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

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Presentation on theme: "MOLECULAR DIAGNOSIS OF GENETIC EPILEPSY Nagi ALHaj Prof of Molecular Biology Assist, Faculty Medicine, Sana’a,University, Yemen Consultant of Genetic Center."— Presentation transcript:

1 MOLECULAR DIAGNOSIS OF GENETIC EPILEPSY Nagi ALHaj 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 similar phenotypes Different mutations within one gene can result in different phenotypes Different mutations in different genes 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

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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 site Mutation identification by linkage analysis 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. Mutational analysis can be used to identify cells or DNA that have genotype and allele frequency differences from the normal genome.

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 genotypephenotype mechanism disease Elucidation of a disease mechanism presents a much more complex set of challenges mRNA protein cell network

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

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

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

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 Chr. 19q13 Generalized epilepsy with febrile seizures plus, type 1 (GEFS+ type 1; GEFSP1) SCN1A Chr. 2q24 GEFS+ type 2; GEFSP2 GABRG2 Chr. 5q33-q34 GEFS+ type 3; GEFSP3 SCN2A Chr. 2q23-q24 Febrile seizures associated with afebrile seizures Severe myoclonic epilepsy in infancy (SMEI) SMEI Benign familial neonatal-infantile seizures (BFNIS)

20 Unknown Chr. 19q12-q13.1 Benign familial infantile convulsions, type 1 (BFIS type 1; BFIC1) Unknown Chr. 16p12-q12 BFIS type 2; BFIC2 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) Paroxysmal kinesigenic choreoathetosis (PKC) Infantile convulsions and paroxysmal choreoathetosis (ICCA)

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

22 Gene-centric nomenclature of inherited epilepsy: Gene 1: Phenotypic range A, B, C, D etc… Gene 2: Gene 3: Gene 4: Phenotypic range A, B, E, F, G Phenotypic range A, S, E Phenotypic range A, E Both “Traditional” and “Gene-centric” nomenclatures have specific advantages and disadvantages Gene 1Gene 2Gene 3Gene 4 A Mutations: B Phenotype: ABCDAACF,GEE E C A

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.

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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,

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29 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.

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

31 Extract and discard plasma, taking care not to remove the buffy coat.

32 Genetic Lab 48 MH

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

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

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

36 COMPARATIVE GENOMIC HYBRIDIZATION (CGH)

37 Identification of a deletion encompassing gene in a male patient. Identification of a hemizygous Xq22.1 deletion with a 370 K SNP microarray 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

38 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

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40 ABI Prism 310 Genetic Analyzer

41 3’-TAAATGATTCC-5’ ATT 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

42 Epilepsy GEFS

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

44 Gen e  Links to Everywhere (almost) Epilepsy GEFS

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

46 Detection of 9 different point mutations of PCDH19 in 11 female patients by direct sequencing. 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. The A of the ATG translation initiation codon in the reference sequence

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. FISH analysis on PBL (C) and fibroblasts (D) of a female control. PCDH19-specific signals (red) are indicated by arrowheads. 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%;

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