1. Nagi ALHaj MOLECULAR DIAGNOSIS OF GENETIC EPILEPSY
Prof of Molecular Biology Assist,
Faculty Medicine , Sana’a ,University, Yemen
Consultant of Genetic Center 48 MH

2. 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.

3. 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)

4. 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.

5. 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).

6. 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)

7. Mutation identification begins with a
phenotype and proceeds toward the genotype
genotype
phenotype

10. The diagram shows
the distribution of all genetic differences that had been mapped to chromosome 1 at the time this diagram was drawn.

11. 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.

12. 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.

13. disease mechanism genotype phenotype
Elucidation of a disease mechanism presents
a much more complex set of challenges
cell
protein
network
disease
mechanism
mRNA
genotype
phenotype

14. disease mechanism genotype phenotype
Not all potential defects arise from each mutation
The number of potential defects increases
exponentially with each emergent stage of complexity
cell
protein
network
disease
mechanism
mRNA
genotype
phenotype

15. disease mechanism T genotype phenotype
Not all potential defects arise from each mutation
The most effective target for therapy ( ) would be
the DNA mutation, but this is currently unfeasible T
cell
protein
network
disease
mechanism
mRNA T
genotype
phenotype

16. T disease mechanism T genotype phenotype
The most effective target for therapy ( ) would be
the DNA mutation, but this is currently unfeasible T T
Proteins are also excellent targets for intervention
cell
protein
network
disease
mechanism
mRNA T
genotype
phenotype

17. 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).

18. 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)

19. SCN1B SCN1A GABRG2 SCN2A 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)

20. Unknown Unknown Unknown Unknown Chr. 19q12-q13.1
Benign familial infantile convulsions, type 1 (BFIS type 1; BFIC1)
Unknown
Chr. 16p12-q12
BFIS type 2; BFIC2
Infantile convulsions and paroxysmal choreoathetosis (ICCA)
Paroxysmal kinesigenic choreoathetosis (PKC)
Unknown
Chr. 16p12-p11.2
Rolandic epilepsy, paroxysmal exercise-induced dystonia, writer's cramp (RE-PED-WC)
Unknown
Chr. 16p13
Autosomal recessive (familial) benign idiopathic myoclonic epilepsy of infancy (FIME)

21. Traditional nomenclature of inherited epilepsy:
Gene 1
Gene 2
Gene 3
Gene 4
Mutations:
Phenotype: B A C D
F,G E
Syndrome “A”:
Gene 1
Gene 2
Gene 3
Gene 4
Syndrome “B”:
Gene 2
Gene 1
Syndrome “C”:
Gene 3
Gene 1
Syndrome “D”:
Gene 1
Gene 4
Gene 2
Gene 3
Syndrome “E”:
Syndrome “F”:
Syndrome “G”:
etc…

22. Gene-centric nomenclature of inherited epilepsy:
Mutations:
Phenotype: B A C A D B A E
F,G A C C E E A
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:
Phenotypic range A, E
etc…
Both “Traditional” and “Gene-centric” nomenclatures have specific advantages and disadvantages

23. Molecular Diagnosis

24. PATIENT HISTORY FOR MOLECULAR GENETIC TESTING

25. (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.

27. (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,

29. Methodology Infantile Epilepsy Panel The Childhood-Onset Epilepsy
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.

30. Individual nucleotides
DNA in the Cell
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotides
Target Region for PCR

31. Extraction of DNA from whole Blood
Extract and discard plasma, taking care not to remove the buffy coat.

32. Genetic Lab 48 MH

33. DNA Amplification with the Polymerase Chain Reaction (PCR)
Separate strands (denature) 5’ 3’
Starting DNA Template 5’ 3’
Add primers (anneal) 5’ 3’
Forward primer
Reverse primer 5’ 3’
Make copies (extend primers)

34. PCR Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Thermal cycle
Thermal cycle
Thermal cycle
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created

35. Deletion analysis of the SCN1A gene by multiplex PCR.
loss of bands
loss of bands
Deletion analysis of the SCN1A gene by multiplex PCR.

36. COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

37. 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

38. Figure 1. Identification of a deletion encompassing PCDH19 in a male patient.
Black horizontal bars represent the gene PCDH19) and pseudogenes comprised in the deleted region
Analysis of the patient and his mother with CGH microarrays, showing that the new deletion occurred.

40. ABI Prism 310 Genetic Analyzer

41. 3’-TAAATGATTCC-5’ A AT ATT ATTT ATTTA ATTTAC ATTTACT ATTTACTA
ATTTACTAA
ATTTACT
ATTTAC
ATTT
ATTTA AT
ATTTACTA
ATTTACTAAG
ATTTACTAAGG A
DNA template 5’ 3’
Primer anneals
Extension produces a series of ddNTP terminated products each one base different in length
Each ddNTP is labeled with a different color fluorescent dye
Figure 10.5 DNA sequencing process with fluorescent ddNTPs. A primer that has been designed to recognize a specific region of a DNA template anneals and is extended with a polymerase. Because a mixture of dNTPs and ddNTPs exist for each of the four possible nucleotides, some of the extension products are halted by incorporation of a ddNTP while other molecules continue to be extended. Each ddNTP is labeled with a different dye that enables each extension product to be distinguished by color. A size-based separation of the extension products permits the DNA sequence to be read provided that sufficient resolution is present to clearly see each base.

42. Epilepsy GEFS

43. OMIM Record  Link to Gene Epilepsy GEFS Coriell Cell Repositories
Human Gene Mutation Database

44. Gene
 Links to Everywhere (almost)
Epilepsy GEFS

45.  Gene
GEFS Genome Maps
GEFS
NM records NT
Gene Model
UniGene
Gene

46. 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.

47. 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.

48. 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.

49. THANK YOU