Yoshiteru Azuma

Long-term Follow-up of Patients with Benign Partial Epilepsy in Infancy

Summary:  Purpose: The aim of this study was to investigate the long‐term outcome of children with benign partial epilepsy in infancy (BPEI).

Mutations in the C-Terminal Domain of ColQ in Endplate Acetylcholinesterase Deficiency Compromise ColQ－MuSK Interaction

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is mostly composed of an asymmetric form in which three tetramers of catalytic AChE subunits are linked to a triple helical collagen Q (ColQ). Mutations in COLQ cause endplate AChE deficiency. We report three patients with endplate AChE deficiency with five recessive COLQ mutations. Sedimentation profiles showed that p.Val322Asp and p.Arg227X, but not p.Cys444Tyr, p.Asp447His, or p.Arg452Cys, inhibit formation of triple helical ColQ. In vitro overlay of mutant ColQ‐tailed AChE on muscle sections of Colq−/− mice revealed that p.Cys444Tyr, p.Asp447His, and p.Arg452Cys in the C‐terminal domain (CTD) abrogate anchoring ColQ‐tailed AChE to the NMJ. In vitro plate‐binding assay similarly demonstrated that the three mutants inhibit binding of ColQ‐tailed AChE to MuSK. We also confirmed the pathogenicity of p.Asp447His by treating Colq−/− mice with adeno‐associated virus serotype 8 carrying mutant COLQ‐p.Asp447His. The treated mice showed no improvement in motor functions and no anchoring of ColQ‐tailed AChE at the NMJ. Electroporation of mutant COLQ harboring p.Cys444Tyr, p.Asp447His, and p.Arg452Cys into anterior tibial muscles of Colq−/− mice similarly failed to anchor ColQ‐tailed AChE at the NMJ. We proved that the missense mutations in ColQ–CTD cause endplate AChE deficiency by compromising ColQ–MuSK interaction at the NMJ.

SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome

The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5′ splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.

IntSplice: prediction of the splicing consequences of intronic single-nucleotide variations in the human genome

Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro–Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from −50 to −3.

Congenital myasthenic syndrome in Japan: Ethnically unique mutations in muscle nicotinic acetylcholine receptor subunits

Congenital myasthenic syndromes (CMS) are caused by mutations in genes expressed at the neuromuscular junction. Most CMS patients have been reported in Western and Middle Eastern countries, and only four patients with COLQ mutations have been reported in Japan. We here report six mutations in acetylcholine receptor (AChR) subunit genes in five Japanese patients. Five mutations are novel, and one mutation is shared with a European American patient but with a different haplotype. Among the observed mutations, p.Thr284Pro (p.Thr264Pro according to the legacy annotation) in the epsilon subunit causes a slow-channel CMS. Five other mutations in the delta and epsilon subunits are splice site, frameshift, null, or missense mutations causing endplate AChR deficiency. We also found a heteroallelic p.Met465Thr in the beta subunit in another patient. p.Met465Thr, however, was likely to be polymorphism, because single channel recordings showed mild shortening of channel openings without affecting cell surface expression of AChR, and the minor allelic frequency of p.Met465Thr was 5.1% in the Japanese population. Lack of shared mutant alleles between the Japanese and the other patients suggests that most mutations described here are ethnically unique or de novo in each family.

Seizure characteristics of epilepsy in childhood after acute encephalopathy with biphasic seizures and late reduced diffusion

The aim of this study was to clarify characteristics of post‐encephalopathic epilepsy (PEE) in children after acute encephalopathy with biphasic seizures and late reduced diffusion (AESD), paying particular attention to precise diagnosis of seizure types.

The effects of co-medications on lamotrigine clearance in Japanese children with epilepsy

PURPOSE Although it has been reported that some antiepileptic drugs have inducing or inhibiting effects on lamotrigine (LTG) clearance, whether they have the same effects in Asian epilepsy patients as in those in other countries has not been clarified, especially in children. The aim of this study was to determine the effects of co-medications on LTG clearance in Japanese children with epilepsy. METHODS A total of 342 routine serum concentration measurements of LTG in 102 Japanese epilepsy patients under 20years of age were reviewed. The dose-corrected concentration (DCC) of LTG was calculated as [concentration]/[dose/(body weight)], and the DCC of LTG was compared by co-medication. The difference in the DCC of LTG was compared between patients with and without valproic acid (VPA) and between those with and without drugs inducing glucuronic acid conjugation (phenytoin (PHT), carbamazepine (CBZ), and phenobarbital (PB)). RESULTS The DCC of LTG was significantly higher in patients on VPA and significantly lower in patients on drugs inducing glucuronic acid conjugation than in patients on LTG monotherapy. The DCC of LTG was significantly higher in patients on CBZ than in patients on PHT or PB. There was no correlation between the DCC of LTG and the concentration of VPA or metabolic inducers within the therapeutic range. Other antiepileptic drugs including clobazam, clonazepam, zonisamide, and levetiracetam had little effect on LTG concentration. CONCLUSION LTG concentration changes dramatically with concomitant antiepileptic drugs in Japanese children, as previously reported from other countries, and special attention is required. Although the dose of LTG should be adjusted when starting or discontinuing VPA or metabolic inducers, no adjustment is needed when changing the dose of VPA or metabolic inducers in the therapeutic range.

MYRF is associated with encephalopathy with reversible myelin vacuolization

Reversible myelin vacuolization is associated with variable conditions including mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), which is characterized by mildly impaired consciousness and transient splenial lesion. Familial and/or recurrent cases with a clinical diagnosis of MERS suggest the presence of genetic factors.

White Matter Abnormality Correlates with Developmental and Seizure Outcomes in West Syndrome of Unknown Etiology.

BACKGROUND AND PURPOSE: West syndrome is an epileptic encephalopathy characterized by epileptic spasms, a specific pattern on electroencephalography of hypsarrhythmia, and developmental regression. Our aim was to assess white matter abnormalities in West syndrome of unknown etiology. We hypothesized that diffusion tensor imaging reveals white matter abnormalities, especially in patients with poor seizure and developmental outcomes. MATERIALS AND METHODS: We enrolled 23 patients with new-onset West syndrome of unknown etiology. DTI was performed at 12 and 24 months of age. Fractional anisotropy images were compared with those of controls by using tract-based spatial statistics. We compared axial, radial, and mean diffusivity between patients and controls in the fractional anisotropy skeleton. We determined correlations of these parameters with developmental quotient, electroencephalography, and seizure outcomes. We also compared DTI with hypometabolism on fluorodeoxyglucose positron-emission tomography. RESULTS: At 12 months of age, patients showed widespread fractional anisotropy reductions and higher radial diffusivity in the fractional anisotropy skeleton with a significant difference on tract-based spatial statistics. The developmental quotient at 12 months of age correlated positively with fractional anisotropy and negatively with radial and mean diffusivity. Patients with seizure and abnormal findings on electroencephalography after initial treatments had lower fractional anisotropy and higher radial diffusivity. At 24 months, although tract-based spatial statistics did not show significant differences between patients and controls, tract-based spatial statistics in the 10 patients with a developmental quotient of <70 had significant fractional anisotropy reduction. In patients with unilateral temporal lobe hypometabolism on PET, tract-based spatial statistics showed greater fractional anisotropy reduction in the temporal lobe ipsilateral to the side of PET hypometabolism. CONCLUSIONS: Diffuse abnormal findings on DTI at 12 months of age suggest delayed myelination as a key factor underlying abnormal findings on DTI. Conversely, asymmetric abnormal findings on DTI at 24 months may reflect underlying focal pathologies.

Cytotoxic edema at onset in West syndrome of unknown etiology: A longitudinal diffusion tensor imaging study

To clarify longitudinal changes in white matter microstructures from the onset of disease in patients with West syndrome (WS) of unknown etiology.