HEMOPHILIA-B CARRIER DETERMINATION BASED ON FAMILY-SPECIFIC MUTATION DETECTION BY DNA SINGLE-STRAND CONFORMATION ANALYSIS

Single-strand conformation (SSC) analysis can distinguish normal from variant DNA fragments containing single point mutations by conformatio n-induced electrophoretic mobility shifts in non-denaturing polyacryla mide gels. We studied 25 hemophilia B kindreds by using SSC analysis a fter polymerase chain reaction (PCR) amplification of the eight factor IX exons and their intron boundaries. Variant SSC fragments were unam biguously identified in 24 kindreds, and direct DNA sequencing of vari ant PCR fragments identified 20 different hemophilia B mutations. This technique was used for rapid and accurate carrier determination in fe male family members without the need for additional sequencing studies , because carriers have both normal and hemophilia family-specific SSC fragments. Of 25 obligate carriers from 15 kindreds, 24 were confirme d to carry variant fragments. The exception, a patient's daughter homo zygous for the normal allele, was demonstrated by subsequent PCR genot yping to be the result of non-paternity. In the additional 32 at-risk females from 16 kindreds studied, 19 were identified as carriers and 1 3 as non-carriers. Eleven of the unique mutations affected restriction enzyme digestion sites, and carriers could then be identified by appr opriate restriction enzyme digestion of amplified DNA. Our study, with hemophilia B as a model system, demonstrates the accuracy and efficie ncy of SSC analysis in screening and tracking unknown mutations in mon ogenic inherited disorders with known gene sequences.