Identification of the antithrombin III Kyoto mutation by restriction fragment length polymorphism analysis

Clarification of the molecular biologic abnormality underlying antithrombin III (AT III) deficiency is important to a complete understanding of the coagulation system. We have reported previously the deficiency of AT III Kyoto in which there has been a single substitution of thymine for guanine at codon 406 of exon VI. This results in the amino acid substitution of methionine for arginine. The nucleotide substitution at codon 406 in the mutant allele eliminates a Hae III restriction site. Accordingly, Hae III restriction analysis of the PCR fragments in this family could segregate the deficient members from the normal members, enabling the detection of the existence of Hae III restriction fragment length polymorphism of the PCR product (PCR-RFLP). Moreover, we confirmed by direct DNA sequencing with the polymerase chain reaction that the tested deficient members in an AT III Kyoto family inherit the same type of mutation in their genes. In conclusion, Hae III PCR-RFLP can be a useful method for screening of this mutant gene in the family.