Partial deletion of an antithrombin III allele in a kindred with a type 1 deficiency

This report details the precise mapping of a partially deleted human antithrombin III (AT-III) allele, found in a kindred with an inherited type 1 AT-III deficiency. Using truncated AT-III probes generated by polymerase chain reaction (PCR) amplification from a full-length AT-III cDNA, as well as other genomic probes specific for the 5' upstream region of the AT-III gene, we were able to characterize a partial deletion on an AT-III allele encompassing exons 1 and 2 of the AT-III gene, and a region 5' to the coding sequences. The absence of the 5' upstream region in the affected AT-III allele was confirmed directly by the PCR amplification of a 1.5-kb polymorphic fragment of genomic DNA samples from family members. The precise determination of the 5' breakpoint of the affected allele was made possible by two different approaches: (1) subcloning plus biotin capture PCR, or (2) inverse PCR. This allowed us to confirm the mapping of the deletion obtained by Southern analysis; to show that the 3' region of the mutant AT-III allele, including exons 3 to 7, was intact; and to sequence approximately 0.7 kb upstream to the breakpoint in the mutant allele. Furthermore, PCR amplification of the region of the breakpoint provided unique products detectable only in affected members of this kindred. The breakpoint in the partially deleted allele is 480 bp upstream from the 5' boundary of exon 3. No significant homology was found between the 0.7-kb sequence upstream to the breakpoint of the mutant allele and known human sequences.