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Genetic Susceptibility Study of Temporal Lobe Epilepsy Targeted Genomic Sequencing Identifies PRRT2Mutations as a Cause of Paroxysmal Kinesigenic Choreoathetosis

Author: LiJingYun
Tutor: LiuYing
School: Beijing Union Medical College
Course: Biochemistry and Molecular Biology
Keywords: temporal lobe epilepsy genetic polymorphism 5-HTT gene KCNMB4gene SLC6A12gene BDNF geneparoxysmal kinesigenic choreoathetosis proline-rich transmembrane protein2gene nextgeneration sequencing mutation
CLC: R742.1
Type: PhD thesis
Year: 2012
Downloads: 86
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Abstract


BackgroundsEpilepsy is one of the most common neurological diseases and characterized by the presence of recurrent and spontaneous seizures, which can bring a series of physical and social psychical issues. In all types of focal epilepsy syndromes, temporal lobe epilepsy (TLE) has the highest incidence and is likely to cause drug resistance. Genetic and environment factors are believed to play important roles in the aetiology of TLE. Genetic variations can influence occurrence, development and prognosis of TLE by several aspects including seizure one-set, clinical endophenotypes, antiepileptic drug response and drug resistance. Candidate gene association studies have been proposed as method choice to understand the genetic background of TLE. Although some studies have reported an association between common variants in specific genes and TLE, the results have been plagued by a lack of replicability. Therefore, This study was untaken to test the hypothesis that one or more variants in serotonin transporter (5-HTT), potassium large conductance calcium-activated channel subfamily M beta member (KCNMB4), betaine/GABA transporter-1(BGT-1or SLC6A12) and brain derived neurophic factor (BDNF) genes were associated with TLE and some clinical symptoms using case-control design. Next, the functional studies including gene expression, protein level and brain imaging were carried out to reveal the pathogenesis of those positive variants.MethodsEligible TLE patients and non-epileptic control subjects from Han Chinese origin were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan(?)methods were used for genotyping. The expression level of SLC6A12gene in peripheral blood mononuclear cells from control samples were detected by real-time PCR. Plasma BDNF levels were measured by enzyme-linked immunosorbent assay (ELISA). Voxel-based morphometry (VBM) on volumetric MRI were used for hippocampus volumetric measurement.Results1. Association of polymorphisms in5-HTT gene with TLEThree functional polymorphisms (5-HTTLPR, rs25531and5-HTTVNTR) in5-HTT gene were genotyping in334TLE patients and487non-epileptic control subjects.10-repeat allele frequency of5-HTTVNTR was moderately higher in patients than in controls (9.1%vs.6.1%, P=0.0187, OR=1.55,95%CI=1.07-2.26).2. Association of tagSNPs in KCNMB4gene with TLEFour tagSNPs (rs710665, rs11178214, rs9634299and rs7301967) in KCNMB4gene were genotyping in358TLE patients and496non-epileptic control subjects. The single-marker analysis did not show any allelic associations with TLE. Haplotypic analysis for four SNPs indicated that the frequency of haplotype C-C-G-T was lower in TLE patients than in controls (28.5%vs.33.6%, P=0.0258, OR=0.79,95%CI=0.64-0.98), but P value did not reach a significant level when Bonferroni correction was applied. When TLE patients were subgrouped according to the presence of hippocampal sclerosis (HS), rs7301967-C allele frequency was higher in TLE-HS(+) than in non-epileptic controls (53.6%vs.45.2%, P=0.041, OR=1.41,95%CI=1.01-1.95).3. Association of polymorphisms in SLC6A12gene with TLEFour tagSNPs (rs542736, rs2284329, rs216243and rs9783494) and one missense SNP (rs557881) were genotyping in358TLE patients and496non-epileptic control subjects. The single-marker analysis did not show any allelic associations with TLE. Haplotypic analysis found rs557881as a compensatory variation might interact epistatically with rs542736. Among individuals carrying rs557881AA homozygote, the rs542736G allele was associated with susceptibility to TLE (P=6.6×10-5), febrile seizure (P=0.012) and increased expression of SLC6A12gene in control subjects (P=1.08×10-8). However, the rs542736G allele did not show any association in rs557881GG carriers.4. Association of polymorphisms in BDNF gene with TLEOne missense SNP (rs6265) was genotyping in360TLE patients and501non-epileptic control subjects. The A-allele frequency was significantly lower in patients than in controls (42.8%vs.49.6%, P=0.005, OR=0.76,95%CI=0.63-0.92). Clinical phenotype analysis showed rs6265-A alllele was associated with febrile seizures (FS) and hippocampal sclerosis (HS)(P=0.003and0.029, respectively). Next, we detected the plasma BDNF levels among17TLE patients and101non-epileptic control subjects. Significantly higher plasma BDNF levels were observed for TLE patients compared with controls (P=4.71×10-7). However, the plasma BDNF levels were not associated with rs6265genotype.Finally, we measured the hippocampus volume and calculated the asymmetry index of hippocampus (AI) among12TLE-HS(+),17TLE-HS(-) and29controls. The AI levels were significantly higher in TLE-HS(+) group than in two another groups (P=2.85×10-5). However, The AI levels were not correlated to rs6265genotype. ConclusionsOur study suggested5-HTT, SLC6A12and BDNF genes variabilities might be associated with TLE susceptibility in Han Chinese, and the role of KCNMB4in the aetiology of TLE may be small or modest. BackgroundParoxysmal kinesigenic choreoathetosis (PKC) is a rare neurological disorder and characterised by recurrent and brief attacks of involuntary movement, inherited as autosomal dominant trait. We previously mapped the disease locus to a region at16p11.2-q12.1that is critical to PKC in four Chinese families. However, the causative gene remains unidentified. Recently, next generation sequencing (NGS) using targeted capture array and high-throughput resequencing platform has proven to be effective for discovering novel causative mutations in inherited diseases. Therefore, we applied NGS to screen the causative gene of PKC.MethodsThe shotgun fragment libraries were hybridized to a custom NimbleGen array targeting this30Mb region and the eluate sequenced on the Illumina HiSeq2000platform in five affected individuals and one healthy control (did not carry the risk haplotype we previously identified) from our four families. Sanger sequencing confirmed the results and screened the mutations in29sporadic PKC individuals.ResultsDeep sequencing showed that a total of149-160nonsynonymous variants, splice-site acceptor or donor variants and coding insertions or deletions (NS/SS/I) were detected in55genes per affected individual. After filtering for variants found in dbSNP129,1000Genome Project and the healthy control sequencing data to remove shared variants, we identified proline-rich transmembrane protein2gene (PRRT2) harbored two heterozygous PRRT2insertions (c.369dupG and c.649dupC), producing frameshifts and premature stop codons (p.S124VfsX10and p.R217PfsX8, respectively) in two different families. Sanger sequencing confirmed these two mutations and revealed a missense PRRT2mutation (c.859G>A, p.A287T) in one of the two remaining families. We also sequenced PRRT2in29sporadic cases affected with PKC and identified mutations in ten cases, including six with the c.649dupC mutation. In a word, we indentified seven types of mutations in PRRT2.Conclusions Our present study identifies PRRT2as the gene mutated in a subset of PKC, and suggests that PKC is genetically heterogeneous.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Brain diseases > Epilepsy
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