broad genomics clinical-grade whole genome...

Broad Genomics Clinical-grade Whole Genome Sequencing

At Scale to Support Research and Translation

Niall Lennon - Broad Institute of MIT and HarvardAnne O'Donnell-Luria - Broad Institute of MIT and Harvard, Boston Children’s HospitalPradeep Natarajan - Massachusetts General Hospital, Broad Institute of MIT and Harvard Sami Amr - Partners HealthCare Personalized Medicine

IntroductionNiall Lennon, PhD

Sr Director, Translational GenomicsBroad Institute, Genomics Platform

Broad Genomics and Broad Genomics Services

Broad Genomics serves four main functions:1. Data generation - sequencing and arrays at scale2. Methods - development of new tools for the community (e.g. GATK)3. Analysis Platform - cloud-based data sharing and analysis4. Engagement - portals, community outreach

Broad Genomics Services is the mechanism through which we make our data generation products available to the community outside Broad.

Broad is known for scale...

The future, we believe, involves clinical sequencing

• Broad Genomics Services encompasses our CLIA/CAP Clinical lab.• We offer germline whole exome sequencing in addition to somatic

tumor-normal exomes, custom panels, and a mass spec-based kidney disease test.

• Most of the work we support is clinical research.

- In anticipation of a growing desire to use the best possible data for translational research, rare disease and routine diagnostics, and to support return of results - we are making our flagship Human Whole Genome Sequencing product CLIA-grade.

Research Application Case Studies Rare Genome Analysis

Anne O’Donnell-Luria, MD, PhDAssociate Director of the Center for Mendelian Genomics, Broad Institute of MIT and Harvard

Clinical and Metabolic Geneticist, Boston Children’s HospitalInstructor in Pediatrics, Harvard Medical School

Rare disease research sequencing at the Broad Institute

Co-Directors: Daniel MacArthur and Heidi Rehm

Center for Mendelian Genomics (CMG) at the Broad Institute

● NIH-funded center launched early 2016 to discover new disease-gene relationships underlying Mendelian disease

● We work with collaborators with existing cohorts of patient samples consented for genetic studies

● CMG covers cost of sequencing; using WES, PCR-free WGS and RNA-seq

https://cmg.broadinstitute.org


● NIH-funded center launched early 2016 to discover new disease-gene relationships underlying Mendelian disease

● We work with collaborators with existing cohorts of patient samples consented for genetic studies

● CMG covers cost of sequencing; using WES, PCR-free WGS and RNA-seq● Collaborative analysis using the seqr platform for rare variant analysis

https://cmg.broadinstitute.org

seqr


● Gene discoveries are pursued and published by the collaborator● Disease areas:

● Commitment to data sharing○ Sequence data in dbGaP and the Broad’s DUOS ○ Variant interpretation shared in ClinVar○ Matchmaker Exchange node to connect with other researchers:

Retinal Cardiology Neurodev Kidney Muscle Orphan

~80 novel candidate disease genes

https://duos.broadinstitute.org

https://raregenomes.org/home

CLIA-confirmed results shared with families and their physicians

Connecting undiagnosed families to research sequencing

Rare Genome Project

As of 10/13/17, 77 families (278 participants) affected by rare and undiagnosed genetic conditions have enrolled

and are undergoing WGS and analysis.

Genomic approaches to rare disease

Reference sequence


Reference sequence

Whole exome sequencing (WES)

Available clinicallyIdentifies a diagnosis in 10-50% of cases depending on phenotype

Limited to targeted sequenceDifficult to capture high GC content regionsChallenging to identify structural variants


Reference sequence

Whole genome sequencing (WGS)

Identification of variants in coding and noncoding sequenceEasier to identify structural variants from WGS than WES data

Challenge to recognize functionally important variants (particularly above variation detected by exome sequencing)

Not widely available clinicallySignificantly higher cost (~3-4x)

Case 1

● Global developmental delay● Moderate intellectual disability● Spasticity● Abnormal cry in infancy● Constipation● Mild microcephaly (-2 SD)

● Normal brain MRI● Trio WES negative

Age 2 Age 6

Mild dysmorphic facial features

○ Hypertelorism (widely spaced eyes)○ Epicanthal folds○ Macrotia (large ears)○ Wide nose○ Long philtrum○ Thin lips Katrin Õunap

Structural variation analysis of WGS identified de novo single exon deletion in CTCF

● Identified with Illumina’s Manta breakpoint caller (Chen et al, Bioinformatics, 2016)

● Paired end reads with unexpectedly large insert size (red, top arrow)● Split read (rainbow, bottom arrow)

Sander Pajusalu

Monkol Lek

Sanger confirmation of CTCF exon 8 deletion breakpoints

Jamie Marshall

Successfully PCR product generated across the deletion, fine mapping the breakpoints

Confirmed de novo event

CTCF HaploinsufficiencyGregor et al., De Novo Mutations in the Genome Organizer CTCF Cause Intellectual Disability, Am J Hum Genet, 2013.

p.Val126Cysfs*14

p.Arg396Lysfs*13

Case 2: Complex structural variation in autism

dupINVdup are among the most common structural variant identified in genomes from patients with autism

dupINVdup

Harrison Brand, Ryan Collins, Mike Talkowski

Paired duplication inversions in autism

dupINVdup

Example of structural variant of uncertain significance: dupINVdup in patient with autism

Harrison Brand, Ryan Collins, Mike Talkowski


Reference sequence



Reference sequence


RNA sequencing

Pair RNA-seq with WGS to evaluate effect on gene expression and splicing

Case 3

Carsten BonnemannFrancesco Muntoni

● Muscle weakness● Wheelchair bound● Muscle biopsy with absence of COLVI

immunostaining ● Clinical sequencing and deletion/duplication

analysis of COL6A1, COL6A2, COL6A3● cDNA sequencing from fibroblasts of COL6 genes● Exome sequencing● Whole genome sequencing

No molecular diagnosis

RNA-seq on muscle biopsy tissue reveals functional impact of non-coding variation

Reference

Control

Patient

Beryl Cummings Cummings et al., Sci Trans Med, 2017

Results in in-frame inclusion of 24 amino acids in the glycine repeat region

Known to have a dominant-negative effect

Non-coding SNV results in intronic splice gain

GC>GT variantCreates a novel splice donor site

Non-coding SNV results in intronic splice gain

GC>GT variantCreates a novel splice donor site

AG acceptor splice site activated

Event confirmed on cDNA studies from muscle biopsy

Jamie Marshall

Novel splice donor site active in muscle but not fibroblasts

Cummings et al., Sci Trans Med, 2017

Recurrent de novo SNV at CpG site is a common cause of COL6 myopathy

● 30 additional patients have been diagnosed

● Estimated to explain 25% of cases who clinically and histologically look like COL6 myopathy but have had normal sequencing

Cummings et al., Sci Trans Med, 2017

For the latest from Beryl, catch her talk in session #73 on Friday at 10:45am in Room 330C

Conclusions: Opportunities provided by WGS

● After exome sequencing, the majority of patients with rare disease remain undiagnosed but this is still an excellent first line test

● Whole genome sequencing is a powerful approach in rare disease diagnosis○ Improved coverage of difficult to sequence or untargeted exomic regions○ Evaluation of non-coding genomic sequence variation○ Detection of copy number variation and other structural variation○ Evaluation of short tandem repeats (STRs)

Conclusions: Challenges remain

● Easier to detect millions of genomic variants through WGS than to identify which variant(s) contribute to rare disease

● Growing but insufficient reference panels to provide population allele frequencies for non-coding and structural variation○ gnomAD contains 15,496 genomes from diverse ancestries○ BRAVO contains 62,784 genomes (unable to share ancestry)○ No WGS structural variation reference panel available yet

● Need for high throughput functional approaches to evaluate the thousands of variants that are unique to each genome

Acknowledgements

Hayley BrooksDuni GetanehMelanie O’LearyIdara NdonClara Williamson

Actively recruiting web/software developers and variant analysts including a Lead Analyst

Looking for collaborators with undiagnosed families for research sequencing

for CMG (already consented) or RGP

Daniel MacArthurHeidi RehmMike Talkowski

Monica WojcikBen WeisburdHarindra ArachchiMoran CabiliCarsten Bonnemann (NIH)Sandra Donkervoot (NIH)Francesco Muntoni (UCL)Sander Pajusalu (U of Tartu)Katrin Õunap (U of Tartu)

Email [email protected] if interested or check out https://macarthurlab.org/jobs/

Beryl CummingsMonkol LekJamie MarshallRyan CollinsHarrison Brand

mailto:[email protected]://macarthurlab.org/jobs/

Translational/Clinical Utility Case StudyPradeep Natarajan, MD MMSc

Director of Preventive Cardiology, Massachusetts General HospitalInstructor of Medicine, Harvard Medical School

Affiliated Scientist, Broad Institute of MIT and Harvard

Introduction

• Me: Preventive cardiologist / cardiovascular geneticist

• My Clinic: individuals with a suspected inherited basis for premature myocardial infarction risk, including those with lipid abnormalities

• My Research: study the genetic basis of premature myocardial infarction and lipids for biological, therapeutic, and clinical insights

Background

• Molecular diagnostic testing for individuals with a suspected disorder has become increasingly sophisticated.

• Current tools: targeted gene sequencing, array CGH, whole exome sequencing.

• Whole genome sequencing can characterize the full allele frequency spectrum across the genome.

Outstanding questions

• Can whole genome sequencing characterize the complete inherited basis of a trait?

• Does that clinically matter?

Familial Hypercholesterolemia

Khachadurian AK. Am J Med. 1964

Gidding SS, et al. Circulation. 2015

Risk distribution of LDLR disruptive coding mutations and early-onset myocardial infarction risk

5,00 early-onset MI cases

5,00 older MI-free cases

Do R*, Stitziel NO*, Won HH*, et al. Nature. 2015

Familial hypercholesterolemia mutations discriminate coronary heart disease risk

Khera AV*, Won HH*, Peloso GM*. J Am Coll Cardiol. 2016

Current guidelines for the management of familial hypercholesterolemia

• Goal LDL cholesterol < 100mg/dl with statins

Separately, polygenic factors influencing LDL cholesterol also influence coronary heart disease risk

Kathiresan S. N Eng J Med. 2008

Current approach for genetic testing for severe hypercholesterolemia

• Sequencing: LDLR, APOB, PCSK9, LDLRAP1• Intragenic deletions/duplications: LDLR

Current diagnostic yield of panel testing

Benn M et al. Eur Heart J. 2016

Whole genome sequencing

• Simultaneous assessment of• Monogenic risk• Polygenic risk

NHLBI TOPMed Lipids Working Group

Whole genome polygenic score

AJHG 2015

Whole genome polygenic score

• 2M SNPs•• LD: 1000G CEU•• Training dataset: HUNT (N~25K)

Genetic effects for LDL cholesterol extremes

High LDL cholesterol

Monogenic carrier

Top 5th percentile of Polygenic score

European Ancestry

10.9(1.6%)

7.7(19.6%)

African Ancestry

7.4(2.8%)

3.2(9.2%)

Low LDL cholesterol

Monogenic carrier

Bottom 5th percentile of Polygenic score

European Ancestry

21.7(2.0%)

10.4(25.9%)

African Ancestry

13.8(4.7%)

3.7(10.7%)

Can polygenic scoring be informative for monogenic mutation carriers?

• APOB p.R3527Q - prevalence 12% in the Amish

• Carriers also with the lowest 5th percentile of polygenic LDL cholesterol score had:• 40 mg/dl lower LDL cholesterol• 70% lower likelihood of severe hypercholesterolemia (LDL cholesterol >

190 mg/dl)

Summary

• Whole genome sequence analysis permits fuller assessment of the genetic basis of clinical traits.

• Diagnostic yield for complex traits with monogenic and polygenic etiologies can be expanded.

• Whole genome sequencing may also provide incremental value for monogenic mutation carriers as well.

Future directions

• Application to other complex traits

• Improvement in identifying monogenic mutations

• Improvement in polygenic risk estimation, including in diverse ethnicities

• Genetic modifier discovery and interpretation

• Refinement of current classification (e.g. “pathogenic”) to effect estimates

Broad WGS Diagnostics Case Study

Sami S. Amr, PhD, FACMG

Instructor of Pathology, BWH/Harvard Medical SchoolDirector, Translational Genomics Core

Assoc. Director, Lab for Molecular Medicine Partners HealthCare Personalized Medicine

Cardiomyopathy• Hypertrophic cardiomyopathy (HCM)• Dilated cardiomyopathy (DCM)• Arrhythmogenic Right Ventricular Cardiomyopathies

(ARVC)• Catecholaminergic polymorphic ventricular tachycardia

(CPVT)• Left ventricular noncompaction (LVNC)• Restrictive cardiomyopathy (RCM)• Fabry Disease• Transthyretin Amyloidosis

Congenital Heart Disease/Defects• Isolated Nonsyndromic CHD (Septal and Cyanotic Defects

with or without Conduction System Disease)• Isolated Supravalvular Aortic Stenosis (SVAS)

Hearing Loss and Related syndromes• Nonsyndromic hearing loss• Auditory neuropathy spectrum disorder• Mitochondrial hearing loss• Pendred syndrome• Usher syndrome• Waardenburg syndrome• Branchio-Oto-Renal syndrome• Wolfram Syndrome• Jervell and Lange-Nielsen syndrome• Perrault syndrome

•

Pulmonary Disorders• Cystic Lung disease• Bronchiectasis• Pulmonary fibrosis• Pulmonary hypertension• Central hypoventilation syndrome

Myopathies• Limb-girdle muscular dystrophy• Emery-Dreifuss muscular dystrophy• Congenital myopathies• Myofibrillar myopathy

Genomic sequencing• Exome Sequencing• Genome Sequencing

Ectodermal Dysplasias• X-Linked Hypohidrotic Ectodermal

Dysplasia (XLHED)• Hidrotic Ectodermal Dysplasia 2

(Clouston Syndrome)• GJB2 - Related Ectodermal Dysplasia• DSP - Related Ectodermal Dysplasia /

Skin Fragility

Connective Tissue Disorders• Familial Thoracic Aortic Aneurysms

and Aortic Dissections (TAAD)• Marfan Syndrome• Loeys-Dietz Syndrome• Ehlers-Danlos syndrome type IV• Lujan-Fryns syndrome• Arterial tortuosity syndrome

Rasopathies (Noonan Spectrum Disorders)• Noonan Syndrome• Cardio-Facio-Cutaneous (CFC)

Syndrome• Costello Syndrome• LEOPARD Syndrome• Neurofibromatosis type 1• Legius syndrome

Laboratory for Molecular Medicine (LMM)

Medical exome analysis: ~7,500 gene with a known disease association

Phenotype-based analysis: disease-specific genes (similar to gene panels)

Clinical labs are not looking at the whole exome/genome

WES WGS Total %

No. cases 21 5 26

Positive 8 2 10 38%

Inconclusive 7 3 10 38%

Negative 6 0 6 23%

LMM WES/WGS testing summary

LMM WES/WGS testing summary

WES WGS Total %

No. cases 21 5 26

Positive 8 2 10 38%

Inconclusive 7 3 10 38%

Negative 6 0 6 23%

FHx Pulmo Cardiac Neuro/ Dev Immuno Onco Metabolic Other

No. cases 12 12 2(1) 4 (2) 1 2 2(2) 1

Positive 2 5 1 3 1

Inconclusive 5 4 1 1 0 1 2 1

Negative 5 3 1 1

What can WGS offer that other molecular diagnostics testing strategies don’t?

Ease and speed of onboarding novel disease genes or disease-associated loci into testing• With gene panels this requires a new validation of the capture kit and sequencing

output for every time genes are added to the kit. Otogenome panel at the LMM: 5 versions in ~10 years19 gene on array (2007-2010) → 19 gene on NGS (2010-2011) → 71 gene on NGS (2011-2014) → 87 genes on NGS (2014-2017) → 109 genes on Exome backbone (current)

No gene panel design limitations• Gene panels may focus on genes associated with a clinically defined diagnosis

and exclude genes associated with diseases that have overlapping features

Pulmonary case

Clinical presentation:• 10 y/o female• Congenital unilateral hearing loss, acquired hearing loss in opposite ear• Bilateral scarred tympanic membranes• Recurrent sinusitis• Recurrent otitis media• Recurrent pneumonias

Lab Findings:

Pulmonary ciliary dyskinesia suspected• Ciliary biopsy has technical limitations and may not capture all

forms of ciliary dysfunction

Immunodeficiency: Initial blood tests within normal range

Variant filtration and analysis strategy

• Patient-specific gene list: Exome testing and variant analysis strategy– 530 genes implicated in recurrent pulmonary and sinus infections

• Includes PCD, immunodeficiency, interstitial pulmonary fibrosis genes

– Screened all variants with MAF 7000 genes implicated in human disease– Screened previously reported and novel LOF variants with MAF G (p.Lys305Arg) • Primary ciliary dyskinesia (AR)• Not previously reported• VUS

Heterozygous ELANE c.598-1G>A (p.?)• Severe congenital or cyclic neutropenia (AD)• Not previously reported• Predicted to disrupt splicing of terminal exon

Mutations in ELANE cause 2 types of neutropenia:

Severe congenital neutropeniaCommon 1st sign: Omphalitis (an infection of the umbilical stump)

Predisposes risk for myelodysplasia (MDS) or acute myelogeneous leukemia (AML) which is exacerbated by granulocyte colony stimulating (G-CSF) factor treatment

Cyclic neutropenia/cyclic haematopoiesisOscillating neutropenia lasting 3-5 days in a 21-day period

Symptoms more severe in children

In both conditions, individuals have fever and recurrent skin and oropharyngeal inflammation (i.e., mouth ulcers, gingivitis, sinusitis, pharyngitis, and cervical adenopathy).

Treatment with granulocyte colony-stimulating factor (G-CSF) is effective in elevating blood neutrophil counts in both congenital neutropenia and cyclic neutropenia.

Limitations of a panel testing strategy:

• ELANE not present on the PulmoGene panel

• Lab results were not indicative of an immunodeficiency

PulmoGene Panel (64 Genes)

Gene content is also offered by exome sequencing…

What advantages does WGS have over exome sequencing?• Exome sequencing is susceptible to the same type of technical issues that panels have (some regions are

difficult to hybridize/enrich for)

• For AR disease: Cis/trans configuration of variants in the same gene

• Detection of intragenic/intronic regions

• Ability to detect a range of structural rearrangements and CNVs

PMP22Distal enhancer

region

PMP2 causative for CMT-type 1A: motor and peripheral neuropathy (AD, prevalence:

OTCc.-366A>G

Ornithine carbamoyltransferase deficiency: most common inherited urea cycle disorder: X-linked disorder (15-20% of females symptomatic)10-15% of causative variants are structural rearrangements and CNVs, and variants in regulatory regions (HGMD)

EPOc.-136G>A

EPO gene associated with familial erythrocytosis: characterized by increased red cell mass and elevated hemoglobin concentration and hematocrit

First report of this gene-disease association from a WGS study: the c.136G>A variant identified in two families and segregated in 8 family members (Taylor JC et al. Nat Genet. 2015 Jul;47(7):717-726)

USH2A c.7595-2144A>G

USH2A is responsible for 30-40% of Usher syndrome casescauses Usher type II: congenital hearing loss and progressive retinitis pigmentosa occurring in the 2-3rd decade, autosomal recessive inheritance

20-30% of patients with USH2 features carry only 1 het USH2A variant or no variant in USH2A or other USH2 genes

c.7595-2144A>G variant reported in 33 unrelated families (LOVD database: https://grenada.lumc.nl/LOVD2/Usher_montpellier/)

https://grenada.lumc.nl/LOVD2/Usher_montpellier/

USH2A case

• 8 week old male• Congenital hearing moderate to severe hearing loss• SNP array at external lab: multi-exon duplication in USH2A• Otogenome testing: 87 hearing loss gene panel, includes Usher genes• A heterozygous pathogenic missense variant identified (p.Thr352Ile)

VisCap: NGS CNV caller NGS data from panel testing picked up the duplication

However, site of inserted sequence of duplicated exons unknown

Thus, cannot predict impact on normal protein product → variant of uncertain significance

Additional evidence needed for classification:• Patient develops additional features of the disorder

• Breakpoint analysis showing that the duplication would disrupt the normal coding sequencing (in-tandem insertion or structural rearrangement that impacts other exons in gene)

Manual sequence review showed one of the breakpoints was captured~60 bp from nearby splice site

Unaligned sequence mapped to intronic region in intron 60

This is not usually the case…

Chr Genomic coordinates Gene Transcript Variant (cDNA) Region Cat Cases

5 g.(?_90144435)_(90159732_?)dup ADGRV1 NM_032119.3 c.(?_17020-19)_(17856+58_?)dup Ex 79-83 Unc Sig, 5 1

21 g.(?_35821543)_(35821932_?)dup KCNE1 NM_000219.3 c.(?_1)_(390_?)dup Ex 03 Unc Sig, 3 1

19 MYH14 c.(?_-3)_(2694+15_?)dup Ex 2-22 Unc Sig, 4 1

6 MYO6 c.(?_1)_(3858_?)dup Ex 2-35 Unc Sig, 4 1

7 g.(?_107312583)_(107312693_?)dup SLC26A4 NM_000441.1 c.(?_305)_(415_?)dup Ex 04 Lik Path (DFN) 1

15 g.(?_43893070)_(43903182_?)dup STRC NM_153700.2 c.(?_3307)_(4844_?)dup Ex 14-25 Unc Sig, 4 3



15 g.(?_43901446)_(43901512_?)dup STRC NM_153700.2 c.(?_3519)_(3557+28_?)dup Ex 16 Unc Sig, 4 12



15 g.(?_43892948)_(43895542_?)dup STRC NM_153700.2 c.(?_4443)_(4845-68_?)dup Ex 23-25 Unc Sig, 4 2


15 g.(?_43892671)_(43892939_?)dup STRC NM_153700.2 c.(?_4845-59)_(4993+61_?)dup In 26 Unc Sig, 4 1

3 g.(?_46742762)_(46752454_?)dup TMIE NM_147196.2 c.(?_-216)_(*1276_?)dup Unc Sig, 3 1

1 g.(?_215914717)_(215933185_?)dup USH2A NM_206933.2 c.11048-?_11711+?dup Ex 57-60 Path (DFN) 2

16 unique duplications 8 gene 14 VUS 32 Cases

Summary

Whole genome has the potential to increase detection rates across broad range of inherited disorders

• More comprehensive disease relevant gene and loci content• Positional information on variants

– Cis/trans configuration of variant in the same gene– Duplication and structural rearrangement

• Avoids technical limitations of enrichment methodology

Limitations in a clinical setting

• Incidental findings• Reporting complexity• Relies on reported gene-disease associations

Acknowledgments

Lab for Molecular MedicineAndrea Oza, MS CGC

Christine Austin-Tse, PhD

MGH Pediatrics DepartmentThomas B. Kinane, MD

Chief, Pediatric Pulmonary Unit

Lael Yonker, MD

Co-Director, Pulmonary Genetics Clinic

Questions and Discussion

broad genomics clinical-grade whole genome...

Documents