Molecular basis of antithrombin type I deficiency: the first large in-frame deletion and two novel mutations in exon 6.

We report three novel mutations accounting for cases of inherited type I antithrombin (AT) deficiency. Using the polymerase chain reaction (PCR) and direct sequencing of the coding sequences of the AT gene, we found one mutation in exon 4 and two in exon 6. A deletion of 105 bp causing an in-frame deletion of 35 amino acids between Tyr 240 and Gly 276 was found in exon 4. In a second kindred, deletion of two adenines in codon 412-413 introduced a frameshift and a stop codon at position 431. The last mutation was an insertion of ACCG in codon 387, generating a frameshift with a stop codon located at the normal position. The finding of a sequence repeat of nine residues located at the 5' and 3' ends of the deleted fragment might explain the 105 bp deletion by slippage and mispairing at the replication fork during DNA synthesis. The second mutation is the fourth described within a region of six amino acids (between Phe 408 and Arg 413), which seems to be a cluster of mutations. In this case, the presence of a double repeat sequence--TTCCT and AACA--flanking this region could be particularly favorable for slipped mispairing. These results confirm that human gene mutations are not random events but are strongly influenced by DNA flanking sequences.

Two novel mutations responsible for hereditary type I protein C deficiency: characterization by denaturing gradient gel electrophoresis.

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.