Familial Breast Cancer

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1 Familial Breast Cancer SEARCHING THE GENES Samuel J. Haryono 1

Issues in HSBOC Spectrum of mutation testing in familial breast cancer

carrier BRCA mutation with TNBC Recurrent mutation in Indonesia

preventive, risk assessment, genetic counselling SAMANDA (

2 Issues in HSBOC Spectrum of mutation testing in familial breast cancer Variant of BRCA vs mutation of BRCA Clinical guideline and management for carrier BRCA mutation with TNBC Recurrent mutation in Indonesia Penetrance, sporadic and modifier genes Sanger, HRM, NGS Options of preventive, risk assessment, genetic counselling SAMANDA ( Syndrome-Adopted Mutation-Assessment- Need-Data-Analysis) risk calculation for Indonesian specific FBC 2

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brca2, ER, lower penetrans, less evidence, is it

8 1866 Paul Broca pedigree in BC varian mutation of BRCA1 RRM =risk reduction mastectomy in Asia is less: brca2, ER, lower penetrans, less evidence, is it appropriate action / option? GWAS (Genome-Wide Association Study) 8

9 Introduction Development in genomic era 9

10 Not 100% Identical!! 10

11 Development in genomic era 11

12 SNP Single Nucleotide Polymorphism 12

13 10 million SNPs Development in genomic era 13

14 Lingkage SNPs International Hapmap Consortium The International HapMap Consortium A haplotype map of the human genome. Nature.437:

15 Development in genomic era 15

16 DNA Microarray Collection of microscopic DNA spots attached to a solid surface Genome-Wide 5.0 Chip (Affymetrix) 16

17 Development in genomic era 17

18 GWAS (Genome-Wide Association Study) GWAS - Whole Genome Association Studies (WGAS) - Correlation between variant vs traits - Variant (e.g SNP) genotype - Trait (major disease in this case : breast cancer) phenotype 18

19 Published Genome-Wide Associations through 07/2012 Published GWA at p 5X10-8 for 18 trait categories NHGRI GWA Catalog es 19

20 Association Study Cross-Sectional Cohor t Case Control Case-control Cohort Cross-Sectional Exposure Past Present Present Disease Present Future Present Case-Control Present (Breast Cancer ) (Breast Cancer -) Genotype Analysis past GENE SNP (marker) SNP (marker) 20

21 Methods Multistage design 21

22 Sample Preparation 50 Cases 50 Control DNA Isolation (Roche) DNA quantity check ([DNA]= ng/μl DNA quantity check (A260/A280 = ) 22

23 Microarray Affymetrix Genome-Wide SNP array 5.0 Sample Preparation (Whole Genome Sampling Assay) Kennedy GC et al Largescale genotyping of complex DNA. Nat Biotechnol. 21:

Signal Measurement Kennedy GC et al. 2003.

24 Signal Measurement Kennedy GC et al Large-scale genotyping of complex DNA. Nat Biotechnol. 21: Data Analysis 24

25 Preliminary Analysis Flowchart Signal from Chip genotyping Genotype Sample (100) SNP (400k) SNP & Sample Filtering 25

26 Flowchart Signal from Chip genotyping Genotype Sample (less than 100) SNP (less than 400k) Haplotype SNPs Tag Genotyping Signal from Chip genotyping Genotype 26

27 Genotyping (BRLMM-P) Software : Genotyping Console 4.0 (Affymetrix) Affymetrix BRLMM-P: a genotype calling method for the SNP 5.0 array. Technical Report. 27

28 Genome-Wide Associated Study (GWAS) on hereditary susceptibility breast cancer in Indonesia -Samuel J Haryono- SNP summary (1) 28

29 SNP summary (2) List of SNPs per-sample and its genotype Probe set ID Allele genotyping confidence Allele genotype contrast strength Sequence genotyping dbsnp rs ID Chromosomal position 29

30 Sample & SNP Filtering Sample (100) SNP (400k) SNP & Sample Filtering Sample (less than 100) SNP (less than 400k) 30

31 Sample Filtering 1. SNP Call Rate Fraction of called SNPs per sample over the total number of SNPs in the datasheet Missing genotype data Software : Genotyping Console 4.0 (Affymetrix) Sample (100) SNP (400k) 31

referred to as completeness) less than some threshold.

32 Sample Filtering Include SNP Exclude SNP 100% SNP Filtering 1. per-snp Call Rate Remove SNP per-snp call rate (sometimes referred to as completeness) less than some threshold. Commonly-used value for the per-snp call rate threshold range from 90-95%. 32

33 Sample (100) SNP (400k) SNP Filtering 2. HWE (Hardy-Weinberg Equilibrium) p-value Allele frequency vs Genotype frequency Remove SNPs significantly out of Hardy Weinberg Equilibrium in cases and/or control. A p-value threshold in the range of 10-7 is commonly used. 33

34 SNP Filtering 3. MAF (Minor Allele Frequency) Frequency at which the less common allele occurs in a given population. Conside removing SNPs with minor allele frequency (MAF) less than certain threshold (for example 1%) Chromosom SNPs before after Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome X total % Exlude SNPs (18.68%) 34

35 Haplotype Analysis Haplotype block Pairwise tagging Linkage Disequilibrium measurement r 2 SNPs tags Linkage Disequilibrium r 2 provides information on the corelation of two loci 35

36 Haplotype Block Software : Haploview

37 Software : Haploview 4.2 Association Study 37

38 Population stratification Marchini J, et al The effects of human population structure on large genetic association studies. Nat Genet. 36:

39 Manhattan Plot Wang K, et al Common genetic variants on 5p14.1 associate with autism spectrum disorders. Nature. 459:

40 Acknowledgement Samuel Haryono Althaf Setyawan IGB Datasena Wahyu Budi Santosa Adrian Salim Raymond Mulyarahardja Dismas A. Chaspuri Uti Nilam Sari Kartika Sari Thank You 40