Splice site mutation in the human protein C gene associated with venous thrombosis: demonstration of exon skipping by ectopic transcript analysis.
Heterozygosity for a G-->C mutation converting the highly conserved Gln184 (CAG) to His (CAC) was identified at the last nucleotide of exon 7 of the protein C gene in two family members with deep vein thrombosis. As the nucleotide is a part of the 5 splice site of intron G, it was examined how the mutation affected splicing of protein C pre-mRNA. Relevant protein C cDNA fragments were amplified with polymerase chain reaction after reverse transcription of ectopic mRNA from peripheral blood lymphocytes. Southern blot analysis and nucleotide sequencing of these fragments showed a fragment (A) corresponding to correctly spliced mRNA originating from the normal allele and a fragment (B) corresponding to a truncated mRNA lacking exon 7, originating from the mutant allele. A third fragment (C) lacking exons 7 and 8 was identified in both affected and unaffected family members, as well as in normal controls. Analysis of human liver protein C mRNA indicated that the ectopic lymphocyte mRNA was qualitatively representative for the tissue-specific mRNA. In conclusion, evidence is provided showing that the mutation abolishes formation of correctly spliced mRNA. This agrees with the observation that the mutation results in a type 1 protein C deficiency.
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
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Lind
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van Solinge
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Schwartz
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Thorsen
D000328:Adult; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D011486:Protein C / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D013924:Thrombophlebitis / Q000235:genetics*
D018076:DNA, Complementary; D011486:Protein C; D012333:RNA, Messenger