Kana Hosoki

Prion-like domains in RNA binding proteins are essential for building subnuclear paraspeckles

Paraspeckles are mammalian subnuclear bodies built on a long noncoding RNA and are enriched in RNA binding proteins with prion-like domains; two of these proteins, RBM14 and FUS, use these domains to hold paraspeckles together.

Epimutation (hypomethylation) affecting the chromosome 14q32.2 imprinted region in a girl with upd(14)mat-like phenotype.

Maternal uniparental disomy for chromosome 14 (upd(14)mat) causes clinically discernible features such as pre- and/or postnatal growth failure, hypotonia, obesity, small hands, and early onset of puberty. The monoallelic expression patterns at the 14q32.2 imprinted region are tightly related to methylation status of the DLK1–MEG3 intergenic differential methylation region (DMR) and the MEG3-DMR that are severely hypermethylated after paternal transmission and grossly hypomethylated after maternal transmission. We examined this imprinted region in a 2 2/12-year-old Japanese patient who was born with a normal birth size (length, +0.2 SD; weight, −0.5 SD) and showed postnatal growth failure (height, −3.1 SD; weight, −3.4 SD), hypotonia, frontal bossing, micrognathia, and small hands. Methylation analysis, genotyping analysis, and deletion analysis were performed with blood samples of the patient and the parents, showing that the DMRs of this patient were grossly hypomethylated in the absence of upd(14)mat and deletion of the DMRs. The results indicate the occurrence of an epimutation (hypomethylation) affecting the normally methylated DMRs of paternal origin, and imply that epimutations should be examined in patients with upd(14)mat-like phenotype.

West syndrome associated with mosaic duplication of FOXG1 in a patient with maternal uniparental disomy of chromosome 14

FOXG1 on chromosome 14 has recently been suggested as a dosage‐sensitive gene. Duplication of this gene could cause severe epilepsy and developmental delay, including infantile spasms. Here, we report on a female patient diagnosed with maternal uniparental disomy of chromosome 14 and West syndrome who carried a small supernumerary marker chromosome. A chromosomal analysis revealed mosaicism of 47,XX, + mar[8]/46,XX[18]. Spectral karyotyping multicolor fluorescence in situ hybridization analysis confirmed that the marker chromosome was derived from chromosome 14. A DNA methylation test at MEG3 in 14q32.2 and microsatellite analysis using polymorphic markers on chromosome 14 confirmed that the patient had maternal uniparental disomy 14 as well as a mosaic small marker chromosome of paternal origin containing the proximal long arm of chromosome 14. Microarray‐based comparative genomic hybridization analysis conclusively defined the region of the gain of genomic copy numbers at 14q11.2‐q12, encompassing FOXG1. The results of the analyses of our patient provide further evidence that not only duplication but also a small increase in the dosage of FOXG1 could cause infantile spasms.

A loss-of-function mutation in the SLC9A6 gene causes X-linked mental retardation resembling Angelman syndrome†

SLC9A6 mutations have been reported in families in whom X‐linked mental retardation (XMR) mimics Angelman syndrome (AS). However, the relative importance of SLC9A6 mutations in patients with an AS‐like phenotype or XMR has not been fully investigated. Here, the involvement of SLC9A6 mutations in 22 males initially suspected to have AS but found on genetic testing not to have AS (AS‐like cohort), and 104 male patients with XMR (XMR cohort), was investigated. A novel SLC9A6 mutation (c.441delG, p.S147fs) was identified in one patient in the AS‐like cohort, but no mutation was identified in XMR cohort, suggesting mutations in SLC9A6 are not a major cause of the AS‐like phenotype or XMR. The patient with the SLC9A6 mutation showed the typical AS phenotype, further demonstrating the similarity between patients with AS and those with SLC9A6 mutations. To clarify the effect of the SLC9A6 mutation, we performed RT‐PCR and Western blot analysis on lymphoblastoid cells from the patient. Expression of the mutated transcript was significantly reduced, but was restored by cycloheximide treatment, indicating the presence of nonsense mediated mRNA decay. Western blot analysis demonstrated absence of the normal NHE6 protein encoded for by SLC9A6. Taken together, these findings indicate a loss‐of‐function mutation in SLC9A6 caused the phenotype in our patient.

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

Clinical phenotype and candidate genes for the 5q31.3 microdeletion syndrome

Array‐based technologies have led to the identification of many novel microdeletion and microduplication syndromes demonstrating multiple congenital anomalies and intellectual disability (MCA/ID). We have used chromosomal microarray analysis for the evaluation of patients with MCA/ID and/or neonatal hypotonia. Three overlapping de novo microdeletions at 5q31.3 with the shortest region of overlap (SRO) of 370 kb were detected in three unrelated patients. These patients showed similar clinical features including severe neonatal hypotonia, neonatal feeding difficulties, respiratory distress, characteristic facial features, and severe developmental delay. These features are consistent with the 5q31.3 microdeletion syndrome originally proposed by Shimojima et al., providing further evidence that this syndrome is clinically discernible. The 370 kb SRO encompasses only four RefSeq genes including neuregulin 2 (NRG2) and purine‐rich element binding protein A (PURA). NRG2 is one of the members of the neuregulin family related to neuronal and glial cell growth and differentiation, thus making NRG2 a good candidate for the observed phenotype. Moreover, PURA is also a good candidate because Pura‐deficient mice demonstrate postnatal neurological manifestations.

MERRF/MELAS overlap syndrome: a double pathogenic mutation in mitochondrial tRNA genes

Background Myoclonic epilepsy with ragged-red fibres (MERRF) and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are established phenotypes of mitochondrial encephalomyopathy. The m.8356T>C transition in the mitochondrial tRNALys gene is a pathogenic mutations of MERRF. The m.3243A>G transition in the mitochondrial tRNALeu gene is detected in most MELAS patients. Although previous analyses of double mutations in mitochondrial DNA (mtDNA) were useful for discussing their nature, many unsolved questions remain. Objective To describe the clinical and genetic features of a family with the above mtDNA double-point mutations and discuss the role of double mtDNA mutations in diverse clinical features in the family. Patients and methods The proband was a 23-year-old woman with MERRF harbouring m.8356T>C and m.3243A>G transitions in mitochondrial tRNA genes. We assessed clinical aspects of her and those of her three relatives and performed mutation analyses on their mtDNA. Results Phenotypes of the four patients were MERRF, MERRF/MELAS overlap syndrome and asymptomatic carrier. We hypothesise that the course of the phenotype of this family begins with MERRF and is followed by MELAS. This double mutation was heteroplasmic in blood of all four patients but with different rates in each patient, while m.8356T>C appeared homoplasmic and m.3243A>G was heteroplasmic in muscle of the two examined cases. No other mutations were detected in the total mtDNA sequence in this family. Conclusions This is the first reported case of a double-point mutation in mtDNA, both of which were heteroplasmic and pathogenic for the established phenotypes.

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.

Successful cochlear implantation in a patient with mitochondrial hearing loss and m.625G>A transition

OBJECTIVE We present a patient with mitochondrial hearing loss and a novel mitochondrial DNA transition, who underwent successful cochlear implantation. CASE REPORT An 11-year-old girl showed epilepsy and progressive hearing loss. Despite the use of hearing aids, she gradually lost her remaining hearing ability. Laboratory data revealed elevated lactate levels, indicating mitochondrial dysfunction. Magnetic resonance imaging showed diffuse, mild brain atrophy. Cochlear implantation was performed, and the patients hearing ability was markedly improved. Whole mitochondrial DNA genome analysis revealed a novel heteroplasmic mitochondrial 625G>A transition in the transfer RNA gene for phenylalanine. This transition was not detected in blood DNA from the patients mother and healthy controls. Mitochondrial respiratory chain activities in muscle were predominantly decreased in complex III. CONCLUSION This case indicates that cochlear implantation can be a valuable therapeutic option for patients with mitochondrial syndromic hearing loss.