Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course.

BACKGROUND: Monitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients' therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. METHODS: We developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G>T, KIT 2446G>T, and KIT 2447A>T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1-RUNX1T1 fusion gene and WT1 by conventional quantitative PCR. RESULTS: The AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1-RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations. CONCLUSIONS: The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored.

[Assembly and secretion of mutant fibrinogens with variant gamma-chain C terminal region (gamma313-gamma345)].

BACKGROUND: It has been reported that the structure of the fibrinogen gamma-chain C terminal (D) region (140-411 residues) has important functions in fibrinogen assembly and/or secretion. Variant fibrinogens, gamma313S>N, gamma336M>I, gamma341A>D, and gamma345N>D have been reported as hypofibrinogenemias or dysfibrinogenemias. To study the assembly and secretion of the variant fibrinogens containing aberrant D regions, we established CHO cells producing these four fibrinogens. METHODS: A fibrinogen gamma-chain expression vector was altered and transfected into CHO cells that expressed normal human fibrinogen Aalpha and Bbeta-chains. Cell lysates and culture media of the selected cell lines were subjected to ELISA and immunoblot analysis. RESULTS: The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen, although these variant fibrinogens were barely secreted into the culture media. In the cell lysates, however, concentrations of these variant fibrinogens were higher than the normal levels. CONCLUSIONS: The present study indicated that the tertiary structure of the fibrinogen gamma-chain C terminal region between 313 and 345 is necessary for fibrinogen secretion. These findings suggest that reduced levels of fibrinogen secretion lead to the hypofibrinogen in the patients and secreted fibrinogens might show dysfibrinogenemia.

In vitro transcription of compound heterozygous hypofibrinogenemia Matsumoto IX; first identification of FGB IVS6 deletion of 4 nucleotides and FGG IVS3-2A>G causing abnormal RNA splicing.

BACKGROUND: We reported a case of hypofibrinogenemia Matsumoto IX (M IX) caused by a novel compound heterozygous mutation involving an FGB IVS6 deletion of 4 nucleotides (Delta4b) (three T, one G; between FGB IVS6-10 and -16) and FGG IVS3-2A/G, which are both identified for the first time. To examine the transcription of mRNA from the M IX gene, we cloned the wild-type and mutant genes into expression vectors. METHODS: The vectors were transfected into CHO cells and transiently produced wild-type, Bbeta- or gamma-mRNA in the cells. The mRNAs amplified with RT-PCR were analyzed by agarose gel electrophoresis and nucleotide sequencing. RESULTS: The RT-PCR product from FGB IVS6Delta4b showed aberrant mRNA that included both introns 6 and 7, and that from FGG IVS3-2G showed two aberrant mRNAs, a major one including intron 3 and a minor in which intron 3 was spliced by a cryptic splice site in exon 4. We speculated that the aberrant mRNAs are degraded before translation into proteins, and/or translated variant chains are subjected to quality control and degraded in the cytoplasm. CONCLUSION: The reduced plasma fibrinogen level of the M IX patient was caused by abnormal RNA splicing of one or both of the FGB and FGG genes.

Analysis of plasmin generation and clot lysis of plasma fibrinogen purified from a heterozygous dysfibrinogenemia, BbetaGly15Cys (Hamamatsu II).

We found a heterozygous dysfibrinogenemia caused by the substitution of BbetaGly15Cys and designated it fibrinogen Hamamatsu II (H-II). Although the propositus suffered an infarction of the medulla oblongata, other thrombotic risk factors, paradoxical cerebral infarction, and arterial dissection were not found. To determine whether the delayed lysis of fibrin clots or not in the context of the BbetaGly15Cys substitution, we examined the clot lysis and plasmin generation of propositus' fibrinogen. Fibrinogen was purified from the propositus' and normal control plasma by immunoaffinity chromatography and was used for the following experiments: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fibrin polymerization, scanning electron microscopic observation of fibrin clot and fibers, clot lysis, and tissue-type plasminogen activator-mediated plasminogen activation. The H-II plasma fibrinogen showed the presence of albumin-binding variant forms, a dimeric molecule of variant fibrinogen, and impairment of lateral aggregation during fibrin polymerization. The H-II fibrin clot showed lower density of bundles and thinner diameters of fibers than in the normal fibrin clot. In the clot lysis experiments with overlaid plasmin, H-II fibrin showed a similar lysis period and lysis rate to the normal control. Moreover, plasmin generation from a mixture of thrombin, tissue-type plasminogen activator, plasminogen, and H-II fibrinogen also showed a similar rate to normal fibrinogen. Although the propositus suffered an infarction, the present study did not observe delayed clot lysis, that is, the clot was not resistant to plasmin degradation. Therefore, we did not clarify an association between the BbetaGly15Cys dysfibrinogenemia and arterial thrombosis.

[Functional analysis of heterozygous plasma dysfibrinogens derived from two families of gammaArg275Cys and three families of gammaArg275His, and haplotype analysis for these families].

We have identified five heterozygous dysfibrinogenemias, two families with variant fibrinogen gammaArg275Cys (CGC > TGC; Matsumoto III and Sendai) and three families with gammaArg275His (CGC > CAC; Otsu II, Iida, and Shizuoka), from PCR-amplified DNA fragments and direct sequence analysis. gammaArg275 is the most important residue in fibrinogen for the so-called "D-D interface" in protofibril elongation. We compared the functions of plasma fibrinogen purified from affected family members with gammaArg275Cys and gammaArg275His. Both fibrinogens showed markedly impaired thrombin-catalyzed fibrin polymerization in comparison with normal controls. The degree of impairment of gammaArg275Cys fibrinogen was greater than that of gammaArg275His. These results were consistent with the fibrinogen concentration ratio (thrombin time method/immunological method). That is, the ratio of gammaArg275Cys was significantly lower than that of gammaArg275His. Moreover, scanning electron microscopy indicated significantly thicker fibers in fibrin clots made from gammaArg275Cys than in those of normal controls or gammaArg275His, and abnormal bundles with tapered ends. Factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chain (in the absence of thrombin) showed a similar delay for gammaArg275Cys and gammaArg275His. We report markedly impaired fibrin polymerization of gammaArg275Cys compared to gammaArg275His, and speculate that the difference is due to the disulfide-linked Cys in gammaArg275Cys, as we have already demonstrated for plasma and recombinant mutant fibrinogens. These results also indicate that an amino acid substitution of gammaArg275 disrupts D:D interactions in fibrin fiber formation. Furthermore haplotype analysis for three families with gammaArg275His suggested that founder of Iida family might be different from that of Otsu II or Shizuoka family.

[Analysis of antibody reactivity for FDP D-dimer fragments by Western blotting].

We evaluated three test kits for fibrin degradation products (FDP) D-dimer. We found that six of 217 plasma sample values obtained by Nanopia test were markedly higher than the values obtained using the other two kits. The regression equation for 211 samples (excluding six) was y=0.64x+3.05 (y: Nanopia, x: LIAS AUTO) and the correlation coefficient was 0.915. Therefore, we classified these samples into three categories, namely correlated(y< 1.0x), incompatible (y= 1.0x-2.9x) and markedly incompatible (y> or =3.0x). Selected samples, eight correlated, four incompatible and four markedly incompatible, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting(WB). WB analysis using anti-fibrinogen antibody showed that both high molecular weight fragments of cross-linked fibrin (HMW-XDP) and DD/E fragments were present in the correlated samples, but there was less HMW-XDP than DD/E in the incompatible samples and mostly DD/E (HMW-XDP was significantly less than DD/E) in the markedly incompatible samples. These data suggest that plasma FDP samples that contain mostly DD/E and little HMW-XDP demonstrated markedly incompatible values using the three D-dimer test kits. These data was reflected by markedly elevated plasmin alpha2-plasmin inhibitor complex values in the incompatible and markedly incompatible samples. Unfortunately, we did not directly demonstrate these phenomena by WB analysis with two anti-D-dimer antibodies used Nanopia or LPIA reagent. In the near future, we expect that standardization of FDP D-dimer assay will be accomplished.