WO2017060727A1

WO2017060727A1 - Epilepsy biomarker

Info

Publication number: WO2017060727A1
Application number: PCT/GB2016/053132
Authority: WO
Inventors: Deb PAL; Lisa Strug
Original assignee: King's College London; The Hospital For Sick Children
Priority date: 2015-10-09
Filing date: 2016-10-07
Publication date: 2017-04-13
Also published as: GB201517873D0

Abstract

The invention relates to a novel biological marker for epilepsy disorders, such as rolandic epilepsy (RE) or benign epilepsy of childhood with centrotemporal spikes (BECTS). The invention provides the use of an SNP as a diagnostic and prognostic marker in assays for detecting such disorders. The invention also relates to methods of treating epilepsy with a therapeutic agent, and apparatus (e.g. panels, arrays or kits) for carrying out the assays and methods.

Description

EPILEPSY BIOMARKER

The present invention relates to a novel biological marker for epilepsy disorders, such as rolandic epilepsy (RE) or benign epilepsy of childhood with centrotemporal spikes (BECTS), and in particular to the use of an SNP as diagnostic and prognostic marker in assays for detecting such disorders. The invention also relates to methods of treating epilepsy with a therapeutic agent, and apparatus (e.g. panels, arrays or kits) for carrying out the assays and methods.

Rolandic epilepsy (RE) or benign epilepsy of childhood with centrotemporal spikes (BECTS) (OMIM #117100) is the most common childhood epilepsy syndrome, occurring in 4-12 year old children, with a prevalence of approximately 1 in 2,500^ ². RE clinically overlaps with more severe syndromes such as atypical benign partial epilepsy (ABPE, OMIM #604827); continuous spikes in slow wave sleep (CSWSS), and Landau- Kleffner (LKS) syndromes (OMIM #245570)3. Rare sequence and structural genomic variants are found in <5% of cases of individuals with Rolandic epilepsy. The genetic model for common forms is unknown.

In rare cases, mutations (eg GRIN2A, KCNQ2, KCNQ3, RBFOXi, GABRG2, DEPDC5) and copy number variation (eg i6pn.i2) play an important etiological role^¹⁰.

However, most cases are presumed to have a complex genetic susceptibility, with a combination of genes, including for the EEG abnormality of centrotemporal spikes (CTS)¹¹, and separate susceptibility loci for the accompanying comorbidity of reading disability most notably chromosome 1q42^12,13. Genomewide studies have reported linkage of CTS to chromosome up 13 but with no identified coding mutation. Thus, it has not been possible to identify individuals who have or are most at risk of developing RE or BECTS.

Hence, there is a need to provide a reliable and accurate means for identifying individuals who suffer from RE or BECTS.

According to a first aspect of the invention, there is provided the use of a genetic polymorphism pattern in the PAX6 gene as a diagnostic or prognostic biomarker for epilepsy. According to a second aspect, there is provided a method for diagnosing an individual who suffers from epilepsy, or has a pre-disposition thereto, or for providing a prognosis of an individual's condition, the method comprising detecting, in a sample obtained from the individual, for the presence of a genetic polymorphism pattern in the PAX6 gene, wherein the presence of the polymorphism pattern means that the individual suffers epilepsy, or has a pre-disposition thereto, or the individual's condition has a negative prognosis.

Advantageously, the use and method of the invention enable the diagnosis or prognosis of an individual (for example, a child) who suffers from epilepsy or has a predisposition thereto. In addition, the inventors have found that PAX6 polymorphisms are robust biomarkers for identifying individuals who suffer from epilepsy. Thus, individuals diagnosed with epilepsy, or that have a predisposition thereto, or have a negative prognosis, according to the use, methods and apparatus of the invention, can benefit from the early therapeutic treatment in order to prevent the onset of an epileptic attack/ seizure.

The human PAX6 gene is located on chromosome 11. The coding DNA sequence of human PAX6 is known and is readily accessible

(GeneBank Acc: AY412986; Entrez Gene: 5080; Ensembl: ENSG00000007372). The

polymorphism pattern in PAX6, which is detected for in the method of the invention, may comprise at least one polymorphism or polymorphic region of the PAX6 gene. The polymorphic region may be in the non-coding region of the PAX6 gene. For example, the non-coding region of PAX6 maybe in the 5'-untranslated region (5'-UTR) or in the 3' untranslated region (3'-UTR). Preferably, the polymorphic region is in the 3'-UTR of the PAX6 gene.

The term "polymorphism" can refer to the co-existence, within a population, of more than one form of a gene or portion thereof (e.g. an allelic variant). A portion of a gene of which there are at least two different forms, i.e. two different nucleotide sequences, is referred to as a "polymorphic region of a gene". A specific genetic sequence at a polymorphic region of a gene is known as an allele. The term "allele" can refer to the different sequence variants found at different polymorphic sites in DNA obtained from a subject. For example, each polymorphic region of the PAX6 gene has at least two different alleles. The sequence variants of each allele maybe single or multiple base changes, including without limitation insertions, deletions, or substitutions, or may be a variable number of sequence repeats. Thus, a polymorphic region may be a single nucleotide (i.e. a single nucleotide polymorphism, or SNP), the identity of which differs in different alleles. A polymorphic region can also be several nucleotides long.

Preferably, the polymorphism pattern in PAX6 comprises a PAX6 rs662702 SNP. The allele of the PAX6 rs662702 SNP (i.e. polymorphism), which may be detected, may be identified as a T-allele. The ancestral allele of PAX6 may be defined as a C-allele. Thus, the SNP at rs662702 may be either C (i.e. the ancestral nucleotide) or T (i.e. the SNP variant). The inventors have surprisingly found that the homozygous form of the T- allele (i.e. the TT genotype) of the PAX6 rs662702 SNP is associated with the development of centrotemporal sharp waves that are a major component of epileptic seizures, particularly in children. Hence, the use and method of the invention may comprise detecting for the presence of the T-allele of the rs662702 SNP.

The epilepsy according to the invention may be Rolandic epilepsy (RE) or benign epilepsy of childhood with centrotemporal spikes (BECTS) or atypical benign partial epilepsy (ABPE, OMIM #604827). Preferably, the epilepsy is RE or BECTS.

Preferably, the sample comprises a biological sample. The sample may be any material that is obtainable from a subject from which genomic DNA or cDNA is obtainable. Furthermore, the sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof.

The term "detecting alleles" refers to the process of genotyping, genetic testing, genetic screening, determining or identifying an allele or polymorphism. The allele actually detected may be a disease-associated polymorphic allele, or a mutation that is in linkage disequilibrium with such an allele. It will manifest in the genomic DNA of an individual, but may also be detectable from RNA or protein sequences transcribed or translated from that region. Accordingly, preferably the sample comprises a nucleic acid. The nucleic acid tested may be RNA or DNA, although DNA is preferred.

Preferably, the sample comprises genomic DNA. Preferably, the nucleic acid encodes at the polymorphic region of PAX6. Techniques for determining the presence of particular alleles are known to the skilled person and include, but are not limited to, nucleic acid techniques based on size or sequence, such as restriction fragment length polymorphism (RFLP), nucleic acid sequencing, or nucleic acid hybridization. These techniques may also comprise the step of amplifying the nucleic acid before analysis. Amplification techniques are known to the skilled person and may include, but are not limited to, cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (PASA), polymerase chain ligation, nested polymerase chain reaction, loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification, rolling circle amplification, helicase dependant amplification, ramification amplification method, and the like.

Thus, the detecting step may comprise amplification of the sample, for example PCR amplification. PCR involves amplifying DNA, preferably small amounts of DNA, to ease subsequent detection of the genetic polymorphic patterns. Many variations of the basic amplification protocol are well-known to those of skill in the art. PCR-based detection means include multiplex amplification of a plurality of polymorphisms or markers, simultaneously. For example, it is well known to select PCR primers to generate PCR products that do not overlap in size and which can be analysed simultaneously.

Alternatively, it is possible to amplify different markers with primers that are differentially labelled and thus can each be differentially detected. Of course, hybridization-based detection means allow the differential detection of multiple PCR products in a sample. Other techniques are known to allow multiplex analysis of a plurality of markers.

The detecting step may comprise conducting a genotyping assay, for example that which is available under the trade name Taqman SNP genotyping assay (Applied Biosystems Europe, Warrington, UK). TaqMan SNP genotyping assays consist of two context sequence amplifying primers and two allele specific TaqMan Minor Groove Binder (MGB) probes. Each allele specific probe preferably contains a reporter dye at the 5' end. For example, the reporter dye for allele ι may be VIC (RTM), and the reporter dye for allele 2 may be FAM (6-carboxy-fluorescein). At the 3' end of each allele specific probe there may be a non-fluorescent quencher, which serves to increase the accuracy of allelic discrimination by suppressing florescence from the reporter dye, whilst the probe is intact. During the assay, each probe anneals to a specific

complimentary sequence between the forward and reverse primer sites. Hybridized probes are cleaved by AmpliTaq Gold® polymerase, separating the reporter dye from the quencher dye. Incomplete hybridization, or mismatches between the probe and the target, are dislodged by AmpliTaq Gold® polymerase without cleaving the probe. Hence, preferably the detecting step comprises use of at least one oligonucleotide operable to be used for amplification of genomic DNA comprising the PAX6 gene. Accordingly, genotyping for C/T polymorphism PAX6 rs662yo2 maybe performed using a Taqman SNP genotyping assay and the relevant primers. Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele specific oligonucleotide (ASO) hybridization, allele specific S' exonuclease detection, sequencing, hybridization and the like.

Polymorphic variations leading to altered protein sequences or structures may also be detected by analysis of the protein itself. Preferably, said detecting comprises subjecting the amplified DNA to size analysis, for example electrophoresis and, preferably comparing the results to a positive control and, preferably a negative control.

Said size analysis may be preceded by restriction enzyme digestion. Said detecting may comprise digesting the amplified DNA with a restriction enzyme, and then, preferably, subjecting the resultant digested DNA to electrophoresis and, preferably, comparing the results to a positive and, preferably, a negative control.

Alternatively, or additionally, the gel may undergo Southern blotting or other hybridization analyses comprising labelling, preferably radio-labelling a probe. Said detecting may comprise sequencing the DNA encoding the polymorphisms to determine the allele or alleles present.

The individual maybe a vertebrate, mammal, or domestic animal. Most preferably, however, the individual is a human being. The individual may be a child or adult. For example, the individual may be less than 20 years old or less than 15 years old. The individual may be less than 10, 5, 3 or 2 years old. The individual may be a baby, i.e. 12 months old, or less, including a foetus. Preferably, the method is carried out in vitro. The inventors have developed an apparatus or kit which can be used to diagnose an individual with epilepsy. Hence, according to a third aspect, there is provided an epilepsy diagnostic or prognostic apparatus for identifying an individual's genetic polymorphism pattern in the PAX6 gene, wherein the presence of the polymorphism pattern is associated with having epilepsy or a predisposition thereto, the apparatus comprising means for determining the presence of a genetic polymorphism pattern in the PAX6 gene.

The apparatus may be used to perform the method of the second aspect. The apparatus may comprise a kit, a panel or an array. The apparatus may comprise a PAX6 negative control, preferably provided in a container in the kit embodiment. The negative control may be total RNA or DNA extracted from a sample of a subject with the C- allele of the PAX6 gene. The apparatus may comprise a PAX6 positive control, preferably provided in a container in the kit embodiment. The positive control may be total RNA or DNA extracted from a sample of a subject with the T-allele of the PAX6 gene. The means for determining the presence of a genetic polymorphism pattern comprises a detection means for detecting the C-allele and/or the T-allele of the PAX6 gene. The detection means may be an oligonucleotide, preferably a primer.

The invention extends to methods of treatment.

Hence, according to a fourth aspect of the invention, there is provided a method of treating an individual having a susceptibility to developing epilepsy, the method comprising:

(i) determining the genotype of an individual to identify the presence of a

polymorphism pattern in the PAX6 gene, which polymorphism pattern is associated with having epilepsy; and

(ii) administering, to the individual, a therapeutic agent that prevents, reduces or delays the development of epilepsy. Examples of suitable therapeutic agents which may be administered include but are not limited to aldehydhes, aromatic allylic alcohols, barbiturates, benzodiazepines, carbamates, carboxamides, fatty acids, fructose derivatives, GABA analogues, hydantoins, oxazolidinediones, propionates, pyrimidinediones, pyrrolidines, succinimides, sulphonamides, triazines and ureas. Fatty acids maybe a valproate, vigabatrin, progabide or tiagabine. A fructose derivative may be topiramate. A GABA analogue may be gabapentin or pregabalin. Hydantoins may be ethotoin, phenytoin, mephenytoin or fosphenytoin. Oxazolidinediones maybe paramethadione,

trimethadone or ethadione. Pyrrolidines may be brivaracetam, levetiracetam or seletracetam. Succinimides may be ethosuxumide, phensuximide or mesuximide.

Sulphonamides may be acetazolamide, sultiame, methazolamide or zonisamide. Ureas maybe pheneturide or phenacemide.

Hence, according to a fifth aspect, there is provided a method of identifying an allele which is associated with the development of epilepsy, the method comprising identifying an allele which is in linkage disequilibrium with the PAX6 rs662702 SNP, wherein said SNP is associated with having epilepsy.

"Linkage disequilibrium" refers to the co-inheritance of two or more alleles at frequencies greater than would be expected from the separate frequencies of occurrence of each allele in a given control population. The expected frequency of occurrence of two alleles that are inherited independently is the frequency of the first allele in that population multiplied by the frequency of the second allele in that population. Alleles that co-occur at expected frequencies are said to be in "linkage equilibrium". The cause of linkage disequilibrium is often unclear. It can be due to selection for certain allele combinations or to recent admixture of genetically heterogeneous populations. In addition, in the case of genetic markers that are very tightly linked to a disease gene, an association of an allele (or group of linked alleles) with the disease gene is expected if the disease mutation occurred in the recent past, so that sufficient time has not elapsed for equilibrium to be achieved through recombination events in the specific

chromosomal region. When referring to allelic patterns or polymorphism patterns that comprise more than one allele, a first allelic pattern is in linkage disequilibrium with a second allelic pattern if at least one of the alleles that comprise the first allelic pattern is in linkage disequilibrium with at least one of the alleles of the second allelic pattern. The skilled person will appreciate how to conduct linkage disequilibrium analysis based on one of more of the SNPs described herein, in order to identify a new allele or SNP which can also be used a prognostic marker for the development of epilepsy.

All of the features described herein (including any accompanying claims, abstract and drawings), and/ or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/ or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:-

Figure 1 shows the association of CTS in the ELP4 Region. (A) LocusZoom¹⁹ plot for the association of 152 CTS cases with 1000 population controls under CTS's l-LOD linkage peak with rs662702 plus two imputed SNPs showing a statistically significant association with CTS. (B) Independent association tests for rs662702 per site. The summary row includes all CTS cases, plus 4 CTS cases from Argentina, and all 1000 population controls.

Figure 2 shows that CTS and RD Associations suggest Cis-Regulatory Mechanisms as Causal. Association of CTS (left) and RD (right) are shown aligned with annotation tracks from the Roadmap Epigenomics and ENCODE projects. rs662702 is within the 3'-UTR of PAX6. Psoas muscle is shown as a negative control. ChromHMM shows rs662702 in an enhancer chromatin state activated in neural cells. The site is accessible for transcription factor binding as shown by DNase hypersensitivity tracks in neural progenitor cells and fetal brain (P < 0.05). MeDIP-Seq shows the site is highly methyated, with some demethylation also detected by MRE-Seq suggesting a heterogenous cell mixture. ChipSeq reveals binding of FOXP2 in PFSK-i cells

(neuroectodermal cell line), FOXAi in T-47D cells (mammary epithelial cell line) and Max in NB4 cells (leukemia cell line). In the association of linkage disequilibrium SNPs with rsi495855 with RD (left), ^10495275 and ^146545453 fall within chromatin states that change throughout the different neurodevelopmental cell stages. This region strongly interacts (as suggested by Hi-C in Hi hESCs) with a region containing a CpG island that is strongly demethylated (as per MRE-Seq and MeDIP-Seq) and accessible (as per DNase). Interestingly, only the adult hippocampal region shows expression of a transcript at this region. Furthermore, rs46545453 shows demethylation and binding by FOXA1 in T-47D cells. DL, dorsolateral; hESC, human embryonic stem cell; HSMM, human skeletal muscle myoblasts.

Figure 3 are the results of a Principal Component Analysis (PCA) of All 457 Rolandic Epilepsy Individuals and 1000 Ontario Science Centre Spit for Science Controls in Reference with Hapmap CEU and TSI Groups. Cases and controls were first analyzed by PCA with Hapmap CEU and TSI individuals. Samples more than 6 standard deviations away of CEU and TSI clusters were excluded. Tracy- Widom test was used to determine the number of principal components needed to correct for population stratification in the association model.

Figure 4 is a LocusZoom Plot for the Association of 152 CTS Cases with 1000

Population Controls Conditional on rs662702. Conditioning on rs662702 abolishes the association with CTS in the region suggesting that all SNPs are marking the same underlying variant.

Examples

In the following Examples, the inventors report replication of the CTS association at the ELP4-PAX6 locus, refinement of the association to a SNP regulating PAX6

developmental expression, and replication and refinement of the reading disability localisation at 1q42.

Samples and genotyping

Probands with RE (Rolandic epilepsy) and their family members were recruited as described previously from the US, Canada, Argentina, France and UK¹⁵, with approval by local institutional review boards. The current study analyzed subjects of European descent with CTS (152 subjects from 126 families), either CTS or SSD (186 subjects from 128 families), and RD (127 subjects from 72 families). Phenotyping for CTS, RD and SSD was conducted as previously reported¹³' ^{15, 16}. Population controls of European descent, determined by principal component analysis (PCA)¹⁷, were selected at random from a pool of 4,491 unrelated subjects from the Ontario Science Centre study (OSC), frequency matched by sex, and self-reported to be unaffected with epilepsy. 561 cases and 4,491 controls were genotyped on the Illumina HumanCoreExome BeadChip, and 84 cases on the HumanOmniExpress BeadChip.

Statistical Analysis and Bioinformatics

The inventors restricted analysis to the genotyped markers at the chromosome np13 CTS locus (chr11:30,862,638-31,815,896, in hg19 coordinates)¹⁵ defined by their i-LOD score interval from the previously reported linkage studies. At each locus the number of independent tests were estimated, and calculated a Bonferroni-corrected critical value for declaring regional statistical significance, using the Genetic Type I Error Calculator (GEC)¹⁸. The -log₁₀P-values required for statistical significance were 2.51 in the ELP4-PAX6, (Table 1). The npi3 region was analysed for association with 152 CTS cases, then 186 cases with either CTS or SSD to assess evidence for pleiotropy.

Generalized was used estimating equations (GEE) to account for the relatedness with an independence correlation structure. SNPs as additive in the GEE model were coded, and adjusted for sex and principal components estimated using KING¹⁷. 1000 OSC population controls of European descent were randomly selected and frequency- matched for sex in all association analyses. Association p-values were plotted using LocusZoom's web interface¹⁹. For finer resolution in post-hoc analysis, all samples were imputed for the two regions of interest (^11:30,862,638-31,815,896; and

chri:209,726,558-232,030,640; NCBI build 37) (Supplementary Methods). The inventors evaluated results with database annotations: UCSC Genome Browser²⁰, Washington University's Epigenome Browser²¹, and the Database of Genomic Variants (DGV)²² to query data from the NIH Roadmap Epigenomics Consortium of 111 reference human epigenomes, the Encyclopedia of DNA Elements (ENCODE)²⁶, Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER)²⁷, and ISCA²⁸ projects. Haploreg²⁹ was queried for

transcription factor (TF) affinity binding predictions.

Table 1- Genetic Type I Error Calculator (GEO⁸ Results on the Effective Number of Independent Tests in the ELP4 and RDG l-LOD Loci

Quality Control of Genotype Data

708 subjects were genotyped on the HumanCoreExome BeadChip (538,448 SNPs), while 228 subjects were genotyped on the HumanOmniExpress BeadChip (730,525 SNPs). 93 of these subjects were genotyped on both platforms. In addition, 4,851 population controls ascertained at the Ontario Science Centre were also genotyped on the HumanCoreExome BeadChip.

PLINK v1.07³⁵ and R statistical software³⁶ were used for quality control of genotype data. Individuals with a genotype missing rate of 10% or greater and SNPs with a call rate less than 90% were removed. Duplicated SNPs for each platform were also identified and the SNP with the highest call rate was kept. Sex was assessed against reported gender, and corrected using heterozygosity from the X chromosome. In addition, samples that were outliers for heterozygosity in autosomal chromosomes were removed. After correction for sex, any identified heterozygous haploid SNPs on the sex chromosomes were removed.

Pairwise relatedness was assessed using PLINK's calculation of the kinship coefficient (--genome option). The pair with the lowest missing genotype rate was kept from identified monozygotic twins and duplicated samples.

Family relationships were recorded, and information from PLINK's kinship coefficient calculations were used in conjunction to build the pedigrees de novo using in-house scripts. The pedigrees were then checked for errors using the kinship2 package in R³⁷.

KING³⁸ was used for PCA with subjects from the International Hapmap Project (Phase 3) from various ethnic backgrounds, while correcting for family relationships. Subjects who clustered close to Hapmap-defined populations other than CEPH (Utah residents with ancestry from northern and western Europe) (CEU) or Toscani in Italy (TSI) were excluded from the analysis. PCA was then re-run with the remaining sample individuals and Hapmap CEU and TSI subjects; sample individuals who were more than six standard deviations from the mean for any of the first three principal components were also excluded from the analysis. All association tests were conducted using the geeglm function specifying an

independent correlation structure in the geepack package in R³⁹.

Forest Plots

The independent association test per continental region for CTS was conducted as follows: for the top SNP associated with CTS, 69 CTS cases from USA and 13 from

Canada were pooled into the "North American" group site and checked for association with 539 population controls; and 59 CTS cases from UK and 7 from France were pooled into the "European" group site and checked for association with 434 non- overlapping population controls. Forest plots were plotted using the forestplot package in R⁴⁰. Imputation

BEAGLE 4.0 version r1399⁴¹ was used to impute all samples in the two regions of interest (chrii:20-50Mb for ELP4 region of interest defined as the region between chr11:30,862,638-31,815,896; and chri:200-240Mb for the l-LOD RD linkage peak mapped as chr 1:209,726,558-232,030,640; NCBI build 37). Whenever available, parent-child relationships were used for imputation. All 2,504 individuals in the 1000 Genomes Project (phase 3) were used as the reference during the phasing and imputation steps. For strand alignment of genotyped SNPs, however, the conform-gt program (version Π174) was used with only the European-identified individuals in the 1000 Genomes Project as reference. Only SNPs with an allelic r² (a measure of estimated correlation between most probable minor allele dose and true minor allele dose) greater than 0.8 were retained for further analysis.

The imputed datasets were merged for each region of interest. For the 93 individuals in common in both platforms, imputed SNPs from the HumanOmniExpress BeadChip were used as the number of imputed SNPs with allelic r² > 0.8 was higher.

Example 1- Association of CTS in the ELP4-PAX6 Region

In the regional analysis of np13, 152 individuals with CTS from Canada, the United States, Argentina and Europe (Table 2), were compared to 1000 ethnically matched controls (Figure 3). One SNP, rs662702, in the 3'-untranslated region (UTR) of PAX6 reached regional significance (-log₁₀P=2.82) with an estimated odds ratio (OR) of 1.97 from an additive model (Figure lA; Table 3). Follow-up fine-mapping with imputation did not reveal other variants with greater evidence of association (Figure lA and 4). The evidence at rs662702 was consistent across continents, with ORs of 1.92 (95% CI: 1.08- 3-43) for North America (69 cases from the US and 13 from Canada) and 2.20 (1.17- 4.13) for the independent sample from Europe (59 from the UK and 7 from France) (Figure lB). This particular variant was not genotyped as part of the original linkage and association study of ELP4¹⁵, but ELP4 associated variants are in LD with rs662702. Because the npi3 locus is pleiotropic for CTS and SSD, the inventors tested the hypothesis that this CTS-associated SNP also contributes to SSD. They included 34 additional individuals with SSD from these 126 RE families, and re-analyzed the corresponding sample of 186 CTS or SSD affecteds. Results argued against a pleiotropic effect with the OR decreasing to 1.84 and the p-value increasing despite the larger sample size (-logi₀P=2.38). The homozygosity frequency of the rs662702 T risk allele among Ontario Science Centre controls is 0.30%, and 0.60% (3/503) in the 1000 genomes European samples³⁰. Among the CTS-affected group, the rs662702 homozygosity frequency is almost 4%, amounting to a 12.29 times greater odds of CTS among rs662702 T allele homozygotes (-logiop=3.589). Table 2 - Distribution of Cases and Controls Used in the Association Studies per Geographic Location

Table 3 - Genotyped SNPs in the ELP4 region (chr11: 30.862.638-31.815,896) Tested for Association in 152 CTS Cases and 1000 Spit for Science Proiect Population Controls Only one genotyped SNP, rs6627Q2, was statistically significant f-log₁₀P > 2.514). This SNP falls in the 3'-UTR region of PAX6 gene. CHR=chromosome; BP=base-pair position; SNP=single nucleotide polymorphism; OR=odds ratio; LogP=-log₁₀(p-value ); "A" symbolizes the minor allele and "a" the major allele; MAF=minor allele frequency; iK=1000 Genomes Proiect (phase 3): MAF lK EUR=MAF in Europeans of the lK proiect.

Example 2 - Bioinformatic Assessment

SNP rs662702 falls in the 3'-UTR of PAX6 and annotation databases suggest this particular region has cis-regulatory elements controlling and or fine-tuning PAX6 transcript levels (Figure 2). Indeed the T allele is associated with a significantly higher PAX6 expression level in a luciferase reporter assay³¹. DNase and chromatin state²⁵ annotations suggest it is a transcription factor binding site in neuronal progenitor cells and fetal brain, and ChlP-seq data²⁶ shows forkhead box protein Ai (FOXAi) and forkhead box protein P2 (FOXP2) as possible transcription factors binding to the region, with increased support for FOXAi due to the presence of its motif in the region. Thus, FOXAi and FOXP2, which are critical for language development, may regulate PAX6 levels. However, there is also strong published evidence of post-transcriptional regulation that is rs662702 allele-specific. SNP rs662702 falls in the predicted micro- RNA-328 (miR-328) 3'-UTR-binding site. miR-328 is expressed in the brain, and previously published experimental evidence has supported PAX6 as a miR-328 target gene with rs662702 allele specific differential binding (Chen et al. Genetics 2012) that could result in altered PAX6 expression levels. The levels of PAX6 may also be affected by the C-to-T allele mismatch of rs662702, which may cause a decrease in

miRNA:mRNA complementarity.

Discussion

RE is associated with a constellation of neurodevelopmental phenotypes that appear to have a complex genetic etiology. The inventors have discovered a strong association between T allele homozygosity at rs662702 and the CTS EEG trait in RE, consistent across North American and European samples. Approximately 4% of cases carried this genotype, a 12-15 times higher frequency than in controls. The T allele may confer risk by cis or post-transcriptional regulation of PAX6 levels in the developing brain, among other possible mechanisms. Although speech sound disorder is linked to the same 1 1p13 locus as CTS, rs662702 does not additionally confer risk for SSD, suggesting that other variants in the ELP4-PAX6 region may mediate SSD risk. The association of RD is localised to an intergenic region of the previous 1q42 linkage peak. These results replicate and refine previous localisation for CTS in ELP4-PAX6.

PAX6 is a developmental control gene crucial for development of the eye, brain, olfactory system and endocrine pancreas. It thus displays complex spatiotemporal and quantitative expression patterns determined by a large array of post-transcriptional and cis-regulatory control elements, some of which are located upstream, within introns, but mostly sited in the ultra-conserved downstream regulatory region (DRR)3². The rs662702 T allele in the DRR increases PAX6 expression, which in turn causes cell- autonomous defects of late cortical progenitor proliferation in fetal mouse brain³³. Specifically, increased Pax6 expression results in abnormalities of cortical thickness and layering in rostral and central regions³³. This impact is strikingly similar to recent findings obtained through longitudinal MRI structural studies in RE³⁴. RE children show areas of reduced frontal, temporal and occipital cortical thickness³⁴. However, detailed experimental modelling is required to confirm the specific developmental and spatial effect of this risk variant on mammalian brain development and cortical excitability. The Examples also suggest either that other undiscovered ELP4-PAX6 variants specifically impair the functional development of the vocal tract to lead to speech sound disorder or that binding of the transcription factor FOXP2, critical for speech dyspraxia, could be compromised. SNP rs662702 TT carriers did not differ in age of onset, seizure or treatment variables, suggesting the existence of modifying factors for the phenotype; these may include variants in GRIN2A, KCNQ2, KCNQ3,

RBFOXi, GABRG2, DEPDC5, recurrent (eg 16p 11.12) or de novo copy number variants. There is less certainty about the role of SNP rs1495855 in the RD locus, which is predicted to affect the binding of several transcription factors in the developing brain, however numerous CNVs associated with developmental delay and speech and language development reported at this iq42 region provide supporting evidence for regional contributions to RE-associated RD.

Summary

PAX6 is an important transcription factor in neurodevelopment sensitive to dosage; the Examples implicate rs662702 homozygosity in the 3'-UTR of PAX6 as contributing to the CTS phenotype by influencing both transcriptional and post-transcriptional regulation.

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Claims

1. A method for diagnosing an individual who suffers from epilepsy, or has a predisposition thereto, or for providing a prognosis of an individual's condition, the method comprising detecting, in a sample obtained from the individual, for the presence of a genetic polymorphism pattern in the PAX6 gene, wherein the presence of the polymorphism pattern means that the individual suffers epilepsy, or has a predisposition thereto, or the individual's condition has a negative prognosis.

2. The method according to claim l, wherein the polymorphism pattern comprises at least one polymorphism or polymorphic region of the PAX6 gene.

3. The method according to claim 2, wherein the polymorphic region is in the 3'- UTR ofthe PAX6 gene.

4. The method according to any preceding claim, wherein the polymorphism pattern comprises a PAX6 rs662702 SNP.

5. The method according to claim 4, wherein the allele of the PAX6 rs662702 SNP is identified as a T-allele.

6. The method according to either claim 4 or claim 5, wherein the method comprises detecting for the homozygous form of the T-allele (i.e. the TT genotype) of the PAX6 rs662702 SNP.

7. The method according to any preceding claim, wherein the epilepsy is Rolandic epilepsy (RE) or benign epilepsy of childhood with centrotemporal spikes (BECTS) or atypical benign partial epilepsy (ABPE, OMIM #604827).

8. The method according to any preceding claim, wherein the epilepsy is rolandic epilepsy, or benign epilepsy of childhood with centrotemporal spikes (BECTS).

9. The method according to any preceding claim, wherein the sample comprises a biological sample.

10. The method according to any preceding claim, wherein the sample comprises a nucleic acid.

11. The method according to claim 9, wherein the nucleic acid encodes at the polymorphic region of PAX6.

12. Use of a genetic polymorphism pattern in the PAX6 gene as a diagnostic or prognostic biomarker for epilepsy.

13. Use according to claim 12, wherein the polymorphism pattern in the PAX6 gene is as defined in any one of claims 2-6.

14. Use according to either claim 12 or 13, wherein the epilepsy is as defined in either claim 7 or claim 8.

15. An epilepsy diagnostic or prognostic apparatus for identifying an individual's genetic polymorphism pattern in the PAX6 gene, wherein the presence of the polymorphism pattern is associated with having epilepsy or a predisposition thereto, the apparatus comprising means for determining the presence of a genetic

polymorphism pattern in the PAX6 gene.

16. The apparatus according to claim 15, wherein the apparatus comprises a PAX6 negative control and/or a PAX6 positive control.

17. The apparatus according to claim 16, wherein the negative control is total RNA or DNA extracted from a sample of a subject with the C- allele of the PAX6 gene.

18. The apparatus according to any one of claims 15 to 17, wherein the positive control is total RNA or DNA extracted from a sample of a subject with the T-allele of the PAX6 gene.

19. The apparatus according to any one of claims 15 to 18, wherein the means for determining the presence of the genetic polymorphism pattern comprises a detection means for detecting the C-allele and/or the T-allele of the PAX6 gene.

20. The apparatus according to claim 19, wherein the detection means is an oligonucleotide, preferably a primer.

21. A method of treating an individual having a susceptibility to developing epilepsy, the method comprising:

(i) determining the genotype of an individual to identify the presence of a

(ii) administering, to the individual, a therapeutic agent that prevents, reduces or delays the development of epilepsy.

22. A method of identifying an allele which is associated with the development of epilepsy, the method comprising identifying an allele which is in linkage disequilibrium with the PAX6 rs662702 SNP, wherein said SNP is associated with having epilepsy.