Presentation on theme: "Nagi ALHaj MOLECULAR DIAGNOSIS OF GENETIC EPILEPSY"— Presentation transcript:
1Nagi ALHaj MOLECULAR DIAGNOSIS OF GENETIC EPILEPSY Prof of Molecular Biology Assist,Faculty Medicine , Sana’a ,University, YemenConsultant of Genetic Center 48 MH
2Overview 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 traitNon-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 DNAChromosomal disorders, in which a gross cytogenetic abnormality is present.
3The ‘common’ non-familial idiopathic epilepsies tend to display ‘complex’ inheritance. They including various forms ofIdiopathic generalised epilepsy (IGE)Juvenile myoclonic epilepsy (JME)Childhood absence epilepsy (CAE)
4Genetics and MutationMutations in over 2,000 genes have now been identified inpatients with more than 3,000 different disease phenotypes.For the clinicians and their patients, it is becoming increasinglyimportant to obtain a genetic diagnosisIdentifying 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.
5The 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).
6Traditional nomenclature of inherited epilepsy: Different mutations in different genes can result in different phenotypesDifferent mutations in different genes can result in similar phenotypesDifferent mutations within one gene can result in different phenotypesAn identical mutation within one gene can result in different phenotypes in different individuals (cause: environment, other genes)
7Mutation identification begins with a phenotype and proceeds toward the genotypegenotypephenotype
10The diagram showsthe distribution of all genetic differences that had been mapped to chromosome 1 at the time this diagram was drawn.
11Mutation 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.mutationsiteGenome scan has been replaced by mutational analysis but in a small number of families in whom the mutation cannot be identifiedRemains the only method for the genetic diagnosis of carriers.
12Mutant 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.
13disease mechanism genotype phenotype Elucidation of a disease mechanism presentsa much more complex set of challengescellproteinnetworkdiseasemechanismmRNAgenotypephenotype
14disease mechanism genotype phenotype Not all potential defects arise from each mutationThe number of potential defects increasesexponentially with each emergent stage of complexitycellproteinnetworkdiseasemechanismmRNAgenotypephenotype
15disease mechanism T genotype phenotype Not all potential defects arise from each mutationThe most effective target for therapy ( ) would bethe DNA mutation, but this is currently unfeasibleTcellproteinnetworkdiseasemechanismmRNATgenotypephenotype
16T disease mechanism T genotype phenotype The most effective target for therapy ( ) would bethe DNA mutation, but this is currently unfeasibleTTProteins are also excellent targets for interventioncellproteinnetworkdiseasemechanismmRNATgenotypephenotype
17The genetic contribution to epilepsy: the known and missing heritability. The Causes of Epilepsy, eds S.D. Shorvon et al, pp 6367. Cambridge University Press, Cambridge, 2011).
18Genes and mutations in neonatal syndromes and GEFS+ KCNQ2Chr. 20q13.3Benign familial neonatal convulsions (BFNC); Benign neonatal epilepsy-1 (EBN1)BFNC/myokymia syndromeKCNQ3Chr. 8q24Benign familial neonatal convulsions (BFNC); Benign neonatal epilepsy-2 (EBN2)
19SCN1B SCN1A GABRG2 SCN2A Chr. 19q13 Generalized epilepsy with febrile seizures plus, type 1 (GEFS+ type 1; GEFSP1)SCN1AChr. 2q24GEFS+ type 2; GEFSP2Severe myoclonic epilepsy in infancy (SMEI)GABRG2Chr. 5q33-q34GEFS+ type 3; GEFSP3SMEISCN2AChr. 2q23-q24Febrile seizures associated with afebrile seizuresBenign familial neonatal-infantile seizures (BFNIS)
20Unknown Unknown Unknown Unknown Chr. 19q12-q13.1 Benign familial infantile convulsions, type 1 (BFIS type 1; BFIC1)UnknownChr. 16p12-q12BFIS type 2; BFIC2Infantile convulsions and paroxysmal choreoathetosis (ICCA)Paroxysmal kinesigenic choreoathetosis (PKC)UnknownChr. 16p12-p11.2Rolandic epilepsy, paroxysmal exercise-induced dystonia, writer's cramp (RE-PED-WC)UnknownChr. 16p13Autosomal recessive (familial) benign idiopathic myoclonic epilepsy of infancy (FIME)
22Gene-centric nomenclature of inherited epilepsy: Mutations:Phenotype:BACADBAEF,GACCEEAGene 1:Phenotypic range A, B, C, DGene 2:Phenotypic range A, B, E, F, GGene 3:Phenotypic range A, S, EGene 4:Phenotypic range A, Eetc…Both “Traditional” and “Gene-centric” nomenclatures have specific advantages and disadvantages
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, includingvoltage-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,
29Methodology Infantile Epilepsy Panel The Childhood-Onset Epilepsy The Adolescent-Onset EpilepsyUsing 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.
30Individual nucleotides DNA in the Cellchromosomecell nucleusDouble stranded DNA moleculeIndividual nucleotidesTarget Region for PCR
31Extraction of DNA from whole Blood Extract and discard plasma, taking care not to remove the buffy coat.
33DNA Amplification with the Polymerase Chain Reaction (PCR) Separate strands (denature)5’3’Starting DNA Template5’3’Add primers (anneal)5’3’Forward primerReverse primer5’3’Make copies (extend primers)
34PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target regionThermal cycleThermal cycleThermal cycleIn 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
35Deletion analysis of the SCN1A gene by multiplex PCR. loss of bandsloss of bandsDeletion analysis of the SCN1A gene by multiplex PCR.
37Identification of a deletion encompassing gene in a male patient. Y-axes representR Log ratioB allele frequencyThe red line (log R ratio profile) corresponds to the median smoothing series.The X-axis indicates the position on the X chromosomeIdentification of a hemizygous Xq22.1 deletion with a 370 K SNP microarray
38Figure 1. Identification of a deletion encompassing PCDH19 in a male patient. Black horizontal bars represent the gene PCDH19) and pseudogenes comprised in the deleted regionAnalysis of the patient and his mother with CGH microarrays, showing that the new deletion occurred.
413’-TAAATGATTCC-5’ A AT ATT ATTT ATTTA ATTTAC ATTTACT ATTTACTA ATTTACTAAATTTACTATTTACATTTATTTAATATTTACTAATTTACTAAGATTTACTAAGGADNA template5’3’Primer annealsExtension produces a series of ddNTP terminated products each one base different in lengthEach ddNTP is labeled with a different color fluorescent dyeFigure 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.
46Detection 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 sequenceSequence 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.
47Alignment 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.
48FISH 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.