Mutsuko Nakahara

Screening for Aetiology of Thrombophilia: A High Prevalence of Protein S Abnormality

We systematically screened for the aetiology of thrombophilia in 115 patients with venous, arterial and small vessel thromboses. Forty-one patients (36% of those we examined) suffering from a variety of thromboses, including deep vein thrombosis, pulmonary embolism, arterial occlusion, cerebral infarction, Moyamoya disease and ulcerative colitis, were characterized either with positive lupus anticoagulants or with decreased activities of protein S, protein C, antithrombin III and/or plasminogen. Eight mutation sites were confirmed in 11 thrombotic patients using gene analysis. Decreased protein S activity was found with a high incidence (23 out of 115) in Japanese patients who suffered from not only venous thrombosis but also arterial and small vessel thrombosis. We emphasize here the important role of protein S in the pathogenesis of thrombosis in the Japanese population.

Four missense mutations identified in the protein S gene of thrombosis patients with protein S deficiency: Effects on secretion and anticoagulant activity of protein S

Four missense mutations, G54R, T589I, K155E, and Y595C, were identified in the protein S (PS) gene of the patients with PS deficiency and venous thrombosis. Three patients were heterozygous for the novel mutations, G54R, T589I, and Y595C, while a remaining one patient was homozygous for the K155E mutation, which is known to be a polymorphism in the Japanese population. A family study revealed that the Y595C mutation was associated with a Type I PS deficiency and the K155E mutation with a Type II PS deficiency, while no family study was performed for the patients with the G54R and T589I mutations. To determine whether these four mutations play a causative role in PS deficiency, the four PS mutants and wild-type PS were stably expressed in human embryo kidney (HEK) 293 cells. Pulse-chase experiments showed intracellular degradation and decreased secretion of the Y595C mutant. In the activated protein C (APC) cofactor assays, the specific activity of the K155E mutant decreased to 58% of that of wild-type PS. The APC cofactor activity of the three mutants, G54R, K155E, and T589I, were inhibited by C4b-binding protein (C4BP) with a dose dependency similar to that of wild-type PS. These results indicate that the Y595C and the K155E mutations are responsible for a secretion defect and a decreased anticoagulant activity of PS, respectively. The remaining two mutations, G54R and T589I, however, did not produce any definite abnormality leading to a low plasma PS activity.

Standardization of Laboratory Data and Establishment of Reference Intervals in the Fukuoka Prefecture: A Japanese Perspective

Abstract Standardization of 22 clinical chemistry analytes and five serum protein constituents has been performed in the Fukuoka Prefecture, which has a population of approximately five million. The standardization project was established to determine reference intervals for these analytes by educating physicians, medical technologists and staff of medical institutions, and by daily or monthly monitoring the use of common control samples through e-mail. Standardization extended to 97％ of the institutions in the prefecture. Results for 14 of the 22 clinical chemistry analytes have become highly reliable and differences between institutions decreased. Standardization of other analytes is now in progress. Regional collaboration based on international guidelines led to a significant improvement in interlaboratory comparability. Areas where further improvements are needed have been identified.

A Novel Splice Acceptor Site Mutation of Protein S Gene in Affected Individuals with Type I Protein S Deficiency: Allelic Exclusion of the Mutant Gene

Sequencing studies of the protein S gene (PROS1) in a Japanese patient suffering from recurrent thrombosis revealed the following. The proband and his first daughter, but not the second daughter, were having the type I protein S (PS) deficiency due to a novel point mutation from A to G at the intronic acceptor splice site in intron 13 of the PROS1. In the affected daughter, exclusion of the aberrant allele was assessed by the BstX1 dimorphism of PROS1 at Pro626 (CCG/CCA). The reduced PS activities in the proband and his first daughter were apparently due to defective production of mRNA from the mutant allele.

Failure in the detection of aberrant mRNA from the heterozygotic splice site mutant allele for protein S in a patient with protein S deficiency.

A 29-year-old male patient with acute arterial obstruction and a medical history including thrombosis in the deep veins and pulmonary infarction presented with a reduced level of both protein S (PS) activity and free PS. Sequencing of the genomic PS gene in this patient revealed that the patient was heterozygous for the mutant PS allele, in which a nucleotide substitution occurred at the donor splice site in intron 12 (GT to GA). The patient was heterozygous for PS genes having dimorphic codons for Pro626 (CCA/CCG) and the aberrant allele in this patient was associated with the CCA form. Allelic exclusion of PS expression was demonstrated by use of Pro626 (CCA/CCG) dimorphism and only a normal mRNA sequence derived from the CCG-allele was identified in the patient. These findings suggested that the mutation at the splice site in the PS gene caused either defective production of mRNA or the gene may have produced extremely unstable RNA products, leading to reduced levels of PS activity and free PS in this patient.