p.E66Q mutation in the GLA gene is associated with a high risk of cerebral small-vessel occlusion in elderly Japanese males.

BACKGROUND AND PURPOSE: GLA is the causative gene of Fabry disease, an X-linked lysosomal storage disorder resulting from α-galactosidase A (α-GAL) deficiency. Stroke is an important manifestation of Fabry disease, and recent epidemiological studies have indicated that up to 4.9% of young male cryptogenic stroke patients have GLA mutations. To determine the importance of GLA mutations in the general stroke population, the frequency of GLA mutations in Japanese male ischaemic stroke (IS) patients with various risk factors and ages was measured. METHODS: A total of 475 male IS patients (mean age 69.7 ± 12.5 years), were enrolled in this study. A blood sample was obtained to produce blood spots for measurement of α-GAL activity. Blood samples with decreased enzymatic activity were reassayed and the entire GLA gene was analyzed by direct DNA sequencing if α-Gal A activity was consistently low. RESULTS: α-Gal A activity was decreased in 10 men, five of whom (1.1%) had the GLA gene mutation, p.E66Q. All IS patients with p.E66Q mutation had substantial residual α-Gal A activity, in contrast to patients with classic-type Fabry disease. Clinically, all patients with p.E66Q mutation were > 50 years old and had multiple small-vessel occlusions (lacunar infarctions). Statistical analysis using Fisher's exact test showed the allele frequency of GLA p.E66Q in patients with small-vessel occlusion to be significantly higher than that in the general Japanese population [odds ratio (OR) = 3.34, P = 0.025). CONCLUSIONS: GLA p.E66Q mutation is a genetic risk factor for cerebral small-vessel occlusion in elderly Japanese males.

Expression of CD64 on polymorphonuclear neutrophils in patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is an autoinflammatory disease characterized by recurrent episodes of fever and serosal or synovial inflammation. We examined the utility of CD64 (FcγRI) expression in polymorphonuclear neutrophils (PMNs) as clinical and biological parameters in patients with FMF. We studied 12 Japanese FMF patients (mean age; 22·8 ± 15·5 years, male/female: 2/10), along with rheumatoid arthritis patients (RA, n = 38 male/female: 6/32, mean age; 52·2 ± 15·3 years), systemic lupus erythematosus (SLE, n = 15 male/female: 0/15, mean age; 38·5 ± 15·9 years) and 12 healthy subjects (male/female: 3/9, mean age; 37·9 ± 17·2 years). CD64 expression on PMNs was determined using flow cytometry. The quantitative expression of CD64 in patients with FMF (2439·6 ± 2215·8 molecules per PMN) was significantly higher than in healthy subjects (547·8 ± 229·5, P = 0·003) or in patients with RA (606·5 ± 228·2, P < 0·0001) and SLE (681·3 ± 281·1, P = 0·004). The increased CD64 expression on PMNs isolated from untreated FMF patients was down-regulated by colchicine treatment. NACHT-LRR-PYD-containing protein 3 (NLRP3) activation using MurNAc-L-Ala-D-isoGln (MDP) resulted in increased CD64 expression on PMNs from healthy subjects. Our results suggest that quantitative measurement of CD64 expression on PMNs can be a valuable tool to discriminate between FMF and autoimmune diseases.

Hypothermia in VGKC antibody-associated limbic encephalitis.

Voltage-gated potassium channel antibody (VGKC-Ab)-associated limbic encephalitis (LE) is a recently described syndrome that broadens the spectrum of immunotherapy-responsive central nervous system disorders. Limbic encephalitis is typically characterised by a sub-acute onset of disorientation, amnesia and seizures, but the clinical spectrum is not yet fully defined and the syndrome could be under-diagnosed. We here describe the clinical profile of four patients with VGKC-Ab-associated LE who had intermittent, episodic hypothermia. One of the patients also described a prodrome of severe neuropathic pain preceding the development of limbic symptoms. Both of these novel symptoms responded well to immunosuppressive therapy, with concurrent amelioration of amnesia/seizures.

An effective chemotherapeutic regimen for acute myeloid leukemia and myelodysplastic syndrome in children with Down's syndrome.

In recent pediatric collaborative studies of acute myeloid leukemia (AML), patients with Down's syndrome (DS) have better outcome than other patients when they were treated according to their intensive AML protocols. This may be attributed to enhanced sensitivity of DS AML cells to selected chemotherapeutic agents. We evaluated a less intensive chemotherapeutic regimen which was specifically designed for children with AML-DS. Remission induction chemotherapy consisted of daunorubicin (25 mg/m2/day for 2 days), cytosine arabinoside (100 mg/m2/day for 7 days), and etoposide (150 mg/m2/day for 3 days). Patients received one to seven courses of consolidation therapy of the same regimen. Thirty-three patients were enrolled on the study and their clinical, hematologic and immunophenotypic features were analyzed. Of the 33 patients, all were younger than 4 years and diagnosed as having acute megakaryoblastic leukemia or myelodysplastic syndrome. All patients achieved a complete remission and estimated 8 year event-free survival rate was 80+/-7%. Three patients relapsed and two died due to cardiac toxicity and one due to septic shock. The results of our study showed that patients with AML-DS constitute a unique biologic subgroup and should be treated according to a less intensive protocol designed for AML-DS.

Neuroserpin mutation S52R causes neuroserpin accumulation in neurons and is associated with progressive myoclonus epilepsy.

Mutations in the Neuroserpin gene have been reported to cause familial presenile dementia. We describe a new family in which the S52R Neuroserpin mutation is associated with progressive myoclonus epilepsy in 2 siblings. The proband presented myoclonus and epilepsy at age 24, his brother and mother presented a similar disorder when they were 25. A clinical diagnosis of progressive myoclonus epilepsy was made on the proband and his brother. Skin and liver biopsies did not reveal the presence of cytological alterations in the proband. His neurological status worsened over the subsequent 19 yr during which he became demented and had uncontrollable seizures. He died at 43 yr of age from aspiration pneumonia. Neuropathologically, eosinophilic bodies, which were positive for periodic acid-Schiff and immunoreactive with antibodies against human neuroserpin, were present in the perikarya and cell processes of the neurons. They were found in large numbers in the cerebral cortex and substantia nigra and to a lesser extent, in most subcortical gray areas, spinal cord, and dorsal root ganglia. By electron microscopy, the intracytoplasmic bodies were contained within the membranes of the rough endoplasmic reticulum. Occasionally neuroserpin immunopositivity was seen throughout the cytoplasm, even without the presence of well-defined bodies. Our study characterizes for the first time the neuropathologic phenotype associated with hereditary progressive myoclonus epilepsy caused by the S52R Neuroserpin mutation.

Complete neurological recovery of an adult patient with type II citrullinemia after living related partial liver transplantation.

Type II citrullinemia is an adult-onset hepatocerebral disease caused by a deficiency of argininosuccinate synthetase in liver. A 25-year-old Japanese man suddenly developed encephalopathy, showing disorientation and flapping tremor. Plasma concentrations of ammonia and citrulline were extremely high, and hepatic argininosuccinate synthetase activity was deficient. The patient's condition deteriorated rapidly in spite of intensive medications. Therefore, we performed a partial liver transplantation using a graft obtained from his healthy 61-year-old father. After surgery, his neurological symptoms soon disappeared and plasma levels of ammonia and citrulline were normalized within 3 months after operation. Type II citrullinemia is one fulminant form of various liver-based metabolic diseases, and immediate liver transplantation is necessary to rescue patients with this disease. As liver transplantation from cadaveric donor is still not possible in Japan, it seems justifiable to use living related partial liver transplantation for our patient.

Detection of bcr/abl fusion transcripts by semiquantitative multiplex RT-PCR combined with a colormetric assay in Ph positive leukemia.

We studied the feasibility of the clinical application of a new bcr/abl analysis system, C-TRAK t(9;22), consisting of a multiplex RT-PCR and a colormetric assay. With this system, bcr/abl transcripts could be detected in all of 24 cytogenetic Philadelphia chromosome (Ph) positive leukemia patients and in none of eight Ph negative patients. Multiple bcr/abl transcripts could be detected in three of the 24 Ph positive patients, the fusion of bcr exon 1 to abl exon 2 (e1a2 junction) dominated that of bcr exon 13 to abl exon 2 (b2a2 junction) in two cases and that of bcr exon 14 to abl exon 2 (b3a2 junction) and b2a2 dominated e1a2 in one case. This system was sensitive enough to be able to detect even one bcr/abl transcript-producing cell in 50000 bcr/abl negative background cells, thus making it suitable for semiquantitative evaluation. Minimal residual disease (MRD) was monitored in one Ph positive leukemia patient who underwent allogenic bone marrow transplantation (allo-BMT). After allo-BMT, a weak positivity of the bcr/abl transcript continued with no clinical relapse; this result was consistent with that of a conventional nested PCR assay using ethidium bromide staining. Including all the procedures for RNA extraction, it took only about 10 h to detect the bcr/abl transcripts. Our findings indicate that this bcr/abl analysis system provides a quick and sensitive method for screening bcr/abl transcripts and possibly for monitoring MRD in Ph positive leukemia patients.

Successful prevention of hematological relapse for a patient with Philadelphia chromosome-positive acute lymphoblastic leukemia after allogeneic bone marrow transplantation by donor leukocyte infusion.

Donor leukocyte infusion (DLI) was carried out on a 12-year-old girl with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) who received allogeneic bone marrow transplantation from an HLA-identical sibling. This is the first report of DLI use before the onset of hematological relapse monitored by the results of RT-PCR. This patient has been in CR for 11 months after BMT, suggesting this alternative treatment is promising for Ph+ ALL with positive reverse transcriptase-polymerase chain reaction (RT-PCR) following BMT.

Generation of HLA-A2 subtype specific cytotoxic T lymphocytes from cord blood used for cord blood stem cell transplantation.

Alloantigen reactive cytotoxic T lymphocytes (CTL) were generated from cord blood (CB) lymphocytes used for cord blood stem cell transplantation (CBSCT). The CTL were cytotoxic against the patient's leukemic cells, as well as the patient's EBV-lymphoblastoid cell line (EBV-LCL), and PHA blasts. The cytotoxicity against patient's EBV-LCL was blocked by anti-HLA-A2 MoAb, and anti-HLA-class I MoAb. The CTL recognized A*0206 positive EBV-LCLs, but not A*0201, A*0204, or A*0207 positive EBV-LCLs, suggesting that this CTL recognizes HLA-A*0206. This case suggests that CB T cells may be competent enough to generate CTL to induce a GVL effect, together with those against A*0206, in patients with CBSCT.

Detection of donor lymphocytes in the cerebrospinal fluid of a patient with metachromatic leukodystrophy following bone marrow transplantation.

An unrelated donor bone marrow transplant (UD-BMT) was carried out on a 10-year-old patient with metachromatic leukodystrophy (MLD). We collected cerebrospinal fluid (CSF) on day +168 and cultured it with recombinant IL-2 and PHA-P to examine the origin of cells in the CSF. Analysis on variable number of tandem repeat (VNTR) of lymphocytes in the CSF amplified by PCR revealed that lymphocytes in the CSF were of donor origin. These data support that BMT at an early stage may prevent deterioration in MLD. Although the patient developed grade III acute GVHD with rash and diarrhea, we successfully treated acute GVHD using rabbit anti-human thymocyte immunoglobulin (ATG). UD-BMT may be an alternative treatment for patients with MLD in the absence of an HLA matched family donor.

A novel minor histocompatibility antigen recognized by HLA-A31 restricted cytotoxic T lymphocytes generated from HLA-identical bone marrow donor lymphocytes.

Bulk cytotoxic T lymphocytes (CTL) were generated by in vitro stimulation of BMT donor lymphocytes with Philadelphia chromosome (Ph)-positive leukemic cells from an HLA-identical sibling patient. CTL were cytotoxic against the patient's leukemic cells as well as the EBV-lymphoblastoid cell line (EBV-LCL) generated from the patient's cells, suggesting that they recognize a minor histocompatibility antigen (mHAg). Subsequently, several CTL lines were established by a limiting dilution method and analyzed. One of these CTL lines, 16C12 CTL which used a single TCRbetaV3S1 for CD8 cells, lysed HLA-A31-positive leukemic cells and EBV-LCL, but not fibroblasts. The cytotoxicity against the patient's leukemic cells and EBV-LCL was blocked by anti-HLA-A31 moAb, anti-HLA-class I moAb, and anti-CD8 moAb, suggesting that this mHAg was presented with HLA-A31. The antigen recognized by 16C12 CTL seemed to be a novel mHAg, since HLA-A31 restricted antigen has not been reported to date and 16C12 CTL showed no cytotoxicity against EBV-LCL which probably express known mHAgs. CTL detecting this mHAg may play an important role in the GVL effect in HLA-A31-positive BMT patients.

Correlation of regional cerebral blood flow with performance on neuropsychological tests in schizophrenic patients.

Regional cerebral blood flow (rCBF) was determined by the 133Xe inhalation technique (Headtome II: ring detection SPECT) in 53 DSM-III schizophrenic patients. The rCBF values were corrected by using end-tidal carbon dioxide concentration values (PECO2). After rCBF measurement, neuropsychological tests--Word Fluency Test, Maze Test and Wisconsin Card Sorting Test--were performed. There were significant correlations between frontal rCBF and scores on each neuropsychological test. In particular, a moderate correlations between the frontal rCBF and the performance on the Wisconsin Card Sorting Test was noted. It seems likely that decrease of rCBF in prefrontal regions at rest reflects a disturbance of frontal lobe function in schizophrenic patients.

Nodular cutaneous amyloidosis and carpal tunnel syndrome due to the amyloidogenic transthyretin His 114 variant.

This is the second report of transthyretin (TTR) amyloidosis in a patient who had ATTR Tyr114His diagnosed by mass spectrometry and gene analysis. This case had some clinical features that differed from those of the first reported cases. The patient, 73-year-old man, complained of generalized cutaneous tubercula that had started at age 68. These tubercula gradually increased in size and became generalized. He felt a slight numbness in his extremities. Clinical and electrophysiological examinations revealed that he had bilateral carpal tunnel syndrome (CTS), whereas there was no clear evidence of sensory and/or motor polyneuropathy. Autonomic symptoms were not present. Biopsy studies revealed that both his tuberculum and his sural nerve contained TTR-related amyloid. In his sural nerve, amyloid deposits were observed mainly in the perineurium, not in the endoneurium, and there was no significant depletion of myelinated fibers. The features of this patient were clinically characterized by generalized cutaneous amyloid deposits and late-onset CTS with a lack of overt polyneuropathy and autonomic dysfunction. The unique clinical features in this case seemed to be consistent with the distribution of amyloid deposits.

Biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia.

The purpose of this study was to investigate the biological characteristics and prognostic value of in vitro three-drug resistance to prednisolone, L-asparaginase, and vincristine in childhood acute lymphoblastic leukemia (ALL). We carried out in vitro tests with a 4-day culture and a methyl-thiazol-tetrazolium assay on bone marrow samples from 209 children newly diagnosed with ALL. After testing the resistance of leukemic cells to 14 drugs, we classified the patients into two groups according to their sensitivity to three drugs (prednisolone, L-asparaginase, and vincristine) used in remission induction therapy. The three-drug resistant group (RR: sensitive to no drugs or to one drug) correlated with both short-term and long-term treatment failure. Three-year event-free survival (95% confidence interval) for the sensitive group (SS: sensitive to two or three drugs) was 0.813 (0.773-0.853) and that of the RR group was 0.616 (0.569-0.669) (P = 0.0001). Univariate analysis showed that Philadelphia-chromosome (Ph1) positivity and immunophenotype of mixed lineage were also prognostic factors in the 209 patients. The prognosis of the SS/RR drug resistance profile within 14 Ph1 patients was marginally significant (P = 0.062). Multivariate Cox regression analysis showed that Ph1 was an overwhelmingly adverse factor in event-free survival, with a relative hazard of 5.37 (2.57-11.21, P < 0.0001), followed by RR, with a relative hazard of 2.98 (1.69-5.25, P = 0.0001). Furthermore, we clarified the characteristics of the RR group by examination of the pattern of drug resistance to other drugs in comparison with the SS group. The leukemic cells of RR patients were more resistant than those of SS patients (P < 0.0001) to all the drugs tested, with resistance ratios of 1.6 to 13.1 (mean 3.4). In conclusion, in vitro three-drug resistance at the initial stage is an important independent predictor of treatment failure for both induction response and long-term outcome in childhood ALL.

Distinctive multidrug sensitivity and outcome of acute erythroblastic and megakaryoblastic leukemia in children with Down syndrome.

We assessed the in vitro chemosensitivity of acute erythroblastic and megakaryoblastic leukemia cells from children with Down syndrome (DS) compared to non-DS children. We conducted in vitro tests using the MTT assay of bone marrow samples from 12 children with DS and 16 children without DS. Patients were newly diagnosed based on the morphology and expression of platelet-specific antigens. Induction failure occurred more frequently in the non-DS group (n = 4) than in the DS group (n = 0, P = .053). Children with DS had a superior event-free survival (EFS) probability of 0.750 at 4 years, compared to an EFS probability of 0.375 for non-DS children (P = .049). Blast cells from DS patients were significantly more sensitive to daunorubicin, melphalan, mitoxantrone, 4-hydroperoxy-cyclophosphamide, vincristine, etoposide, bleomycin, and pirarubicin than those from non-DS patients. Four of the 16 non-DS patients were found to have acquired an extra chromosome 21 in their leukemia cells: blasts from these patients also tended to have greater chemosensitivity than those from patients without an extra chromosome 21. Blast cells from DS patients are markedly sensitive to various drugs. These results suggest that the fragility of blast cells derived from DS patients may be related to an increased susceptibility to apoptosis.

Mutation of potential N-linked glycosylation sites in the Alzheimer's disease amyloid precursor protein (APP).

In order to study the mechanism of intracellular sorting and processing of the Alzheimer's disease amyloid precursor protein (APP), we deleted two potential N-linked glycosylation sites of APP by site-directed mutagenesis. Substitution of alanines for the critical asparagine residues Asn467 and Asn496 was performed. Wild-type and mutant APPs were expressed in COS-1 cells by cDNA transfection and the expressed of the protein and secretion of N-terminal large fragment was observed. The initial secretion of the mutant APP appeared to be slow compared with wild-type. In addition, we found that a distinct APP fragment, the cytosolic form, is transiently increased in the cytosol fraction of COS-1 cells. These results suggest that aberrant processing occurs following the expression of a mutant APP with Ala substituted for Asn, and that glycosylation may modulate the intracellular sorting of APP.

A novel splicing mutation in the ceruloplasmin gene responsible for hereditary ceruloplasmin deficiency with hemosiderosis.

Hereditary ceruloplasmin deficiency with hemosiderosis (aceruloplasminemia) is a newly recognized autosomal recessive disorder of copper-iron metabolism due to mutations in the ceruloplasmin (Cp) gene. We report here a novel mutation in the Cp gene in a 54-year-old Japanese woman with this disease. She showed clinical triad; diabetes mellitus, retinal degeneration and neurological disorder in her middle age. Laboratory findings were characteristic for no detectable serum ceruloplasmin and increased serum ferritin. Liver biopsy revealed excessive storage of iron in hepatocytes and magnetic resonance imaging of the brain was indicative of increased iron content in the basal ganglia, thalamus and dentate nucleus. The a-->g substitution at the splice acceptor site of the intron 6 (1209-2) caused a 8-bp deletion in Cp mRNA by defective splicing, resulting in a premature termination codon at the amino acid position 388. Truncation of Cp, even if effectively translated, may cause loss of its normal function because of drastic change in its triangular structure.

Familial frontotemporal dementia with a P301L tau mutation in Japan.

We have reported the family line with frontotemporal dementia (FTD) in Japan. This family line has so far included four patients. Patient II-1 (man) had a 10 year history of slowly progressive personality and behavioral changes and died at the age of 56. His neuropathological examination showed severe atrophy of the bilateral frontal and temporal cortices with neuronal loss, gliosis and superficial spongiosis. Pick bodies were not found. The neuropathological diagnosis was atypical Pick's disease without Pick bodies or Pick-type in FTD. Patient III-2 is patient II-1's oldest daughter and was taken ill with personality change at the age of 52. She died at the age of 68. Patient III-4 is patient II-1's second daughter. Her onset with strange speech and behavior was at the age of 59. Patient III-5 is patient II-1's oldest son. He also had onset with personality change at the age of 54 and had the P301L mutation in tau. In all III generation cases CT/MRI revealed circumscribed frontotemporal atrophy. Patient III-5's PET/SPECT showed signs of hypoperfusion or hypometabolism in the bilateral frontotemporal areas. This is the first report of familial FTD with the P301L mutation in Japan.

Life-threatening orthostatic hypotension in a case with bulbo-myelo-radiculo-neuropathy.

A 59-year-old female developed acute autonomic failure accompanied by life-threatening orthostatic hypotension. Reduced plasma noradrenaline levels and enhanced pressure response to noradrenaline infusion were compatible with a diagnosis of acute pan-dysautonomia. However, nerve conduction tests clearly revealed motor and sensory nerve involvement and abnormal F-responses. A sural nerve biopsy and catecholamine fluorescence study of the rectal mucosa revealed relatively preserved postganglionic unmyelinated nerve fibers. Six weeks later, the patient developed another episode of bulbar palsy and right hemiparesis; the MRI showed lesions in the medulla oblongata and right cervical spinal cord. The prognosis of acute pan-dysautonomia is usually unsatisfactory, but the present patient showed good steroid-responsiveness probably because impaired preganglionic sympathetic myelinated fibers and medulla oblongata recovered quickly.

An 80-year-old mitochondrial disease patient with A3243G tRNA(Leu(UUR)) gene presenting cardiac dysfunction as the main symptom.

MELAS is characterized by mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes, but cardiac involvement also frequently occurs. An 80-year-old female patient had been suffering from insulin-dependent diabetes mellitus and neurosensory hearing loss. At the age of 79 she suffered metabolic acidosis with persistent drowsiness and was subsequently found to have severe cardiac dysfunction. Muscle biopsy disclosed the presence of abnormal mitochondria, and the MELAS gene mutation (A3243G of the tRNA(Leu(UUR))) was demonstrated. It is noteworthy that this mitochondrial disease patient has survived until a great age, which shows the wide clinical spectrum of MELAS, especially in the age of onset.