Isamu Sugiura

A phenotypically neutral dimorphism of protein S: The substitution of Lys155 by Glu in the second EGF domain predicted by an A to G base exchange in the gene

During the course of structural gene analysis of a family with type III protein S deficiency, we found a novel DNA polymorphism: an A or G variation at nucleotide 732 in exon 6 of the PS-alpha gene. This A to G mutation would lead to a substitution of Lys155 by Glu in the second EGF domain. Linkage study with restriction enzyme analysis using mutagenic PCR strategy showed that the same mutation was also present in three other members of the patients family and two individuals from an unrelated kindred, while they all had normal amounts of both immunological and functional PS levels. Restriction enzyme analysis of 182 normal Japanese genomic samples showed that 1.65% of normal population were heterozygotes for this variant allele. These findings suggest that this substitution in exon 6 is not responsible for the type III protein S deficiency but a phenotypically neutral polymorphism. Hereby we designate this polymorphism as PS-732.

Direct carrier detection in hemophilia B kindreds: Use of modified primers (mutagenic primers) for enzymatic amplification of the factor IX gene

Rapid direct detection of point mutations in hemophilia B kindreds was performed by analyzing the restriction fragments of the factor IX gene amplified by polymerase chain reaction (PCR). The family members of the two patients, HB 5 and HB 6, whose mutant factor IX gene had previously been defined by sequence analysis, were examined in this study. Since there are no restriction endonucleases available for detecting each mutation directly, we designed modified primers which were substituted one nucleotide near the mutated positions. Following PCR with these primers, new Mbo I or Alu I cleavage sites for normal alleles were created. In each family, the restriction fragment revealed the carriership of each patients mother. This simple methodology to detect mutations of interest is useful for genetic counseling in sporadic cases of hemophilia B.