Human Fibrinogen Polymorphic Site Analysis by Restriction Endonuclease Digestion and Allele-Specific Polymerase Chain Reaction Amplification: Identification of Polymorphisms at Positions Aα312 and Bβ448

In the fibrinogen molecule, a total of seven sites have been tentatively identified as polymorphic; however, disagreements about these sites have been observed among the various protein and DNA sequence data published. To allow examination of the potential polymorphic sites at the DNA level, human genomic DNA samples were prepared from 110 unrelated, healthy individuals. Either allele-specific polymerase chain reaction (ASPCR) amplification or PCR amplification followed by restriction endonuclease digestion was used to detect the presence of possible polymorphisms. Two polymorphic sites were confirmed, one at Aα312 (Thr/Ala) by RsaI restriction analysis, and a second at Bβ448 (Arg/Lys) by MnlI restriction analysis. Mendelian inheritance of both polymorphisms was demonstrated and allele frequencies were estimated as 0.76/0.24 and 0.85/0.15 for the Aα312 and Bβ448 sites, respectively. The sites at Aα47, Aα296, Bβ162, Bβ296, and γ88 showed no evidence of variation in any of our samples. The amino acid polymorphisms at Aα312 and Bβ448 reflect conservative residue changes with unknown effects on fibrinogen structure or function. An additional, previously unrecognized DNA sequence variant was detected in a single individual in the second intron of the Aα chain using HinfI restriction analysis.