Neurology Research InternationalVolume 2012 (2012), Article ID 876234, 18 pagesdoi:10.1155/2012/876234

Review Article

From Genetics to Genomics of Epilepsy

Silvio Garofalo, Marisa Cornacchione, and Alfonso Di Costanzo

Dipartimento di Medicina e Scienze per la Salute (Me.S.pe.S.), Università del Molise, Via De Sanctis snc, 86100 Campobasso, Italy

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.

Abstract.

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 of these genes due to the multisubunit composition of the molecules have greatly overestimated the role of their mutations in the disease.

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

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.

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 V0 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 1
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 1
Bartter syndrome

KCNJ10
Potassium inwardly rectifying channel, subfamily J, member 10
Seizures, deafness, ataxia, mental retardation

KIAA1279
Kinesin family member 1 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 1
Neurofibromatosis

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

PANK2
Pantothenate kinase 2
Neurodegeneration with brain iron accumulation

PI12
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

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 midface 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 1A
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 1
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
GLUT1 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

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

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)

ASPM
Asp (abnormal spindle) homolog, microcephaly associated (Drosophila)
Microcephaly

ATR
Ataxia telangiectasia and Rad3 related
Microcephaly

BUB1B
Budding uninhibited by benzimidazoles 1 homolog beta (yeast)
Microcephaly

CASK
Calcium/calmodulin-dependent serine protein kinase (MAGUK family)
Microcephaly

CDK5RAP2 [Microcephaly]
CDK5 regulatory subunit associated protein 2
Microcephaly

CENPJ
Centromere protein J
Microcephaly

CEP152
Centrosomal protein 152 kDa
Microcephaly

LIG4
Ligase IV, DNA, ATP-dependent
Microcephaly

MCPH1
Microcephalin 1
Microcephaly

MED17
Mediator complex subunit 17
Microcephaly

NHEJ1
Nonhomologous end-joining factor 1
Microcephaly

PCNT
Pericentrin
Microcephalic osteodysplastic Dwarfism

PNKP
Polynucleotide kinase 3'-phosphatase
Microcephaly

PQBP1
Polyglutamine binding protein 1
X-linked mental retardation

RARS2
Arginyl-tRNA synthetase 2, mitochondrial
Pontocerebellar hypoplasia

SLC25A19
Solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), member 19
Microcephaly

STIL
SCL/TAL1 interrupting locus
Microcephaly

TSEN2
tRNA splicing endonuclease 2 homolog (S. cerevisiae)
Pontocerebellar hypoplasia

TSEN34 [Pontocerebellar Hypoplasia]
tRNA splicing endonuclease 34 homolog (S. cerevisiae)
Pontocerebellar hypoplasia

TSEN54 [Pontocerebellar Hypoplasia]
tRNA splicing endonuclease 54 homolog (S. cerevisiae)
Pontocerebellar hypoplasia

VRK1
Vaccinia related kinase 1
Pontocerebellar hypoplasia

WDR62
WD repeat domain 62
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

(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

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, 26 kDa
Leukodystrophy

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

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

NOTCH3
Notch3
CADASIL

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)

NOTCH3
Notch 3
CADASIL

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
GLUT1 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

GPHN
Gephyrin
Hyperekplexia with epilepsy

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

(e) Neuronal migration disorders (31 genes)

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

ARX
Aristaless-related homeobox
Early infantile epileptic encephalopathy

COL18A1
Collagen, type XVIII, alpha 1
Polymicrogyria

COL4A1
Collagen, type IV, alpha 1
Porencephaly

CPT2
Carnitine palmitoyltransferase 2
Polymicrogyria

DCX
Doublecortin
Lissencephaly

EMX2
Empty spiracles homeobox 2
Schizencephaly

EOMES
Eomesodermin
Polymicrogyria

FGFR3
Fibroblast growth factor receptor 3
Polymicrogyria

FKRP
Fukutin related protein
Walker-Warburg syndrome

FKTN
Fukutin
Walker-Warburg syndrome

FLNA
Filamin A, alpha
Periventricular heterotopia

GPR56
G protein-coupled receptor 56
Polymicrogyria

LAMA2
Laminin, alpha2
Merosin deficiency

LARGE
Like-glycosyltransferase
Walker-Warburg syndrome

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

PAX6
Paired box 6
Polymicrogyria

PEX7
Peroxisomal biogenesis factor 7
Polymicrogyria

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

PQBP1
Polyglutamine binding protein 1
X-linked mental retardation

RAB3GAP
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
Voltage-dependent anion channel 1
Polymicrogyria

VDAC1
Voltage-dependent anion channel 1
Polymicrogyria

WDR62
WD repeat domain 62
Microcephaly, cortical malfor, mental retardatation

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
Aminoacylase1 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

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

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 Q10 deficiency

COQ2
Coenzyme Q2 homolog, prenyltransferase (yeast)
Coenzyme Q10 deficiency

COQ9
Coenzyme Q9 homolog (S. cerevisiae)
Coenzyme Q10 deficiency

COX10
COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase (yeast)
Leigh syndromeCOX10

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]

POLG
Polymerase (DNA directed), gamma
Mitochondrial DNA 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
MPS 6 (Maroteaux-Lamy syndrome)

GALNS
Galactosamine (N-acetyl)-6-sulfate sulfatase
MPS 4A (Morquio syndrome)

GLB1
Galactosidase, beta 1
GM1-gangliosidosis

GNPTAB
N-acetylglucosamine-1-phosphate transferase, alpha and beta subunits
Mucolipidosi 2 (I cell disease) and 3A

GNPTG
N-acetylglucosamine-1-phosphate transferase, gamma subunit
Mucolipidosi 3C

GNS
Glucosamine (N-acetyl)-6-sulfatase
MPS 3D (Sanfilippo D syndrome)

GUSB
Glucuronidase, beta
MPS 7 (Sly syndrome)

HGSNAT
Heparan-alpha-glucosaminide N-acetyltransferase
MPS 3C (Sanfilippo C syndrome)

HYAL1
Hyaluronoglucosaminidase 1
MPS 9

IDS
Iduronate 2-sulfatase
MPS 2 (Hunter syndrome)

IDUA
Iduronidase, alpha-L-
MPS 1H (Hurler syndrome)

MCOLN1
Nucolipin 1
Mucolipidosi 4

NAGLU
N-acetylglucosaminidase, alpha
MPS 3B (Sanfilippo B syndrome)

SGSH
N-sulfoglucosamine sulfohydrolase
MPS 3A (Sanfilippo A syndrome)

SUMF1
Sulfatase modifying factor 1
Multiple sulfatase deficiency

(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

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 lamotrigine, 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.

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 of individuals affected by Dravet syndrome and epilepsy and mental retardation limited to females [16]. Considering 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]. Array-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 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.

Figure 1

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 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 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 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.

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 of end-blocked fluorescent dNTPs

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

Progression
Unlabeled dNTPS added in base-specific fashion
Chemical cleavage removes dye and oligoNTP
Chemical cleavage fluorescent dye and blocking group

Errors
Insertion/deletion
End of read
End of read

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: Giga base pair (10⁹ bp), dNTP: deoxynucleotide-tri-phosphate, PPi: pyrophosphate.

Other third-generation platforms under development make use of nanophotonic visualization chamber, ion semiconductor, electron microscopy, a variety of nanotechnologies like nanopores (Oxford Nanopore Technologies), nanochannels (BioNanomatrix/nanoAnalyzer), nanoparticles (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 by NGS 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 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 sub-panels. At the present the first diagnostic panels for epilepsy that can analyze up to 400 different genes (CeGaT GmbH) are commercially available [47].

Table 9: Epilepsy diagnostic panels.

Subpanels With Homogeneous Clinical Entities
Table
Number of genes

Myoclonic epilepsy, febrile seizures, absences
1
37

Encephalopathies
3
30

X-linked mental retardation (XLMR)
4(a)
25

Joubert syndrome
4(b)
10

Lissencephaly and polymicrogyria
4(c)
18

Severe Microcephaly and pontocerebellar hypoplasia
4(d)
22

Walker-Warburg syndrome
4(e)
6

Holoprosencephaly
4(f)
8

Leukodystrophies
5(a)
20

Migraine
5(b)
6

Disorders of the Ras-MAPK pathway
5(c)
13

Hyperekplexia for defective glycine neurotransmission
5(d)
5

Neuronal migration disorders
5(e)
31

Inherited errors of metabolism
6
49

Congenital disorder of glycosylation (CDG)
7(a)
23

Neuronal ceroid lipofuscinosis (NCL)
7(b)
8

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

Mucopolisaccaridosis and mucolipidosis
7(d)
15

Peroxisome biogenesis disorders (PBD)
7(e)
9

Syndromic epilepsy
2
47

5. Future Perspectives and Conclusions

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

Acknowledgments

This work was supported by grants from Telethon, MIUR, Università del Molise (DISPeS), Università 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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