Kenji Iijima

Carbohydrate-deficient glycoprotein syndrome: electrophoretic study of multiple serum glycoproteins

The serum glycoproteins in three patients with the carbohydrate-deficient glycoprotein (CDG) syndrome were examined using isoelectric focusing in polyacrylamide gels, SDS-PAGE, and a two-dimensional electrophoretic technique. Increases in isoforms with higher isoelectric points (pIs) were observed in all asparagine-N-linked glycoproteins tested. Abnormal components of transferrin, antithrombin III and orosomucoid had smaller molecular weights, about 3000 and 6000 Da, than normal ones. Antithrombin III gave an increased cathodal band. Its pI and molecular weight were the same as those of a physiological variant of antithrombin III (beta fraction), which is known to lack the asparagine-N-linked oligosaccharide chain at asparagine-135. The evidence suggests that a deficiency of asparagine-N-linked oligosaccharide transfer occurs in multiple serum glycoproteins in patients with CDG syndrome. Thus, examination of serum by isoelectric focusing in polyacrylamide gels and SDS-PAGE should be very useful for differentiating the CDG syndrome type I from clinical and biochemical variants.

Hemostatic studies in patients with carbohydrate-deficient glycoprotein syndrome

The carbohydrate deficient glycoprotein (CDG) syndrome is a newly described disorder characterized by impaired glycosylated molecules. It has been reported that transient stroke-like episodes appear in half of the patients. We performed hemostatic studies on three CDG syndrome patients belonging to two unrelated families. The most characteristic findings were decreases in antithrombin III (AT III), protein C and alpha 2 plasmin inhibitor to nearly half normal levels. Protein S was reduced in two (siblings) patients. Isoelectric focusing of AT III in native plasma revealed decreased intensity of the major band and increased intensity of a minor cathodal band. These minor AT III molecules were considered to lack an oligosaccharide sidechain. A 12-year-old girl defective not only for AT III but also protein C and protein S developed disseminated intravascular coagulation accompanied by arterial thrombosis in her left hand following dyspnea associated with bronchial asthma. These findings suggest that thrombotic predisposition in patients with CDG syndrome is due to decreased levels of major coagulation inhibitors, particularly as a result of impaired glycosylation of AT III.

A homozygous protein C deficiency (Lys 192 del) who developed venous thrombosis for the first time at adulthood.

Protein C is the zymogen of a vitamin K-dependent serine protease, which plays an important role in the regulation of the blood coagulation pathways [1,2]. Protein C is physiologically activated by thrombin binding to its membrane cofactor protein thrombomodulin and protein C receptor at the endothelial cell surface. ActivatedproteinCproteolytically degrades factors Va and VIIIa in the presence of protein S [3]. Individuals with mutation in the protein C gene tend to have an increased risk of thromboembolism. A considerable number of heterozygous mutants have been reported in patients who develop deep vein thrombosis in the lower extremities or fatal pulmonary embolism during adulthood [4]. Homozygous or compound heterozygous mutant in patients who develop purpura fulminans in the neonatal period is rare [5,6]. We report here a patient with homozygous protein C deficiency (PC Tottori), who developed venous thrombosis for the first time at the age of 23. Genetic analysis demonstrated a 3 nucleotide deletion (AAG) that caused one amino acid deletion (Lys192 or Lys193) at the C-terminal region of the light chain. We described the methionine encoded by the initiation codon as number 1 in amino acid alignment. The proband was a 23-year-old Japaneseman. He developed swelling and tenderness of the left leg the morning after he had slept in a car after drinking. Ultrasonography confirmed to the presence of thrombus in the left femoral vein. His parents were asymptomatic and there was no consanguinitywithinhis knownancestry. Blood sampleswere taken from the proband and his parents after informed consent was obtained. This study was approved by the Ethical Committee of the Faculty of Medicine, Tottori University. Anticoagulant activity of protein C was measured by prolongation of activated partial thromboplastin time using STA Staclot protein C (Diagnostica Stago,Asnières, France). In thismethod,proteinC is activated by snake venom (ProtacTM) and then activated protein C proteolytically degrades the physiological substrate, factors Va and VIIIa, in the presence of calcium ions, phospholipids and protein S. Amidolytic activity was measured by amidolysis of synthesized chromogenic substrate using STA StachromproteinC(Diagnostica Stago,Asnières, France), inwhichprotein C is activated by ProtacTM and then activated protein C hydrolyzes the small synthetic substrate. The total protein C antigen level and normal carboxylated protein C antigen level pretreated with barium citrate absorption andMes elution [7] weremeasured by the ELISA system using polyclonal antibody (Diagnostica Stago, Asnières, France). Barium citrate does not absorb the protein C molecule with dysfunctional Gla domain, resulting in a reduction of the recovery rate of protein C level in Mes elution. Genomic DNAwas prepared from peripheral blood leukocytes. The protein C gene was amplified using polymerase chain reaction (PCR) with oligonucleotide primers designed to cover all exons and intronexon junctions of the protein C gene [6]. Cycle sequencing was

A factor XI deficiency associated with a nonsense mutation (Trp501stop) in the catalytic domain.

We identified a novel mutation in an asymptomatic 65‐year‐old Japanese man with severe factor XI deficiency. Sequence analysis after polymerase chain reaction single‐stranded conformation polymorphism (PCR‐SSCP) analysis of his factor XI gene revealed a G→A transition in codon 501 of exon 13, resulting in a substitution of Trp501 (TGG) by a stop codon (TAG) in the catalytic domain. This mutation abolished a FokI restriction site. The PCR product from normal subjects was digested with FokI and yielded two fragments, one of 223 bp and one of 47 bp. The PCR product from the patient gave a single 270‐bp fragment, demonstrating possible homozygosity.

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

We identified 3 mutations in the factor V (FV) gene(F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.

A new hereditary abnormal protein C (protein C Yonago) with a dysfunctional Gla-domain

A familial abnormal protein C most probably with the dysfunctional Gla domain was found in a 60-year-old man with recurrent thrombosis. Namely, the anticoagulant activity as measured by the APTT method and the antigen level by an ELISA utilizing a calcium-dependent antibody were reduced to nearly half of normal, 43.5% and 2.1 micrograms/ml (normal range: 2.8-5.0 micrograms/ml), respectively. On the other hand, the amidolytic activity determined on a synthetic chromogenic substrate, S-2366, and the total antigen measured by an ELISA utilizing a polyclonal antibody were both in the normal range, 74.1% and 83% of normal, respectively. Crossed immunoelectrophoresis showed more anodal migration than the normal control in the presence of calcium ions, and adsorption of protein C to barium citrate was insufficient. These data altogether indicated that a half population of protein C in the patients plasma was dysfunctional in the Gla domain or its related structures. Four other members of his immediate family were found to have the same abnormality of protein C, although they had been all asymptomatic. We thus conclude that the dysfunctional protein C is hereditary, and that the abnormalities noted in several tests are most likely due to a structural defect residing in the Gla or its related regions. We hereby designate this abnormal protein C as protein C Yonago.

A case of atypical hemolytic uremic syndrome due to anti-factor H antibody in a patient presenting with a factor XII deficiency identified two novel mutations.

A 9-year-old boy with pallor and macrohematuria showed hemolytic anemia, thrombocytopenia and renal failure. There was no history of diarrhea and the stool culture was negative. A diagnosis of atypical hemolytic uremic syndrome (HUS) was confirmed; however, the cause of the prolonged activated partial thromboplastin time (APTT) was unknown. Plasma exchange and hemodialysis were performed because of progressive hemolytic anemia and renal dysfunction. Fresh frozen plasma was administered frequently to correct the prolonged APTT after hemolysis was controlled and C3 levels had recovered. Factor H (FH) and factor I (IF) levels were normal and we did not detect mutations of FH, IF and membrane cofactor protein. Further investigation revealed the presence of anti-FH antibody in the patient’s plasma and a deficiency of coagulation factor XII. Analysis of the patient’s coagulation system displayed <3% functional activity of factor XII, whereas levels of other coagulation factors were within the normal range. Two novel mutations (W222G and R447S) were identified upon analysis of the factor XII gene in this patient. Moreover, further investigation revealed that compound heterozygous mutations were present in two of the patient’s three siblings, while the third sibling only had a mutation at W222G. The patient was treated for atypical HUS; however, no treatment was required for factor XII deficiency as he did not display a hemorrhagic tendency. We report here a rare case of atypical HUS due to anti-FH antibody presenting with a coagulation factor XII deficiency.

Reduction in protein S activity during normal pregnancy

We investigated the serial changes in blood protein S (PS) and related proteins in 11 normal pregnant women. The PS activity decreased significantly in the third trimester and reached minimum levels (23.3%) one hour after delivery. Although the PS activity was reduced markedly below the normal limits, all the women delivered safely. The mechanisms that cause the reduction in PS activity and the clinically dangerous conditions involving PS activity during pregnancy warrant further investigation.

Two Novel Gene Mutations in Type I Antithrombin Deficiency

We studied the molecular basis of type I antithrombin (AT) deficiency in 2 Japanese families, in which affected persons had histories of recurrent venous thrombosis and low (about 50% of normal) levels of AT protein according to measurements by both functional and antigen assays. Southern blotting of DNA isolated from peripheral leukocytes revealed no abnormalities in all the cases examined. Direct sequencing of the polymerase chain reaction (PCR) products from case 1 suggested a novel heterozygous nonsense mutation in exon 4 (GAG→TAG at nucleotide position 7627, leading to Glu306 stop). The sequencing of the subclones of the patient’s exon 4 products confirmed the nonsense mutation. No other sequence abnormalities were detected in the rest of the PCR products.The same mutation was detected in this patient’s brother, who had a history of recurrent venous thrombosis and a reduced level of AT activity. In case 2, the direct sequencing of PCR products suggested a novel heterozygous 9-bp deletion in exon 3a (—CACTTC at nucleotide position 5354-5362, leading to the deletion of 3 amino acids, His120, Phe121, and Phe122). The 9-bp deletion mutation in the region of a unique quasi palindrome was confirmed by sequencing several of the subclones of the patient’s exon 3a from the PCR products. No other mutations were found by direct sequencing of the rest of the coding regions. The 2 mutations found in this study are novel. The use of PCR and the sequencing of the PCR product subclones has simplified and confirmed the detection and characterization of the various AT mutations.

Molecular characterization of four alpha-1-antitrypsin variant alleles found in a Japanese population: a mutation hot spot at the codon for amino acid 362

In this study alpha-1-antitrypsin (AAT) phenotypes at the protease inhibitor (PI) locus were determined by isoelectric focusing of native and desialylated serum samples from 236 Japanese subjects living in the western part of Japan. The shifts in relative mobility between some PI types were observed before and after desialylation. This technique was useful in distinguishing between some PI M subtypes and variants. The molecular basis of four variant alleles, including two new alleles found in this study, was characterized: PI E(tokyo) [Lys(335)(AAG)--> Glu(GAG)] and PI N(nagato) [Leu(276)(CTG)-->Pro(CCG)] arose from PI M1(Val(213)) and PI M2, respectively. A new PI P(yonago) [Asp(19)(GAT)-->Ala(GCT)] originated from PI M1(Val(213)). A new PI M5(gunma) [Pro(362)(CCC)-->Ser(TCC)], arising from PI M3, was the sixth allele involving a mutation at codon 362, which is suggested to be a mutation hot spot. PI M5(gunma) was likely to show normal AAT levels and function although the mutations occurred near codon 358 for Met(358). The molecular basis of PI variant alleles found in Japanese was different from that reported in previous studies.