Michiyo Urata

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.

Paediatric presentation and outcome of congenital protein C deficiency in Japan

Severe heritable protein C (PC) deficiency is quite rare, although heterozygous PROC mutation is the second leading cause of genetic predisposition to thrombosis in Japanese adults. The aim of the study was to search the optimal management, the paediatric onset and outcomes of PC deficiency were characterized in Japan. The genetic study, postmarketing survey of activated PC (aPC) concentrate (Anact®C) and intensive review in Japan for 20 years enabled the analysis of the disease onset, genotype, treatment and prognosis. Symptomatic PC deficiency was determined in 27 Japanese children. All but two patients presented within 16 days after birth (three prenatal and six neonatal onsets). Postnatal‐onset cases had normal growth at full‐term delivery. Of the 27 patients, 19 suffered intracranial thrombosis or haemorrhage (ICTH) (three foetal hydrocephalies), 16 developed purpura fulminans (PF) and 10 had both at the first presentation. ICTH preceded PF in both affected cases. Low PC activities of 18 mothers and/or 12 fathers indicated 20 inherited PC deficiencies (2 homozygotes, 11 compound heterozygotes and 7 heterozygotes) and seven unidentified causes of PC deficiency. Nine of 11 patients studied had PROC mutations. Four unrelated patients (50%) carried PC nagoya (1362delG). No PC‐deficient parents had experienced thromboembolism. Of the 18 patients with aPC therapy, two died and eight evaluable survivors had neurological sequelae. This first comprehensive study of paediatric PC deficiency suggested that perinatal ICTH was the major presentation, occurring earlier than neonatal PF. PC nagoya was prevalent in paediatric, but not adult, patients in Japan. Early maternal screening and optimal PC therapy are required for newborns at risk of PC deficiency.

Platelet contamination causes large variation as well as overestimation of mitochondrial DNA content of peripheral blood mononuclear cells

Background Alterations in the copy number of mitochondrial DNA (mtDNA) play a role in the pathogenesis of mitochondrial diseases and other many common diseases. Recently, the copy number of leukocyte mtDNA has been considered to serve as a biomarker to monitor or chase such diseases. Therefore, reproducible mtDNA measurement is required. Methods Peripheral blood mononuclear cells were prepared by a density-based method. The mtDNA/cell was measured by quantitative realtime polymerase chain reaction. Results The degree of platelet contamination varied to a large extent among preparations. The mtDNA copy numbers per mononuclear cell were 269 ± 51 and 146 ± 14 in the samples before and after the platelet depletion, respectively. Conclusion A density-based mononuclear cell preparation causes heavy platelet contamination. The platelet depletion from a sample is particularly important for comparing the mtDNA contents between different dates or between different patients.

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.

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 -

Fulminant sepsis/meningitis due to Haemophilus influenzae in a protein C-deficient heterozygote treated with activated protein C therapy.

A 13-month-old Japanese female with Haemophilus influenzae type b meningitis presented with unusually severe septic shock and cerebral infarction in half a day of fever. The initial therapy of plasma-derived activated protein C (Anact C®) led to an impressive effect on the aggressive condition. However, purpura fulminans and the consistent decline of plasma protein C activity (<20%) required prolonged activated protein C therapy and gene analysis. The patient carried a novel heterozygous mutation of PROC (exon 4; 335 GAC>TAC, Asp46Tyr). This is the first report of infectious purpura fulminans in a protein C-deficient heterozygote. The clinical onset and treatment course adequately corroborated the aggravated immune/hemostatic reactions and the cytoprotective effects of activated protein C replacement in human heterozygous protein C deficiency. The monitoring of plasma protein C activity and sufficient administration of activated protein C product could improve the outcome of severe sepsis in children.

Successful therapy with argatroban for superior mesenteric vein thrombosis in a patient with congenital antithrombin deficiency

Abstract:  A 38‐year‐old woman was admitted with superior mesenteric vein (SMV) thrombosis, which was refractory to anticoagulation therapy. The plasma antithrombin activity was decreased and hardly compensated by concentrated antithrombin preparation due to high consumption rate. However, successful anticoagulation was achieved by administration of direct thrombin inhibitor, argatroban. Family studies of antithrombin activity revealed that she had type I congenital antithrombin deficiency. A novel heterozygous mutation in the gene for antithrombin (single nucleotide T insertion at 7916 and 7917, Glu 272 to stop in exon 4) was identified. Argatroban administration would be effective in the treatment of congenital antithrombin deficiency with SMV thrombosis.

Fetal hydrocephalus and neonatal stroke as the first presentation of protein C deficiency

Severe protein C-deficiency is a rare heritable thrombophilia of the newborn. Infants with biallelic PROC mutations present purpura fulminans and intracranial thromboembolism, while the prenatal onset of mutated heterozygotes remains unclear. We herewith present the first case of fetal ventriculomegaly and neonatal stroke associated with heterozygous PROC mutation. The infant was born to a healthy mother at 38 gestational weeks. The fetal growth had been normal, but the routine ultrasound screening had indicated mild hydrocephalus at 28 weeks of gestation. He developed convulsions two days after birth. Computed tomography of the brain revealed multiple hemorrhagic infarctions and ventriculomegaly. Dissociated levels of the plasma activity between protein C (21%) and protein S (42%) reached to determine the heterozygote of PROC c.574_576delAAG, a common thrombophilic predisposition in Asian ancestries. PC-mutant heterozygotes may have a limited high risk of cerebral thromboembolism during the perinatal course.

A novel mutation in protein C gene (PROC) causing severe phenotype in neonatal period.

Homozygous protein C deficiency is among rare causes of thrombophilia. Herein, we present a neonate with purpura fulminans, disseminated intravascular coagulation and severe intracranial hemorrhage who was found to have plasma protein C level of 4%. The molecular work‐up revealed a novel homozygous mutation of T903C (amino acid position Leu 270 Pro) located in a catalytic domain region of PROC gene. Asymptomatic course in patients with low but measurable levels of protein C levels has been reported, which is different than observed in our patient who had a very severe course despite plasma protein C level of 4%. Pediatr Blood Cancer 2014;61:763–764.