MOLECULAR DIAGNOSIS OF
GENETIC EPILEPSY
Nagi ALHaj
Prof of Molecular Biology Assist,
Faculty Medicine , Sana’a ,University, Yemen
Consultant of Genetic Center 48 MH
Overview on Genetic Epilepsy
A genetic contribution to etiology has been estimated to be
present in about 40% of patients with epilepsy..
Three major groups:
• Mendelian disorders, in which a single major locus can
account for segregation of the disease trait
• Non-mendelian or 'complex' diseases, in which the
pattern of familial clustering can be accounted for by the
interaction of the maternal inheritance pattern of
mitochondrial DNA
• Chromosomal disorders, in which a gross cytogenetic
abnormality is present.
• The ‘common’ non-familial idiopathic
epilepsies tend to display ‘complex’
inheritance.
•
They including various forms of
• Idiopathic generalised epilepsy (IGE)
• Juvenile myoclonic epilepsy (JME)
• Childhood absence epilepsy (CAE)
Genetics and Mutation
• Mutations in over 2,000 genes have now been identified in
patients with more than 3,000 different disease phenotypes.
For the clinicians and their patients, it is becoming increasingly
important to obtain a genetic diagnosis
• Identifying the genetic aetiology of a disease may influence
clinical management and will provide information regarding
risk to future pregnancies.
•
With the advent of high-throughput capillary sequencers
and sequence analysis software, direct sequencing provides
an accurate method for single gene analysis.
The inheritance pattern can be
autosomal dominant, autosomal recessive, or X-linked.
Mutations in a single gene may be associated with
different types of seizures (clinical heterogeneity),
and, conversely, mutations in different genes can
cause the same epilepsy phenotype (genetic
heterogeneity).
Traditional nomenclature of inherited epilepsy:
Different mutations in different genes can
result in different phenotypes
Different mutations in different genes can
result in similar phenotypes
Different mutations within one gene can
result in different phenotypes
An identical mutation within one gene can
result in different phenotypes in different
individuals (cause: environment, other genes)
Mutation identification begins with a
phenotype and proceeds toward the genotype
genotype
phenotype
The diagram shows
the distribution of all genetic differences that had been mapped to
chromosome 1 at the time this diagram was drawn.
Mutation identification by linkage analysis
Mutational analysis can be used to identify cells or DNA that have
genotype and allele frequency differences from the normal genome.
mutation
site
• Genome scan has been replaced by mutational
analysis but in a small number of families in whom the
mutation cannot be identified
• Remains the only method for the genetic diagnosis of
carriers.
Mutant alleles generating defects in
particular proteins could disrupt the dance .
Cells homozygous for a mutant allele
might
be
unable
to
complete
chromosome duplication or mitosis or
cytokinesis
because
a
required
component of the molecular machinery
is missing or unable to function.
A genetic map of part of the human
X chromosome.
Elucidation of a disease mechanism presents
a much more complex set of challenges
cell
protein
network
disease
mechanism
mRNA
genotype
phenotype
Not The
all potential
number defects
of potential
arisedefects
from each
increases
mutation
exponentially with each emergent stage of complexity
cell
protein
network
disease
mechanism
mRNA
genotype
phenotype
The
Not most
all potential
effective
defects
target arise
for therapy
from each
( T ) mutation
would be
the DNA mutation, but this is currently unfeasible
cell
protein
network
disease
mechanism
mRNA
T
genotype
phenotype
The
Proteins
mostare
effective
also excellent
target fortargets
therapyfor( Tintervention
) would be
the DNA mutation, but this is currently unfeasible
cell
protein
T
network
disease
mechanism
mRNA
T
genotype
phenotype
The genetic contribution to epilepsy: the known and missing
heritability.
The Causes of Epilepsy, eds S.D. Shorvon et al, pp 63 67.
Cambridge University Press, Cambridge, 2011).
Genes and mutations in neonatal
syndromes and GEFS+
KCNQ2
Chr. 20q13.3
Benign familial neonatal convulsions (BFNC);
Benign neonatal epilepsy-1 (EBN1)
BFNC/myokymia syndrome
KCNQ3
Chr. 8q24
Benign familial neonatal convulsions (BFNC);
Benign neonatal epilepsy-2 (EBN2)
SCN1B
Chr. 19q13
Generalized epilepsy with febrile seizures plus,
type 1 (GEFS+ type 1; GEFSP1)
SCN1A
Chr. 2q24
GEFS+ type 2; GEFSP2
Severe myoclonic epilepsy in infancy (SMEI)
GABRG2
Chr. 5q33-q34
GEFS+ type 3; GEFSP3
SMEI
SCN2A
Chr. 2q23-q24
Febrile seizures associated with afebrile seizures
Benign familial neonatal-infantile seizures (BFNIS)
Unknown
Chr. 19q12-q13.1
Benign familial infantile convulsions, type 1
(BFIS type 1; BFIC1)
Chr. 16p12-q12
Unknown
BFIS type 2; BFIC2
Infantile convulsions and paroxysmal
choreoathetosis (ICCA)
Paroxysmal kinesigenic choreoathetosis (PKC)
Chr. 16p12-p11.2
Unknown
Rolandic epilepsy, paroxysmal exercise-induced
dystonia, writer's cramp (RE-PED-WC)
Chr. 16p13
Unknown
Autosomal recessive (familial) benign idiopathic
myoclonic epilepsy of infancy (FIME)
Traditional nomenclature of inherited epilepsy:
Gene 1
Mutations:
Phenotype: B A C A D
Syndrome “A”:
Syndrome “B”:
Syndrome “C”:
Syndrome “D”:
Syndrome “E”:
Syndrome “F”:
Syndrome “G”:
etc…
Gene 2
B A E F,G
Gene 1
Gene 2
Gene 3
Gene 1
Gene 4
Gene 2
Gene 2
Gene 3
Gene 4
A CC E
E
A
Gene 2 Gene 3 Gene 4
Gene 1
Gene 1
Gene 3
Gene-centric nomenclature of inherited epilepsy:
Gene 1
Mutations:
Phenotype: B A C A D
Gene 2
B A E F,G
Gene 3
Gene 4
A CC E
E
Gene 1:
Phenotypic range A, B, C, D
Gene 2:
Phenotypic range A, B, E, F, G
Gene 3:
Phenotypic range A, S, E
Gene 4:
etc…
Phenotypic range A, E
A
Both “Traditional” and “Gene-centric” nomenclatures
have specific advantages and disadvantages
Molecular Diagnosis
PATIENT HISTORY FOR MOLECULAR
GENETIC TESTING
(1) The Infantile Epilepsy includes sequencing and
deletion/duplication analysis of 38 genes causing
Mendelian forms of epilepsy with onset of seizures
during the first year of life.
38 gene activity, including
• voltage-gated sodium channels,
• the voltage-gated calcium channels,
• and gamma-aminobutyric acid (GABAA) receptors.
• (2) The Childhood-Onset Epilepsy 40 Genes
(3)The Adolescent-Onset Epilepsy 21 Genes
Includes sequencing and deletion/duplication
analysis of 40 and 21 genes respectively that causing
Mendelian forms of epilepsy.
• Genes that encode nicotinic acetylcholine receptors and
calcium channels,
Methodology
(1) Infantile Epilepsy Panel
(2) The Childhood-Onset Epilepsy
(3) The Adolescent-Onset Epilepsy
Using genomic DNA obtained from blood, ~ 570 coding exons
and the flanking splice junctions of 38 genes are sequenced
simultaneously by (next-generation sequencing).
• The sequence is assembled and compared to published
genomic reference sequences.
• Sanger sequencing is used to compensate for low coverage
and refractory amplifications in regions where pathogenic
mutations have been previously published.
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
Extraction of DNA from whole
Blood
Extract and discard plasma, taking care
not to remove the buffy coat.
Genetic Lab 48 MH
DNA Amplification with the
Polymerase Chain Reaction
(PCR) 3’
5’
5’
3’
3’
3’
5’
5’
Starting DNA
Template
Separate
strands
(denature)
Forward primer
5’
3’
5’
3’
Make copies
Add
primers
(extend
primers)
5’
(anneal)
3’
3’
5’
Reverse primer
PCR Copies DNA Exponentially
through Multiple Thermal
Cycles
Original DNA target region
Thermal cycle
In 32 cycles at 100% efficiency, 1.07 billion copies of
targeted DNA region are created
M 1
2
3
4
5
6
7
8
9
10 11
loss of bands
loss of bands
Deletion analysis of the SCN1A gene by
multiplex PCR.
COMPARATIVE GENOMIC
HYBRIDIZATION (CGH)
Identification of a deletion encompassing gene in a male patient.
Y-axes represent
R Log ratio
B allele frequency
The red line (log R ratio profile) corresponds
to the median smoothing series.
The X-axis indicates
the position on the X
chromosome
Identification of a hemizygous Xq22.1 deletion with a 370 K SNP microarray
Figure 1. Identification of a deletion encompassing PCDH19 in a male patient.
Analysis of the patient and his mother
with CGH microarrays, showing that
the new deletion occurred.
Black horizontal bars
represent the gene PCDH19)
and pseudogenes comprised
in the deleted region
ABI Prism 310 Genetic Analyzer
DNA template
3’-TAAATGATTCC-5’
5’
3’
A
Primer
Extension produces a series of ddNTP
AT
anneals
terminated products each one base
ATT
different in length
ATTT
ATTTA
ATTTAC
ATTTACT
ATTTACTA
ATTTACTAA
ATTTACTAAG
ATTTACTAAGG
Each ddNTP is labeled with
a different color fluorescent
dye
Epilepsy GEFS
 Link to Gene
OMIM Record
Epilepsy GEFS
Coriell Cell Repositories
Human Gene Mutation Database
Gen  Links to Everywhere (almost)
e
Epilepsy GEFS
GEFS Genome Maps

NT
Gene
Model
UniGene
Gene
NM
records
Gene
GEFS
Detection of 9 different point mutations of PCDH19 in 11 female patients by direct sequencing.
The A of the ATG
translation initiation
codon in the reference
sequence
Sequence electropherograms of the mutations and the missense
variant (c.3319C>G/p.Arg1107Gly) identified in association with the
c.859G>T/p.Glu287X nonsense mutation.
Alignment of the regions surrounding the mutations (indicated by an arrow) in
orthologous and paralogous proteins,
showing the high conservation of each affected amino-acid in
vertebrates and in the delta protocadherin paralogous genes.
FISH analysis of the gene deletion in the male patient showing somatic mosaicism in fibroblasts.
A) Absence of the specific
Xq22.1 probe site on
metaphase chromosomes
(PBL);
(B) In fibroblasts, presence of
one hybridization spot in 53% of
the cells and absence of signal
in the remaining 47%;
FISH analysis on PBL (C) and fibroblasts (D) of a female control.
PCDH19-specific signals (red) are indicated by arrowheads.