Impaired secretion of the elongated mutant of protein C (protein C-Nagoya). Molecular and cellular basis for hereditary protein C deficiency.

Abstract

Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue ("'7G) among four consecutive guanine nucleotides [3Trp(TGG)-31Gly(GGT)j in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids ("1Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new Aval site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this. mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and P-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with I35Slmethionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma. (J. Clin. Invest. 1992.90:2439-2446

A case of purpura fulminans is caused by homozygous Δ8857 mutation (protein C‐Nagoya) and successfully treated with activated protein C concentrate

“…All six clones from the patient contained this mutation. The mutation was identical to that reported by our group as protein C‐Nagoya ( Yamamoto et al , 1992 ; Katsumi et al , 1996 ). The 8857 deletion causes a frameshift of the transcribed protein C gene, replacing the carboxy‐terminal 39 amino acids with 81 abnormal amino acid sequences ( Yamamoto et al , 1992 ; Katsumi et al , 1996 ) .…”

“…B. DNA analysis of the family of the patient with homozygous protein C deficiency. A 234‐bp PCR fragment was amplified from each DNA sample with the mutagenic primers, as described previously ( Yamamoto et al , 1992 ). The fragment was digested by Ava I and subjected to 2% agarose gel electrophoresis.…”

“…The far left lane is a molecular weight DNA marker with a 50‐bp ladder. Lane 1, patient; lane 2, patient's father; lane 3, patient's mother; lane 4, the propositus of the original protein C‐Nagoya family ( Yamamoto et al , 1992 ); lane 5, normal individual. Only the PCR fragment from the normal allele is digested, yielding two fragments of 207 bp and 27 bp; the 27‐bp fragment is not visible.…”

Definite diagnosis in Japanese patients with protein C deficiency by identification of causative PROC mutations

“…All mutations were previously reported in Japanese patients with protein C deficiency, except for c.439G [ A which had been already reported in 2 Dutch families [11][12][13].…”

Hereditary Protein C Deficiency: The Australian Experience

“…This could result in impaired transport across intracellular membranes by &dquo;chaperone&dquo; proteins(28,29). Indeed, several groups have performed in vitro protein translation studies with mutated protein C expression vectors and have found loss of secretion of the aberrant proteins(30,31). Presumably, in families C and H with type II deficiency there is still adequate protein secretion, but the amino acid substitution interferes with protein function.…”