ECTOPIC TRANSCRIPT ANALYSIS INDICATES THAT ALLELIC EXCLUSION IS AN IMPORTANT CAUSE OF TYPE-I PROTEIN-C DEFICIENCY IN PATIENTS WITH NONSENSEAND FRAMESHIFT MUTATIONS IN THE PROC GENE

Nonsense mutations, deletions and splice site mutations are a common c ause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these lesions generate or may generate pre mature stop codons and could therefore be expected to result in premat ure termination of translation. In this study, the possibility that su ch mutations could instead exert their pathological effects at an earl ier stage in the expression pathway, through ''allelic exclusion'' at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for tw o nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymp hocytes were analysed by RT-PCR and direct sequencing of amplified PRO C cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allel e had been expressed. Similarly for the splice site mutations, only no rmal PROC cDNAs were obtained. In one case, exclusion of the mutated a llele could be confirmed by polymorphism analysis. In contrast to thes e six mutations, the 2bp insertion was not associated with loss of mRN A from the mutated allele. In this case, cDNA analysis revealed the ab sence of 19 bases from the PROC mRNA consistent with the generation an d utilization of a cryptic splice site 3' to the site of mutation, whi ch would result in a frameshift and a premature stop codon. It is conc luded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons.