Chips and next-generation sequencing tools for diagnosis in NMDs

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1 Chips and next-generation sequencing tools for diagnosis in NMDs Madhuri Hegde, PhD, FACMG Associate Professor Scientific Director, Emory Genetics Laboratory Emory University

2 Genomic Variation Microarray (~5 Mb) FISH G-banded karyotype Scherer et al. (2007) Nat Genet 39:s7-s15

3 Gene mutation spectrum Other NMD genes 80% small mutations 20% deletions/duplications Duchenne Muscular Dystrophy (DMD) 30% small mutations 65-70% deletions/duplications Small mutations (nucleotide substitutions, small indels) Larger mutations (exon and multi-exon dels and dups) Some common del- -NMD genes Cystic Fibrosis (CFTR) >90% small mutations <10% deletions/duplications Krabbe disease (GALC) 35-50% deletions/duplications 50-65% small mutations Figure 1

4 Deletion/Duplication Detection Methodology in clinical diagnostics acgh FISH MLPA Real-Time PCR

5 Deletion Ex8-13 DMD gene: MLPA vs CGH

6 MLPA

7 MLPA (DMD gene)

8 Ex79 DMD Array CGH (385k): Males Ex1 del Ex17 Ex44 del Ex48 Ex52 dup Ex2 Ex4

9 DMD Array CGH (385k): Females Ex79 Ex1 del Ex46 Ex55 del Ex49 Ex50 dup Ex18 Ex38

10 Other sample types

11 Using blood spots as a starting material

12 Data from dried blood spots- Newborn/ Infant screening

13 Data from dried blood spots- Newborn/ Infant screening

14 Data from dried blood spots- Newborn/ Infant screening

15 DMD (Archived sample) A young woman (K.M.) in early pregnancy She knew of a history in her mother s family of DMD. Three maternal uncles (2 were identical twins) had been diagnosed with DMD in the 1960s, and died in the 1970s-80s Don Love, Auckland, NZ

16 tissue found!

17 Exon 63 dup

18 NMD design similar to multi gene design 48 diseases genes NMD chip design

19 11DSGCG07789 Dup Ex1-2 LGMD 2C - SGCG del/dup

20 11DLAM Inv Ex1? MDC1A - LAMA2 Del/Dup

21 11DCMDP SEPN1 Dup Ex3 CMD del/dup panel

22 11DEMDX Del Ex2-6 Emery-Dreifuss MD, X-Linked - EMD del/dup

23 Gene mutation spectrum Other NMD genes 80% small mutations 20% deletions/duplications Duchenne Muscular Dystrophy (DMD) 30% small mutations 65-70% deletions/duplications Small mutations (nucleotide substitutions, small indels) Larger mutations (exon and multi-exon dels and dups) Some common del- -NMD genes Cystic Fibrosis (CFTR) >90% small mutations <10% deletions/duplications Krabbe disease (GALC) 35-50% deletions/duplications 50-65% small mutations

24 Lifecycle of a single gene test - Evidence based testing Gene reported in literature Mutation spectrum, clinical phenotype reported in literature Clinical laboratory decision Mutation, UV and benign changes database Determine gene transcript, exons, scan literature Clinical validation (CLIA/ CAP) Test launch

25 Why move to NGS? Sanger sequencing Used to detect mutations in disease genes has limited throughput and costly Mutation identification is of paramount importance for diagnosis confirmation genetic counseling risk assessment carrier screening NGS allows many genes in a patient to be analyzed at same time Allowing mutation detection when there are many genes for a specific phenotype Allows deep intronic and promoter mutations to be analyzed Bioinformatic tools mature to implement in a clinical laboratory

26 Target Enrichment Individual PCR Not viable to be done routinely in the laboratory for large fragment libraries Microarray Agilent SureSelect, Nimblegen SeqCap were evaluated. SeqCap did not give satisfactory results - unequal coverage, inability to select all exons, allele dropout, genes/ pseudogenes. Microfluidic chip Fluidigm & Raindance

27 Validation testing PCR (all coding exons/gene) 1 day PCR purification PCR quantification 4 days Pooling Solid epcr & Run 16 days Sequence Match / Analysis 22 days Confirm all changes 27 days False +ve & -ve rate Raindance (all coding exons/gene) 1 day 2 days Droplet PCR PCR purification Solid epcr & Run 14 days Sequence Match / Analysis 20 days Confirm all changes 30 days False +ve & -ve rate SureSelect (Coding exons/gene) 3 days 4 days Hybridization & Capture Amplification Solid epcr & Run 16 days Sequence Match / Analysis 22 days Confirm all changes 32 days False +ve & -ve rate

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29 Rules for library design Highly stringent Primers average length Overlapping fragments (100 bp) No primers on SNP s and repeat regions No allele dropout Gene with pseudogenes not included

30 Single genes>>>>>> multi gene panels using next gen sequencing

31 Nextgen sequencing panels X-linked Intellectual disability 92 genes, 1500 amplicons, 898 kb Syndromic and non- syndromic Availability of positive controls Mainly males Hemizygous XLID CDG CMD Congenital Disorders of Glycosylation All types of genes: metabolic, ID 25 genes, 288 amplicons, 101 kb Availability of positive controls Biochem test (first) Molecular confirmation Congenital Muscular Dystrophy 13 genes, 383 amplicons, 65 kb Genes well characterized Availability of positive controls Immunohistochemistry - Painful muscle biopsy and no definitive results - Secondary effect

32 COG7 homozygous c.323dupt Fluidigm Raindance ID Gene Mutation Coverage Ins(%) CDG_0327 COG7 c.323_324dupt ID Gene Mutation Coverage Ins(%) CDG_0327 COG7 c.323_324dupt Sanger

33 RainDance vs SureSelect

34 Total variants detected using panels Panel Number of genes Number of pseudogenes Average variants found XLID CDG CMD Internal Mutation database for continuous reclassification of variants

35 Mutation Database

36 Lifecycle of a single gene test - Evidence based testing Gene reported in literature Mutation spectrum, clinical phenotype reported in literature Clinical laboratory decision Mutation, UV and benign changes database Determine gene transcript, exons, scan literature Clinical validation (CLIA/ CAP) Test launch

37 Genome web article 25 May 2010 When it costs $1,000 to sequence a genome, why would you take a test that costs $5,000? Callum Bell, program lead at the NCGR

38 Whole ex/ge ome analysis Challenges - Data interpretation- mapping, SNP and indel detection - Who will do the analysis? Bioinformatics - Time needed to do analysis - Reclassification of VOUS - Gene discovery in a clinical setting? -Identification of mutation (truncating vs. missense) in new gene of unknown function (e.g. biochemical) - CLIA / CAP Certification - Reagent cost vs. cost of the test - How will the reports be written?

39 Future of Sequencing in a clinical laboratory - Use experience from single gene and panel sequencing - Clinical testing (CLIA/CAP) based on known disease associated genes, positive controls available - Single gene (SS) >> Panels (SS or NGS) >> Exome >> Genome - Logical interpretation of the data - Exome / Genome analysis involves a TEAM - Clinical & Laboratory Geneticists, Genetic Counselor, and other healthcare specialist - More appropriate for familial cases vs. isolated cases - Clinical presentation Strategy

40 Sequencing panels Exome Genome What is complete test? What is purpose of the test? Test interpretation What will be reported? Databasing of sequence variation - Core laboratories - Clinically curated variants

41 Exome

42 Emory Ephrem Chin Brenda Billotte Sharon Langley Martin Littlejohn Lisa Kuang Shruti Bhide Melanie Jones Devin Rhodenizer Miao He Alice Tanner Lori Bean Brad Coffee Stephen Warren Acknowledgement Softgenetics John Fosnacht Jonathan Liu Megan Manion Greenwood Genetic Laboratory Mike Friez Roger Stevenson Burham Sanford Institute Hudson Freeze Bobby Ng Funding: Muscular Dystrophy Association TRIG grant Parent Project Muscular Dystrophy NINDS RC1 (MH) NIH ORDR CETT (MH) U 41 Grant Heidi Rehm Sherri Bale Patrick Willems David Ledbetter Christa Martin Robert Nussbaum