8/23/2014

Unmasking the Genetic Diagnosis: Updates on Whole Exome Sequencing and Inherited Cancer Testing

Contact Information Jackie Tahiliani, MS, CGC [email protected] Kristen Vogel Postula, MS, CGC [email protected] Erica Vaccari, MS, CGC [email protected]

2

Conflict of Interest  We are employees of GeneDx, a laboratory that performs multi-gene testing panels and Whole Exome Sequencing (WES), which will be highlighted in this presentation. This session is sponsored by GeneDx.  GeneDx is a wholly owned subsidiary of BioReference Laboratories, a publicly traded company.

3

1

8/23/2014

Humble Beginnings

Sherri Bale and John Compton, two scientists from the National Institutes of Health (NIH), founded GeneDx in the year 2000 to address the needs of patients and clinicians concerned with rare inherited disorders. 4

Milestones

>100 Tests

5

The GeneDx Difference Clinical Expertise Technical Expertise

• More than 30 MDs/PhDs and 50 Genetic Counselors • Expertise in next-generation sequencing, whole genome CGH array, exon-level CGH array, etc.

Innovation

• First to launch clinical next-gen sequencing panels, first to launch oligo-based array CGH

Provider and Patient Resources

• Customer service genetic counselors, variant testing program, patient-friendly result reports

Extensive Test Offering

• More than 400 tests on the menu for both common and rare genetic disorders

6

2

8/23/2014

Learning Objectives  Describe the inherited cancer genetic testing and Whole Exome Sequencing (WES) offerings provided by GeneDx, Inc. and discuss testing strategies.  Review variant classification methods and the Variant Testing Program.  Examine the clinical utility and genetic counseling considerations of inherited cancer NGS gene panels, WES, and XomeDxSlice. 7

Unmasking the Genetic Diagnosis: Updates on Whole Exome Sequencing Jackie Tahiliani, MS, CGC September 19th, 2014

8

AGENDA

Whole Exome Sequencing (WES) XomeDxSlice Case Examples

9

3

8/23/2014

Whole Exome Sequencing 

The coding region of the nuclear genome = ~200,000 exons



~1-2% of the genome (30Mb) ~20,000 genes



85% of mutations known to cause disease are in exons



Coding regions of interest are targeted and “captured” for sequencing



Sequenced using Next Generation technology



Generates a massive amount of data which needs to be filtered to find the genetic diagnosis 10

Next Generation Sequencing C>G het



High-throughput system that allows sequencing of multiple genes simultaneously



Traditionally used to sequence multiple genes of interest



Many overlapping “reads” are obtained for each DNA base



Reads are aligned to the reference sequence

Reference

Black arrow indicating the reference sequence. Red arrow pointing to the nucleotide change. Blue color= reverse reads; Green color= forward reads

NGS & WES Technical Benefits and Limitations Benefits

Limitations

 NextGen technology sequences thousands of targeted regions simultaneously



With NGS, some genes or portions of genes are not amenable to sequencing and alignment

 Cost effective way to analyze a large number of genes



Challenging to sequence difficult areas of the genome



Not designed to detect exon-level deletions or duplications



With WES, it is not technically possible to capture and sequence the entire exome at present



The scientific knowledge available about the function of all genes in the human genome is incomplete

 Turn around time for results  Coverage: – Inherited Cancer Panels: >500x coverage – WES: >95% of the targeted region of an individual’s exome will be assessed at 10x coverage

12

4

8/23/2014

WES Methodology Variant detection in the lab

Bioinformatic pipeline

Analysis

Variant Interpretation

Report Writing

Who is undergoing whole exome sequencing?  Patients who have undergone an extensive testing, with no molecular basis identified – Microarray negative – Individual gene tests negative – Targeted panels negative

 Patients with a clinical phenotype that could be explained by one of many genes – Neurological disorders, eye disorders, etc. – Multiple congenital anomalies – Disorders for which testing doesn’t exist 14

WES Testing Options XomeDx Whole exome sequence analysis

XomeDxPlus Whole exome sequence analysis Mitochondrial genome sequencing and deletion analysis

Proband and trio options available

Proband and trio options available

N/A

Maternal status of mitochondrial genome variants

1 report

2 reports

15

5

8/23/2014

Testing Strategies for WES  Clinical records and prior genetic testing results are reviewed prior to analysis  WES is most effective when other family members are included in the analysis – Parents to help determine inheritance of variants

Positive Rates Singleton Analysis

~25%

Duo Analysis

~26%

Trio Analysis

~31%

– Other affected relatives to help assess segregation of variants

16

WES Reports  Reportable variants are selected based on molecular and clinical strength  Variants not associated with the patient’s reported clinical presentation are not reported

Variant Classifications: – Pathogenic – Variant, Likely Pathogenic – Variant of Uncertain Significance

ACMG Incidental Findings – Opt in – Opt out 17

What to do with a WES result?  Results of genetic testing can have important implications for patient management  Genetic counseling is always recommended  Inconclusive results may require additional follow-up – Research Analysis – Segregation analysis within families

18

6

8/23/2014

What is XomeDxSlice?  Client-generated custom gene or panel test XomeDxSlice - Analysis driven by

XomeDx - Analysis driven by phenotype

the gene list - Sequence whole exome

- Proband Only - No ACMG IF

- Proband/Trio Options - Shorter TAT - ACMG IF

When to order XomeDxSlice?  XomeDxSlice is best suited for: – Individuals with a clearly defined, oligogenic phenotype where a comprehensive gene panel is not available – Individuals with a single gene disorder for which clinical testing is not currently available

21

7

8/23/2014

How to order XomeDxSlice?  Our XomeDxSlice Tool found on the GeneDx website http://www.genedx.com/xomedx-slice-tool/

How to order XomeDxSlice?  All XomeDxSlice Tool submissions are reviewed by the WES clinical team at GeneDx  Once a submission is approved, an approval email is sent to the client with instructions on how to order the testing

23

Generating the Gene List  Tools to help generate the Gene List – OMIM – GeneReviews® Please review your XomeDxSlice Tool Request

– Pubmed – Google – XomeDxSlice Tool

24

8

8/23/2014

XomeDxSlice Benefits

Limitations



Sequence genes that are not currently available in a CLIA laboratory





Create a personalized panel for a patient’s clinical presentation



Option to reflex to WES analysis is available



Shorter turn around time

Complete coverage of requested genes is not guaranteed – Gene specific coverage available on the XomeDxSlice Tool – No fill ins for genes with lower coverage



Some sequence variations are not amendable to capture



Analysis is limited to the gene list requested and not full exome



Proband only analysis requiring parental follow up

25

Summary  WES – WES is a powerful tool in genetic diagnostics – The power of WES is increased when additional familial specimens are sent for concurrent analysis

 XomeDxSlice – XomeDxSlice is a custom client-generated genetic test – When utilizing XomeDxSlice, it is necessary to understand and consider the benefits and limitations of this test

26

Acknowledgements Bethany Friedman, MS, CGC Heather Pierce, MS, CGC Elizabeth Butler, MS, CGC Kyle Retterer, MS Jane Juusola, PhD, FACMG Patrik Vitazka, MD, PhD, FACMG Eden Haverfield, PhD, FACMG Julie Neidich, MD, FACMG, FAAP Kristin Monaghan, PhD, FACMG Gabriele Richard, MD, FACMG Sherri Bale, PhD, FACMG

GeneDx WES clinical directors and genetic counseling team

27

9

8/23/2014

28

AGENDA

Inherited Cancer Test Offerings and Technology Variant Classification Panel testing - Outcomes Name That Gene! Case Examples

29

30

10

8/23/2014

Testing for cancer susceptibility is evolving…

31

Options are expanding… High Risk Panel

Single Syndrome

Large Panel Specific to Cancer Type Comprehensive Panel

32

And while options may lead to:

33

11

8/23/2014

Our top priority remains:

34

Other Testing Options Single Gene Testing

Custom Panels

 BRCA1/BRCA2

 Build your Own Panel

 PTEN

 1-29 genes

 TP53  MEN1/MEN2  VHL  Among others…

36

12

8/23/2014

Vetting Process  Comprehensive literature reviews  Expert consultation from PhD and MD Geneticists  Genetic Counseling Consultant contributions  Comparable to market availability

37

Turn-Around-Times  BRCA seq/del/dup: 8-10 days  Breast High Risk: 3 weeks  Lynch/Colorectal High Risk: 3 weeks  All other Panels: 4-5 weeks  RUSH status available

38

39

13

8/23/2014

NGS Expertise

40

NGS Panels: HiSeq NGS  Sureselect target capture – covers all exons plus splice site junctions (20bp) – 5’ and 3’ UTRs (50bp and 20bp) – PTEN promoter

 NextGen sequencing on Illumina HiSeq – Can detect up to ~20bp dels/~10bp ins

 Average depth of coverage = ~500X  Low quality amplicons (