Genetic variability and diversity of intracellular genome-length hepatitis C virus RNA in long-term cell culture.

Hepatitis C virus (HCV) is known to circulate persistently in vivo as a complex population of different but closely related viral variants. To understand the quasispecies nature of HCV, we performed genetic analysis of intracellular HCV RNAs obtained in long-term cell culture of genome-length HCV-RNA-replicating cells. The results revealed that genetic mutations in HCV RNAs accumulated in a time-dependent manner, and that the mutation rates of HCV RNAs were 3.5-4.8 x 10(-3) base substitutions/site/year. The mutation rates of nonstructural regions that are essential for RNA replication were lower than those of structural regions. The genetic diversity of HCVs was also enlarged in a time-dependent manner. Furthermore, we found that the GC content of HCV RNA was increased in a time-dependent manner. These results suggest that an HCV-RNA-replicating cell culture system would be useful for analysis of the evolutionary dynamics and variations of HCV.

Molecular characterization of the ERGIC-53 gene in two Japanese patients with combined factor V-factor VIII deficiency.

Combined deficiency of factor V and factor VIII is a distinct clinical entity and is an autosomal recessive disorder. Recently identification of the gene, the endoplasmic reticulum-Golgi intermediate compartment (ERGIC-53), responsible for combined factor V-factor VIII deficiency and mutations of the ERGIC-53 gene in affected patients have been reported. In this report we analyzed two Japanese patients with combined factor V-factor VIII deficiency by genomic polymerase chain reaction and sequencing analysis. In one patient we found a point mutation of C to T at nucleotide 604 in exon 5, resulting in a transition of arginine to stop codon, which was reported in previous reports. The DdeI digestion study demonstrated that this patient is homozygous for this nonsense mutation. In the other patient we found no mutation in the ERGIC-53 gene in analysis of the entire coding region and the intron/exon junctions, which is also consistent with the previous reports, suggesting the possibility of defects at other genetic loci.

Therapy-related myelodysplastic syndrome/acute myeloid leukemia M2 and translocation (8;21).

An 80-year-old woman developed therapy-related myelodysplastic syndrome with translocation (8;21), which was successfully treated with an acute myeloid leukemia oriented chemotherapy. Five years before admission she had received cyclophosphamide, epirubicin, and carboplatin for endometrial cancer. The leukemia cell morphology alerted us to the possibility of the presence of t(8;21) before cytogenetic results were obtained, and AML1/ETO fusion transcript was detected by reverse transcription polymerase chain reaction. She achieved complete remission after one course of idarubicin and cytosine arabinoside. She has remained in complete remission for 6 months. Our experience suggests that recognition of typical morphological features for de novo M2 acute myeloid leukemia with t(8;21) would be important in diagnosis of therapy related myelodysplastic syndrome/acute myeloid leukemia with this translocation, which could respond to an intensive chemotherapy.

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 I 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, Phel21, 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.

Dominant negative isoform of the Ikaros gene in patients with adult B-cell acute lymphoblastic leukemia.

Gene targeting studies in mice have shown that the transcription factor Ikaros plays an essential role in lymphoid development and as a tumor suppressor in T cells, whereas the related gene Aiolos functions as a tumor suppressor in B cells. We analyzed the expression levels of the Ikaros gene family, Ikaros and Aiolos, in human bone marrow samples from patients with adult acute lymphoblastic leukemia [ALL (n = 46; B-cell ALL = 41; T-cell ALL = 5)]. Overexpression of the dominant negative isoform of Ikaros gene Ik-6 was observed in 14 of 41 B-cell ALL patients by reverse transcription-PCR, and the results were confirmed by sequencing analysis and immunoblotting. None of the other dominant negative isoforms of the Ikaros gene were detected by reverse transcription-PCR analysis. Southern blotting analysis with PstI digestion revealed that those patients with the dominant negative isoform Ik-6 might have small mutations in the Ikaros locus. We did not detect any overexpression of dominant negative isoforms of Aiolos in adult ALL patients. These results suggest that Ikaros plays a key role in human B-cell malignancies through the dominant negative isoform Ik-6.

Additional translocation (8;21)(q22;q22) in a patient with Philadelphia-positive chronic myelogenous leukaemia in the blastic phase.

We report a case of Philadelphia-positive chronic myelogenous leukaemia in blastic phase with the additional translocation (8;21)(q22;q22), which is frequent in acute myeloid leukaemia but not in chronic myelogenous leukaemia. The t(8;21) was not detected in the chronic phase, and was the only additional chromosomal anomaly in the blastic clone. Reverse transcription-polymerase chain reaction revealed the AML1/ETO fusion transcript in the cells of blastic phase but not in those of chronic phase. Regarding t(9;22), the breakpoint on chromosome 22 occurred in the mu-BCR region of the BCR gene, resulting in hybrid BCR/ABL mRNA with an e19a2 junction. Our findings provided molecular evidence that t(8;21) can occur as an additional genetic change in Philadelphia-positive chronic myelogenous leukaemia.

Molecular characterization of total kininogen deficiency in Japanese patients.

Kininogens are multifunctional plasma glycoproteins. There are two forms of human kininogen: low molecular weight kininogen (LK) and high molecular weight kininogen (HK). Both are derived from the same gene by alternative splicing. Some patients with kininogen deficiency have been reported to be deficient only in HK while others are deficient in both HK and LK (total kininogen deficiency). We analyzed three Japanese patients with total kininogen deficiency by the Csp45I digestion study of exon 5 as previously reported in Williams trait and found that two had the same point mutation of C to T at base 22 of exon 5, resulting in a transition of CGA (Arg) codon to TGA (Stop) codon. This is the first report of molecular characterization of total kininogen deficiency in the Japanese population.

Effects of chemical modification of arginine residues outside the active site cleft of ricin A-chain on its RNA N-glycosidase activity for ribosomes.

Ricin A-chain, a protein that inactivates ribosomes by a specific RNA N-glycosidase activity, has been shown to be inactivated by chemical modification of a few arginine residues. When two or fewer arginine residues in the A-chain were modified with [14C]phenylglyoxal, arginines at positions of 193, 196, 213, and 234/235 were found to be modified, from amino acid compositions and radioactivities of the modified peptides that were obtained by cyanogen bromide cleavage followed by tryptic and chymotryptic digestion. All these arginines have side chains outside the active site cleft; the side chain of Arg213 is adjacent to the edge of the cleft, while other modified arginines are located on the opposite side of the cleft. Kinetic analysis showed that the modification of two arginine residues caused a 8-fold loss in kcat with a 3-fold increase in Km, suggesting that this modification mainly decrease the rate of depurination with an additional effect on the affinity for ribosomes. Neither the environment of tryptophan 211 at the bottom of the cleft nor an interaction of adenine with the cleft was changed by this modification, as judged by fluorescence spectroscopy, suggesting that a conformational change of the catalytic site does not occur upon the modification. These results, taken together with other works, suggest that some of the above arginine residues outside the active site cleft may additively contribute to the catalysis of depurination and/or the initial formation of the A-chain/ribosome complex.