Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HCII Tokushima

Kanagawa, Y Shigekiyo, T Aihara, K Akaike, M Azuma, H Matsumoto, T

Y. Kanagawa et al., Molecular mechanism of type I congenital heparin cofactor (HC) II deficiency caused by a missense mutation at reactive P2 site: HCII Tokushima, THROMB HAEM, 85(1), 2001, pp. 101-107

36

INGLESE

Article

Cardiovascular & Hematology Research

THROMBOSIS AND HAEMOSTASIS

0340-6245 → ACNP

85

1

2001

101 - 107

ISI

0340-6245(200101)85:1<101:MMOTIC>2.0.ZU;2-I

We found a 66-year-old Japanese patient with type I congenital heparin cofa ctor (HC) II deficiency manifesting multiple atherosclerotic lesions. To in vestigate the molecular pathogenesis of our patient, we performed sequencin g analysis and expressed recombinant human wild-type and mutant HC II molec ules in COS-1 and CHO-KI cells. Sequencing analysis following amplification of each of all 5 exons and its flanking region showed a single C to T tran sition at nucleotide position 12,854 in exon 5, which changed a Pro(443) co don (CCG) to Leu codon (CTG). Because this mutation generates a new Bbv I s ite, the Bbv I digestion pattern of the PCR-amplified exon 1 fragments from each family member was analyzed. In all cases, the patterns were consisten t with the activities and antigen levels of plasma HC II in those members. Transient transfection, metabolic labeling and purse-chase experiments foll owed by immunoprecipitation analysis showed that the recombinant mutant HC II molecules were secreted from COS-1 cells in reduced amounts compared wit h the wild-type, and that an enhanced intracellular association of the muta nt molecules with a chaperone, GRP78/BiP, was observed in CHO-KI cells, Nor thern blot analysis indicated that the mutant HC II mRNA was transcribed at a similar level as that of wild-type. Immunohistochemical staining of the transfected cells revealed that COS-I c ells expressing the mutant HC II molecules were stained mainly in the perin uclear area. We conclude that the impaired secretion of the mutant BC Il mo lecules, due to intracellular degradation, is the molecular pathogenesis of type I congenital HC II deficiency caused by a Pro(443) to Leu mutation at reactive P2 site.