Kyoko Takano

Application of magnetoencephalography in epilepsy patients with widespread spike or slow-wave activity.

Summary:  Purpose: To examine whether magnetoencephalography (MEG) can be used to determine patterns of brain activity underlying widespread paroxysms of epilepsy patients, thereby extending the applicability of MEG to a larger population of epilepsy patients.

Germline mosaicism of a novel mutation in lysosome‐associated membrane protein‐2 deficiency (Danon disease)

We identified a family with lysosome‐associated membrane protein‐2 deficiency (Danon disease) associated with a novel 883 ins‐T mutation in the lysosome‐associated membrane protein‐2 gene located at Xq24. Although the affected son and daughter carried the same mutation, it was not detected in their mothers peripheral blood or buccal cells; this indicated germline mosaicism. This is the first molecular evidence for germline mosaicism in Danon disease and has important implications for genetic counseling.

Cerebellar and brainstem involvement in familial juvenile nephronophthisis type I

We report on an 11-year-old boy with familial juvenile nephronophthisis type I associated with cerebellar ataxia and nystagmus, but not with ocular motor apraxia. An MRI revealed hypoplasia of the brainstem and vermis, and an enlargement of the fourth ventricle. A molecular genetic analysis demonstrated a homozygous deletion including the NPHP1 gene. These findings suggest that NPHP1 may play an important role in the normal development of the brainstem and the cerebellum as well as renal tissue.

Mosaic paternally derived inv dup(15) may partially rescue the Prader-Willi syndrome phenotype with uniparental disomy.

To the Editor: Inv dup(15) can be associated with Prader– Willi syndrome (PWS) with maternal uniparental disomy (UPD), but it is not thought to have a significant phenotypic contribution because it does not contain the PWS chromosome region (PWCR) in most cases (1). There have been only two reports describing supernumerary marker chromosomes (SMCs) of paternal origin containing a PWCR in PWS patients with maternal UPD (2, 3). Here, we describe a girl showing a PWS-like mild phenotype who is mosaic for cells with maternal heterodisomy of chromosome 15 (80%) and for cells with additional paternally derived inv dup(15) containing a PWCR (20%). The relatively mild phenotype of the patient may indicate partial rescue of the PWS phenotype by expression of imprinted genes from the paternally derived inv dup(15). The 16-month-old girl was referred to our hospital because of mild developmental delay. She had no family history of neurological or chromosomal disorders. She was born at 41 weeks gestation, measured 49 cm (10.3 SD) in length, weighed 2712 g (20.7 SD), and had an occipitofrontal circumference of 35 cm (20.2 SD). No floppiness or failure to thrive was noted from neonate to infant. She lifted her head at 6 months and sat at 10 months of age. She had a happy disposition, thin eyebrows, almond-shaped palpebral fissures, narrow bifrontal diameter and a thin upper lip but no other PWS-specific anomalies such as fine light-brown hair or small hands and feet. She walked and spoke several meaningful words at 21 months. Her intelligent quotient was 65 at 48 months. Chromosomal analysis revealed the karyotype 46,XX [80%]/47,XX,1inv dup(15) [20%] [Fig. 1(a)]. Fluorescence in situhybridization analysis using an SNRPN probe demonstrated two signals on inv dup(15) as well as on both chromosome 15s (data not shown). A polymerase chain reaction (PCR)-based DNA methylation test for SNURF-SNRPN was performed using the bisulfite method (4). Microsatellite polymorphism analyses using PCR were performed for five loci located in 15q11–q13 (D15S11, D15S128, D15S817, D15S97 and GABRB3), one locus in 15q13-qter (ACTC) and one locus in 11q13.5 (D11S527). Expression of SNURF-SNRPN and GAPDH was examined by reverse transcription-PCR as described previously (5). This study was approved by the ethics committee of Hokkaido University Graduate School of Medicine. The SNURF-SNRPN methylation test revealed a faint unmethylated (paternal) signal as well as a methylated (maternal) signal of normal intensity [Fig. 1(b)]. Microsatellite polymorphism analysis of 15q11–q13 demonstrated inheritance of both maternal alleles plus one paternal allele of reduced signal intensity [Table 1 and Fig. 1(c)]. These findings indicated that the patient had uniparental maternal heterodisomy with paternally derived inv dup(15) in a mosaic fashion. Reverse transcription-PCR demonstrated expression of SNURF-SNRPN in lymphoblastoid cells isolated from the patient, although the expression level was lower than that in cells from control individuals [Fig. 1(d)]. SMCs containing PWCRs of paternal origin are of most interest because they should express the imprinted genes that are normally silenced in PWS patients. PWS is caused by loss of expression of the imprinted genes that are active only in the paternal allele (6). If the SMCs of paternal origin express the imprinted genes, they might ameliorate the PWS phenotype. The present patient is the third reported case with maternal UPD of chromosome 15 accompanied by mosaic paternal SMCs containing a PWCR (2, 3). The case reported by Baumer et al. (2) had a PWS phenotype and 8% of cells had paternal inv dup(15) containing two copies of the PWCR. The

Possible involvement of the tip of temporal lobe in Landau–Kleffner syndrome

Landau-Kleffner syndrome (LKS) is a childhood disorder of unknown etiology characterized by an acquired aphasia and epilepsy. We have performed comprehensive neurofunctional studies on an 8-year-old girl with typical LKS, with the aim of identifying lesions that may be responsible for her condition. 18F-fluoro-D-glucose (FDG) positron emission computed tomography (PET), 11C-Flumazenil (FMZ) PET, 99mTc-hexamethylpropyleneamine oxime single photon emission computed tomography (SPECT) and magnetoencephalography were performed before and after changes to the patients medication led to a clinical improvement. Interictal SPECT showed hypoperfusion in the left frontal, left temporal, and left occipital lobes. 18F-FDG PET demonstrated a decrease in glucose metabolism medially in both temporal lobes and superiorly in the left temporal lobe. 11C-FMZ PET revealed a deficit in benzodiazepine receptor binding at the tip of the left temporal lobe. Magnetoencephalography demonstrated equivalent current dipoles located superiorly in the left temporal lobe. Our results suggest that the tip of the left temporal lobe plays an important role in the pathogenesis of LKS in our patient.

Uniparental disomy and imprinting defects in japanese patients with Angelman syndrome

We examined 54 patients with deletion-negative Angelman syndrome (AS) using DNA methylation testing and microsatellite polymorphism analysis, and identified three patients with paternal uniparental disomy (UPD) and seven patients with imprinting defects (ID). The three patients with UPD were shown to have paternal isodisomy 15, which we hypothesized to have arisen from duplication of chromosome 15. Two of the patients with ID were siblings and carried microdeletions of the imprinting center (IC), while the remaining five patients had no evidence of deletions and represented sporadic cases. Two of the three patients with UPD and two of the seven patients with ID had not developed seizures. The only patients displaying microcephaly were those with ID who had microdeletions at the IC. These data support the previous findings that indicate that patients with UPD and ID may have a milder phenotype of AS.

Germline mosaicism of a novel UBE3A mutation in Angelman syndrome.

Angelman syndrome (AS) (OMIM 105830) is a neurodevelopmental disorder that occurs with a frequency of approximately 1 in 15,000 births [Clayton-Smith and Laan, 2003]. AS is characterized by severemental retardation, profound speech impairment, ataxic gait, seizures, and characteristic behaviors including easily evoked laughter [Clayton-Smith and Laan, 2003]. AS is related to genomic imprinting of 15q11-q13, and a loss of function of maternally expressedUBE3A causes the AS phenotype [Kishino et al., 1997;Matsuura et al., 1997;Nicholls et al., 1998]. Although themajority of AS cases are caused by a common maternally derived 5 Mb deletion of 15q11-q13, with some cases caused by paternal uniparental disomy of chromosome 15 or imprinting defects, approximately 10% of the AS cases are caused by a mutation inUBE3A [Lossie et al., 2001]. UBE3A encodes an E3 ubiquitin ligase but the targets and specific functions of UBE3A remain to be elucidated. The UBE3A gene shows tissue-specific imprinting and only the maternally derived allele is expressed in certain areas of the brain, including the hippocampus and cerebellum [Vu and Hoffman, 1997]. A recent in vitro study showed thatUBE3A is imprinted only inneurons andnot in glial cells, suggesting cellspecific imprinting [Yamasaki et al., 2003]. More than 50 mutations ofUBE3A have been described, and these are either maternally inherited or have arisen de novo [Malzac et al., 1998; Fang et al., 1999; Lossie et al., 2001; Rapakko et al., 2004].Here, we report on a familial case of AS in siblingswith a novel UBE3A mutation that was transmitted via maternal germline mosaicism. This study was approved by the ethical committee of Hokkaido University Graduate School of Medicine. The two affected siblings were males, aged 10and 5-years. Both patients demonstrated typical AS phenotypes including severe mental retardation, absence of speech, epilepsy, ataxic gait, inappropriate laughter, and characteristic facies including prominent chin and largemouth. EEGswere characteristic for AS in both siblings. The affected siblings had a non-affected 7-year-old male sibling. Their parents were phenotypically normal and no other family history of AS or neurological disorders was present. Chromosomal analysis including FISH and the SNURF-SNRPN DNA methylation test excluded a 15q11-q13 deletion, uniparental disomy, and imprinting defects in the affected siblings. Lymphoblastoid cell lineswere established from the affected siblings, a non-affected sibling, and the parents. GenomicDNA andRNAwere extractedusing standardprocedures. All coding exons and flanking sequences of UBE3A were amplified by PCR and directly sequenced on an ABI PRISM 310 machine with primers described by Lossie et al. [2001]. We identified a previously unreported base substitution at a splice acceptor consensus site, IVS14-2A>G that was shared by the two affected siblings. This substitution was not present in the mother or father, although we could only examine DNA derived from the parental peripheral leukocytes or lymphoblastoid cells. To confirm if this base substitution altered splicing,we performedRT-PCRusing a forward primer in exon 14 and a reverse primer in exon 16 (primer sequences available on request). RT-PCR using RNA from lymphoblastoid cells, where UBE3A was not imprinted, revealed the skipping of exon 15, which resulted in the deletion of 48 amino acid residues (Fig. 1). Therefore, we concluded that this base substitution was indeed a novel splicing mutation. To begin to analyze the parental origin of this region, we then examined microsatellite polymorphisms at loci D15S11, GABRB3, and D15S97 (15q11-q13) and ACTC (15q13-ter) on an ABI PRISM 310 machine using GeneScan software (Applied Biosystems, Foster City, CA). Microsatellite analysis revealed that the two affected siblings inherited the same haplotypes from each of their parents. Thus, this analysis could not be used to help identify the parental origin of themutation chromosome in the affected boys. The non-affected sibling inherited different haplotypes from the parents (data not shown). To establish the parental origin of themutation,we searched the JSNPdatabase tofindamaternal-specific single nucleotide polymorphism (SNP) in the vicinity of the mutation. We initially found three SNPsnearUBE3A exon 15 from the JSNP database, however, all of the SNPs were non-informative for the family. Then, we sequenced the IMP-JST162148 PCR product (http://snp.ims.u-tokyo.ac.jp) [Haga et al., 2002], which contained one of the SNPs and was located 3.5 kb downstream ofUBE3A exon 15. Sequence analysis identified a novel SNP in IMP-JST162148 that coulddiscriminate parental alleles in the family. The family was tested for this novel SNP. The affected siblings were heterozygous for the SNP (T/C), as was their father; themother had T/T alleles (Fig. 2). Therefore, the T allele of the SNP in the affected siblings must be derived from the mother. To identify which allele of the SNP was associated with the novel UBE3A splicing mutation, longrange PCR covering both the SNP and the mutation, was carried out using LA-PCR (TaKaRa, Shiga, Japan) with the IMP-JST162148 forward primer and a primer located in UBE3A intron 14 (primer sequences available on request). LA-PCR products from one of the affected siblings were cloned into the TA-vector (Invitrogen, Carlsbad, CA), and six clones were selected and sequenced. Three clones contained the Grant sponsor: Ministry of Health, Labor andWelfare of Japan; Grant number: (15B-4) The Research Grant for Nervous and Mental Disorders.

Comparison of three methods for localizing interictal epileptiform discharges with magnetoencephalography.

Purpose To compare three methods of localizing the source of epileptiform activity recorded with magnetoencephalography: equivalent current dipole, minimum current estimate, and dynamic statistical parametric mapping (dSPM), and to evaluate the solutions by comparison with clinical symptoms and other electrophysiological and neuroradiological findings. Methods Fourteen children of 3 to 15 years were studied. Magnetoencephalography was collected with a whole-head 204-channel helmet-shaped sensor array. We calculated equivalent current dipoles and made minimum current estimate and dSPM movies to estimate the cortical distribution of interictal epileptiform discharges in these patients. Results The results for four patients with localization-related epilepsy and one patient with Landau–Kleffner Syndrome were consistent among all the three analysis methods. In the rest of the patients, minimum current estimate and dSPM suggested multifocal or widespread activity; in these patients, the equivalent current dipole results were so scattered that interpretation of the results was not possible. For 9 patients with localization-related epilepsy and generalized epilepsy, the epileptiform discharges were wide spread or only slow waves, but dSPM suggested a possible propagation path of the interictal epileptiform discharges. Conclusion Minimum current estimate and dSPM could identify the propagation of epileptiform activity with high temporal resolution. The results of dSPM were more stable because the solutions were less sensitive to background brain activity.

DNA demethylation reactivation of imprinted genes in cell lines from patients with Prader-Willi syndrome and a mouse model.

Genomic imprinting refers to a parent-of-origin specific gene modification, resulting in preferential or exclusive gene expression either from the paternal or the maternal allele. Prader-Willi syndrome (PWS) is a representative disorder related to genomic imprinting [Nicholls et al., 1998]. PWS is a neurodevelopmental disorder characterized by neonatal hypotonia, obesity secondary to hyperphagia, mental retardation, and genital hypoplasia. Imprinted genes associated with PWS are clustered on 15q11q13, which harbors a series of paternally expressed genes including SNURF-SNRPN, NDN, MKRN3, and a cluster of snoRNA genes, as well as two maternally expressedgenes,UBE3A andATP10C. PWS is caused by loss of paternally expressed genes on 15q11-q13, although the causative genes are yet to be determined. The loss of function of UBE3A causes Angelman syndrome (AS), which is a distinct disorder characterized by severe mental retardation with lack of speech, ataxic movement, and seizures [Nicholls et al., 1998]. Gene silencing and genomic imprinting involve DNA methylation, histone modifications, and chromatin structure [Brannan and Bartolomei, 1999; Jenuwein and Allis, 2001]. Allele-specific DNA methylation is found throughout 15q11-q13, and the most striking difference is located in the CpG island of SNURF-SNRPN. The CpG island of SNURFSNRPN is totally unmethylated on the paternally derived active allele, whereas it is completely methylated on the maternally derived inactive allele [Nicholls et al., 1998]. Moreover, the promoter of SNURF-SNRPN displays parent-of-origin specific histone modification. Histone H3 and H4 are acetylated on the active paternal allele and hypoacetylated on the inactive maternal allele [Saitoh and Wada, 2000; Fulmer-Smentek and Francke, 2001]. Histone H3 lysin4 (H3 Lys4) is methylated on the active paternal allele, whereas histone H3 lysin9 (H3 Lys9) is methylated on the inactive maternal allele [Xin et al., 2001]. We have previously demonstrated that inactive SNURF-SNRPN could be reactivated by the DNA methyltransferase inhibitor 5-azadeoxycytidine (5aza-dC), but not by the histone deacetylase inhibitor trichostatin A (TSA) [Saitoh and Wada, 2000]. We therefore decided to extend our experiments to encompass other imprinted genes on 15q11-q13, and to also examine fibroblasts from a mouse model of PWS to investigate potential therapeutic strategies for PWS and other imprinting-related disorders. Ebstein–Barr virus-transformed lymphoblastoid cell lines (LCLs) were established from peripheral blood leukocytes of patients with PWS caused by a paternal deletion of 15q11-q13, and from normal controls using the standard methods. For mouse experiments, we used fibroblasts established from a PWS/AS mouse model (TgPWS/AS) that carried a transgene-mediated deletion in 7C, corresponding to human 15q11-q13, and fibroblasts from a wild-type mouse [Gabriel et al., 1999]. We treated the cell lines with 5-aza-dC and TSA to reactivate the silenced alleles of imprinted genes. Approximately 3 10 cells from the human LCLs were synchronized with