Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin

Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20 LD families from the Indian population. We identified 12 distinct mutations in 15 LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210 K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype.     

Lafora progressive myoclonus epilepsy mutation database‐EPM2A and NHLRC1 (EMP2B) genes

Progressive Myoclonus Epilepsy (PME) of the Lafora type is an autosomal recessive disease, which presents in teenage years with myoclonia and generalized seizures leading to death within a decade of onset. It is characterized by pathognomonic inclusions, Lafora bodies (LB), in neurons and other cell types. Two genes causing Lafora disease (LD), EPM2A on chromosome 6q24 and NHLRC1 (EPM2B) on chromosome 6p22.3 have been identified, and our recent results indicate there is at least one other gene causing the disease. The EPM2A gene product, laforin, is a protein tyrosine phosphatase (PTP) with a carbohydrate‐binding domain (CBD) in the N‐terminus. NHLRC1 encodes a protein named malin, containing a zinc finger of the RING type in the N‐terminal half and 6 NHL‐repeat domains in the C‐terminal direction. To date 43 different variations in EPM2A and 23 in NHLRC1 are known, including missense, nonsense, frameshift, and deletions. We have developed a human LD mutation database using a new generic biological database cross‐referencing platform. The database, which currently contains 66 entries is accessible on the World Wide Web ( http://projects.tcag.ca/lafora ). Entries can be submitted via the curator of the database or via a web‐based form. © 2005 Wiley‐Liss, Inc.     

Clinical and genetic data on Lafora disease patients of Serbian/Montenegrin origin

Lafora disease (LD) is an autosomal recessive, progressive disorder characterized by myoclonus and seizures, inexorable neurologic deterioration, cognitive decline and poor prognosis. LD is caused by mutations either in the EPM2A or in NHLRC1 genes. Here we report clinical and genetic findings on 14 LD patients from 10 families of Serbian/Montenegrin origin. Molecular diagnostics was performed by sequencing the coding regions of the EPM2A and NHLRC1 genes. In addition, haplotype analysis of the chromosomes carrying the two most frequent mutations (c.1048‐1049delGA and deletion of the whole NHLRC1 gene) using eight different markers flanking the NHLRC1 gene was conducted. We identified one new mutation (c.1028T>C) along with the 3 previously reported mutations (c.1048‐1049delGA, c.990delG, deletion of the whole NHLRC1 gene), all of which were located on the NHLRC1 gene. The two predominant mutations (c.1048‐1049delGA and complete NHLRC1 gene deletion) appear to be founder mutations. In addition to documenting the genetic heterogeneity observed for LD, our study suggests that mutations in the NHLRC1 gene may be a common cause of LD in the Serbian/Montenegrin population, primarily because of a founder effect.     

Lafora progressive myoclonus epilepsy: A meta‐analysis of reported mutations in the first decade following the discovery of the EPM2A and NHLRC1 genes

Lafora disease (LD) is an autosomal recessive and fatal form of progressive myoclonus epilepsy. LD patients manifest myoclonus and tonic–clonic seizures, visual hallucinations, and progressive neurologic deterioration beginning at 12 to 15 years of age. The two genes known to be associated with LD are EPM2A and NHLRC1. Mutations in at least one other as yet unknown gene also cause LD. The EMP2A encodes a protein phosphatase and NHLRC1 encodes an ubiquitin ligase. These two proteins interact with each other and, as a complex, are thought to regulate critical neuronal functions. Nearly 100 distinct mutations have been discovered in the two genes in over 200 independent LD families. Nearly half of them are missense mutations, and the deletion mutations account for one‐quarter. Several reports have provided functional data for the mutant proteins and a few also provide genotype–phenotype correlations. In this review we provide an update on the spectrum of EPM2A and NHLRC1 mutations, and discuss their distribution in the patient population, genotype–phenotype correlations, and on the possible effect of disease mutations on the cellular functions of LD proteins. Hum Mutat 0, 1–9, 2009. © 2009 Wiley‐Liss, Inc.     

Loss of function of the cytoplasmic isoform of the protein laforin (EPM2A) causes Lafora progressive myoclonus epilepsy

Lafora disease is the most severe teenage-onset progressive epilepsy, a unique form of glycogenosis with perikaryal accumulation of an abnormal form of glycogen, and a neurodegenerative disorder exhibiting an unusual generalized organellar disintegration. The disease is caused by mutations of the EPM2A gene, which encodes two isoforms of the laforin protein tyrosine phosphatase, having alternate carboxyl termini, one localized in the cytoplasm (endoplasmic reticulum) and the other in the nucleus. To date, all documented disease mutations, including the knockout mouse model deletion, have been in the segment of the protein common to both isoforms. It is therefore not known whether dysfunction of the cytoplasmic, nuclear, or both isoforms leads to the disease. In the present work, we identify six novel mutations, one of which, c.950insT (Q319fs), is the first mutation specific to the cytoplasmic laforin isoform, implicating this isoform in disease pathogenesis. To confirm this mutation's deleterious effect on laforin, we studied the resultant protein's subcellular localization and function and show a drastic reduction in its phosphatase activity, despite maintenance of its location at the endoplasmic reticulum.     

Low density lipoprotein receptor-related protein gene polymorphisms and risk for late-onset Alzheimer's disease in a Japanese population

Low density lipoprotein (LDL) receptor-related protein (LRP) gene polymorphisms located in the 5' region and in exon 3, and the apolipoprotein E (APOE) genotype were determined in 100 Japanese patients affected by late-onset Alzheimer's disease (AD). We matched 246 controls for age and found no association between the polymorphism located in the 5' region of the LRP gene. The distribution of LRP exon 3 genotypes and alleles did not differ between AD and the control groups. However, the frequency of T allele in the Alzheimer's group having APOE-epsilon4 was lower than that in the control group having APOE-epsilon4, but it was only marginally significant (p = 0.022). Age of onset was significantly younger in the patients with CC genotype than those carrying the T allele (p = 0.03), and this trend was more evident among non-APOE-epsilon4 carriers (p = 0.008). These results support the possibility that ApoE and LRP may contribute to the development of AD.     

Absence of mutation in the NOD2/CARD15 gene among 483 Japanese patients with Crohn's disease

 Chronic inflammatory bowel diseases (IBDs), specifically Crohn's disease (CD) and ulcerative colitis (UC), have increased significantly in western countries and Japan over the last decade, but very little is known about their pathogenesis. A candidate-gene approach recently identified NOD2/CARD15 as one susceptibility gene from the IBD1 locus on chromosome 16. Alterations in this gene were found in many Caucasian patients with CD; in particular, two nonsynonymous substitutions (R702W and G908R) and a frameshift mutation (1007fs) were shown to be independent risk factors for CD. We investigated DNA from 483 Japanese CD patients to detect those three mutations in NOD2/CARD15 by appropriate genotyping techniques, but found only an R702Q substitution in a single patient. Direct sequencing of DNA from 96 of our patients in the regions containing the three reported major mutations detected no sequence alterations of consequence. Our findings indicate that the NOD2/CARD15 gene is not a major contributor to CD susceptibility in the Japanese population. Received: May 15, 2002 / Accepted: May 21, 2002     

HLA-DR gene frequencies in the Japanese population obtained by oligonucleotide genotyping

ABSTRACT: We determined HLA-DRB types of 375 randomly chosen healthy Japanese donors using a set of 29 different sequence-specific oligonucleotide (SSO) probes directed against various DRB alleles. Except for a few cases, these SSOs enabled us to identify 33 different DRB types including those detectable only by SSO genotyping. Gene frequencies were calculated for each of the DRB types identified. The "blank" frequency calculated by our SSO typing was essentially zero, in contrast to the considerably high 'blank" frequencies reported at serological HLA-DR or cellular HLA-D workshops. This indicates that almost all of the DRB types in the Japanese population are positively detectable by our SSO typing. By comparing the gene frequencies for each of the DR types obtained by our SSO typing with those obtained by immunological typing at workshops, significant differences were observed for several of the DR types.     

Variations in the FTO gene are associated with severe obesity in the Japanese

Variations in the fat-mass and obesity-associated gene (FTO) are associated with the obesity phenotype in many Caucasian populations. This association with the obesity phenotype is not clear in the Japanese. To investigate the relationship between the FTO gene and obesity in the Japanese, we genotyped single nucleotide polymorphisms (SNPs) in the FTO genes from severely obese subjects [n = 927, body mass index (BMI) > or = 30 kg/m2] and normal-weight control subjects (n = 1,527, BMI < 25 kg/m2). A case-control association analysis revealed that 15 SNPs, including rs9939609 and rs1121980, in a linkage disequilibrium (LD) block of approximately 50 kb demonstrated significant associations with obesity; rs1558902 was most significantly associated with obesity. P value in additive mode was 0.0000041, and odds ratio (OR) adjusted for age and gender was 1.41 [95% confidential interval (CI) = 1.22-1.62]. Obesity-associated phenotypes, which include the level of plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure were not associated with the rs1558902 genotype. Thus, the SNPs in the FTO gene were found to be associated with obesity, i.e., severe obesity, in the Japanese.     

Mutations of the puratrophin-1 (PLEKHG4) gene on chromosome 16q22.1 are not a common genetic cause of cerebellar ataxia in a European population

Autosomal dominant cerebellar ataxia (ADCA) is a genetically heterogeneous group of neurodegenerative disorders with overlapping clinical presentation. Recently, a single nucleotide substitution in the 5-untranslated region (UTR) of the puratrophin-1 (PLEKHG4) gene on chromosome 16q22.1 has been shown to be associated with ADCA in 52 unrelated Japanese families. As this mutation has so far not been investigated in other populations, we have screened 537 European patients with a clinical diagnosis of cerebellar ataxia for this specific nucleotide substitution. The mutation was not identified in our cohort. In addition, we screened the complete 5-UTR as well as the entire coding region of this gene in 120 patients for variations that might account for their clinical symptoms. Several new rare variations were found. For none of the variations could an obvious pathogenetic relevance be postulated at this point, albeit some findings should be followed up in additional populations and by functional assays. We conclude that mutations of the puratrophin-1 gene are not a common cause of hereditary ataxia in our Caucasian population.     

HapMap coverage for SNPs in the Japanese population

The coverage of human genomic variations is known to substantially affect the success of genome-wide association studies. We therefore assessed the SNP coverage in the HapMap database for a total of 1,304 subjects from the Japanese population by combining resequencing and high-density genotyping approaches. First, we resequenced 48 Japanese subjects in 86 genes (572 kb in total), and we then genotyped the subset of tag SNPs and also imputed genotypes for all of the detected SNPs in an additional panel of 1,256 subjects. Subsequently, we genotyped 555,352 tag SNPs selected from the HapMap in 72 Japanese subjects (from the panel of 1,256 subjects) and further imputed genotypes for all SNPs currently included in the HapMap. Of 738 common genic SNPs (1.3 per kb) that we detected by resequencing, 58% had already been genotyped in the HapMap, and 31% were not genotyped but had a proxy SNP in the HapMap with a linkage disequilibrium coefficient r ² ≥ 0.8, whereas 11% were not represented in the current HapMap database. Thus, the HapMap coverage appears to be high although not thorough for SNPs in the Japanese population as compared to its coverage reported in Caucasians, and this needs to be considered when we interpret association results. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mutations in VPS26A are not a frequent cause of Parkinson's disease

VPS35 mutations have been identified as a cause of autosomal dominantly inherited Parkinson's disease (PD). VPS35 interacts with VPS26A in the retromer complex that links mitochondrial and lysosomal pathways, which have both been shown to be dysfunctional in PD. Thus, mutations in VPS26A may be associated with PD. To test this hypothesis, we screened 245 idiopathic PD patients and 185 control subjects for mutations in the retromer subunit VPS26A. We found 2 novel missense variants in patients and 2 known missense variants in control subjects. The missense variants were unlikely to be disease causing, suggesting that VPS26A mutations are not a frequent cause of PD.     

Mutations in the profilin 1 gene are not common in amyotrophic lateral sclerosis of Chinese origin

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease primarily involving the corticospinal tract, brainstem, and anterior cells of the spinal cord. Mutations in the profilin 1 gene (PFN1) were recently described in ALS families. To investigate the spectrum and frequency of PFN1 mutations further, we sequenced all 3 exons of the PFN1 gene in 20 familial ALS index cases, 324 sporadic ALS patients, and 355 healthy control subjects. No nonsynonymous coding variants were identified. Our findings suggest that mutations in the PFN1 gene are not a common cause of ALS in the Chinese population.     

Detecting gene mutations in Japanese Alzheimer's patients by semiconductor sequencing

Alzheimer's disease (AD) is the most common form of dementia. To date, several genes have been identified as the cause of AD, including PSEN1, PSEN2, and APP. The association between APOE and late-onset AD has also been reported. We here used a bench top next-generation sequencer, which uses an integrated semiconductor device, detects hydrogen ions, and operates at a high-speed using nonoptical technology. We examined 45 Japanese AD patients with positive family histories, and 29 sporadic patients with early onset (<60-year-old). Causative mutations were detected in 5 patients in the familial group (11%). Three patients had a known heterozygous missense mutation in the PSEN1 gene (p.H163R). Two patients from 1 family had a novel heterozygous missense mutation in the PSEN1 gene (p.F386L). In the early onset group, 1 patient carrying homozygous APOEε4 had a novel heterozygous missense mutation in the PSEN2 gene (p.T421M). Approximately 43% patients were APOEε4 positive in our study. This new sequencing technology is useful for detecting genetic variations in familial AD.     

Two novel mutations in the <Emphasis Type="Italic">EPM2A </Emphasis>gene in a Korean patient with Lafora's progressive myoclonus epilepsy

 The progressive myoclonus epilepsy of the Lafora type (LD; MIM 254780) is a rare autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration, and the presence of periodic acid-Schiff-positive polyglucosan inclusions (Lafora bodies). Mutations in the EPM2A gene have recently been found to cause LD and about 30 or more mutations have been reported thus far. LD is relatively common in countries of the Mediterranean Basin, the Middle East, India, and Pakistan. Although a few sporadic cases with the typical LD phenotype have also been reported in the Far East including Korea and Japan, a recent effort to find mutations in Japanese LD families was not successful. In the present study, we report two novel mutations in a Korean girl with LD; a 1-bp insertion mutation (c.223insC; G75fsX107) in exon 1 and a missense mutation (c.559A>G; T187A) in exon 3 of the EPM2A gene. To our knowledge, this is the first report of a genetically confirmed case of LD in Koreans and also in the Far East. Received: September 2, 2002 / Accepted: November 8, 2002 Acknowledgments This work was supported by the fund from the Center for Functional Analysis of Human Genome (FG-2–1-02), Korea. The third and sixth authors contributed equally to this work. Correspondence to:J.-W. Kim     

Mutations in the COL1A2 gene of type I collagen that result in nonlethal forms of osteogenesis imperfecta

Although virtually all mutations that result in osteogenesis imperfecta (OI) affect the genes that encode the chains of type I procollagen, the effects of mutations in the COL1A2 gene have received less attention than those in the COL1A1 gene. We have characterized mutations in 4 families that give rise to different OI phenotypes. In three families substitutions of glycine residues by cysteine in the triple helical domain (a single example at position 259 and 2 families in which substitution of glycine at 646 by cysteine) have been identified, and in the fourth a G for A transition at position + 4 in intron 33 led to use of an alternative splice site and inclusion of 6 amino acids (val-gly-arg-ile-leu-phe) between residues 585 and 586 of the normal triple helix. The relation between position of substitution of glycine by cysteine in the COL1A2 gene does not follow the pattern developed in the COL1A1 gene. To determine how COL1A2 mutations produce OI phenotypes, we have produced a full-length mouse cDNA into which we plan to place mutations and examine their effects in stably transfected osteogenic cells and in transgenic animals.     

No evidence for association of the ENPP1 (PC-1) K121Q variant with risk of type 2 diabetes in a Japanese population

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, also known as PC-1) inhibits insulin signal transduction pathway(s). Previous studies have demonstrated the K121Q variant of the ENPP1 gene to have a significant functional role in determining susceptibility to insulin resistance and type 2 diabetes (T2D). To assess whether the K121Q variant has any impact on T2D in Japanese, we undertook an extensive case-control association study using a total of 911 unrelated Japanese T2D patients and 876 control subjects. No significant difference was observed in either genotype distribution (P=0.95) or allele frequency (P=0.83) between T2D and control groups. Notably, the frequency of the ancestral Q121 allele, which is also present in other primates, was quite high in African-Americans, and showed a marked ethnic variation (77.3% in African-Americans, 16.7% in European Americans, 10.5% in Japanese and 4.2% in Han Chinese). Consequently, the pairwise F_ST value (a classic measure of genetic distance between pairs of population) showed highly significant differentiations between African-American and non-African-American populations (F_ST>0.3). Our results indicated that the K121Q variant of the ENPP1 gene has very little, if any, impact on T2D susceptibility in Japanese, but may play a role in the inter-ethnic variability in insulin resistance and T2D.An erratum to this article can be found at     

Molecular basis of fabry disease: Mutations and polymorphisms in the human α-galactosidase A gene

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the α-galactosidase A gene at Xq22.1. Studies of the mutations in unrelated Fabry families have identified a variety of lesions indicating the molecular genetic heterogeneity underlying the disease. Forty-nine different mutations have been described including five partial gene deletions, one partial gene duplication, nine small deletions and insertions, three splice junction consensus site alterations, and 31 coding region single base substitutions. Most mutations resulted in the classical disease phenotype; however, five missense mutations were detected in atypical hemizygotes who were asymptomatic or had symptoms confined to the heart, including N215S, which was described in three unrelated atypical males. Most mutations were confined to a single pedigree with the exception of N215S, R227Q, R227X, R342Q, and R342X, which were each found in several unrelated families. Five of the 14 coding region CpG dinucleotides were sites of point mutations including the CpGs in codons 227 and 342, which were each mutated in both orientations. The identification of the mutation in a given Fabry family permits precise prenatal diagnosis and heterozygote detection of other family members with this X-linked recessive disease. Studies of additional Fabry families will provide information on the nature and frequency of the mutations causing this disease as well as potential insights into the structure/ function relationships of this lysosomal hydrolase. © 1994 Wiley-Liss, Inc.