Scholarly article on topic 'From Genetics to Genomics of Epilepsy'

From Genetics to Genomics of Epilepsy Academic research paper on "Clinical medicine"

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Neurology Research International
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Academic research paper on topic "From Genetics to Genomics of Epilepsy"

Hindawi Publishing Corporation Neurology Research International Volume 2012, Article ID 876234, 18 pages doi:10.1155/2012/876234

Review Article

From Genetics to Genomics of Epilepsy

Silvio Garofalo, Marisa Cornacchione, and Alfonso Di Costanzo

Dipartimento di Medicina e Scienzeper la Salute (, Universita del Molise, Via De Sanctis snc, 86100 Campobasso, Italy Correspondence should be addressed to Silvio Garofalo, Received 13 December 2011; Accepted 17 February 2012 Academic Editor: Alexander Rotenberg

Copyright © 2012 Silvio Garofalo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The introduction of DNA microarrays and DNA sequencing technologies in medical genetics and diagnostics has been a challenge that has significantly transformed medical practice and patient management. Because of the great advancements in molecular genetics and the development of simple laboratory technology to identify the mutations in the causative genes, also the diagnostic approach to epilepsy has significantly changed. However, the clinical use of molecular cytogenetics and high-throughput DNA sequencing technologies, which are able to test an entire genome for genetic variants that are associated with the disease, is preparing a further revolution in the near future. Molecular Karyotype and Next-Generation Sequencing have the potential to identify causative genes or loci also in sporadic or non-familial epilepsy cases and may well represent the transition from a genetic to a genomic approach to epilepsy.

1. Introduction

In the last decades a large number of gene discoveries have changed our views of idiopathic and symptomatic epilepsy [1]. Indeed, idiopathic epilepsy has the considerable genetic advantage to be found very often in informative autosomal dominant families that have been of great relevance to map and to positional clone the causative gene, opening insight into the biology and molecular pathology of this condition [2,3].

The search of epilepsy genes has allowed the identification of several genes in idiopathic generalized epilepsy (Table 1), the vast majority of which are channelopathies [4, 5] or affect the activity of excitatory or inhibitory neurotransmitters in central nervous system [6]. It is possible that the dominant nature ofthese genes due to the multisubunit composition of the molecules have greatly overestimated the role of their mutations in the disease.

Other important insights came from the discoveries of causative genes of syndromic epilepsy (Table 2) [7] and other disorders where epilepsy is associated with encephalopathies (Table 3) [8], mental retardation with brain malformation (Table 4) [9, 10], other neurologic conditions including neuronal migration disorders (Table 5) [11], and inborn errors of metabolism (Tables 6 and 7) [12, 13]. Without any doubt,

these discoveries have been great advances in the field; however, their impact on the management of epileptic patients was limited because of the failure to collect significant genetic information from each patient to distinguish the large number of genetic defects that can lead to the disease. Therefore, genetic testing was possible only for few or selected family cases.

Technical improvements in human chromosomes recognition and better definition of chromosome regions realized by increasing the number of detectable chromosome bands have provided higher resolution of normal and pathological karyotype. It is today well established an association between epileptic seizures and chromosome abnormalities recognized by high-resolution chromosome banding [14, 15]. However, the type and the size of the chromosome defects are not always easy to detect even by the highest-resolution cytogenetic techniques available for light microscopes.

The identification of the specific genetic defect in a patient with epilepsy may clarify the diagnosis (diagnostic testing), suggest the prognosis, assist with treatment and management (e.g., the use of a ketogenic diet in glucose transporter type 1 deficiency syndrome or the avoidance of lamotrig-ine, phenytoin, and carbamazepine in Dravet syndrome), elucidate the risk of a disease in family members and future children, and save the patient from further diagnostic evaluation and potentially invasive testing.

Table 1: Disease genes identified in generalized myoclonic epilepsy, febrile seizures, absences (37 genes).

Gene Symbol Gene name and description

ALDH7A1 Aldehyde dehydrogenase 7 family, member A1

BRD2 Bromodomain containing 2

CACNA1A Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit

CACNA1H Calcium channel, voltage-dependent, T type, alpha 1H subunit

CACNB4 Calcium channel, voltage-dependent, beta 4 subunit

CASR Calcium-sensing receptor

CHRNA2 Cholinergic receptor, nicotinic, alpha 2 (neuronal)

CHRNA4 Cholinergic receptor, nicotinic, alpha 4

CHRNB2 Cholinergic receptor, nicotinic, beta 2 (neuronal)

CLCN2 Chloride channel 2

CSTB Cystatin B (stefin B)

EFHC1 EF-hand domain (C-terminal) containing 1

EPM2A Epilepsy, progressive myoclonus type 2A, Lafora disease (laforin)

GABRA1 Gamma-aminobutyric acid (GABA) A receptor, alpha 1

GABRB3 Gamma-aminobutyric acid (GABA) A receptor, beta 3

GABRD Gamma-aminobutyric acid (GABA) A receptor, delta

GABRG2 Gamma-aminobutyric acid (GABA) A receptor, gamma 2

GPR98 G protein-coupled receptor 98

GRIN2A Glutamate receptor, ionotropic, N-methyl D-aspartate 2A

GRIN2B Glutamate receptor, ionotropic, N-methyl D-aspartate 2B

KCNMA1 Potassium large conductance calcium-activated channel, subfamily M, alpha member 1

KCNQ2 Potassium voltage-gated channel, KQT-like subfamily, member 2

KCNQ3 Potassium voltage-gated channel, KQT-like subfamily, member 3

KCTD7 Potassium channel tetramerisation domain containing 7

MBD5 Methyl-CpG-binding domain protein 5

ME2 Malic enzyme 2, NAD(+)-dependent, mitochondrial

NHLRC1 NHL repeat containing 1

PCDH19 Protocadherin 19

PRICKLE1 Prickle homolog 1 (Drosophila)

PRICKLE2 Prickle homolog 2 (Drosophila)

SCARB2 Scavenger receptor class B, member 2

SCN1A Sodium channel, voltage-gated, type I, alpha subunit

SCN1B Sodium channel, voltage-gated, type I, beta subunit

SCN2A Sodium channel, voltage-gated, type II, alpha subunit

SCN9A Sodium channel, voltage-gated, type IX, alpha subunit

SLC2A1 Solute carrier family 2 (facilitated glucose transporter), member 1

TBC1D24 TBC1 domain family, member 24

In asymptomatic subjects with increased risk of seizures because of a family history, genetic test may predict onset of epilepsy (predictive testing) [16, 17]. Despite such potential benefits, genetic testing has also potential harms, such as its ethical, legal, and social implications, and the potential for stigma, distress, adverse labeling, and nonconfidentiality that exists in the setting of inadequate safeguards against discrimination [18]. Considering that our understanding of the epidemiology and clinical utility of genetic testing in the epilepsies is incomplete, the assessment of these potential benefits and harms is particularly complex and is closely linked to the clinical scenario.

The International League Against Epilepsy (ILAE) Genetic Commission presented a tool in the approach to specific tests for epilepsy [16]. According to ILAE report, the diagnostic genetic testing is "very useful" in individual affected by early-onset spasms, X-linked infantile spasms, Dravet and related syndromes, Ohtahara syndrome, epilepsy and mental retardation limited to females, early-onset absence epilepsy, autosomal dominant nocturnal frontal lobe epilepsy, and epilepsy with paroxysmal exercise-induced dyskinesia; the predictive testing is "very useful" in unaffected relatives ofin-dividuals affected by Dravet syndrome and epilepsy and mental retardation limited to females [16]. Considering

Table 2: Disease genes identified in syndromic epilepsy (47 genes).

Gene symbol Gene name and description Syndrome

ARFGEF2 ADP-ribosylation factor GEF2 Periventricular heterotopia

ARHGEF9 Cdc42 GEF 9 Hyperekplexia with epilepsy

A2BP1 Ataxin 2-binding protein 1 (RNA binding protein fox-1 homolog 1) Mental retardation and epilepsy

ASPA Aspartoacylase Canavan syndrome

ATP1A2 ATPase, Na/K transporting, alpha 2 polypeptide Familial hemiplegic migraine

ATP2A2 ATPase, Ca transporting, cardiac muscle, slow twitch 2 Darier-White syndrome

ATP6V0A2 ATPase, H+ transporting, lysosomal VO subunit a2 Cutis laxa with epilepsy and mental retardation

CACNA1A Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit Familial hemiplegic migraine

CCDC88C Coiled-coil domain containing 88C Hydrocephalus with medial diverticulum

CLCNKA Chloride channel Ka Bartter syndrome

CLCNKB Chloride channel Kb Bartter syndrome

COH1 Cohen syndrome protein 1—vacuolar protein sorting 13 homolog B Cohen syndrome

DLGAP2 Discs, large (Drosophila) homolog-associated protein 2 Progressive epilepsy with mental retardation

GFAP Glial fibrillary acidic protein Alexander disease

GLI3 GLI family zinc finger 3 Pallister-hall syndrome

GLRA1 Glycine receptor, alpha f Hyperekplexia

GLRB Glycine receptor, beta Hyperekplexia

GPHN Gephyrin Hyperekplexia

KCNA1 Potassium voltage-gated channel, shaker-related Episodic ataxia

KCNJ1 Potassium inwardly rectifying channel, subfamily J, member f Bartter syndrome

KCNJ10 Potassium inwardly rectifying channel, subfamily J, member f 0 Seizures, deafness, ataxia, mental retardation

KIAA1279 Kinesin family member f binding protein Goldberg-Shprintzen

LAMA2 Laminin, alpha 2 Merosin deficiency

LBR Lamin B receptor Pelger-Huet syndrome

LGI1 Leucine-rich, glioma inactivated 1 Autosomal dominant lateral temporal lobe epilepsy

MLC1 Megalencephalic leukoencephalopathy with subcortical cysts 1 Megalencephalic leukoencephalopathy with cysts

MLL2 Myeloid/lymphoid or mixed-lineage leukemia 2 Kabuki syndrome

NF1 Neurofibromin f Neurofibromatosis

NIPBL Nipped-B homolog (Drosophila) Cornelia de Lange syndrome

PANK2 Pantothenate kinase 2 Neurodegeneration with brain iron accumulation

PI 12 Serpin peptidase inhibitor, clade I (neuroserpin), member 1 Encephalopathy with neuroserpin inclusion bodies

PIGV Phosphatidylinositol glycan anchor biosynthesis, class V Hyperphosphatasia with mental retardation

PLA2G6 Phospholipase A2, group VI (cytosolic, calcium independent) Infantile neuroaxonal dystrophy

Table 2: Continued.

Gene symbol Gene name and description Syndrome

RAI1 Retinoic acid induced 1 Smith Magenis syndrome

SCN8A Sodium channel, voltage gated, type VIII, alpha subunit Cerebellar atrophy, ataxia, and mental retardation

SETBP1 SET binding protein 1 Schinzel-Giedion midtace retraction syndrome

SHH Sonic hedgehog Holoprosencephaly

SLC4A10 Solute carrier family 4, sodium bicarbonate transporter, member 10 Epilepsy with mental retardation

SLC6A5 Solute carrier family 6 (neurotransmitter transporter, glycine), member 5 Hyperekplexia

SMC1A Structural maintenance of chromosomes f A Cornelia de lange syndrome

SMC3 Structural maintenance of chromosomes 3 Cornelia de lange syndrome

SYNGAP1 Synaptic Ras GTPase activating protein 1 Epilepsy and mental retardation

TBX1 T-box f Di George syndrome

TSC1 Tuberous sclerosis 1 Tuberous sclerosis

TSC2 Tuberous sclerosis 2 Tuberous sclerosis

VPS13A Vacuolar protein sorting 13 homolog A Neuroacanthocytosis

ZEB2 Zinc finger E-box binding homeobox 2 Mowat-Wilson syndrome

Table 3: Disease genes identified in epileptic encephalopathies (30 genes).

Gene symbol Gene Name and Description Diseases

ARHGEF9 Cdc42 guanine nucleotide exchange factor (GEF) 9 Early infantile epileptic encephalopathy

ARX Aristaless related homeobox Early infantile epileptic encephalopathy

CDKL5 Cyclin-dependent kinase-like 5 Early infantile epileptic encephalopathy

CNTNAP2 Contactin associated protein-like 2 Pitt Hopkins syndrome

FOXG1 Forkhead box G1 Rett syndrome

GABRG2 Gamma-aminobutyric acid (GABA) A receptor, gamma 2 Early infantile epileptic encephalopathy

GRIN2A Glutamate receptor, ionotropic, N-methyl D-aspartate 2A Early infantile epileptic encephalopathy

GRIN2B Glutamate receptor, ionotropic, N-methyl D-aspartate 2B Early infantile epileptic encephalopathy

MAPK10 Mitogen-activated protein kinase 10 Lennox Gastaut syndrome

MECP2 Methyl CpG binding protein 2 Rett syndrome

NRXN1 Neurexin 1 Pitt Hopkins Syndrome

PCDH19 Protocadherin 19 Early infantile epileptic encephalopathy

PNKP Polynucleotide kinase 3'-phosphatase Early infantile epileptic encephalopathy

RNASEH2A Ribonuclease H2, subunit A Aicardi-Goutieres syndrome

RNASEH2B Ribonuclease H2, subunit B Aicardi-Goutieres syndrome

RNASEH2C Ribonuclease H2, subunit C Aicardi-Goutieres syndrome

SAMHD1 SAM domain and HD domain 1 Aicardi-Goutieres syndrome

SCN1A Sodium channel, voltage-gated, type I, alpha subunit Early infantile epileptic encephalopathy

SCN1B Sodium channel, voltage-gated, type I, beta subunit Early Infantile epileptic encephalopathy

SCN2A Sodium channel, voltage-gated, type II, alpha subunit Early infantile epileptic Encephalopathy

SCN9A Sodium channel, voltage-gated, type IX, alpha subunit Early infantile epileptic encephalopathy

SLC2A1 Solute carrier family 2 (facilitated glucose transporter), member 1 GLUTI deficiency syndrome

SLC25A22 Solute carrier family 25 (mitochondrial carrier: glutamate), member 22 Early infantile epileptic encephalopathy

SLC9A6 Solute carrier family 9 (sodium/hydrogen exchanger), member 6 Angelman syndrome

SPTAN1 Spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) Early infantile epileptic encephalopathy

STXBP1 Syntaxin binding protein 1 Early infantile epileptic encephalopathy

TCF4 Transcription factor 4 Pitt Hopkins syndrome

TREX1 Three prime repair exonuclease 1 Aicardi-Goutieres syndrome

UBE3A Ubiquitin protein ligase E3A Angelman syndrome

ZEB2 Zinc finger E-box binding homeobox 2 Mowat-Wilson syndrome

the potential harms, genetic testing should always be performed with the patient's consent or parental consent in the case of minors. A team approach, including a genetic counselor, a psychologist, and a social worker, is recommended throughout the process of evaluation.

In the last years a number of new molecular genetic technologies became available and they promise to change genetic testing for epilepsy, allowing to extend genetic analysis also to sporadic or nonfamilial cases. Two are the major new technologies that can affect the management of epileptic patients: Oligonucleotide Arrays Comparative Genomic Hybridization (Array-CGH) and Next-Generation Sequencing (NGS).

2. Molecular Karyotype

During the last 50 years cytogenetics has evolved from simple chromosome counting or banded chromosome morphological identification under light microscope to a molecular

approach where chromosomes are analyzed through sophisticated computer system for their ability to hybridize to specific oligonucleotides spanning the entire genome [19]. Ar-ray-CGH is nowadays a basic diagnostic tool for clinical diagnosis of several types of developmental delays [20], intellectual disabilities [21, 22], and congenital abnormalities [23]. Epilepsy is also enjoying several advantages from the use of this technology that significantly improves diagnostic resolution of classic cytogenetics [24, 25].

Chromosomes did not become individually identifiable before the discovery that several procedures could create reproducible, permanent, and specific banding patterns [26, 27]. This was fundamental for gene mapping and positional cloning of disease genes and also revealed a large number of rare and subtle pathological conditions that disturbed the normal band patterning of chromosomes. The improvements of high-resolution banding techniques allowed the identification of several subtle chromosomal abnormalities associated with epilepsy [14, 15]. The possibility to study

Table 4: Epilepsy with mental retardation and brain malformations.

Gene symbol Name Disease

(a) Mental retardation (25 genes)

ARHGEF9 Cdc42 guanine nucleotide exchange factor (GEF) 9 Early infantile epileptic encephalopathy

ARX Aristaless related homeobox Early infantile epileptic encephalopathy

ATP6AP2 ATPase, H+ transporting, lysosomal accessory protein 2 Epilepsy with XLMR*

ATRX Alpha thalassemia/mental retardation syndrome X-linked Epilepsy with XLMR*

CASK Calcium/calmodulin-dependent serine protein kinase (MAGUK family) Mental retardation and microcephaly

CDKL5 Cyclin-dependent kinase-like 5 Early infantile epileptic encephalopathy

CUL4B Cullin 4B Epilepsy with XLMR*

CXORF5 Oral-facial-digital syndrome 1 Simpson-Golabi-Behmel syndrome

DCX Doublecortin Lissencephaly

FGD1 FYVE, RhoGEF and PH domain containing 1 Aarskog-Scott syndrome

GPC3 Glypican 3 Simpson-Golabi-Behmel syndrome

GRIA3 Glutamate receptor, ionotrophic, AMPA 3 Epilepsy with XLMR*

HSD17B10 Hydroxysteroid (17-beta) dehydrogenase 10 Epilepsy with XLMR*

JARID1C Lysine (K)-specific demethylase 5C Epilepsy with XLMR*

OPHN1 Oligophrenin 1 Epilepsy with XLMR*

PAK3 P21 protein (Cdc42/Rac)-activated kinase 3 Epilepsy with XLMR*

PHF6 PHD finger protein 6 Borjeson Forssmann Lehmann syndrome

PLP1 Proteolipid protein 1 Pelizaeus-Merzbacher disease

PQBP1 Polyglutamine binding protein 1 Epilepsy with XLMR*

RAB39B RAB39B, member RAS oncogene family Epilepsy with XLMR*

SLC9A6 Solute carrier family 9 (sodium/hydrogen exchanger), member 6 Angelman-Like syndrome

SMC1A Structural maintenance of chromosomes 1A Cornelia De Lange syndrome

SMS Spermine synthase Epilepsy with XLMR*

SRPX2 Sushi-repeat containing protein, X-linked 2 Rolandic epilepsy

SYP Synaptophysin Epilepsy with XLMR*

*XLMR: X-linked mental retardation

(b) Joubert syndrome (10 genes)

AHI1 Abelson helper integration site 1 Joubert syndrome

ARL13B ADP-ribosylation factor-like 13B Joubert syndrome

CC2D2A Coiled-coil and C2 domain containing 2A Joubert syndrome

CEP290 Centrosomal protein 290 kDa Joubert syndrome

CXORF5 Oral-facial-digital syndrome 1 Joubert syndrome

INPP5E Inositol polyphosphate-5-phosphatase, 72 kDa Joubert syndrome

NPHP1 Nephronophthisis 1 (juvenile) Joubert syndrome

RPGRIP1L Retinitis pigmentosa GTPase regulator interacting protein 1 like Joubert syndrome

TMEM67 Transmembrane protein 67 Joubert syndrome

TMEM216 Transmembrane protein 216 Joubert syndrome

(c) Lissencephaly and polymicrogyria (18 genes)

COL18A1 Collagen, type XVIII, alpha 1 Polymicrogyria

CPT2 Carnitine palmitoyltransferase 2 Polymicrogyria

DCX Doublecortin Lissencephaly

EOMES Eomesodermin Polymicrogyria

FGFR3 Fibroblast growth factor receptor 3 Polymicrogyria

FLNA Filamin A, alpha Periventricular heterotopia

GPR56 G protein-coupled receptor 56 Polymicrogyria

Gene symbol Name Disease

PAFAH1B1 Platelet-activating factor acetylhydrolase 1b, regulatory subunit 1 (45kDa) Lissencephaly

PAX6 Paired box 6 Polymicrogyria

PEX7 Peroxisomal biogenesis factor 7 Polymicrogyria

RAB3GAP1 RAB3 GTPase activating protein subunit 1 (catalytic) Warburg microsyndrome

RELN Reelin Lissencephaly

SNAP29 Synaptosomal-associated protein, 29 kDa Cerebral dysgenesis

SRPX2 Sushi-repeat containing protein, X-linked 2 Rolandic epilepsy

TUBA1A Tubulin, alpha 1a Lissencephaly

TUBA8 Tubulin, alpha 8 Polymicrogyria

TUBB2B Tubulin, beta 2B Polymicrogyria

VDAC1 Voltage-dependent anion channel 1 Polymicrogyria

(d) Severe microcephaly and pontocerebellar hypoplasia (22 genes)











Asp (abnormal spindle) homolog, microcephaly associated (Drosophila) Ataxia telangiectasia and Rad3 related

Budding uninhibited by benzimidazoles 1 homolog beta (yeast) Calcium/calmodulin-dependent serine protein kinase (MAGUK family)

CDK5 regulatory subunit associated protein 2

Centromere protein J Centrosomal protein 152 kDa Ligase IV, DNA, ATP-dependent Microcephalin 1 Mediator complex subunit 17 Nonhomologous end-joining factor 1 Pericentrin

Polynucleotide kinase 3 -phosphatase Polyglutamine binding protein 1 Arginyl-tRNA synthetase 2, mitochondrial

Solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), member 19

SCL/TAL1 interrupting locus

tRNA splicing endonuclease 2 homolog (S. cerevisiae) tRNA splicing endonuclease 34 homolog (S. cerevisiae)

tRNA splicing endonuclease 54 homolog (S. cerevisiae)

Vaccinia related kinase 1 WD repeat domain 62

Microcephaly Microcephaly Microcephaly Microcephaly


Microcephaly Microcephaly Microcephaly Microcephaly Microcephaly Microcephaly

Microcephalic osteodysplastic Dwarfism Microcephaly

X-linked mental retardation Pontocerebellar hypoplasia


Microcephaly Pontocerebellar hypoplasia

Pontocerebellar hypoplasia

Pontocerebellar hypoplasia

Pontocerebellar hypoplasia Microcephaly, cortical malformations and mental retardation

(e) Walker-Warburg syndrome (WWS) or muscle, eye and brain disease (6 genes) anomalies type A2 (MDDGA2)

FKRP Fukutin-related protein Walker-Warburg syndrome

FKTN Fukutin Walker-Warburg syndrome

LARGE Like-glycosyltransferase Walker-Warburg syndrome

POMGNT1 Protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase Walker-Warburg syndrome

POMT1 Protein-O-mannosyltransferase 1 Walker-Warburg syndrome

POMT2 Protein-O-mannosyltransferase 2 Walker-Warburg Syndrome

Gene symbol Name Disease

(f) Holoprosencephaly (HPE) (8 genes)

FGF8 Fibroblast growth factor 8 (androgen-induced) Holoprosencephaly

GLI2 GLI family zinc finger 2 Holoprosencephaly 9

GLI3 GLI family zinc finger 3 Greig cephalopolysyndactyly syndrome

PTCH1 patched 1 Holoprosencephaly 7

SHH Sonic Hedgehog Holoprosencephaly 3

SIX3 SIX homeobox 3 Holoprosencephaly 2

TGIF1 TGFB-induced factor homeobox 1 Holoprosencephaly 4

ZIC2 Zic family member 2 Holoprosencephaly 5

these chromosomes regions with specific hybridization DNA probes through fluorescence in situ hybridization (FISH) greatly improved sensitivity to detect small chromosomal aberrations in specific regions [28].

Today molecular karyotyping is rapidly replacing conventional cytogenetics and FISH. This name refers to the analysis of all chromosomes using hybridization to standard DNA sequences arranged on a "chip" rather than microscope observation. The technological development of this approach allows now clinicians to evaluate the entire genome for copy number variants (CNVs, duplications, deletions) in a single test. The high resolution of this approach is however limited by the difficulty to identify balanced chromosome translocations or inversions, even if this powerful technique recognizes in many of them microdeletions or cryptic anomalies at the chromosomal breakpoints. Detection of deletions or duplications is based on the comparison of two genomes (Figure 1). Labelled patient DNA is cohybridized with control DNA to an array spotted with oligonucleotide DNA probes spanning the entire genome at critical intervals. The distance between these oligonucleotide sequences in the genome marks the resolution of the technique and can be as low as 1000 bp. The intensity of the signal from patient and control are then read and normalized by an electronic scanning device coupled with a software that generates a graphic plot of intensities for each probe.

A search of Online Mendelian Inheritance in Man (OMIM) clinical synopsis with the term "seizure" reveals that there are at least 754 mendelian disorders in which epilepsy is or can be part of the clinical condition, but not the main feature. Many of these disorders can be associated with DNA sequence mutations or subtle chromosomal anomalies that can be conveniently detected by array-CGH. Some will be private or sporadic cases and others will be familial. With the widespread use of CGH in both circumstances, many more genetic events will be reported in patients and the genetic aetiology will be recognized making possible over the time to saturate the genome with all possible loci and events that have an epileptogenic role.

3. Next-Generation Sequencing

From the publication of the draft of human genome sequence in Nature, on 15th February 2001 issue, our view and

Array-CGH Patient's DNA Reference DNA



Loss of DNA Gain of DNA

(deletion) (CNV)

Figure 1

knowledge of human genome has considerably changed [29] and the technologies to sequence DNA are today of common use in diagnostic practice and much cheaper. Chain termination or Sanger's method [30] was largely used for the Human Genome Project and has dominated the past decades. The logic of this technology was to create by synthesis a population of DNA fragments of different size each one terminated at all possible positions by one of the four labelled dideoxynucleotides (ddNTPs) terminators. Separation of these fragments by polyacrylamide gel or capillary electrophoresis allowed the reading of the sequence through the first developed sequencing machines that could distinguish the fluorescence emitted by the blocking ddNTP [31]. Therefore, these are considered the "first generation" of DNA sequencing technologies.

The need to reduce the cost of large sequencing projects has stimulated the development of a variety of cheaper sequencing technologies that are generally called "Next-Generation Sequencing" (NGS) [32, 33]. The final goal of this new field is to reduce the cost of human genome sequencing till or lower than $1,000 per genome to make it available

Table 5: Epilepsy with other neurological problems.

Gene symbol Name Disease

(a) Leukodystrophies (20 genes)

ARSA Arylsulfatase A Leukodystrophy metachromatic (MLD)

ASPA Aspartoacylase Canavan disease

EIF2B1 Eukaryotic translation initiation factor 2B, subunit 1 alpha, 26kDa Leukodystrophy

EIF2B2 Eukaryotic translation initiation factor 2B, subunit 2 beta, 39 kDa Leuko dystrophy

EIF2B3 Eukaryotic translation initiation factor 2B, subunit 3 gamma, 58 kDa Leukodystrophy

EIF2B4 Eukaryotic translation initiation factor 2B, subunit 4 delta, 67 kDa Leukodystrophy

EIF2B5 Eukaryotic translation initiation factor 2B, subunit 5 epsilon, 82 kDa Leukodystrophy

GALC Galactosylceramidase Leukodystrophy globoid cell (GLD)

GFAP Glial fibrillary acidic protein Alexander disease

MLC1 Megalencephalic leukoencephalopathy with subcortical cysts 1 Megalencephalic leukoencephalopathy


PLP1 Proteolipid protein 1 Leukodystrophy hypomyelinating type 1 (HLD1)

PSAP Prosaposin Leukodystrophy metachromatic

RNASEH2A Ribonuclease H2, subunit A Aicardi-Goutieres syndrome type 4 (AGS4)

RNASEH2B Ribonuclease H2, subunit B Aicardi-Goutieres syndrome type 2 (AGS2)

RNASEH2C Ribonuclease H2, subunit C Aicardi-Goutieres syndrome type 3 (AGS3

SAMHD1 SAM domain and HD domain 1 Aicardi-Goutieres syndrome type 5 (AGS5)

SDHA Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) Leigh syndrome

SUMF1 Sulfatase modifying factor 1 Multiple sulfatase deficiency (MSD)

TREX1 Three prime repair exonuclease 1 Aicardi-Goutieres syndrome type 1 (AGS1)

(b) Migraine (6 genes)

ATP1A2 ATPase, Na+/K+ transporting, alpha 2 polypeptide Migraine familial hemiplegic type 2 (FHM2)

CACNA1A Calcium channel, voltage-dependent, P/Q type, alpha 1A subunit Spinocerebellar ataxia type 6 (SCA6)


POLG Polymerase (DNA directed), gamma Progressive external ophthalmoplegia

SCN1A Sodium channel, voltage-gated, type I, alpha subunit Migraine familial hemiplegic type 3 (FHM3)

SLC2A1 Solute carrier family 2 (facilitated glucose transporter), member 1 GLUTI deficiency type 1 (GLUT1DS1) syndrome

(c) Disorders of Ras-MAPK pathway with epilepsy (13 genes)

BRAF V-raf murine sarcoma viral oncogene homolog B1 Cardiofaciocutaneous (CFC ) syndrome

CBL Cas-Br-M (murine) ecotropic retroviral transforming sequence Noonan syndrome-like disorder (NSL)

HRAS V-Ha-ras Harvey rat sarcoma viral oncogene homolog Faciocutaneoskeletal (FCSS) syndrome

KRAS V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog Noonan type 3 (NS3) syndrome

MAP2K1 Mitogen-activated protein kinase kinase 1 cardiofaciocutaneous (CFC) syndrome

MAP2K2 Mitogen-activated protein kinase kinase 2 cardiofaciocutaneous (CFC) syndrome

NF1 Neurofibromin 1 Neurofibromatosis type 1

NRAS Neuroblastoma RAS viral (v-ras) oncogene homolog Noonan type 6 (NS6) syndrome

PTPN11 Protein tyrosine phosphatase, non-receptor type 11 LEOPARD type 1 (LEOPARD1) syndrome

RAF1 V-raf-1 murine leukemia viral oncogene homolog 1 Noonan type 5 (NS5) syndrome

SHOC2 Soc-2 suppressor of clear homolog (C. elegans) Noonan syndrome-like with loose anagen hair

SOS1 Son of sevenless homolog 1 (Drosophila) Noonan type 4 (NS4) syndrome

SPRED1 Sprouty-related, EVH1 domain containing 1 Neurofibromatosis type 1-like syndrome

(d) Hyperekplexia (5 genes)

ARHGEF9 Cdc42 guanine nucleotide exchange factor (GEF) 9 Hyperekplexia with epilepsy

GLRA1 Glycine receptor, alpha 1 Hyperekplexia with epilepsy

GLRB Glycine receptor, beta Hyperekplexia with epilepsy

Gene symbol Name Disease

GPHN SLC6A5 Gephyrin solute carrier member 5 Hyperekplexia with epilepsy family 6 (neurotransmitter, transporter, glycine), TT , , . Hyperekplexia with epilepsy

(e) Neuronal migration disorders (31 genes)

ARFGEF2 ADP-ribosylation factor guanine nucleotide-exchange factor 2 (brefeldin A-inhibited)

ARX Aristaless-related homeobox

COL18A1 Collagen, type XVIII, alpha 1

COL4A1 Collagen, type IV, alpha 1

CPT2 Carnitine palmitoyltransferase 2

DCX Doublecortin

EMX2 Empty spiracles homeobox 2

EOMES Eomesodermin

FGFR3 Fibroblast growth factor receptor 3

FKRP Fukutin related protein

FKTN Fukutin

FLNA Filamin A, alpha

GPR56 G protein-coupled receptor 56

LAMA2 Laminin, alpha2

LARGE Like-glycosyltransferase

PAFAH1B1 Platelet-activating factor acetylhydrolase 1b, regulatory subunit 1 (45kDa)

PAX6 Paired box 6

PEX7 Peroxisomal biogenesis factor 7

POMGNT1 Protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase

POMT1 Protein O-mannosyltransferase 1

POMT2 Protein O-mannosyltransferase 2

PQBP1 Polyglutamine binding protein 1

RAB3GAP RAB3 GTPase activating protein subunit 1 (catalytic)

RELN Reelin

SNAP29 Synaptosomal-associated protein, 29 kDa

SRPX2 Sushi-repeat containing protein, X-linked 2

TUBA1A Tubulin, alpha 1a

TUBA8 Tubulin, alpha 8

TUBB2B Voltage-dependent anion channel 1

VDAC1 Voltage-dependent anion channel 1

WDR62 WD repeat domain 62


Early infantile epileptic encephalopathy








Walker-Warburg syndrome

Walker-Warburg syndrome

Periventricular heterotopia


Merosin deficiency

Walker-Warburg syndrome


Polymicrogyria Polymicrogyria Walker-Warburg syndrome Walker-Warburg syndrome Walker-Warburg syndrome X-linked mental retardation Warburg microsyndrome Lissencephaly Cerebral dysgenesis Rolandic epilepsy Lissencephaly Polymicrogyria Polymicrogyria Polymicrogyria

Microcephaly, cortical malfor, mental retardatation

for common medical practice and diagnostic use [34]. The development of further third-generation sequencing technologies should make possible to sequence single DNA molecules in real time with a cost that it is projected to be very close to the goal [35].

The development of NGS platforms was a major progress in the technology because, differently from Sanger method, rather than producing about one thousand nucleotides for run, they are able to produce orders of magnitude more sequence data using massive parallel process, resulting in substantial increase of data at a lower cost per nucleotide [36, 37].

Several commercial platforms are today available, including Roche/454 [38], Illumina/Solexa and Life Technologies/SOLiD (Table 8(a)). In very general terms these platforms follow similar process that includes: (a) template preparation by breaking large DNA macromolecule to generate short fragment libraries with platform-specific synthetic DNA adapters at the fragment ends, (b) massive and parallel clonal amplification of individual DNA fragment molecules on glass slide or microbeads by PCR [39] to generate a sufficient copy number of the labelled fragment to be detected by the machine optical system, and (c) sequencing by several cycles of extensions that are repeated and detected

Table 6: Inherited errors of metabolism with epilepsy (49 genes).

Gene symbol Defective enzyme name Disease

ABCC8 ATP-binding cassette, subfamily C (CFTR/MRP), member 8 Hypoglcemia

ACY1 Aminoacylase1 Aminoacylasel deficiency

ADSL Adenylosuccinate lyase Adenylosuccinase deficiency

AGA Aspartylglucosaminidase Aspartylglucosaminuria

ALDH4A1 Aldehyde dehydrogenase 4 family, member A1 Hyperprolinemia

ALDH5A1 Aldehyde dehydrogenase 5 family, member A1 Succinic Semialdehyde dehydrogenase deficiency

ALDH7A1 Aldehyde dehydrogenase 7 family, member A1 Pyridoxine deficiency

ARG1 Liver arginase Argininemia

ARSA Arylsulfatase A Metachromatic leukoodystrophy

ASPA Aspartoacylase Canavan disease

ATIC 5-aminoimidazole-4-carboxamide ribonucleotide (AICAr) formyltransferase/IMP cyclohydrolase AICAr transformylase/IMP cyclohydrolase deficiency (ATIC Deficiency)

BTD Biotinidase Biotinidase deficiency

CPT2 Carnitine palmitoyltransferase 2 Carnitine palmitoyltransferase II deficiency

CTSA Cathepsin A Galactosialidosis

DPYD Dihydropyrimidine dehydrogenase Dihydropyrimidine dehydrogenase deficiency

ETFA Electron-transfer-flavoprotein, alpha polypeptide Glutaraciduria

ETFB Electron-transfer-flavoprotein, beta polypeptide Glutaraciduria

ETFDH Electron-transferring-flavoprotein dehydrogenase Glutaraciduria

FH Fumarate hydratase Fumarase deficiency

FOLR1 Folate receptor 1 (adult) Cerebral folate transport deficiency

FUCA1 Fucosidase, alpha-L- 1, tissue Fucosidosis

GALC Galactosylceramidase Krabbe disease

GAMT Guanidinoacetate N-methyltransferase Guanidinoacetate N-methyltransferase deficiency

GCDH Glutaryl-CoA dehydrogenase Glutaraciduria

GCSH Glycine cleavage system protein H (aminomethyl carrier) Glycine encephalopathy

GCST Glycine cleavage system protein T (aminomethyltransferase) Glycine encephalopathy

GLB1 Galactosidase, beta 1 Gangliosidosis

GLDC Glycine dehydrogenase (decarboxylating) Glycine encephalopathy

GNE Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase Sialuria

HEXA Hexosaminidase A (alpha polypeptide) Gangliosidosis

HEXB Hexosaminidase B (beta polypeptide) Gangliosidosis

HPD 4-Hydroxyphenylpyruvate dioxygenase Tyrosinemia

L2HGDH L-2-Hydroxyglutarate dehydrogenase L-2-Hydroxyglutaric aciduria

LAMA2 Laminin, alpha 2 Muscular dystrophy

MOCS1 Molybdenum cofactor synthesis 1 Molybdene cofactor deficiency

MOCS2 Molybdenum cofactor synthesis 2 Molybdene cofactor deficiency

NEU1 Sialidase 1 (lysosomal sialidase) Neuraminidase deficiency

NPC1 Niemann-Pick disease, type C1 Niemann-Pick disease

NPC2 Niemann-Pick disease, type C2 Niemann-Pick disease

PGK1 Phosphoglycerate kinase 1 GAMT deficiency

PRODH Proline dehydrogenase (oxidase) 1 Hyperprolinemia

PSAP Prosaposin Krabbe disease

QDPR Quinoid dihydropteridine reductase Hyperphenylalaninemia

Gene symbol Defective enzyme name Disease

SLC17A5 Solute carrier family 17 (anion/sugar transporter), member 5 Sialuria

SLC25A15 Solute carrier family 25 (mitochondrial carrier; ornithine transporter) member 15 Ornithine translocase deficiency

SLC46A1 Solute carrier family 46 (folate transporter), member 1 Folate malabsorption

SMPD1 Sphingomyelin phosphodiesterase 1, acid lysosomal Niemann pick disease

SUMF1 Sulfatase modifying factor 1 Sulfatidosis

SUOX Sulfite oxidase Sulfitoxidasis

automatically to create short reads [40]. The data of these reads are then collected by the device, and the alignment of the short reads with specific software allows to rebuild the initial template sequence. Helicos and Pacific Biosystem platforms (Table 8(b)) are substantially different because they use a more advanced laser-based detection system that does not require massive parallel amplification with the considerable advantages to simplify preparation process, to eliminate PCR-induced bias and errors, and to make easy data collection. Ion Torrent developed an entirely new approach to sequencing based on hydrogen ion release when a nucleotide is incorporated into a DNA strand by polymerase (Table 8) [41]. An ion sensor can detect hydrogen ions and convert this ion chemical signal to digital sequence information eliminating the need of optical reading at each dNTP incorporation.

Other third-generation platforms under development make use of nanophotonic visualization chamber, ion semiconductor, electron microscopy, a variety of nanotech-nologies like nanopores (Oxford Nanopore Technologies), nanochannels (BioNanomatrix/nanoAnalyzer), nanoparti-cles (GE Global Research), nanoballs (Complete Genomics), nanowells (CrackerBio), nanoknifes (Renveo), and specially engineered sensor DNA polymerase (VisiGen Biotechnologies) [42]. They promise even larger and faster data production although they are still under development and a few years away from commercial use. In principle also DNA microarrays could allow sequencing by hybridization using ultrafast nonenzymatic methods (Genizon Biosciences) and somebody even suggests that mass spectrometry might be used to determine mass differences between DNA fragments produced by chain termination [43].

The beginning of several individual genome projects has gradually decreased the cost of sequencing an individual genome, and it is likely that the $1,000 cost per person will be reached in few years. In medicine, the "personal genome" age made possible byNGS will be an important milestone for the entire genomic field and will mark a transition from single gene testing to whole genome evaluation [44].

It is impossible to predict today which NGS will eventually dominate genomic research, but it is sure that cost reductions, sequencing speed, and better accuracy will make NGS an essential molecular tool in several areas of biology and medicine.

Although the cost of whole genome sequencing has dropped significantly, it remains a major obstacle since it can reach $100,000 for a single individual. However, targeting

sequencing of specific regions of interest can decrease the overall cost and improve efficiency of NGS making this technology ready for diagnostic use [45].

Also the field of epilepsy is potentially affected by NGS. Indeed too many genes and genetic conditions can be associated with epilepsy to make impossible for the clinicians a general use of specific monogene test for the vast majority of nonsyndromic or idiopathic epileptic patients. NGS is changing this situation by targeting several genome regions where known epilepsy genes are located and using enrichment techniques to significantly reduce the cost and improve efficiency. Targeted sequencing usually tests all protein-coding exons (functional exosome) which only requires roughly 5% as much sequencing than whole genome. This strategy will reduces the cost to about $3,000 or even less per single individual. Targeted selection technologies have been marketed and successfully used in different NGS projects and are becoming the tool of choice in several conditions, including epilepsy [46].

4. Targeting Sequencing and Epilepsy Gene Panels

A diagnostic panel is the contemporaneous targeted sequencing of a number of known genes that have already been identified as cause of a particular disease. A diagnostic panel is very different from whole genome or exome sequencing. Only genes clearly associated with a disease are examined. The genes included in the panel can be decided by the prescribing physician or by ad hoc committees of experts that can reach a consensus on the number and type of genes to test making commercially available diagnostic panel kits for specific diseases. This strategy should make easier to detect genetic variants that after validation by Sanger sequencing can be interpreted as the cause of the disease. Of course diagnostic panels and targeted sequencing make sense only if the condition is caused by several or very large number of genes. Many genetic disorders fall in this condition and are excellent candidate for the development of diagnostic panels. Epilepsy is an excellent example of such situation since it is a relative frequent disease affecting 1% of the population in a variety of forms, at different ages, with different progression. A genetic cause of epilepsy can be reasonably supposed in sporadic cases if trauma, tumor, or infection can be ruled out. In such circumstance all genetic information about epilepsy genes identified over the years in familial cases can

Table 7: Other inherited errors of metabolism with epilepsy.

Gene symbol Defective enzyme name Disease

(a) Congenital Disorder of Glycosylation (CDG) (23 genes)

ALG1 N-linked glycosylation 1, beta-1,4-mannosyltransferase homolog CDG

ALG2 N-linked glycosylation 2, alpha-1,3-mannosyltransferase homolog CDG

ALG3 N-linked glycosylation 3, alpha-1,3-mannosyltransferase homolog CDG

ALG6 N-linked glycosylation 6, alpha-1,3-glucosyltransferase homolog CDG

ALG8 N-linked glycosylation 8, alpha-1,3-glucosyltransferase homolog CDG

ALG9 N-linked glycosylation 9, alpha-1,3-glucosyltransferase homolog CDG

ALG12 N-linked glycosylation 12, alpha-1,3-glucosyltransferase homolog CDG

B4GALT1 UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1 CDG

COG1 Component of oligomeric golgi complex 1 CDG

COG7 Component of oligomeric golgi complex 7 CDG

COG8 Component of oligomeric golgi complex 8 CDG

DOLK Dolichol kinase CDG

DPAGT1 Dolichyl-phosphate (UDP-N-acetylglucosamine) N-acetyl glucosamine phosphotransferase 1 (GlcNAc-1-P transferase) CDG

DPM1 Dolichyl-phosphate mannosyltransferase polypeptide 1, catalytic subunit CDG

DPM3 Dolichyl-phosphate mannosyltransferase polypeptide 3 CDG

MOGS Mannosyl-oligosaccharide glucosidase CDG

MGAT2 Mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase CDG

MPDU1 Mannose-P-dolichol utilization defect 1 CDG

MPI Mannose phosphate isomerase CDG

PMM2 Phosphomannomutase 2 CDG

RFT1 Requiring fifty three 1 homolog CDG

SLC35A1 Solute carrier family 35 (CMP-sialic acid transporter), member A1 CDG

SLC35C1 Solute carrier family 35, member C1 CDG

(b) Neuronal ceroid lipofuscinosis (NCL) (8 genes)

CLN3 Ceroid-lipofuscinosis, neuronal 3 NLC

CLN5 Ceroid-lipofuscinosis, neuronal 5 NLC

CLN6 Ceroid-lipofuscinosis, neuronal 6 NLC

CLN8 Ceroid-lipofuscinosis, neuronal 8 NLC

CTSD Cathepsin D NLC

MFSD8 Major facilitator superfamily domain containing 8 NLC

PPT1 Palmitoyl-protein thioesterase 1 NLC

TPP1 Tripeptidyl peptidase I NLC

(c) Defects of mitochondrial metabolism including coenzyme Q deficiency (35 genes)

APTX Aprataxin Coenzyme Q10 Deficiency

ATPAF2 ATP synthase mitochondrial F1 complex assembly factor 2 ATPase deficiency

BCS1L BCS1-like Leigh syndrome

C12ORF65 Chromosome 12 open reading frame 65 Leigh syndrome

C8ORF38 Chromosome 8 open reading frame 38 Leigh syndrome

CABC1 Chaperone activity of bc1 complex-like, mitochondria Coenzyme Ql0 deficiency

COQ2 Coenzyme Q2 homolog, prenyltransferase (yeast) Coenzyme Ql0 deficiency

COQ9 Coenzyme Q9 homolog (S. cerevisiae) Coenzyme Ql0 deficiency

COX10 COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase (yeast) Leigh syndromeCOXlO

Gene symbol Defective enzyme name Disease

COX15 COX15 homolog, cytochrome c oxidase assembly protein (yeast) Leigh syndrome

DLD Dihydrolipoamide dehydrogenase Leigh syndrome

GCSH Glycine cleavage system protein H (aminomethyl carrier) Glycine encephalopathy

GCST Aminomethyltransferase (glycine cleavage system protein T) Glycine encephalopathy

GLDC Glycine dehydrogenase (decarboxylating) Glycine encephalopathy

HSD17B10 Hydroxysteroid (17-beta) dehydrogenase 10 HSD17B10 deficiency

LRPPRC Leucine-rich PPR-motif containing Leigh syndrome

NDUFA2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2,8 kDa Leigh syndrome

NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75 kDa Leigh syndrome

NDUFS3 NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30 kDa Leigh syndrome

NDUFS4 NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18 kDa Leigh syndrome

NDUFS7 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20 kDa Leigh syndrome

NDUFS8 NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23 kDa Leigh syndrome

NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51 kDa Leigh syndrome

PC Pyruvate carboxylase Leigh syndrome

PDHA1 Pyruvate dehydrogenase (lipoamide) alpha 1 Leigh syndrome

PDSS1 Prenyl (decaprenyl) diphosphate synthase, subunit 1 Coenzyme Q10 deficiency

PDSS2 Prenyl (decaprenyl) diphosphate synthase, subunit 2 Coenzyme Q10 deficiency]

Polymerase (DNA directed), gamma Mitochondrial DNA

POLG depletion Syndrome

RARS2 Arginyl-tRNA synthetase 2, mitochondrial Pontocerebellar hypoplasia

SCO2 SCO cytochrome oxidase deficient homolog 2 (yeast) Leigh syndrome

SDHA Succinate dehydrogenase complex, subunit A, flavoprotein (Fp) Leigh syndrome

SURF1 Surfeit 1 Leigh syndrome

TACO1 Translational activator of mitochondrially encoded cytochrome c oxidase I Leigh syndrome

TMEM70 Transmembrane protein 70 Encephalocardiomyopathy

VDAC1 voltage-dependent anion channel 1 VDAC deficiency

(d) Mucopolysaccharidosis (MPS) and mucolipidosis (MLP) (15 genes)

ARSB Arylsulfatase B

GALNS Galactosamine (N-acetyl)-6-sulfate sulfatase

GLB1 Galactosidase, beta 1

GNPTAB N-acetylglucosamine-1-phosphate transferase, alpha and beta subunits

GNPTG N-acetylglucosamine-1-phosphate transferase, gamma subunit

GNS Glucosamine (N-acetyl)-6-sulfatase

GUSB Glucuronidase, beta

HGSNAT Heparan-alpha-glucosaminide N-acetyltransferase

HYAL1 Hyaluronoglucosaminidase 1

IDS Iduronate 2-sulfatase

IDUA Iduronidase, alpha-L-

MCOLN1 Nucolipin 1

NAGLU N-acetylglucosaminidase, alpha

SGSH N-sulfoglucosamine sulfohydrolase

SUMF1 Sulfatase modifying factor 1

MPS 6 (Maroteaux-Lamy


MPS 4A (Morquio



Mucolipidosi 2 (I cell

disease) and 3A

Mucolipidosi 3C

MPS 3D (Sanfilippo D


MPS 7 (Sly syndrome) MPS 3C (Sanfilippo C syndrome) MPS 9

MPS 2 (Hunter syndrome) MPS 1H (Hurler syndrome) Mucolipidosi 4 MPS 3B (Sanfilippo B syndrome)

MPS 3A (Sanfilippo A syndrome) Multiple sulfatase deficiency

Gene symbol Defective enzyme name Disease

(e) Peroxisome biogenesis disorders (PBD) (9 genes): Zellweger syndrome (ZWS): neonatal adrenoleukodystrophy (NALD): infantile refsum disease (IRD): rhizomelic chondrodysplasia punctata type 1 (RCDP1)

PEX1 Peroxisomal biogenesis factor 1 ZWS-NADL-IRD

PEX2 Peroxisomal biogenesis factor 2 ZWS-IRD

PEX3 Peroxisomal biogenesis factor 3 ZWS

PEX5 Peroxisomal biogenesis factor 5 ZWS-NADL

PEX6 Peroxisomal biogenesis factor 6 ZWS

PEX7 Peroxisomal biogenesis factor 7 RCDP1

PEX12 Peroxisomal biogenesis factor 12 ZWS

PEX14 Peroxisomal biogenesis factor 14 ZWS

PEX26 Peroxisomal biogenesis factor 26 ZWS-NADL-IRD

Table 8: Comparison of commercially available sequencing platforms.

(a) Massive parallel clonal amplification with optical detection

Roche 454 Life Technologies SOLiD Illumina

Library amplification emPCR* emPCR* On glass

Sequencing Incorporation of unlabeled dNTPs Ligase-mediated addition of fluorescent oligoNTPs (2bp) Incorporation ofend-blocked fluorescent dNTPs

Detection Light emission from release ofPPi Fluorescence emission from ligated dye-labeled oligoNTPs Fluorescence emission from incorporated labeled oligoNTPs

Progression Unlabeled dNTPS added in Chemical cleavage removes dye and Chemical cleavage fluorescent dye

base-specific fashion oligoNTP and blocking group

Errors Insertion/deletion End of read End ofread

Length 400 bp 75 bp 150 bp

Overall yield/run 500 Mbp >100 Gbp 200 Gbp

(b) Fluorescent and semiconductor single molecule sequencing

Helicos Pacific biosystem Iontorrent

Library amplification N/A-tSMS** N/A-SMRT*** sequencing Optional PCR

Sequencing Incorporation of fluorescent labeled dNTPs Polymerase incorporation terminal phosphate labeled dNTPs Polymerase incorporation of dNTPs releases H+

Detection Laser-induced emission from incorporated dNTP Real time detection of fluorescent dye in polymerase active site Semiconductor ion sensor detects H+ released during dNTPs incorporation

Progression Chemical cleavage of dNTP fluorescent group N/A fluorescent dyes are removed as PPi with dNTPs incorporation H+ signal during each dNTP incorporation is converted in voltage signal

Errors Insertion/deletion Insertion/deletion Insertion/deletion

Length 35 bp 1000 bp 200-400 bp

Overall yield/run 21-37 Gbp >100 Gbp 1 Gbp

* emPCR (emulsion PCR) is an amplification method where DNA library fragments are mixed with beads and PCR reagents in an oil emulsion that allows massive amplification of bead-DNA in a single reaction. **tSMS: true Single Molecule Sequencing. ***SMRT (Single Molecule Real Time)

bp: base pair, Mbp: Mega base pair (106 bp), Gbp: Gigabase pair (109 bp), dNTP: deoxynucleotide-tri-phosphate, PPi: pyrophosphate.

be used to identify the causative gene through an epilepsy diagnostic panel. Indeed in the case of epilepsy the identified genes are so many that they can be classified in subpanels of genes that underline a common clinical entity (Table 9). Clinical considerations may suggest the clinician to include in a diagnostic panel other genes or genes from different subpanels. At the present the first diagnostic panels for epilepsy

that can analyze up to 400 different genes (CeGaT GmbH) are commercially available [47].

5. Future Perspectives and Conclusions

The genetics of epilepsy has evolved from ion channel and neurotransmitter receptor subunits to newly discovered

Table 9: Epilepsy diagnostic panels.

Subpanels With Homogeneous Clinical Entities Myoclonic epilepsy, febrile seizures, absences Encephalopathies

X-linked mental retardation (XLMR)

Joubert syndrome

Lissencephaly and polymicrogyria

Severe Microcephaly and pontocerebellar hypoplasia

Walker-Warburg syndrome




Disorders of the Ras-MAPK pathway Hyperekplexia for defective glycine neurotransmission Neuronal migration disorders Inherited errors of metabolism Congenital disorder of glycosylation (CDG) Neuronal ceroid lipofuscinosis (NCL)

Defects of mitochondrial metabolism including coenzyme Q deficiency Mucopolisaccaridosis and mucolipidosis Peroxisome biogenesis disorders (PBD) Syndromic epilepsy

genes highlighting the importance of different pathways in the epileptogenesis. Furthermore, it has been demonstrated that copy number variations collectively explain a larger portion of idiopathic epilepsy than any single gene. These studies have identified structural genomic variations associated with idiopathic epilepsy, representing a change from the conventional knowledge that chromosome microarray analysis is useful only for patients with intellectual disability or dysmorphism [48]. Genetic testing techniques are rapidly evolving and whole exome or whole genome sequencing, performing at increasingly cheaper costs, will allow rapid discovery of other pathogenic mutations, variants in non-coding DNA, and copy number variations encompassing several genes. This rapidly accumulating genetic information will expand our understanding of epilepsy, and will allow more rational and effective treatment. However, along with the ability to identify genetic variants potentially associated with epilepsy it is imperative to validate genetic associations and analyze their clinical significance.

Is it worth? The main objective of a diagnostic panel for epilepsy is to discover the molecular defect in all possible cases to create a specific and personalized treatment of the disease than can be pharmacologically different for different types of molecular defects. Personalized therapy will be possible only within a genomic medicine. But genomic medicine at the same time will raise other questions: what to do if more than one genetic variant is identified in the same epileptic patient? Can we understand how genetic interactions will modulate the disease severity and prognosis? Can interaction of specific genetic variants and environmental factors modulate the clinical spectrum of the disorder? What

Table Number of genes

4(a) 25

4(b) 10

4(c) 18

4(d) 22

4(e) 6

4(f) 8

5(a) 20

5(b) 6

5(c) 13

5(d) 5

5(e) 31

7(a) 23

7(b) 8

7(c) 35

7(d) 15

7(e) 9

to do if the diagnostic panel is inconclusive? Are the costs affordable? These are only few questions that the genomics of epilepsy will raise. The answers will require time, a lot of sequencing, and probably the development of new and cheaper sequencing technologies.


This work was supported by grants from Telethon, MIUR, Universita del Molise (DISPeS), Universita di Genova (DOBIG), Regione Molise, and Assomab to S. Garofalo. The authors thank Dr. Saskia Biskup for kindly sharing information before publication and for inspiring with her work the authors.


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