Masayoshi Yanagisawa

A point mutation in the mitochondrial tRNALeu(UUR) gene in melas (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes)

Mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episode (MELAS) is a major group of heterogeneous mitochondrial disorders. To identify the defective gene, mitochondrial DNA from a patient with MELAS was sequenced by using amplified DNA fragments as sequencing templates. In 14.1 kbp determined out of 16.6 kbp of the whole mitochondrial gene, at least 21 nucleotides were different from those of a control human mitochondrial DNA. One of the substitutions was a transition of A to G in the tRNA(Leu) (UUR) gene at Cambridge nucleotide number 3,243. This nucleotide is conserved not only in many mitochondrial tRNAs but in most cytosolic tRNA molecules. An Apa I restriction site was gained by the substitution of this nucleotide. The Apa I digestion of the amplified DNA fragment revealed that all independent 6 patients had G at nucleotide number 3,243 in their mitochondrial DNAs, but none of 11 control individuals had G at this position. This result strongly suggests that the mutation in the mitochondrial tRNALeu gene causes MELAS.

Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children

We examined mRNA expression and internal tandem duplication of the Fms‐like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and genomic PCR followed by sequencing. By RT‐PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B‐precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B‐ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia

We investigated the alterations of the p53, p21, p16, p15 and RAS genes in childhood T-cell acute lymphoblastic leukemia (T-ALL) and T-ALL cell lines by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Mutations of the p53 gene were found in three of 57 (5%) patients at diagnosis, one of 14 (7%) patients at relapse and in 12 of 18 (67%) cell lines. In these 12 cell lines, four had more than two mutations of the p53 gene. The p53 mutations were found in four of five cell lines whose original fresh leukemic cells were simultaneously examined original fresh leukemic cells. However, only one of the four fresh leukemic cells had the same mutation. All patients with p53 mutations in the course of disease died. Mutations of the p21 gene were not identified in 71 fresh samples and in 18 cell lines. N-RAS mutations were found in two of 57 (4%) fresh T-ALL patients at diagnosis, and four of 18 cell lines (22%), whereas no mutations were detected in any samples at relapse. Alterations of the p16 gene were found in 18 of 47 (38%) patients at diagnosis and in seven of 14 (50%) at relapse. These differences were not statistically significant. There were no differences in the frequency of alteration of the p16 and p15 genes between event-free patients and the remaining patients. Furthermore, we found the methylation of p16 gene in three of seven patients lacking homozygous deletions, suggesting higher frequency of p16 inactivation than previous reports in T-ALL. Interestingly, we found that one allele is inactivated by methylation and another allele had nonsense mutation in one cell line (KOPT-KI), resulting in loss of protein expression of p16. This type of p16 inactivation has not been so far reported in leukemia. We conclude that, (1) p53 mutations are infrequent at diagnosis but tend to be associated with poor clinical outcome; (2) RAS and p21 mutations may not be involved in the pathogenesis of T-ALL; (3) not only frequent alterations of p16 and p15 genes but also methylation of p16 gene are involved in initiating the leukemogenesis of T-ALLs, and (4) these 5 genes are independently involved in T-ALL.

Consistent detection of CALM-AF10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts

The t(10;11)(p13‐14;q14‐21) is a rare but recurring translocation associated with acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML). Recently the CALM gene was cloned from the t(10;11) breakpoint of U937 and fused to AF10, a putative transcription factor, which had been identified as one of the fusion partners of the MLL gene. In order to define the involvement of these genes in primary leukaemias and cell lines with t(10;11), we analysed the expression of fusion transcripts by reverse transcriptase‐polymerase chain reaction (RT‐PCR) in five patient samples including ALL, AML and lymphoblastic lymphoma, and three monocytic cell lines (P31/Fujioka, KP‐Mo‐TS and U937). The CALM‐AF10 fusion transcript was detected in all samples; however, the AF10‐CALM fusion was not detected in two patient samples and one cell line. In RT‐PCR analysis there were six isoforms of the CALM‐AF10 fusion transcripts and five of AF10‐CALM fusion transcripts. We also detected novel transcripts in U937. Sequence analysis revealed that all these isoforms had in‐frame junctions and that some of them resulted from alternative splicing at different exons of CALM and others from different breakpoints at CALM and/or AF10. There were at least two different breakpoints of CALM and three of AF10 gene. Our results suggest that the CALM‐AF10 fusion gene is a constant feature and is involved in the pathogenesis of haematological malignancies with t(10;11)(p13‐14;q14‐21), showing various and often multilineage phenotypes. Thus, t(10;11) needs to be investigated by RT‐PCR for identification of the genes involved.

Zonisamide - induced urinary lithiasis in patients with intractable epilepsy

We report here three patients with intractable epilepsy who developed urinary lithiasis during zonisamide (ZNS) treatment. Abdominal pain due to left-sided hydronephrosis was the initial symptom in the first patient, and it was resolved after the excretion of a stone. The second patient, who had no specific symptoms, was found to have a thick sludge of calcium phosphate in the bladder when he suffered from aspiration pneumonia and dehydration. The third patient, who had a history of recurrent urinary obstruction, was also found to have a thick sludge of calcium oxalate in the bladder. The urinalysis of the three patients revealed alkaline urine and hypercalciuria. Although their urinary lithiasis was resolved by discontinuation of ZNS and supportive therapy, routine examination of urine parameters such as pH and sediments, and daily urine-output checks are thought to be necessary during treatment with ZNS, especially for patients who are bedridden for a long time and receive multiple antiepileptic drugs.

Novel missense mutations in the glutamate dehydrogenase gene in the congenital hyperinsulinism-hyperammonemia syndrome

OBJECTIVES The objectives of this study were to clarify the involvement of the glutamate dehydrogenase gene in congenital hyperinsulinemia-hyperammonemia syndrome (CHHS) and the relationships between the mutation of the gene and clinical severity. STUDY DESIGN Five unrelated Japanese patients (3 girls and 2 boys) with CHHS were investigated. All patients had convulsions or loss of consciousness resulting from hypoglycemia at less than 1 year of age. We examined mutations of the glutamate dehydrogenase gene using genomic or reverse-transcriptase polymerase chain reactions, followed by direct sequencing. RESULTS We identified heterozygous missense mutations in all patients. Three patients had a previously identified mutation (C-->T at nt 1506) at codon 445 in the allosteric domain. Two novel missense mutations were identified in the other patients. These mutations included a change of A-->C at nt 1059 and a change of G-->A at nt 966, within the catalytic domain of the glutamate dehydrogenase gene. The locus of the mutations was not associated with the severity of hypoglycemia. CONCLUSIONS Our results suggest that structural aberrations of not only the allosteric domain but also the catalytic domain of the glutamate dehydrogenase protein, caused by missense mutations, can result in the development of CHHS.

Mutations of the RAS genes in childhood acute myeloid leukemia, myelodysplastic syndrome and juvenile chronic myelocytic leukemia

Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108 fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examined for mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 11q23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. All mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies.

The use of intravenous immunoglobulin in Miller Fisher syndrome

We report a patient with Miller Fisher syndrome who was treated with an intravenous high-dose of immunoglobulin. This syndrome is considered to be a benign variety of acute inflammatory demyelinating polyneuropathy (Guillain-Barré syndrome). However, there have been several reports of the need for ventilatory support and a few cases have had a fatal outcome. We observed a case of progressive Miller Fisher syndrome in a 3-year-old boy. Following 2 episodes of apnea lasting about 50 s each, he was treated with intravenous immunoglobulin (400 mg/kg/day) for 5 consecutive days. His respiratory state, general muscle strength, truncal ataxia and emotional state improved remarkably after this therapy.

Does CSF copper level in Wilson disease reflect copper accumulation in the brain

The levels of copper and ceruloplasmin in the cerebrospinal fluid (CSF) of patients with Wilson disease were investigated. Ceruloplasmin concentrations in the CSF of all patients were almost the same but were lower than those of the controls. CSF copper concentrations in patients without neurologic signs were within the normal range, 22 +/- 6 ng/ml. In contrast, CSF copper concentrations in patients with neurologic signs (69-98 ng/ml) were significantly higher than the normal levels before and at the beginning of the treatment with D-penicillamine; it gradually decreased in response to treatment. These results suggest that the appearance of neurologic manifestations in Wilson disease is not related to the CSF ceruloplasmin concentration. The CSF copper concentration in this disease appears to reflect copper accumulation in the brain and may be useful as a marker for monitoring therapy.

Changes of the expression and distribution of retinoic acid receptors during neurogenesis in mouse embryos

The expression and distribution of three retinoic acid receptors, alpha, beta, and gamma, were investigated in the CNS of mouse embryos during development. mRNAs and protein of RAR-beta that were expressed in the spinal cord of the 12.5-day mouse embryo decreased during development but they were not decreased in the brain. The RAR-beta-positive cells were already present in the ventral region of the spinal cord of 10.5-day mouse embryos, gradually appeared in the dorsal region during development and then disappeared from the spinal cord after birth. In the brain, RAR-beta-positive cells were detected in the mesencephalon and rhombencephalon but not in the telencephalon of the 12.5-day mouse embryos. RAR-beta-positive cells were present in the hippocampus and cingulum but not in the neocortex of 14.5-day mouse embryos. Most neurons in the hippocampus of 16.5-day mouse embryos and the cortex of newborn mice were RAR-beta-positive. In the spinal cord, RAR-alpha mRNAs and proteins also decreased during development but more gradually than RAR-beta mRNAs and proteins. During development, the distributions of RAR-alpha and -beta in the spinal cord and brain did not differ substantially. The main difference was the appearance of a subtypes of RAR-alpha, a 52-kDa protein, in the brain of newborn mice. On the other hand, RAR-gamma proteins were only faintly detected in the spinal cord and the brain of the mice during the embryonal stages but these increased after birth. The distribution of RAR-alpha- or -beta-positive cells were consistent with the neurogenesis during development in the spinal cord and brain.