Hereditary Thrombophilia Caused by Abnormality of the Anticoagulant Protein C Pathway: Prenatal Diagnosis of Compound Heterozygous Protein C Deficiency by Direct Detection of the Mutation Sites

Summary

In the present study, we report the molecular characterization of a hereditary compound heterozygous protein C (PC) deficiency observed in a male infant with neonatal purpura fulminans, and its prenatal diagnosis done by direct detection of the mutation sites in the PC gene. DNA-sequence analysis of the patient’s and his mother’s PC gene disclosed the presence of a deletion of one of the four consecutive G nucleotides encoding Trp³⁸⁰(TGG)-G1y³⁸¹(GGT) in exon IX, resulting in a frameshift mutation, and an abnormal sequence of the 81 amino acid residues following Val³⁸¹; this mutation was the same as previously reported as PC-Nagoya. The patient’s and his father’s PC gene had a missense mutation (G to A) in exon III with a substitution of Lys for G1u²⁶; this mutation was named PC-Mie. This mutation may be responsible for the reduced immunological PC levels detected in the patient and the father, as measured by a monoclonal antibody that recognizes the Gla-domain of PC in a Ca²⁺-dependent manner (3.8% and 57%, respectively). Abnormal PC/activated PC (APC) purified from the father’s plasma showed a decreased binding ability to phospholipids, thrombomodulin, and to endothelial cell PC receptor, suggesting that Gla²⁶-dependent conformation is required for these bindings. The proband died at 3 years old. Thereafter, the parents insisted on having a healthy baby. The second pregnancy resulted in spontaneous abortion. During the third and fourth pregnancies, prenatal screening of PC abnormalities was performed. We detected the above-mentioned mutations using two methods; one was based on the development of new restriction enzyme sites using mutagenic primer and the other was the single nucleotide primer extension. In the third pregnancy, the fetus presented both paternal and maternal mutations and thus the pregnancy was terminated by artificial abortion. In the fourth pregnancy, the fetus was free from both mutations, the pregnancy continued, and the woman underwent a cesarean section. The plasma level of Gla-PC antigen in the cord of the female baby was within the normal range. Postnatal analysis of her PC gene disclosed identical results to those done in the prenatal period.