TY - JOUR
T1 - Molecular characterization of mild-to-moderate hemophilia A
T2 - Detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis
AU - Higuchi, Miyoko
AU - Antonarakis, Stylianos E.
AU - Kasch, Laura
AU - Oldenburg, Johannes
AU - Economou-Petersen, Effrosini
AU - Olek, Klaus
AU - Arai, Morio
AU - Inaba, Hiroshi
AU - Kazazian, Haig H.
PY - 1991/10/1
Y1 - 1991/10/1
N2 - To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, we analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, we determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, we mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations in the factor VIII gene were studied, and all 26 mutations were easily distinguishable from normal. After analyzing the 29 patients with unknown mutations, we identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.
AB - To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, we analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, we determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, we mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations in the factor VIII gene were studied, and all 26 mutations were easily distinguishable from normal. After analyzing the 29 patients with unknown mutations, we identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.
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U2 - 10.1073/pnas.88.19.8307
DO - 10.1073/pnas.88.19.8307
M3 - Article
C2 - 1924291
AN - SCOPUS:0025989429
SN - 0027-8424
VL - 88
SP - 8307
EP - 8311
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19
ER -