Hiroko Iida

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.

Identification of simultaneous mutation of fibrinogen α chain and protein C genes in a Japanese kindred

Summary. Afibrinogenaemia usually induces a bleeding tendency during infancy, whereas protein C deficiency increases susceptibility to thrombosis in children or adolescence. Mutations of these genes have been, therefore, established as independent risk factors for coagulation disorders. We describe the homozygous mutation of the fibrinogen α chain gene and additional heterozygous mutation of the protein C gene in a male infant who showed prolonged umbilical bleeding after birth. On examination, the plasma fibrinogen was undetectable, and the activity and antigen level of protein C were reduced. The patient showed no fibrinogen Aα chain as well as Bβ and γ chains by Western blotting. The sequencing analysis showed the homozygous deletion of 1238 bases from intron 3 at position 2008 to intron 4 at position 3245 in the fibrinogen α chain gene. Both parents were heterozygous carriers of this mutation. In this patient, an additional mutation was also detected in the protein C gene: the heterozygous deletion of exon 7 at position 6161–6163 or 6164–6166, resulting the deletion of one amino acid (Lys150 or 151). His mother was also a carrier of this mutation. As the simultaneous mutation of the fibrinogen α chain and protein C genes has not been previously reported, the influence of the interaction between these two mutations on the clinical manifestations of this patient should be carefully monitored for a long period.

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.

Fluorescence derivatizing procedure for 5-hydroxytryptamine and 5-hydroxyindoleacetic acid using 1,2-diphenylethylenediamine reagent and their sensitive liquid chromatographic determination

A pre-column derivatization method using a fluorogenic reagent, 1,2-diphenylethylenediamine (DPE) was studied for the sensitive HPLC determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), which are biosubstances used in the diagnosis of several diseases. For the quantitative determination, the biogenic indole compounds were converted to their corresponding fluorescent derivatives with DPE in the presence of potassium hexacyanoferrate (III) at room temperature, and then the derivatives were separated by reversed-phase liquid chromatography with fluorescence detection. The chromatographic detection limits of the fluorescent peaks at a signal-to-noise ratio of 3 were 0.3 fmol for 5-HT and 0.2 fmol for 5-HIAA. The proposed method permits the simultaneous quantification of 5-HT and 5-HIAA at concentrations higher than 2.4 nM in human urine without a clean-up procedure.

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.

Analytical goals for coagulation tests based on biological variation

Abstract Allowable imprecision and bias reference limits for laboratory data can be calculated based on measurements of biological variation. Although biological variation of clinical chemical data has been reported from many laboratories, there have been few reports of biological variation in coagulation tests. In this study, we calculated the biological variation of 13 coagulation tests in the clinical laboratory of Kyushu University Hospital and determined allowable imprecision and bias limits of variation. The participating subjects were 17 healthy individuals: three males and two females in their 20s, two males and two females in their 30s, one male and four females in their 40s, and two males and one female in their 50s. Monthly measurements were performed before breakfast 12 times from June 2001 to May 2002 and allowable imprecision and bias limits were calculated. Taken together with coefficient of variation of control plasma used in daily laboratory work at the hospital, the allowable imprecision limits of intra-laboratory variation determined in this study appear to be in attainable ranges.

Factor XII gene (F12) –4C/C polymorphism in combination with low protein S activity is associated with deep vein thrombosis

Factor XII gene (F12) –4C/C polymorphism in combination with low protein S activity is associated with deep vein thrombosis -

Single nucleotide polymorphisms and haplotypes of protein C and protein S genes in the Thai population.

Protein C (PC) and protein S (PS) play key roles in an anticoagulant pathway in order to control the haemostatic system. We identified single nucleotide polymorphisms (SNPs) and/or haplotypes in the promotor and exons of the whole PC and PS genes and in the 3′-untranslated region of the PS gene in 55 Thai individuals. The PC gene revealed 10 haplotypes. One synonymous SNP at 2196 was found in the normal Thai population with a minor allele frequency of 4.90%. One homozygous mutation in exon 7, R147W, co-segregated with the synonymous SNP 2196 (homozygote) of the PC gene, resulting in decreased PC activity and antigenic levels. The PS gene revealed three haplotypes with two frequent dimorphisms in exon 15 and the 3′-untranslated region. The most frequent haplotype in the PS gene was H3 (wild type). There was no correlation between the haplotypes of PC and PS genes with functional and antigenic levels of PC and PS.