Molecular characterization of mild-to-moderate hemophilia A: Detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis

Journal Article · Tue Oct 01 04:00:00 EDT 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.19.8307· OSTI ID:5821627

Higuchi, Miyoko; Antonarakis, S E; Kazazian, Jr, H H ^[1]; Oldenburg, J; Olek, K ^[2]; Arai, Morio; Inaba, Hiroshi ^[3]

---

1. Johns Hopkins Univ., Baltimore, MD (United States)
2. Univ. of Bonn (West Germany)
3. Tokyo Medical College (Japan)

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, the authors 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, they 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, they 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 were easily distinguishable from normal. After analyzing the 29 patient with unknown mutations, they 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.

• OSTI ID: 5821627
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 88:19; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
DISEASES
DNA BASE TRANSITIONS
DNA SEQUENCING
ELECTROPHORESIS
GENE AMPLIFICATION
GENE MUTATIONS
HEMIC DISEASES
HEMOPHILIA
HEREDITARY DISEASES
MOLECULAR BIOLOGY
MUTATIONS
PATIENTS
STRUCTURAL CHEMICAL ANALYSIS