Mc. Poon et al., HEMOPHILIA-B CARRIER DETERMINATION BASED ON FAMILY-SPECIFIC MUTATION DETECTION BY DNA SINGLE-STRAND CONFORMATION ANALYSIS, The Journal of laboratory and clinical medicine, 122(1), 1993, pp. 55-63
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.