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Two novel mutations responsible for hereditary type I protein C deficiency: Characterization by denaturing gradient gel electrophoresis
Author(s) -
Gandrille S.,
Vidaud M.,
Aiach M.,
AlhencGelas M.,
Fischer A. M.,
GouaultHeilman M.,
Toulon P.,
Fiessinger J. N.,
Goossens M.
Publication year - 1992
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1380010607
Subject(s) - exon , biology , genetics , temperature gradient gel electrophoresis , microbiology and biotechnology , nonsense mutation , intron , gene , mutation , polymerase chain reaction , denaturing high performance liquid chromatography , stop codon , gel electrophoresis , transition (genetics) , missense mutation , 16s ribosomal rna
Hereditary protein C (PC) deficiency is usually associated with a high risk of thrombosis. We report the results of a study undertaken to screen for molecular defects in families with hereditary quantitative PC deficiency. Using a strategy combining polymerase chain reaction amplification of selected gene fragments, denaturing gradient gel electrophoresis of the amplification products, and direct sequencing of fragments with altered melting behavior, we studied the PC gene exons and exon/intron junctions of subjects with hereditary type I PC deficiency. Computer simulation of DNA melting was used to design several sets of primers, each containing a GC‐clamp, permitting the complete analysis of each amplified exon sequence. Using this procedure, we identified two previously undescribed mutations located in exon VII: a C‐to‐T substitution generating a nonsense codon in place of Arg 157 in the mature PC and a G‐to‐A substitution converting Arg 178 to GIn. The two mutations were detected in, respectively, 3 and 2 apparently independent families. This strategy is therefore a valuable tool for screening patients, and the results emphasize its advantages over plasma assays in individuals with a family history of thrombosis. © 1992 Wiley‐Liss, Inc.