Genetic analysis of adult leukoencephalopathy patients using a custom-designed gene panel.

Leukoencephalopathies encompass all clinical syndromes that predominantly affect brain white matter. Genetic diagnosis informs clinical management of these patients, but a large part of the genetic contribution to adult leukoencephalopathy remains unresolved. To examine this genetic contribution, we analyzed genomic DNA from 60 Japanese patients with adult leukoencephalopathy of unknown cause by next generation sequencing using a custom-designed gene panel. We selected 55 leukoencephalopathy-related genes for the gene panel. We identified pathogenic mutations in 8 of the 60 adult leukoencephalopathy patients (13.3%): NOTCH3 mutations were detected in 5 patients, and EIF2B2, CSF1R, and POLR3A mutations were found independently in 1 patient each. These results indicate that cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most frequent adult leukoencephalopathy in our cohort. Moreover, brain imaging analysis indicates that CADASIL patients who do not present typical phenotypes may be underdiagnosed if not examined genetically.

Homozygous c.14576G>A variant of RNF213 predicts early-onset and severe form of moyamoya disease.

OBJECTIVE: RNF213 was recently reported as a susceptibility gene for moyamoya disease (MMD). Our aim was to clarify the correlation between the RNF213 genotype and MMD phenotype. METHODS: The entire coding region of the RNF213 gene was sequenced in 204 patients with MMD, and corresponding variants were checked in 62 pairs of parents, 13 mothers and 4 fathers of the patients, and 283 normal controls. Clinical information was collected. Genotype-phenotype correlations were statistically analyzed. RESULTS: The c.14576G>A variant was identified in 95.1% of patients with familial MMD, 79.2% of patients with sporadic MMD, and 1.8% of controls, thus confirming its association with MMD, with an odds ratio of 259 and p < 0.001 for either heterozygotes or homozygotes. Homozygous c.14576G>A was observed in 15 patients but not in the controls and unaffected parents. The incidence rate for homozygotes was calculated to be >78%. Homozygotes had a significantly earlier age at onset compared with heterozygotes or wild types (median age at onset 3, 7, and 8 years, respectively). Of homozygotes, 60% were diagnosed with MMD before age 4, and all had infarctions as the first symptom. Infarctions at initial presentation and involvement of posterior cerebral arteries, both known as poor prognostic factors for MMD, were of significantly higher frequency in homozygotes than in heterozygotes and wild types. Variants other than c.14576G>A were not associated with clinical phenotypes. CONCLUSIONS: The homozygous c.14576G>A variant in RNF213 could be a good DNA biomarker for predicting the severe type of MMD, for which early medical/surgical intervention is recommended, and may provide a better monitoring and prevention strategy.

Evaluation of a chromogenic agar medium for the detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae.

AIM: To compare the performance of a new chromogenic agar medium CHROMagar ESBL (KC-ESBL) to chromID ESBL (SB-ESBL) for the detection and presumptive identification of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae directly from clinical specimens. METHODS AND RESULTS: A total of 256 specimens were screened for ESBL producers. Also, the genotypes of the ESBLs and plasmid-mediated AmpC ß-lactamases (pAmpCBLs) were characterized by PCR and sequencing. Among the 256 specimens, 17 (6.6%) ESBL producers were isolated on both media. The sensitivity, specificity, positive predictive value and negative predictive value were higher for KC-ESBL (100, 93.3, 51.5 and 100%, respectively) than for SB-ESBL (88.2, 92.9, 46.9 and 99.1%, respectively) (P = 0.72). Enterobacteriaceae harbouring pAmpCBL genes as well as chromosomal cephalosporinase- and penicillinase-hyperproducing Enterobacteriaceae and Pseudomonas aeruginosa accounted for the false-positive results. CONCLUSION: KC-ESBL can detect ESBL producers from clinical specimens with good selectivity and rapid presumptive identification by means of colony colour at 24 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that has evaluated the performance of KC-ESBL that enables the detection and presumptive identification of ESBL producers from clinical specimens.

Paradoxical absence of nuclear inclusion in cerebellar Purkinje cells of hereditary ataxias linked to CAG expansion.

Degeneration of cerebellar cortex is one of the principal features of hereditary ataxias linked to expansion of CAG repeat. In an attempt to clarify possible correlation between neuronal depletion and neuronal intranuclear inclusions, both triggered by the pathological expansion of CAG repeat, cerebellar sections from SCA1, SCA2, SCA3, and DRPLA cases were immunostained with anti-ubiquitin or anti-expanded polyglutamine antibody (1C2) and were screened for the presence of neuronal intranuclear inclusions. Although the degree of cerebellar degeneration varied greatly, cerebellar Purkinje cells were uniformly characterised by the absence of neuronal intranuclear inclusion. Complete absence of neuronal intranuclear inclusion in Purkinje cells is apparently paradoxical and hardly explained if neuronal intranuclear inclusion formation is positively correlated to a mechanism accelerating neuronal death. It may, otherwise, suggest an intrinsic link between neuronal intranuclear inclusion formation and neurodegeneration in opposite directions in human Purkinje cells, more or less affected in these CAG repeat disorders.

Modulation of event-related potentials in normal human subjects by visual divided attention to spatial and color factors.

We investigated how visual event-related potentials (ERPs) are modulated by visual divided attention using an S1-S2 paradigm. Stimulus S2 consisted of non-target stimuli (Stimulus 1, 2, 3) and a target stimulus (Stimulus 4). The spatial/color factor was compared between S1 and S2: same/same (Stimulus 1); same/different (Stimulus 2); different/same (Stimulus 3); and different/different (Stimulus 4). The P1/N1 (90 approximately 150 ms) showed significantly greater amplitude in Stimulus 3 than in Stimuli 1 and 2. The N2 (230 approximately 290ms) showed significantly greater amplitude in Stimulus 2 than in Stimuli 1 and 3. We assumed that the P1/N1 was related to spatial attention, enhanced by alterations to the spatial factor, and that the N2 was related to color attention, enhanced by alterations to the color factor.

Preferential recruitment of ataxin-3 independent of expanded polyglutamine: an immunohistochemical study on Marinesco bodies.

In an immunohistochemical study of Marinesco bodies--a neuronal intranuclear inclusion often seen in neurons of the substantia nigra of patients with hepatic encephalopathy--it was shown that one of the polyglutamine proteins, ataxin-3, is preferentially recruited into this inclusion, whereas other polyglutamine proteins (ataxin-2 and TATA box-binding protein) are not. This suggests that recruitment of each of the polyglutamine proteins may be differently regulated. Because this nuclear inclusion is thought to be formed in response to cellular stress, as occurs in hepatic encephalopathy, even in the absence of an expanded CAG/polyglutamine repeat, recruitment of ataxin-3 and ubiquitin into Marinesco bodies may represent a cellular response to noxious external stimuli unrelated to expanded CAG/polyglutamine.

Non-expanded polyglutamine proteins in intranuclear inclusions of hereditary ataxias--triple-labeling immunofluorescence study.

Neuronal intranuclear inclusions (NIIs) found in CAG/polyglutamine-expansion disorders contain both expanded polyglutamine and the gene product without the CAG repeat. The gene product containing expanded polyglutamine has, therefore, been considered to be a major component of NIIs. In this immunohistochemical study, we showed recruitment of ataxin-2, ataxin-3 and TATA box binding protein (TBP) into NIIs of the pontine neurons of spinocerebellar ataxia type (SCA) 1, SCA2, SCA3 and dentatorubral-pallidoluysian atrophy brains. Triple-labeling immunofluorescence demonstrated colocalization of ataxin-2 and ataxin-3 in NIIs containing expanded polyglutamine, irrespective of the disease examined. These in vivo findings indicate that polyglutamine proteins recruited into NIIs are not restricted to their expanded form. Among these proteins, recruitment of ataxin-2 was least frequent in every case examined, suggesting that the rate of recruitment partly depends on the protein transported into NIIs. Because other proteins lacking polyglutamine motif were not detected in NIIs, it is suggested that the presence of polyglutamine is a prerequisite for these proteins to be recruited into nucleus and to form NIIs. Interaction between expanded and non-expanded polyglutamine may play roles during these processes.

[A case of Vernet's syndrome due to varicella-zoster virus infection].

We report a 73-year-old man who suffered from an acute onset of dysphagia, cough, hoarseness and left facial and occipital pain. On the 44 days of illness, he was admitted to our clinic. A neurological examination revealed left IX, X and XI cranial nerve palsy. The diagnosis of Vernet's syndrome due to varicella-zoster virus (VZV) infection was made, based on the high titers of VZV antibody in serum. Magnetic resonance imaging revealed a unique nodular lesion with gadolinium enhancement at the medial side of the left jugular foramen. Clinical symptoms improved with intravenous high dose pulse methylprednisolone therapy. The clinical course suggests that the inflammation extended from the left X cranial nerve ganglion.

Ataxin-3 is translocated into the nucleus for the formation of intranuclear inclusions in normal and Machado-Joseph disease brains.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is one of the dominantly inherited cerebellar ataxias. The gene responsible for the disease, a novel gene of unknown function, encodes ataxin-3 containing a polyglutamine stretch. Although it has been known that ataxin-3 is incorporated into neuronal intranuclear inclusions (NIIs) in neurons of affected regions, the relationship between NII formation and neuronal degeneration still remains uncertain. In the present study we show two different conditions in which ataxin-3 is recruited into the nucleus and suggest a process to form nuclear inclusions. In normal brains, wild-type ataxin-3 localizes within the ubiquitin-positive nuclear inclusion, the Marinesco body, indicating that ataxin-3 is recruited into the nuclear inclusion even in the absence of pathologically expanded polyglutamine. In MJD/SCA3 brains, immunohistochemical analyses with anti-ataxin-3 antibody, anti-ubiquitin antibody, and monoclonal antibody 1C2 known to recognize expanded polyglutamine revealed differences in frequency and in diameter among NIIs recognized by each antibody. These results were confirmed in the same inclusions by double immunofluorescent staining, suggesting that expanded ataxin-3 forms a core, thereby recruiting wild-type ataxin-3 into the nucleus around the core portion, and then followed by activation of the ubiquitin/ATP-dependent pathway. Recruitment of ataxin-3 into the nucleus and formation of nuclear inclusion under two different conditions suggest that ataxin-3 may be translocated into the nucleus under certain conditions stressful on neuronal cells such as aging and polyglutamine neurotoxicity.

Protein-loss into retroperitoneal lymphangioma: demonstration by lymphoscintigraphy and blood-pool scintigraphy with Tc-99m-human serum albumin.

A rare, benign congenital lymphangioma has been reported to occur frequently in the neck and axilla, but rarely in the retroperitoneal space. We report a case of a retroperitoneal lymphangioma associated with hypoproteinemia caused by protein-loss into the tumor. In this case, lymphoscintigraphy with subcutaneously injected Tc-99m-human serum albumin (HSA) disclosed the communication between the tumor and the lymphatic system, and sequential abdominal scintigraphy with intravenously injected Tc-99m-HSA revealed the protein loss into the tumor. Abdominal scintigraphy with Tc-99m-HSA injected intravenously or subcutaneously is occasionally useful for determining the etiology of hypoproteinemia.

Neuronal intranuclear inclusions in spinocerebellar ataxia type 2: triple-labeling immunofluorescent study.

Spinocerebellar ataxia type 2 (SCA2) is associated with an expansion of CAG/polyglutamine-repeat of a gene of unknown function. We performed an immunohistochemical study to identify the immunolocalization of the disease protein ataxin-2 in normal and SCA2 patients. Although normal and expanded ataxin-2 were ubiquitously localized to the cytoplasm of neurons, ubiquitinated intranuclear inclusions were observed selectively in 1-2% of neurons of affected brain regions except the cerebellum. Triple-labeling immunofluorescence revealed that ataxin-2, expanded polyglutamine and ubiquitin were colocalized to these neuronal intranuclear inclusions (NIs), indicating that SCA2 shares morphological characteristics common to other neurological disorders associated with an expansion of CAG/polyglutamine-repeat. Lack of NIs in the cerebellar lesion, however, suggests the discrepancy between formation of NIs and neuronal degeneration in SCA2.

A novel Jun N-terminal kinase (JNK)-binding protein that enhances the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1.

We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.

[Correlation of clinichopathological features and CAG repeats in SCA2].

Spinocerebellar ataxia type 2 (SCA2) is an inherited neurodegenerative disorder characterized clinically by cerebellar ataxia, slow eye movement, hyporeflexia, involuntary movement, dementia and sensory disturbance and neuropathologically by neuronal loss, mainly in the cerebellar cortex involving all three layers, the pontine nucleus, the inferior olivary nucleus, anterior horn, substantia nigra and thalamus. For making one's diagnosis, it is necessary to give careful consideration to two factors, (age at onset, disease duration). A distinctive neuropathological feature is having both simple atrophy (without degeneration) and numerical atrophy. SCA2 is associated with an expanded CAG repeat that encodes polyglutamine of a gene and a larger number of the repeat is associated with earlier onset and more severe symptoms and more severe neuronal degenerations.

Encephalopathy associated with haemophagocytic lymphohistiocytosis following rotavirus infection.

UNLABELLED: A 2-year-old Japanese boy with a haemophagocytic lymphohistiocytosis (HLH) associated encephalopathy which developed after rotavirus infection is described. The neurological symptoms consisted of coma, seizures and spastic quadriplegia. On therapy with steroids, etoposide and cyclosporin A, the patient recovered without any neurological deficits. The interferon-gamma levels in serum and CSF were elevated at onset of the disease but had returned to normal at the time of clinical remission. Brain MRI revealed diffuse white matter abnormalities and parenchymal volume loss. Proton magnetic resonance spectroscopy revealed elevated lactate in the abnormal lesions observed on MRI, indicating that macrophages not exhibiting aerobic metabolism had infiltrated the CNS. At the time of clinical remission, the white matter abnormalities and brain lactate had disappeared. These findings suggested that the neurological symptoms resulted from the overproduction of cytokines by activated T-cells and macrophages. The pathophysiology of a HLH associated encephalopathy was considered to be a local immune response within the CNS, because interferon-gamma can induce the expression of major histocompatibility complex class I and II antigens on glial cells in the CNS. CONCLUSION: Haemophagocytic lymphohistiocytosis associated encephalopathy should be considered early in the differential diagnosis of cases with acute onset neuropathy.

[A rapid determination of allantoin by high-performance liquid chromatography using tris(hydroxymethyl)aminomethane-HCl buffer as a mobile phase].

A rapid and simple method for the determination of allantoin in pharmaceuticals by reversed-phase ion-pair high-performance liquid chromatography using an ODS column was presented. In general, it is difficult to retain allantoin to the ODS column owing to its very low hydrophobicity. We solved these problems by the use of a Tris-HCl buffer (pH 7.5) containing tetra-n-hexyl-ammonium bromide (THAB) as an ion-pair reagent for the mobile phase. Comparatively low concentrations of Tris-HCl buffer (0.9 mM) and THAB (0.5 mM) gave a high capacity factor (k'). As a results of the examination of the chromatographic behavior, it is confirmed that the retention mechanism of allantoin to the ODS column on the present method was not the ion-pair mode, but the ion-exchange mode. Calibration curves for allantoin showed a good linearity in the range of 10 to 400 micrograms/ml (r = 0.9999). The reproducibility (R.S.D., n = 6) was invariably good (0.37%). The lowest concentration of allantoin for the determination was 200 ng per 20 microliters of injection. The present method was successfully applied to the determination of allantoin in commercial eyedrops with good recovery (99.4%). It was found that allantoin in pharmaceuticals could be determined by the present method in short time and without any complicated derivatization.

Stereoselective renal secretion of carbenicillin in rabbits: role of the organic anion transporter at the renal brush border membrane.

Stereoselectivity in the renal secretion of carbenicillin (CBPC) was studied in rabbits. Significant renal secretion of CBPC was observed in vivo, with the secretion of the S-epimer being greater than that of the R-epimer. Stereoselective transport of CBPC was further studied in vitro using basolateral and brush border membrane vesicles prepared from rabbit kidneys. The transport of CBPC by the organic anion transporter into the basolateral membrane vesicles (BLMV) was not stereoselective. In contrast, a distinct stereoselectivity was observed in the transport of CBPC by the organic anion transporter into the brush border membrane vesicles (BBMV), with the transport of the S-epimer being more favorable. Significant epimer-epimer interactions were also observed in the transport into BBMV. The stereoselectivity of the transport of CBPC was calculated from the kinetic parameters with consideration of epimer-epimer interactions and was similar to that observed in vivo. It was concluded that the observed stereoselectivity in the renal secretion of CBPC in vivo reflected that of transport via the organic anion transporter located at the brush border membrane.

Lack of renal secretion of carbenicillin in rats: poor affinity to the organic anion transporter at renal brush border membrane.

The renal secretion of carbenicillin (CBPC) was studied in rats. The results obtained in the in vivo study indicated very poor renal secretion of CBPC in rats, which was entirely different from those observed in humans and rabbits. In humans and rabbits, significant and stereoselective renal secretion of CBPC was observed in vivo. In order to verify the poor renal secretion of CBPC in rats, the transport characteristics of the organic anion transporters were studied in vitro using basolateral and brush border membrane vesicles. Transport of p-aminohippuric acid (PAH) into the basolateral membrane vesicles (BLMVs) was inhibited by CBPC, indicating that the organic anion transporter located at the BLM may have affinity to CBPC. In contrast, the transport of PAH into the brush border membrane vesicles (BBMVs) was not inhibited by CBPC, suggesting that the organic anion transporter located at the BBM may not have affinity to CBPC. Similar results were obtained for sulbenicillin (SBPC). Since CBPC and SBPC exist as di-anions at physiological pH, the organic anion transporter located at the rat renal BBM may not exhibit affinity to water-soluble di-anions, which in turn will result in poor renal secretion of these compounds.

Stereoselective disposition of sulbenicillin in humans.

Stereoselective disposition of sulbenicillin (SBPC) epimers in healthy human volunteers was studied in order to clarify the differences in pharmacokinetic behavior between the epimers. Stereospecific high-performance liquid chromatography was used for the determination of SBPC epimers. Plasma protein binding was measured in vitro with an ultrafiltration method. The binding was stereoselective, with the unbound fraction (fu) of the R-epimer being approximately 1.3-fold greater than that of the S-epimer. SBPC was administered intravenously to human volunteers, and concentrations of SBPC in plasma and urinary excretion rates were measured. Renal clearance (CLR) for the unbound drug (approximately 400 ml/min) was greater than the glomerular filtration rate (GFR) (approximately 109 ml/min) for both epimers, suggesting that both epimers are secreted at the renal tubules. Renal tubular secretion appeared to be greater for the S-epimer. When probenecid was coadministered, the CLR values of both epimers were significantly reduced and were approximately equal to the GFR values. CLR was greater for the S-epimer (37.5 and 49.8 ml/min for R-SBPC and S-SBPC, respectively), which was simply due to the greater fu of the S-epimer in plasma. In contrast, total body clearance was greater for the R-epimer (67.8 and 56.3 ml/min for R-SBPC and S-SBPC, respectively) because of the stereoselective degradation of the R-epimer in plasma. It was revealed that stereoselective degradation in the body had significant influence on the disposition of SBPC epimers.