The quest for Juvenile Myoclonic Epilepsy genes - Epilepsy & Behavior

Epilepsy & Behavior 28 (2013) S52–S57

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Review

The quest for Juvenile Myoclonic Epilepsy genes Antonio V. Delgado-Escueta a, b, c,⁎, Bobby P.C. Koeleman e, Julia N. Bailey a, b, d, Marco T. Medina b, f, Reyna M. Durón a, b, f a

Epilepsy Genetics/Genomics Laboratories, Neurology and Research Services, VA GLAHS-West Los Angeles, USA GENESS International Consortium, USA Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA d Department of Epidemiology, Fielding School of Public Health at UCLA, Los Angeles, CA, USA e Utrecht — Universitair Medisch Centrum Utrecht, Department of Medical Genetics, Division of Biomedical Genetics, The Netherlands f Neurology Training Program, National Autonomous University of Honduras, Tegucigalpa, Honduras b c

a r t i c l e

i n f o

Article history: Accepted 29 June 2012 Keywords: Linkage Genome-wide associations Juvenile myoclonic epilepsy genes

a b s t r a c t Introduced into a specific population, a juvenile myoclonic epilepsy (JME) mutation generates linkage disequilibrium (LD). Linkage disequilibrium is strongest when the JME mutation is of recent origin, still “hitchhiking” alleles surrounding it, as a haplotype into the next thousands of generations. Recombinations decay LD over tens of thousands of generations causing JME alleles to produce smaller genetic displacements, requiring other genes or environment to produce an epilepsy phenotype. Family-based linkage analysis captures rare epilepsy alleles and their “hitchhiking” haplotypes, transmitted as Mendelian traits, supporting the common disease/multiple rare allele model. Genome-wide association studies identify JME alleles whose linkage disequilibrium has decayed through thousands of generations and are sorting out the common disease/common allele versus rare allele models. Five Mendelian JME genes have been identified, namely, CACNB4, CASR, GABRa1, GABRD, and Myoclonin1/EFHC1. Three SNP alleles in BRD2, Cx-36, and ME2 and microdeletions in 15q13.3, 15q11.2, and 16p13.11 also contribute risk to JME. This article is part of a supplemental special issue entitled Juvenile Myoclonic Epilepsy: What is it Really? © 2012 Published by Elsevier Inc.

1. Introduction The epilepsies affect about 3 million people in the USA and 65 million people in the world [1]. Amongst the heritable epilepsies, juvenile myoclonic epilepsy (JME) is the most common. Juvenile myoclonic epilepsy is the most common idiopathic or genetic generalized epilepsy and the most common cause of hereditary grand mal seizures in people with epilepsy in the population at large [2,3]. Juvenile myoclonic epilepsy has both Mendelian inheritance and complex genetic inheritance and accounts for 3% (population-based prevalence) to 12% (hospital/clinic-based prevalence) of all epilepsies [4,5]. Forty-nine percent of our JME families have clinical and EEG traits that are ‘vertically’ inherited over several generations suggesting an autosomal dominantly inherited disease. In the other 51%, variants of JME genes, with small to modest effects, contribute to risk/susceptibility and to its complex genetics. Presently, Mendelian JME genes and non-Mendelian risk alleles have not been defined in over 90% of affected patients. Consequently, nobody has found a cure for JME [6]. ⁎ Corresponding author at: Epilepsy Genetics/Genomics Laboratories, Comprehensive Epilepsy Program, David Geffen School of Medicine at UCLA and VA GLAHS-West Los Angeles, Room 3049 (127B), Building 500, West Los Angeles VA Medical Center, 11301 Wilshire Boulevard, Los Angeles, CA 90073, USA. Fax: +1 310 268 4937. E-mail address: [email protected] (A.V. Delgado-Escueta). 1525-5050/$ – see front matter © 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.yebeh.2012.06.033

How, then, are Mendelian genes that cause monogenic JME captured within various populations of specific countries worldwide? How do you identify the many single nucleotide polymorphisms (SNPs)/variants that increase risk for non-Mendelian complex JME? When and how did these epilepsy alleles arise? Why is it so important to identify these epilepsy alleles? 2. When did Mendelian or non-Mendelian complex JME genes first mutate? How do such alleles increase in populations? Were epilepsy alleles already present in the human genome during the first successful migration of modern humans (Homo Sapiens Sapiens) out of the Horn of Africa 60 to 100 thousand years (ka) ago? Mitochondrial and Y chromosome relics and haplogroups have recently revealed an ancient ancestry within the Arabian Peninsula that spread from Africa and then through the Gulf oasis region toward the Near East and Europe 55–24 ka ago [7]. Did epilepsy mutations arise during this spread through the Gulf oasis to the Near East and Europe? Or did these epilepsy alleles arise during the dispersal of modern humans during late glacial and postglacial periods into Southwest Asia, Australasia, Southeast Asia, Central Europe, Siberia, and the Americas 50 to 20 ka ago [8–10]? Alternately, are epilepsy alleles Biblical, colonial, or modern, having mutated in the last 10 ka or last 600 years?

A.V. Delgado-Escueta et al. / Epilepsy & Behavior 28 (2013) S52–S57

Consider then the quest for these JME genes in our present time, when one or more of these epilepsy mutation(s) or JME alleles have mutated during dispersal from Africa (60–70 ka or 1800–2000 generations ago) or during Biblical times (8–10 ka or 300 generations ago) or during the Colonial era (0.5 ka or 16–20 generations ago) or during more recent modern periods of Homo Sapiens Sapiens. Once introduced into a specific population, an epilepsy mutation generates linkage disequilibrium followed by subsequent drift and/or genetic selection. In this specific population, the co-occurrence of specific DNA markers and a JME mutation at a higher frequency than would be predicted by random chance constitute linkage disequilibrium. In other words, DNA microsatellites or SNPs (single nucleotide polymorphisms) and the JME mutation (allele) are physically close on the DNA strand and occur together more often than can be accounted for by chance. Once a JME mutation is introduced in a specific population, how does it end up in greater numbers in such populations? Genetic selection, through inbreeding, famine, wars, and conquest such as the colonization of the Americas and genocide like the Holocaust, could produce a population bottleneck that increases the frequency of the newly created epilepsy allele within a specific population [11]. Migration of a small proportion of individuals from the population to homestead a sparsely inhabited area would have a similar selecting effect. When this new epilepsy allele is transmitted to the next generation, it brings the haplotype of other alleles linked to it in a segment of a chromosome, along for the “ride” to the next generations, like a “hitchhiking effect” [12] (Fig. 1). As mentioned above, the new epilepsy mutation/allele does not occur independently of surrounding alleles, microsatellites, and SNPs, and these chromosome loci are, therefore, in linkage disequilibrium. As time goes on and transmissions into more and more thousands of generations occur, linkage disequilibrium decays through recombinations. The chromosome segment near the epilepsy allele is exchanged and crosses over with homologous segments of other chromosomes which in turn carry different alleles at nearby sites of the exchanging chromosome [11]. With more and more recombinations, the hitchhiked haplotype is eventually lost, and the epilepsy allele has smaller and smaller genetic effects.

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Linkage disequilibrium is strongest and covers the widest region of a chromosome when the epilepsy allele is of recent origin (Biblical/ 300 generations ago or Colonial/16–20 generations ago), has not yet decayed over many thousands of generations, and has large genetic effects, e.g., Mendelian dominant or recessive effects. With more and more thousands of generations and more migrations into outbred populations, more recombinations decay the linkage disequilibrium over the course of time, and epilepsy alleles over hundreds and thousands of generations decrease their genetic effects, produce smaller genetic displacements, and require other epilepsy alleles or environment to produce the epilepsy phenotype. Here, in this Special Issue on Juvenile Myoclonic Epilepsy, we present an introduction/summary on Genetics of JME. B.P.C. Koeleman, then R. Buono and then Helbig, Hartmann and Mefford discuss the two methods and their results that are used to harness these epilepsy alleles. B.P.C. Koeleman used family based studies to capture the more rare photoparoxysmal epilepsy alleles and their "hitchhiking" haplotypes that are still transmitted as Mendelian traits (common JME disease/multiple rare epilepsy allele model). R. Buono used Genome-wide association studies to identify the many epilepsy alleles whose linkage disequilibrium has decayed through thousands of generations and that are likely to be involved in the complex genetics of the more common outbred JME population (common JME disease/ common epilepsy allele model versus rare epilepsy allele model). 3. Expanding the JME genome: massive parallel deep sequencing — whole genome or exome sequencing To date, five Mendelian JME genes are listed in OMIM or the “Online Mendelian Inheritance in Man” (http://omim.org and http://www.ncbi. nlm.nih.gov/omim/). These are CACNB4 (calcium channel beta4 subunit) [13], CASR (calcium channel sensor receptor) [14], GABRa1 (GABA receptor alpha one subunit) [15], GABRD (GABA receptor delta subunit) [16], and Myoclonin1/EFHC1 (myoclonin1/one EF-hand containing gene) [17] (Table 1). Three SNP susceptibility alleles of putative JME genes in epistasis, namely, bromodomain-containing 2 (BRD2) [18], connexin 36 (Cx-36) [19], and malic enzyme2 (ME2) [20] have been reported to be major susceptibility alleles that contribute to the complex genetics

Fig. 1. The haplotype “hitchhiking effect”.

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Table 1 Juvenile myoclonic epilepsy genes and chromosome loci. Chromosome locus

HGNC gene symbola or phenotype

Gene/encoded protein

Country/ethnic group/ancestral origin

Mode of inheritance

1p36.22

Absence and JME

Suggestive linkage

Europe (Germany, Netherlands, UK, France, Italy, and Greece)

1p36.33 2q23.3

EJM7 EJM6

GABRDb CACNB4b

2q34

JME

Unknown

2q23.3 and 2q24.1 2q33–36 3p14.2

JME EJM9 JME/absence

Unknown Unknown Suggestive linkage

Australian families incl GEFS+ One family (father and son with absences and tonic–clonic) from Germany; one woman with JME and a daughter with 3-Hz spike waves Europe (Germany, Netherlands, UK, France, Italy, and Greece) Tunisia — one large consanguineous family India — one large 4 generation family Europe(Germany, Netherlands, UK, France, Italy, and Greece)

AD/oligogenic: meta analysis of 235 absence families and 118 JME families AD AD

3q21.1

JME

5q12–14 5q35.3

One large Indian family; 5 of 96 unrelated JME patients from Bangalore, India India Netherlands

5q34

EJM4 JME with PPR (photosensitivity) EJM5

CASR (calcium sensor receptor gene)b Unknown Unknown GABRA1b

5q34

(Absence and JME)

Unknown

French Canadian (Quebec, Canada) — one family; one absence patient from Germany; one absence patient from France Europe — Germany, Netherlands, UK, France, Italy, and Greece

6p12

EJM1

Myoclonin1/EFHC1b

6p12 6p21.3

JME EJM3

Unknown BRD2c (bromodomain-containing gene)

6p20 6p21.2 6q24 7q32

8q21.13 10q25–q26 13q13 13q31.3 13q31

JME JME JME Photoparoxysmal response 3 (PPR3), with or without myoclonic epilepsy JME with PPR (photosensitivity) JME with PPR JME with PPR (photosensitivity) CAE

Xp11.4–11.3

JRK, glutamate receptor GRIN2A are suspected – – –

JME with PPR (photosensitivity)

15q14 15q14 18q21 19q13

– – – Unknown

EJM2 JME Generalized epilepsy with febrile seizures plus, including JME (GEFS + 1) JME

Cx36c (connexin36) or a gap junction protein2 or GJP2 CHRNA7 is candidate gene ME2c (malic enzyme2) SCN1B

EFHC2

Hispanics [LA, California] Hispanics [Belize] Hispanics [Mexicoc] Hispanics [Honduras and Mexico] Japan Italy and Brazil Tennessee Austria Netherlands European descent [Los Angeles, California] European descent [NYc]; Germany Germany Germany Saudi Arabia Netherlands 16 multiplex families

AD/oligogenic: meta analysis of 118 JME families AR AD AD/oligogenic: meta analysis of 235 absence families and 118 JME families AD AD AD AD

AD/oligogenic: meta analysis of 235 absence families and 118 JME families AD

AD AD/oligogenic

AD AR AR AD

Netherlands

AD

India (New Delhi), 5 families Netherlands

AD AD

Europe and Australia (Germany, Netherlands, UK, France, Italy and Greece) Netherlands, United Kingdom, Denmark, France, Greece, Portugal, Sweden European descent (New York)

AR/meta analysis of 235 absence families AD

UK, Sweden families European descent (New York) Europe and Australia (Germany, Netherlands, UK, France, Italy, and Greece)

AR Association studies with JME Meta analysis of 235 absence and 118 JME families AR

Germany, 81 patients

Association study with JME

AR

a

Human Genome Nomenclature Committee gene symbol in bold letters. b Mutation segregate with epilepsy affected members across 2 to 4 generation families or in singletons. c SNP-associated variants of BRD2, Cx36 and ME2; AD, autosomal dominant; AR, autosomal recessive; JME, juvenile myoclonic epilepsy; and pCAE, pyknoleptic childhood absence epilepsy.

of JME [4,6]. Meanwhile, over 22 chromosome loci linked to JME have yet to be unraveled for their epilepsy genes (Table 1) [6]. The declining per unit cost and high throughput of deep sequencing of all human genes for discovering allelic variants are now expanding the JME genome. Massively parallel DNA sequencing technologies have rendered whole exome sequencing (WES) or genome sequencing (WGS) of

individual epilepsy patients increasingly practical [21–23]. However, around 24,000 individual genetic variations in a single individual's exome (all exonic sequences of all known human genes) are on average observed after deep sequencing. Therefore, extensive filtering of detected variation has been devised specifically for the epilepsy genome, rendering a more manageable number of putative disease variants for


follow-up. Typically, putative JME disease genes are identified through evidence from familial segregation or association with disease. With identification of JME genes by deep sequencing, proof for causality by replication of epilepsy phenotypes in knockout or knockin mice models of the putative epilepsy gene and public health evidence of their clinical importance should decide which epilepsy genes have priority for studies of cures and repairs [6]. Two general approaches have been used to maximize the power of deep sequencing and harness it for efficient identification of JME genes [21–27]: (1) Family-based approach where linkage data, homozygosity mapping and LOD score peaks in multiplex, multigeneration (Mg) families focus the search for the JME-causing SNP variants to specific chromosome loci while family relations (sequencing affected siblings and affected first degree cousins and affected distant relatives) reduce the number of nonsynonymous SNP variants to be filtered, excluding irrelevant parts of the exome or genome prior to application of computational filters. Chromosome regions that are identical by descent (IBD) can be inferred based on the exome sequences of affected individuals using methods such as Hidden Markov Model-based algorithms [21]. In consanguineous families, epilepsy-affected individuals share two identical by descent (IBD) haplotypes inherited from a common ancestor. The putative JME gene must be located within the IBD haplotype block. (2) Individual-based approach where affected individuals from independent kindreds with the same JME syndrome have WG or WE sequenced. In both approaches, SNP variants are filtered against public SNP databases, and SNP variants commonly shared by affected persons further identified for their potential disease mechanisms by neurobiological functions, evolutionary conservation, and mutation impact (candidate genes) [27]. An individual exome usually has 20,000 to 30,000 variants. About 10,000 of these variants lead to missense mutations, changes in conserved splice sites, or are small deletions or insertions. Almost 90% of these variants are in the dbSNP public SNP database, then the 5000 Genome Project and at private “in house” exon data bases. Assuming that common variants are unlikely to cause rare Mendelian diseases, such common variants are filtered out. Variants that are computationally predicted to be benign and not pathogenic are also filtered out [27]. The remaining variants then, must be rare, expressed in the brain, and potentially epilepsy-causing. A candidate epilepsy gene should show at least one variant per affected individual in autosomal dominant JME. In autosomal recessive JME, each candidate epilepsy gene should show homozygous mutations or compound heterozygous mutations [21–23]. The individual-based approach works if the JME phenotype is caused by one or two genotypes (minor amount of genetic heterogeneity), and a search for the discovered JME gene can then be found in individuals with the same JME phenotype. Until a second unrelated individual with JME or a second family with JME is found with a mutation in the same putative epilepsy gene, one can never be entirely certain that a candidate epilepsy gene is in fact the sought after JME gene. It is generally argued that with deep sequencing and the “individual-based approach,” described above, large families are no longer needed to define epilepsy genes. However, the individualbased approach is only now being tested in heritable epilepsies where phenotypic variability, misdiagnosis, genetic heterogeneity, and incomplete penetrance are most common. The efficiency of these two study designs, the time efficiency of their analysis methods, and success rates in isolating JME-causing genes need to be compared in the studies of genetic epilepsies. Both the “family-based approach” and the “individual-based approach” should be validated and fine-tuned in genetic studies of JME.

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3.1. De novo mutations Studies of copy number variations (CNVs) have demonstrated the contributions of de novo mutations in the epilepsies (see Section 3). Copy number variations contribute to genetic generalized epilepsies with complex inheritance, including JME. Recent experience with Dravet syndrome, epilepsy combined with mental retardation with or without congenital anomalies/dysmorphisms, or epilepsy combined with autism show that de novo mutations have been underappreciated in the epilepsies. The per generation mutation rate for de novo mutations is between 7.6 × 10 to 9th and 2.2 × 10 to the 8th or about one in a hundred million positions in the haploid genome. This corresponds to 0.86 de novo mutations per newborn. Sequencing trios (parents and affected proband) could identify pathogenic de novo mutations in the exome sequence of JME patients [22,23]. 3.2. Whole exome sequencing (WES) versus whole genome sequencing (WGS) Because the majority of disease-causing mutations, characterized to date, are located in exons, it is reasonable (it is also less costly) to concentrate sequencing efforts on the approximate 1% of the human genome that codes for protein sequences or WES. However, the cost of WGS continues to fall with each year, and data on the more common non-coding sequences and variants in distant enhancers and other regulatory elements are becoming available and associated with hereditary diseases. Various laboratories are claiming success rates of at least 50% in identifying novel non-epilepsy disease genes using WGS or WES [23]. 4. Proving a putative mutation is pathogenic and a putative epilepsy gene is sufficient by itself to cause epilepsy The first step in proving that a Mendelian gene may be epilepsycausing is to show its mutations segregate in affected members across 2 or more generations. This step was taken in the 5 five Mendelian JME genes listed in OMIM or the “Online Mendelian Inheritance in Man” database (http://omim.org) and cited above (Table 1). Usually, the potential pathogenicity of a mutation is surmised from (a) the domain sites of mutations and their potential deleterious effects on functions of the encoded protein as predicted by “in Silico analysis” and (b) location of mutations in evolutionarily conserved domains. However, these prediction programs are not infallible, and most investigators will show that a mutation may be epilepsy-causing by actually evaluating the functional consequences of the mutation. The second step, therefore, in proving that a Mendelian gene may be epilepsy-causing, is to actually assess the purported function of the putative epilepsy gene and show that the mutation deleteriously affects function of its encoded protein, a deleterious effect in a disease biochemical pathway that is potentially epileptogenic. This was done in 4 of the 5 Mendelian JME genes listed in OMIM, namely, CACNB4, GABRD, GABRA1, and Myoclonin1/EFHC1. This has yet to be done for CASR. The altered biophysical properties of CACNB4, GABRD, and GABRA1 that contained nucleotide mutations and transfected in cell lines support a possible causal role in the JME phenotype observed in the patient/proband [13,28,29]. In the case of Myoclonin1/EFHC1, functional studies of individual missense mutations supported a role in apoptosis and R-type VDCC. Overexpression of EFHC1 in mouse hippocampal primary culture neurons induced apoptosis that was significantly decreased by each of five EFHC1 missense mutations tested in hippocampal cells in culture. These missense mutations were double heterozygous 229C > A and 662> A; 685T >C; 628 G> A; 757G >T; and 545G >A (4). In patch clamp analysis in HEK cell cultures, EFHC1 specifically increased R-type Ca2+ currents that could increase apoptosis. The increased

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R-type Ca2+ currents were partly reversed by each of five missense mutations [17]. To investigate if a putative JME gene is sufficient by itself to produce an epilepsy phenotype, the third and final step is to target a gene in embryonic stem cells and create strains of mice lacking the putative epilepsy gene. This was done for GABRD, GABRA1, Myoclonin1/EFHC1, and one of the susceptibility genes, BRD2. Spontaneous seizures were not present in GABRD−/− mice [30] indicating that GABRD was not sufficient by itself to cause seizures and possibly acts with other genes or environment to produce a JME phenotype. GABRD−/− mice did have reduced pro-absence seizure effects (e.g., ganaxalone failed to prolong PTZ-induced absence-like freezing) suggesting it had a susceptibility role in absence seizures [30]. In contrast, ethosuximide-sensitive spike-wave discharges and absence-like seizures appeared in heterozygous GABRA1+/− mice and exhibited a female sex dependent effect on the spike-wave frequency in C57BL/6J strain [31]. In the first generation of breeding homozygous null mutants efhc1−/− mice and heterozygous efhc1 +/− mice, spontaneous myoclonias with single or 2 diffuse rapid spikes, low threshold to PTZ seizures, and large lateral ventricles were observed [32]. After seven generations of breeding, spontaneous massive myoclonias and grand mal clonic-tonic-clonic seizures with lower threshold to PTZ seizures as shown by death during convulsive status, are present in efhc1−/− mice and efhc1+/− mice (unpublished observations). Nature provided its own experiment when an inherited defect (a four-nucleotide insertion into a splice donor site resulting in exon skipping, frameshift, and truncation with loss of alpha1 binding sites) in Cchb4 in mouse chromosome 2, the mice homologue of human CACNB4, produced absence-like seizures and ataxia. These mice are called Tottering mice [33]. In the case of the bromodomain-containing gene BRD2, a null mutation of the homologous BRD2 locus results in embryonic lethality, while heterozygous BRD2 +/− males have decreased clonic and females have decreased tonic-clonic seizure threshold to flurothyl. Spontaneous seizures also appear in BRD2+/− female mice [34]. In summary, mutations in Cchb4, the mouse homologue of human CACNB4 or mutations in GABRA1 are sufficient by themselves to produce the absence phenotype while mutations in Myoclonin1/efhc1 or BRD2 are sufficient by themselves to produce a convulsive phenotype (myoclonic or clonic or tonic-clonic seizures). 5. Why is it important to identify JME alleles? Identifying epilepsy alleles that cause JME is important for the following reasons: (1) Targeted designer antiepileptic drugs: A sea change is occurring in the development of antiepileptic drugs (AEDs) with the realization that drugs developed against genetic epilepsies work against both genetic generalized epilepsies and symptomatic lesional epilepsies but not the other way around. In the past, development of AEDs relied on the use of animal models of seizures and animal models of animal epilepsies. Only recently has an AED been developed against a mouse model of human genetic epilepsy, namely, the seizure threshold-1 (Szt1), a kcnq2 knockout mice. Retigabine, an M-channel enhancer, decreased seizure sensitivity of mice carrying the szt1 mutation which deletes the C-terminus of mouse kcnq2 [35]. The myoclonin/efhc1 KO model and the tottering mice can be used as a test model for developing AEDs against JME. (2) Advanced brain imaging in myoclonin/efhc1 KO mice, BRD2 KO mice, and tottering mice can decide if striato-thalamic-frontal cortical MRI changes in human JME are part of the JME genotype, the JME network for seizures or the consequences of seizures. Woermann [see elsewhere in this Supplement] showed

brain imaging abnormalities in the dorsolateral prefrontal cortex, premotor cortex, basal frontal cortex, thalamus, and putamen in human JME [36]. What remains unclear is — which anatomical networks are part of the genotype, which anatomical networks are effects of the myoclonias and tonic-clonic convulsions, and which anatomical networks subserve seizures and epilepsy? Since rarely, if ever, do we have access to JME human brains to answer these questions, MRI studies and neuropathological studies of efhc1−/− mice brains, brd2−/− mice brains, and tottering (cacnb4−/−) mice brains could help. (3) Identifying mutations in epilepsy genes can lead to genotyping of epilepsy genes, improved and early diagnosis, and early curative treatment [37]. Following this introduction/summary of JME genetics, B.P.C. Koeleman discusses epilepsy alleles that are still transmitted with "hitchhiking haplotypes" in families with EEG photoparoxysmal response and/or clinical sensitivity to light. Epilepsy alleles that have survived transmission through thousands of generations but may have reduced the size of or lost their “hitchhiking haplotypes” are captured by GWAS in R.J. Buono's studies. The unexpected contribution of copy number variations to JME risk is highlighted by Helbig, Hartmann, and Mefford. Of the five JME Mendelian genes, Myoclonin1/EFHC1 has received special attention because it appears to have been genetically selected in specific populations, responsible for 3 to 9% of JME in various populations worldwide. Myoclonin1/EFHC1 has also assumed more importance now that it is clear that a single copy of its mutation, transmitted as an autosomal dominant gene, produces adolescent myoclonias and grandmal convulsions (JME), while two copies of its mutation, transmitted as an autosomal recessive gene, produce a severe intractable epilepsy of infancy with death at 3 to 96 months. Thus, the functions of myoclonin1/EFHC1 in brain development are discussed by de Nijs et al. and Yamakawa and Suzuki. Conflict of interest The authors declare that there are no conflicts of interest. References [1] Hauser WA, Hesdorffer DC. Epilepsy-frequency, causes and consequences. Landover MD: Demos; 1990. [2] Janz D. Die Epilepsien. Stuttgart, Germany: Georg Thieme Verlag; 1969. [3] Delgado-Escueta AV, Enrile-Bacsal F. Juvenile myoclonic epilepsy of Janz. Neurology 1984;34:285–94. [4] Delgado-Escueta AV. Advances in genetics of juvenile myoclonic epilepsies. Epilepsy Curr 2007;7:61–7. [5] Nicoletti A, Reggio A, Bartoloni A, et al. Prevalence of epilepsy in rural Bolivia: a door-to-door survey. Neurology 1999;53(9):2064–9. [6] Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV. The next decade of research in the basic mechanisms of the epilepsies. In: Noebeles JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, editors. Jasper's basic mechanisms of the epilepsies. Oxford University Press; 2012. p. 3–11. [7] Fernandes V, Alshamali F, Alves ML, et al. The Arabian cradle: mitochondrial relicts of the first steps along the Southern route out of Africa. AJHG 2012;90:347–55. [8] Soares P, Alshamali F, Pereira JB, et al. The expansion of mtDNA haplogroup L3 within and out of Africa. Mol Biol Evol 2012;29(3):915–27. [9] Perego UA, Achilli A, Angerhofer N, et al. Distinctive Paleo-Indian migration routes from Beringia marked by two rare mtDNA haplogroups. Curr Biol 2009;19:1–8. [10] Barker G, Barton H, Bird M, et al. The ‘human revolution’ in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Dave (Sarawak, Borneo). J Hum Evol 2007;52:243–61. [11] Terwilliger JD, Göring HH. Gene mapping in the 20th and 21st centuries: statistical methods, data analysis, and experimental design. Hum biology, 72(1). Detroit, Michigan: Wayne State University Press; 2000. p. 63–132. [12] Kaplan NL, Hudson RR, Langley CH. The “hitchhiking effect” revisited. Genetics 1989;123:887–99. [13] Escayg A, De Waard M, Lee DD, et al. Coding and noncoding variation of the human calcium-channel beta4-subunit gene CACNB4 in patients with idiopathic generalized epilepsy and episodic ataxia. Am J Hum Genet 2000;66(5): 1531–9. [14] Kapoor A, Satishchandra P, Ratnapriya R, et al. An idiopathic epilepsy syndrome linked to 3q13.3-q21 and missense mutations in the extracellular calcium sensing receptor gene. Ann Neurol 2008;64(2):158–67.

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