133 osteoporotic subjects with femur fractures, 7 cases of ss homozygosity were consistently detected both by direct DNA sequencing and by the ARMS-PCR method. This, in contradistinction to the mismatched-primer method by which 3 of these 7 cases were inaccurately diagnosed as Ss heterozygosites.
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