Identification of six novel mutations in type I antithrombin deficient Italian families.

Antithrombin (AT) is the most important inhibitor of thrombin and factor X and one of the major physiologic inhibitors of hemostasis. Hereditary AT deficiency is an autosomal dominant disease occurring in up to 6% of thromboembolic patients. Type I AT is defined by concordantly low circulating levels of both functional and immunological AT, whereas type II AT deficiency corresponds to the presence of an abnormal protein in the bloodstream. Identification of gene mutations occurring in AT-deficient patients has given important information on the structurefunction relationships of this hemostatic protein. We investigated 8 Caucasian subjects, belonging to 8 unrelated families, with a confirmed diagnosis of AT deficiency looking for gene alterations within the AT gene. We also investigated the subjects’ first-degree relatives, when available. Blood samples were collected and tested for antiphospholipid antibodies, antithrombin activity and immunoreactivity (ELISA, Diagnostica Stago, Asnières, France), protein C (amidolytic and immunological assays; Behring, Marburg, Germany) and total and free protein S antigen (ELISA, Diagnostica Stago, Asnières, France), as reported elsewhere. Isolation of DNA and FV Leiden and FII A20210 mutation analysis were done as previously described. All coding regions of the AT gene and intron/exon boundaries were amplified using sense and antisense oligonucleotides designed on the basis of known sequences of the AT gene locus (Genbank accession number X68793). Amplified DNA fragments were purified and subjected to direct cycle sequence analysis using the Taq dye-deoxy terminator method and an ABI PRISM 3100 Genetic Analyzer sequencer (PE Biosystems, USA). The 8 different heterozygous mutations we identified within the AT gene are reported in Table 2. We found 2 missense mutations, 2 nonsense mutations, 1 insertion of two nucleotides, 1 deletion of a single nucleotide, and 2 mutations that alter the pattern of mRNA processing. None of the mutations identified was found in unaffected relatives. To exclude the possibility that one or more of mutations identified could be a polymorphism, all mutations were further investigated in another 100, apparently healthy, subjects. None of these subjects was found to carry any of the mutations. As for other inherited or acquired (antiphospholipid antibodies) thrombophilic risk factors, only the proband of family 4 was found to carry the factor V Leiden mutation. Of the 8 mutations identified, only the G→A transition at nucleotide 9788 and the nonsense mutation at the R132 residue have been previously reported. The G→A transition 14 bp before the end