Ayumi Narisawa

A genome-wide association study identifies RNF213 as the first Moyamoya disease gene.

Moyamoya disease (MMD) shows progressive cerebral angiopathy characterized by bilateral internal carotid artery stenosis and abnormal collateral vessels. Although ∼15% of MMD cases are familial, the MMD gene(s) remain unknown. A genome-wide association study of 785 720 single-nucleotide polymorphisms (SNPs) was performed, comparing 72 Japanese MMD patients with 45 Japanese controls and resulting in a strong association of chromosome 17q25-ter with MMD risk. This result was further confirmed by a locus-specific association study using 335 SNPs in the 17q25-ter region. A single haplotype consisting of seven SNPs at the RNF213 locus was tightly associated with MMD (P=5.3 × 10−10). RNF213 encodes a really interesting new gene finger protein with an AAA ATPase domain and is abundantly expressed in spleen and leukocytes. An RNA in situ hybridization analysis of mouse tissues indicated that mature lymphocytes express higher levels of Rnf213 mRNA than their immature counterparts. Mutational analysis of RNF213 revealed a founder mutation, p.R4859K, in 95% of MMD families, 73% of non-familial MMD cases and 1.4% of controls; this mutation greatly increases the risk of MMD (P=1.2 × 10−43, odds ratio=190.8, 95% confidence interval=71.7–507.9). Three additional missense mutations were identified in the p.R4859K-negative patients. These results indicate that RNF213 is the first identified susceptibility gene for MMD.

Increased expression of serum Matrix Metalloproteinase-9 in patients with moyamoya disease.

BACKGROUND Moyamoya disease is a chronic occlusive cerebrovascular disease with unknown etiology characterized by an abnormal vascular network at the base of the brain, which can manifest both as ischemic stroke and as cerebral hemorrhage. It was also reported that the patients with moyamoya disease are more vulnerable to cerebral hyperperfusion such as postoperative hemorrhagic complication after extracranial-intracranial bypass surgery despite its low flow revascularization. However, the underlying mechanisms of its pathologic angiogenesis and the occurrence of hemorrhage are undetermined. Excessive degradation of the vascular matrix by MMPs, proteolytic enzymes that degrade all the components of extracellular matrix, can lead to instability of the vascular structure and can thereby cause bleeding. The MMPs also play an important role in tissue remodeling including angiogenesis in both physiologic and pathologic condition. METHODS We examined the serum levels of MMP-2 and MMP-9 in 16 cases with definitive moyamoya disease by enzyme-linked immunosorbent assay and compared them with those from healthy controls. RESULTS The serum MMP-9 level was significantly higher in moyamoya disease (40.18 ng/mL) than in healthy controls (13.75 ng/mL, P = .0372). There was no difference in serum MMP-2 level between moyamoya disease (646.65 ng/mL) and healthy control (677.60 ng/mL). Immunohistochemistry on the surgical specimens showed significant increase in MMP-9 expression within the arachnoid membrane of moyamoya disease. CONCLUSION The increased expression of MMP-9 may contribute to pathologic angiogenesis and/or to the instability of the vascular structure and could thereby cause hemorrhage in moyamoya disease.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt−/− mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Genomic deletion within GLDC is a major cause of non-ketotic hyperglycinaemia

Background: Non-ketotic hyperglycinaemia (NKH) is an inborn error of metabolism characterised by accumulation of glycine in body fluids and various neurological symptoms. NKH is caused by deficiency of the glycine cleavage multienzyme system with three specific components encoded by GLDC, AMT and GCSH. Most patients are deficient of the enzymatic activity of glycine decarboxylase, which is encoded by GLDC. Our recent study has suggested that there are a considerable number of GLDC mutations which are not identified by the standard exon-sequencing method. Methods: A screening system for GLDC deletions by multiplex ligation-dependent probe amplification (MLPA) has been developed. Two distinct cohorts of patients with typical NKH were screened by this method: the first cohort consisted of 45 families with no identified AMT or GCSH mutations, and the second cohort was comprised of 20 patients from the UK who were not prescreened for AMT mutations. Results:GLDC deletions were identified in 16 of 90 alleles (18%) in the first cohort and in 9 of 40 alleles (22.5%) in the second cohort. 14 different types of deletions of various lengths were identified, including one allele where all 25 exons were missing. Flanking sequences of interstitial deletions in five patients were determined, and Alu-mediated recombination was identified in three of five patients. Conclusions:GLDC deletions are a significant cause of NKH, and the MLPA analysis is a valuable first-line screening for NKH genetic testing.

Direct Correlation Between Ischemic Injury and Extracellular Glycine Concentration in Mice With Genetically Altered Activities of the Glycine Cleavage Multienzyme System

Background and Purpose— Ischemia elicits the rapid release of various amino acid neurotransmitters. A glutamate surge activates N-methyl-d-aspartate (NMDA) glutamate receptors, triggering deleterious processes in neurons. Although glycine is a coagonist of the NMDA receptor, the effect of extracellular glycine concentration on ischemic injury remains controversial. To approach this issue, we examined ischemic injury in mice with genetically altered activities of the glycine cleavage multienzyme system (GCS), which plays a fundamental role in maintaining extracellular glycine concentration. Methods— A mouse line with increased GCS activity (340% of C57BL/6 control mice) was generated by transgenic expression of glycine decarboxylase, a key GCS component (high-GCS mice). Another mouse line with reduced GCS activity (29% of controls) was established by transgenic expression of a dominant-negative mutant of glycine decarboxylase (low-GCS mice). We examined neuronal injury after transient occlusion of the middle cerebral artery in these mice by measuring extracellular amino acid concentrations in microdialysates. Results— High-GCS and low-GCS mice had significantly lower and higher basal concentrations of extracellular glycine than did controls, respectively. In low-GCS mice, the extracellular glycine concentration reached 2-fold of control levels during ischemia, and infarct volume was significantly increased by 69% with respect to controls. In contrast, high-GCS mice had a significantly smaller infarct volume (by 21%). No significant difference was observed in extracellular glutamate concentrations throughout the experiments. An antagonist for the NMDA glycine site, SM-31900, attenuated infarct size, suggesting that glycine operated via the NMDA receptor. Conclusions— There is a direct correlation between ischemic injury and extracellular glycine concentration maintained by the GCS.

A novel KCNQ4 one-base deletion in a large pedigree with hearing loss: implication for the genotype–phenotype correlation

AbstractAutosomal-dominant, nonsyndromic hearing impairment is clinically and genetically heterogeneous. We encountered a large Japanese pedigree in which nonsyndromic hearing loss was inherited in an autosomal-dominant fashion. A genome-wide linkage study indicated linkage to the DFNA2 locus on chromosome 1p34. Mutational analysis of KCNQ4 encoding a potassium channel revealed a novel one-base deletion in exon 1, c.211delC, which generated a profoundly truncated protein without transmembrane domains (p.Q71fsX138). Previously, six missense mutations and one 13-base deletion, c.211_223del, had been reported in KCNQ4. Patients with the KCNQ4 missense mutations had younger-onset and more profound hearing loss than patients with the 211_223del mutation. In our current study, 12 individuals with the c.211delC mutation manifested late-onset and pure high-frequency hearing loss. Our results support the genotype-phenotype correlation that the KCNQ4 deletions are associated with later-onset and milder hearing impairment than the missense mutations. The phenotypic difference may be caused by the difference in pathogenic mechanisms: haploinsufficiency in deletions and dominant-negative effect in missense mutations.

Efficacy of the revascularization surgery for adult-onset moyamoya disease with the progression of cerebrovascular lesions

OBJECT In moyamoya disease, despite its progressive nature of the occlusive lesions in pediatric patients, the prevalence of the progression in adult patients is undetermined. Furthermore, the optimal timing of the revascularization surgery for progressive cases is controversial. To address these issues, we retrospectively investigate four cases with the adult-onset moyamoya disease manifesting as progression before revascularization surgery. METHODS From March 2004 to May 2007, 49 patients with adult-onset moyamoya disease aged from 19 to 62 years old (mean 40.5) underwent superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis on 63 hemispheres. All patients were strictly followed up by magnetic resonance (MR) imaging/angiography postoperatively. Twenty-seven hemispheres of 15 adult patients without surgery were also followed up at outpatient service during the same period. If the patients manifest as the progression of the steno-occlusive lesion on the hemisphere without surgery, they undergo revascularization surgery after the confirmation of hemodynamic compromise. RESULTS During this period, 47 hemispheres including those of outpatient cases were conservatively followed up after initial diagnosis. Among them, six hemispheres (12.8%) of four patients had been proven to show apparent progression of steno-occlusive lesion and were subjected to revascularization surgery. Postoperative courses were uneventful in all four cases, and no patient suffered cerebrovascular event on the operated hemisphere after surgery. CONCLUSION Adult-onset moyamoya disease, either bilateral or unilateral, has a substantial risk for progression, and careful follow-up is necessary for asymptomatic hemisphere. Once the patient manifests as the progression of cerebrovascular occlusive lesions or ischemic symptoms, we recommend revascularization surgery after the confirmation of the hemodynamic compromise.

Rapid diagnosis of glycine encephalopathy by 13C-glycine breath test.

It is currently problematic to confirm the clinical diagnosis of glycine encephalopathy, requiring either invasive liver biopsy for enzymatic analysis of the glycine cleavage system or exhaustive mutation analysis. Because the glycine cleavage system breaks down glycine generating carbon dioxide, we suppose that the glycine cleavage system activity could be evaluated in vivo by measuring exhaled 13CO2 after administration of [1‐13C]glycine.

Shunt placement after cyst fenestration for middle cranial fossa arachnoid cysts in children

OBJECT Some pediatric patients with middle cranial fossa arachnoid cysts present with symptoms of increased intracranial pressure (ICP) and require shunt placement after a cyst fenestration. However, factors concerning increased ICP after fenestration followed by shunt placement have not been elucidated. This study evaluated factors that are associated with shunt placement following cyst fenestration in pediatric patients with middle cranial fossa arachnoid cysts. METHODS Twenty-six pediatric patients with middle cranial fossa arachnoid cysts who were surgically treated at a single institution between 2004 and 2013 were retrospectively identified. The surgical indications for middle cranial fossa arachnoid cysts were as follows: 1) arachnoid cysts associated with symptoms such as headache and abnormally enlarging head circumference; 2) progressively expanding arachnoid cysts; and 3) large arachnoid cysts such as Galassi Type III. A cyst fenestration was performed as a first-line treatment, and shunt placement was required if symptoms associated with increased ICP were found following fenestration. The risk factors evaluated included age, sex, presenting symptoms, the presence of head enlargement, progressive cyst expansion, and subdural hematoma/hygroma. RESULTS Four patients (15.4%) required shunt placement after cyst fenestration. Younger age, abnormal head enlargement, and progressive cyst expansion before fenestration were significantly associated with the need for shunt placement following fenestration. Arachnoid cysts decreased in size in 22 patients (84.6%) after fenestration and/or shunt placement. The presence of symptoms was not associated with postoperative cyst size in this study. CONCLUSIONS In this study, younger age, abnormal head enlargement, and progressive cyst expansion were risk factors for shunt placement after cyst fenestration in pediatric patients with middle cranial fossa arachnoid cysts. It is important to consider that cyst fenestration may not be effective because of a latent derangement of CSF circulation in patients with these risk factors.

Model Mice for Mild-Form Glycine Encephalopathy: Behavioral and Biochemical Characterizations and Efficacy of Antagonists for the Glycine Binding Site of N-Methyl D-Aspartate Receptor

Glycine encephalopathy (GE) is caused by an inherited deficiency of the glycine cleavage system (GCS) and characterized by accumulation of glycine in body fluids and various neurologic symptoms. Coma and convulsions develop in neonates in typical GE while psychomotor retardation and behavioral abnormalities in infancy and childhood are observed in mild GE. Recently, we have established a transgenic mouse line (low-GCS) with reduced GCS activity (29% of wild-type (WT) C57BL/6) and accumulation of glycine in the brain (Stroke, 2007; 38:2157). The purpose of the present study is to characterize behavioral features of the low-GCS mouse as a model of mild GE. Two other transgenic mouse lines were also analyzed: high-GCS mice with elevated GCS activity and low-GCS-2 mice with reduced GCS activity. As compared with controls, low-GCS mice manifested increased seizure susceptibility, aggressiveness and anxiety-like activity, which resembled abnormal behaviors reported in mild GE, whereas high-GCS mice were less sensitive to seizures, hypoactive and less anxious. Antagonists for the glycine-binding site of the N-methyl-D-aspartate receptor significantly ameliorated elevated locomotor activity and seizure susceptibility in the low-GCS mice. Our results suggest the usefulness of low-GCS mice as a mouse model for mild GE and a novel therapeutic strategy.