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Autosomal recessively inherited progressive myoclonic epilepsy of Lafora, which is also known as Lafora disease (LD; MIM# 254780) is a fatal neurodegenerative disorder. It affects individuals in late childhood or early adolescence and presents with symptoms of progressive mental and physical deterioration. Progressively increasing myoclonic jerks, seizures (myoclonic, tonic–clonic, or focal occipital), evocative visual symptoms and cognitive decline with fatal outcome within 5–10 years of diseases onset are typical clinical features of LD. Periodic acid–Schiff (PAS) positive cytoplasmic inclusions called Lafora bodies composed of dense aggregates of polyglucosan fibrils are found in most tissues of the affected individuals. LD is genetically heterogeneous and is caused by mutations in either EPM2A gene (OMIM 607566) on chromosome 6q24 encoding laforin or NHLRC1/EPM2B gene (OMIM 608072) on chromosome 6p22 encoding malin [
Lafora progressive myoclonus epilepsy: a meta-analysis of reported mutations in the first decade following the discovery of the EPM2A and NHLRC1 genes.
]. In the current study, we performed whole exome sequencing (WES) to identify pathogenic variant underlying LD in a consanguineous Pakistani family.
2. Case report
Approval of the study was obtained from the Institutional Review Board of Quaid-i-Azam University, Islamabad. Guardians signed an informed consent form. Clinical findings and diagnostic tests were carried out at Pakistan Institute of Medical Sciences Islamabad. Peripheral blood (5 ml) was drawn from the subjects and stored in EDTA tubes at 4 °C. The patient VI-6, a 17 years old male, at the time of the study presented with myoclonic jerks, generalized-tonic-clonic seizures, dysarthria including confusion and visual hallucination as aura of the seizures. He had these symptoms presented at the age of 11, which became progressively intractable. EEG showed generalized background slowing with abnormal spikes. MRI studies did not reveal any brain malformations; muscle biopsies (with PAS staining) from eccrine and apocrine regions showed normal muscular structure and were negative for Lafora bodies. During three years of follow up study, the patient underwent a sharp mental decline and speech impairment along with losing control of daily functions, swallowing, walking and other motor movements. In the pedigree (Fig. 1), the members VI-2,-3,-4,-5 all showed the identical clinical phenotypic course of LD and died around the ages of 14, 15, 15, and 17, respectively. Even though their DNA was not available for analyses, the phenotypic correlations were made based on the medical records.
Fig. 1Pedigree structure and autosomal recessive segregation of EPM2A sequence variant c.262 T > G (p.F88V) in a Pakistani family affected with Lafora disease phenotype. The filled symbols indicate the affected individuals. Whole Exome Sequencing was performed for the two individuals marked with asterisk.
DNA extraction of affected VI-6 and normal V-4, using the QIAamp DNA blood Maxi kit (Qiagen, Hilden, Germany) was preceded to WES using the SureSelect V4 kit (Agilent Technologies, Santa Clara, CA, USA) and sequenced as 100-bp paired-end on an Illumina HiSeq2000 sequencer (Illumina, San Diego, CA, USA). Raw FASTQ files were assembled onto hg19 assembly (NCBI build GRCh37) with the help of SeqMan NGen and annotated with ArrayStar according to dbSNP142. The genotype of the candidate variant was screened in five family members and 100 healthy controls from local population by Sanger sequencing. In silico analysis using MutationTaster, SIFT, PolyPhen and InterVar classification system were applied to evaluate pathogenic effect of the candidate variants [
WES of the patient VI-6 and normal V-4 gave an average of 97% of reads with >20 Phred score totaling 9.2 Gb in size. Filtering of 37,858 variants based on their functional significance, low frequency (<0.01 MAF) and recessive pattern of inheritance identified a novel homozygous missense variant (c.262 T > G) in EPM2A. This variant changed the amino acid phenylalanine with valine at position 88 (p.F88V) in translated protein. The variant segregated within the family in autosomal recessive fashion and was not found in 100 healthy controls from local population (Fig. 2). The variant was not listed in dbSNP, ExAC, 1000 Genomes and gnomAD. In silico prediction for the EPM2A variant p.F88V were ‘disease causing’ by MutationTaster, ‘damaging’ by SIFT, ‘possibly damaging’ by PolyPhen and likely pathogenic’ by InterVar classification system. The MutationTaster-based amino acid conservation analysis showed that p.F88 amino acid of EPM2A protein was highly conserved among vertebrate and non-vertebrate species.
Fig. 2Sequence analysis of the EPM2A gene (c.262 T > G). Sanger sequencing results of the normal (IV-8, VI-8 with normal T/T allele, V-1, -3, -4 and VI-1 with one mutated allele T/G) and affected (VI-6 with two mutated alleles G/G) individuals.
Laforin is a dual-specificity protein phosphatase containing an N-terminal carbohydrate-binding module (CBM) that binds to glycogen. Its concentration is regulated by malin, an E3 ubiquitin ligase, by means of polyubiquitin-dependent degradation. Laforin-Malin complex is involved in the regulation of glycogen metabolism through an unknown mechanism [
Lafora progressive myoclonus epilepsy: a meta-analysis of reported mutations in the first decade following the discovery of the EPM2A and NHLRC1 genes.
This study reports a homozygous missense variant c.262 T > G (F88V) in a Pakistani family affected with Lafora disease, which lies in the β4 chain of CBM of laforin protein. To date, 74 different mutations are reported in the EPM2A gene according to The Human Gene Mutation Database (http://www.hgmd.cf.ac.uk). Several mutations in the CBM are known with myriad of functional effects on the protein; like W32G, K87A and W99A result in 57%–96% decrease in glycogen phosphatase activity without bringing any structural or binding bias to laforin for glycogen. CBM domain of laforin engages the substrate glycogen and therefore, sequence variants affecting either the stability of CBM or its binding to glycogen could render the protein ineffective [
]. This study further supports the evidence for the importance of CBM domain in laforin expanding the spectrum of EPM2A mutations. Functional studies are needed to elucidate the molecular mechanisms of the novel EPM2A variant c.262 T > G (F88V) underlying Lafora disease.
Ethical publication statement
We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.
Disclosure of conflict of interest
None.
Acknowledgements
This study was supported to CK by the intramural grant from Sungshin Women’s University (2017-1-21-006/1). ZA was supported by Indigenous Ph.D. Fellowship of Higher Education Commission of Pakistan.
References
Singh S.
Ganesh S.
Lafora progressive myoclonus epilepsy: a meta-analysis of reported mutations in the first decade following the discovery of the EPM2A and NHLRC1 genes.
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