Background: Lafora disease is a progressive myoclonus epilepsy with polyglucosan accumulations and a peculiar neurodegeneration with generalised organellar disintegration. It causes severe seizures, leading to dementia and eventually death in early adulthood.
Methods: One Lafora disease gene, EPM2A, has been identified on chromosome 6q24. Locus heterogeneity led us to search for a second gene using a genome wide linkage scan in French-Canadian families.
Results: We mapped a second Lafora disease locus, EPM2B, to a 2.2 Mb region at 6p22, a region known to code for several proteins, including kinesins. Kinesins are microtubule dependent motor proteins that are involved in transporting cellular components. In neurones, they play a major role in axonal and dendritic transport.
Conclusion: Analysis of the present locus in other non-EPM2A families will reveal whether there is further locus heterogeneity. Identification of the disease gene will be of major importance towards our understanding of the pathogenesis of Lafora disease.
- LB, Lafora bodies
- LD, Lafora disease
- RER, rough endoplasmic reticulum
- homozygosity mapping
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Lafora disease (LD) is the principal progressive myoclonus epilepsy with onset in adolescence. Onset is characterised by the appearance of sporadic myoclonus, occipital lobe seizures (visual hallucinations, photoconvulsive seizures), and/or generalised seizures, with intervening insiduous behavioral changes and cognitive decline. Within 3 years, myoclonus is almost constant, seizures frequent, and dementia evident. Symptoms then progress slowly in a protracted fashion often over more than 10 years. Most patients die in status epilepticus.1,2
In a number of organs, pathological analysis reveals cytoplasmic inclusions known as Lafora bodies (LB). These consist of dense aggregates of polyglucosans, which are malformed glycogen molecules lacking normal regular branching.3 Polyglucosan formation appears to initiate in association with the rough endoplasmic reticulum (RER);4 in brain, LB are found in the same neuronal compartments as RER,5 namely perikarya and dendrites, but not axons.6 Neuronal degeneration in LD occurs through an unusual non-apoptotic generalised organellar disintegration as documented in a murine model.7
In ∼70% of patients, the disease is caused by mutations in the EPM2A gene.8 Laforin, the protein product of EPM2A, localises to the RER,9,10 and contains a carbohydrate binding module11 with which it appears to bind glycogen.12 Laforin also contains a dual specificity phosphatase domain,9,10 the phosphoprotein substrate of which is still unknown.
The remaining LD families do not genetically link to the EPM2A locus.13 The number of loci containing genes that cause LD is not known, but the mode of inheritance is consistent with autosomal recessive in all families studied to date. Here, we report the identification of a second LD locus.
PATIENTS AND METHODS
The genome scan was based on four families of French-Canadian (F-C) origin with eight affected members. The parents in three families (LD7, LD28, LD27) were first degree cousins. Although the exact pedigree structure is unknown in the fourth family, LD6, consanguinity was confirmed through a review of church records. Linkage to the EPM2A locus on 6q24 was excluded in these families,13 and sequencing of the EPM2A gene revealed no mutations.
All patients followed a typical clinical course with disease onset within a year around the 12th birthday. All had LB on biopsy or at autopsy (fig 1). As an illustrative example, patient A (fig 2) developed normally until the age of 12 years, when he had his first generalised tonic-clonic seizure, followed soon after by the appearance of myoclonic jerks. EEG showed multiple spike and polyspike-slow wave generalised discharges enhanced by photic stimulation. With seizure intractability and mental decline, a liver biopsy was performed (fig 1A), and the diagnosis of LD was made. By the age of 17 years, the patient was bedridden, tube fed and demented. He died at 20 years of age. Brain autopsy was declined, but histology of the spinal cord revealed numerous pathognomonic neuronal LB (shown in reference 2). Patient A had a first cousin who died from LD at 19 years of age. Their fathers are brothers, their mothers sisters, and the fathers are first degree cousins of the mothers (fig 2).
The genome wide linkage scan was performed using Weber panel version 8A (www.marshfieldclinic.org/research/genetics). A total of 387 fluorescently labelled, highly polymorphic microsatellite markers were included in the screening set, with an average heterozygosity of 0.77 calculated from CEPH families. Markers had an average intermarker spacing distance of approximately 10 centimorgans (cM). Chromosomal regions that generated a maximum two point lod score of >1.0 with the disease were genotyped using additional flanking markers. Linkage analysis was performed, specifying an autosomal recessive mode of inheritance with no phenocopies allowed, and a disease allele frequency of 0.001. Marker allele frequencies for the genome wide linkage scan were calculated using the pedigrees being analysed (n=9-18), and a panel of ninety F-C control individuals were genotyped for five markers at the 6p22 region. Pairwise linkage analysis was calculated using the programs MLINK and ILINK.14–16 In the analysis, inbreeding loops were broken according to Terwilliger and Ott.17 The lod scores were calculated for recombination fractions ranging from 0 to 0.5 and under the assumption that there was no sex difference in recombination rates. Multipoint linkage analysis was performed using GeneHunter (version 2.1_r3 beta)18,19 with inbreeding loops kept intact. Intermarker, sex averaged distances were obtained from published maps.20
The genome wide scan of 387 markers was initiated in the F-C families, and after 303 markers were genotyped, linkage to the D6S1969 microsatellite on chromosome 6p22 was detected with a two-point lod score of 2.84 at a recombination fraction of 𝛉=0.00. To fine map the putative LD locus, 25 additional microsatellites spanning a region of 16 cM were genotyped in the four families. This resulted in maximum two-point lod scores ranging from 0.89–6.35 for 25 microsatellites at 𝛉=0.001–0.219 (table 1). The EPM2B markers are new microsatellites identified in this study (Genbank accession numbers: EPM2B-1, BVO12563; −2, BVO12564; −3, BVO12565; −3, BVO12566; −4, BVO12567; BVO12568). Multipoint linkage analysis was analysed by GeneHunter using the 26 markers spanning a genetic distance of 16 cM. This generated the same maximum homogeneity and heterogeneity lod scores of 5.37 across 13 markers in the critical interval (fig 3) and produced a heterogeneity parameter, α, of 1.00 across this region.
Haplotype analysis revealed a critical recombinant between D6S1567 and EPM2B-1 in the paternal chromosome of individual B, establishing the telomeric boundary of the disease gene region at D6S1567 (fig. 2). This person is unaffected and is 15 years older than the age of disease onset in these families. The centromeric boundary of the critical interval is defined by a recombination event observed in the maternal chromosome of individual C between D6S1588 and D6S1686. Regions of homozygosity shared by affected individuals in all four families allowed further fine mapping of the centromeric border. A nine marker haplotype (D6S1567, EPM2B-1, EPM2B-2, D6S1688, EPM2B-3, GATA137G09, D6S285, EPM2B-4, EPM2B-5) is identical and homozygous in all patients. Loss of homozygosity in the affected individuals in family LD6 beyond EPM2B-5 places the centromeric boundary at the next marker, EPM2B-6. The final critical interval is therefore between D6S1567 (telomeric) and EPM2B-6 (centromeric), spanning a distance of 2 235 308 bases. A five marker haplotype present in affected individuals of families LD7, LD27 and LD28 at markers D6S1567, D6S285, D6S1959, D6S1665, and GAAT3A06 spanning our critical interval was found to be absent in 180 F-C control chromosomes; no significant pairwise linkage disequilibrium was observed between these markers (P>0.17, alleles<20% frequency were pooled).
We have identified a second LD gene locus, named EPM2B, at 6p22 based on a study of LD families from a F-C isolate. Affected individuals from these families are probably segregating a single mutation. All four families are of exclusively F-C descent, and all originate from the same region of east Quebec (lower Saint-Lawrence, southern shore; P. Rioux et al., in preparation), their linkage to the new locus is highly significant (two-point maximum lod score=5.2), and all share the same nine marker disease haplotype. All but two of the patients in this study have died since they were recruited.
The current genetic mapping data localises the EPM2B locus to a 2.2 Mb critical region. Known proteins encoded in this region include the acute myeloid leukaemia associated protein, DEK,21 thiopurine s-methyl transferase (TPMT), responsible for the TPMT deficiency trait,22 and KIF13A, a kinesin heavy chain protein similar to Drosophila kinesin-73.23 Kinesins are microtubule dependent motor proteins that are involved in transporting cellular components. In neurones, they play a major role in axonal and dendritic transport.24 Hypothetical proteins in the region include a zinc finger, RING domain containing protein (AL832329) and an amino oxidase domain containing protein (FLJ34109). Mutation analysis of all known and putative genes in the region is in progress.
EPM2A disclosed a novel RER associated glycogen binding tyrosine phosphatase and recently its first interacting partner.25 The EPM2B protein should further unravel this biological pathway and elucidate the cellular mechanisms underlying the unique RER associated disturbance of glycogen metabolism, the non-apoptotic neurodegeneration, and the florid neuronal hyperexcitability and intractable epilepsy of LD.
Analysis of the present locus in other non-EPM2A families will reveal whether there is further locus heterogeneity. Identification of the EPM2B gene will improve patient diagnosis, prenatal diagnosis and genetic counselling for this devastating epilepsy.
We would like to thank Dr P Rioux and C Truchon for sharing the genealogical data prior to publication, the Quebec Lafora Disease Association families, patients and president (Mrs O Malenfant) for their unwavering support despite very trying times, and the Centre for Applied Genomics (http://tcag.bioinfo.sickkids.on.ca/) at The Hospital for Sick Children for technical support. This work was funded by the Canadian Institute of Health Research (CIHR) and Genome Canada. ADP holds a Canada Research Chair in Genetics of Complex Diseases. SWS is a Scholar of CIHR and International Scholar of the Howard Hughes Medical Institute.