INTRODUCTION NEW GENETIC TECHNIQUES IN METABOLIC DISEASES 26/01/2016 FROM ONE GENERATION TO THE NEXT. Image challenge of the week D.

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1 NEW GENETIC TECHNIQUES IN METABOLIC DISEASES D. RYMEN, MD, PHD Pentalfa session Leuven, January 21 st 2016 INTRODUCTION Image challenge of the week 1

2 INTRODUCTION But what about Hypotonia Facial dysmorphism Moderate ID Obesity INTRODUCTION But what about heterogeneity? One phenotype MANY DISEASES One disease MANY GENES 2

3 INTRODUCTION But what about heterogeneity? CLINICAL AWARENESS HETEROGENEITY SCREENING TEST DIAGNOSTIC TEST BIOCHEMISTRY GENETICS INTRODUCTION But what about heterogeneity? 3

4 HOW TO SOLVE Image challenge of the week HOW TO SOLVE Image challenge of the week 4

5 HOW TO SOLVE But what about Hypotonia Facial dysmorphism Moderate ID Obesity HOW TO SOLVE A diagnostic odyssey CLINICAL AWARENESS HETEROGENEITY SCREENING DIAGNOSIS??? 5

6 HOW TO SOLVE A diagnostic odyssey Biochemistry vs genetics Screening BIOCHEMISTRY Diagnosis Confirmation Diagnosis GENETICS Confirmation THE GENETICMAZE From genome to library Library Genome Bookcase (22 x 2) + XY Chromosome Book Gene Word 20 Amino Acid Letter 4 Base Pair 6

7 THE GENETICMAZE From genome to library Library Genome FISH Bookcase (22 x 2) + XY Chromosome Karyotype Array CGH Sanger sequencing Book Gene??? Word 20 Amino Acid Letter 4 Base Pair THE GENETICMAZE From genome to library CLINICAL AWARENESS SCREENING: IEF of serum transferrin (type 2 pattern) DIAGNOSIS??? 7

8 THE GENETICMAZE From genome to library MASSIVE PARALLEL SEQUENCING TECHNIQUES THE GENETICMAZE From genome to library Library Genome FISH Bookcase (22 x 2) + XY Chromosome Karyotype Array CGH Sanger sequencing Targeted gene panels Book Gene WES WGS Word 20 Amino Acid Letter 4 Base Pair 8

9 THE GENETICMAZE A genomic revolution Screening BIOCHEMISTRY Diagnosis Confirmation Screening Diagnosis GENETICS Confirmation THE GENETICMAZE A genomic revolution 9

10 Sanger sequencing (the first generation) Targeted approach Genomic DNA Exon1 Exon2 Exon3 SNPs Small deletions/insertions Sanger sequencing (the first generation) Dideoxy chain termination method TAGCT datp dttp dgtp dctp ddatp ddttp ddgtp ddctp TAGCTGA TAGCTGACT TAGCTG TAGCTGAC TAGCTGACTCA TAGCTGACTCAGT TAGCTGACTCAG TAGCTGACTC TAGCTGACTCAGTTCTTGA TAGCTGACTCAGTT TAGCTGACTCAGTTCTTG TAGCTGACTCAGTTC 10

11 Sanger sequencing (the first generation) Dideoxy chain termination method Genome wide approach Exome (+ intronic boundaries) Known protein coding regions (1 2% of the total genome) 85% of mutations in Mendelian disorders SNPs small deletions/insertions Copy number variations (WGS) Targeted panel Mendeliomes WES WGS 11

12 Library preparation 1. Fragmentation of template DNA 2. Ligation of adaptor molecules 3. Denaturation of template DNA Clonal amplification 12

13 Sequencing Alignment and data analysis 13

14 Alignment and data analysis Alignment and data analysis Quality filters Population databases Predictive software Inheritance pattern Clinical fit 14

15 Success rate Hypotonia Facial dysmorphism Moderate ID Obesity 12% 20% 40% 46% 42% 47% 25% 43% 18% 8% 32% 38% 27% 29% 36% 45% 1% 5% 26% 30% 19% 24% 50% 44% 41% 15% 35% 3% 2% 16% 10% 49% 33% 7% 34% 0% 14% 28% 21% 37% 23% 4% 13% 17% 6% 11% 31% 39% 48% 9% Success rate: 15 45% Targeted panel Mendeliomes WES WGS 15

16 Success rate: 15 45% Targeted panel Mendeliomes WES WGS Success rate: 15 45% Annotation Number of genes Targeted panel Mendeliomes WES WGS 16

17 Success rate: 15 45% Annotation Ease of interpretation Targeted panel Mendeliomes WES WGS Success rate: 15 45% Targeted panel Mendeliomes WES WGS 17

18 WHAT ABOUT CDG II Hypotonia Facial dysmorphism Moderate ID Obesity Diagnostics Research Targeted panel Mendeliomes WES WGS 18