Gene Mapping in Families with Inherited Epilepsy Syndromes

Abstract

Epilepsy is a mysterious problem of nervous system. It has been reported in earliest times of human history. The epilepsy syndromes are hard to define; they are so heterogeneous phenotypically that diagnosis becomes a problem for clinicians. Two or more unprovoked seizures are criteria to diagnose epilepsy in any patient. Along with environmental factors, genetics plays a significant role in causation of epilepsy. Inherited epilepsy when reported with other anomalies is called an epilepsy syndrome. In inherited epilepsy syndromes, efforts have been made to identify the disease-causing gene mutations in humans. In this study, seven families were identified with affected individuals showing neurological symptoms including epilepsy. They include two families (Families A & B) with Lafora Disease (LD), One family (Family C) with Hereditary Spastic Paraplegia 26 (HSP26), One family (Family D) with Bainbridge-Ropers Syndrome (BRS), One family (Family E) with Chorea-Acanthocytosis (ChAC) and two families (Families F & G) with epilepsy and neurological signs. By using different next generation sequencing platforms, causative mutations were identified in four families. Lafora epilepsy disease (LD; MIM 254780) is myoclonic epilepsy having autosomal recessive inheritance. The onset is from late childhood to early adolescent. EPM2A, NHLRC1 and PRDM8 genes have been identified previously to be mutated in affected individuals with LD. The families, A and B are unrelated consanguineous families belong to Pakistan showing features of LD. Affected members in both families have, generalized tonic clonic seizures, cognitive decline, and intellectual disability with ataxia. Lafora Disease was diagnosed by both histo-pathological analysis of the skin biopsy and electroencephalogram. Illumina TruSight One Abstract x Sequencing Panel covering 4813 OMIM genes was done for family A. We identified a homozygous mutation c.T94G; p.W32G of EPM2A gene which was found co-segregated in this family through Sanger sequencing. Bi-directional sequencing was done for both EPM2A and NHLRC1 genes in family B but no mutation was identified. Hereditary Spastic Paraplegias (HSPs) is a group of heterogeneous disorders characterized with progressive spasticity and weakness of the lower limbs sometimes combined with additional neurological like ataxia, epilepsy, neurodegenerative and neurodevelopmental disorders. HSPs have more than seventy types. Family C belongs to Kingdom of Saudi Arabia (KSA) having consanguineous union. The affected individuals have episodic ataxia with myokemia and microcephaly. The index case presented with episodic ataxia with myokemia, proximal muscle weakness, preserved DTR and intact cognition. Genetic analysis identified a novel missense homozygous mutation in B4GALNT1, c.C1358G:p.P453R by using whole exome sequencing of the two affected individuals in the family (HSP26; MIM 609195). The mutation was co-segregated in the family by bi-directional sequencing. Bainbridge-Ropers syndrome (BRS; MIM 615485) is a rare genetically inherited disorder with severe developmental delay, feeding problems, short stature, epileptic seizures in some patients, characteristic facial appearance. Family D is non-consanguineous family belonging to Republic of South Korea. The index case is affected male who was presented to clinic with global developmental delay, myoclonic seizures and dysmorphic features. His myoclonic seizures made it suspicious case of Lafora body’s disease. Whole exome sequencing was done, and data analysis found a de-novo substitution deletion mutation in ASXL3 gene: c.1314_1316delinsA:p.S439Rfs*7/+ that was confirmed by bi-directional Sanger sequencing and was absent in his parents. Abstract xi Chorea-Acanthocytosis (MIM#200150) is a rare progressive disorder associated with neurodegeneration. The mutations in VPS13A gene are reported to cause the disease. The clinical features are loss of cognitive and locomotor functions with severe tics. In Family E, a three generation pedigree from Pakistan, clinical diagnosis of the affected members revealed phenotypes of episodes of seizures, tics, hyperactive behavior, tongue and lip bites with psychiatric problems. Through whole genome Copy Number Variation (CNV) data analysis, a ~1.2 Kb deletion was identified in VPS13A gene already implicated in Chorea-Acanthocytosis, and found co-segregated with disease phenotype in this family through Sanger sequencing. Two other families with multiple affected individuals (Families F and G), showed multiple neurological deficits including epilepsy. Whole exome sequencing and extensive data analysis was unable to find segregation of potential selected variants in respective families. These results show the limitations of WES for identification of disease gene causing mutations in complex epilepsy syndromes. In future, whole genome sequencing would be used to resolve these cases. At present, bioinformatics tools are playing critical roles in prediction and analysis of the disease associated variants. We selected 32 epilepsy associated risk loci (available online) from six Genome Wide Association Studies (GWAS) to predict proxy Single Nucleotide Polymorphisms (SNPs), from SNAP tool based on linkage disequilibrium. These results were then used as input data for RegulomeDB; a software for understanding of regulatory elements in human genome to predict their potential functional roles in epilepsy. Only 10 SNPs returned with significant scores, indicating the regulatory function. Structural insights of the human B4GALNT1 protein were predicted by In-silico studies. One signal peptide and one trans membrane domain predicted in human wild type B4GALNT1 protein with aliphatic Abstract xii index of 92.76 and theoretical (iso-electric point) pI of 8.93. The protein showed interaction with different proteins including ST8SIA5, SLC33A1 and GLB1. Impact of all reported missense mutations in B4GALNT1 (in cHSP26 phenotype) has shown the decrease in stability of protein. Next generation sequencing along with bioinformatics analysis is growing field of clinical genetics to precisely diagnose the heterogenous inherited disorders like epilepsy syndromes. The present study will help genetic counseling of the families. It also has impact on establishment of clinical genetics studies in families with complex epilepsy syndromes and to understand molecular mechanisms involved in these disorders.