Translating Rapid Whole Genome Sequences into Precision Medicine for Infants in Intensive Care Units Stephen F. Kingsmore, MB, ChB, DSc, FRCPath, President, Rady Children’s Institute for Genomic Medicine, San Diego

"Tonight, I'm launching a new Precision Medicine Initiative....to give all of us access to the personalized information we need to keep ourselves and our families healthier….a new era of medicine….that delivers the right treatment at the right time."

National Adoption of Genomic Medicine

14% of US newborns admitted to a NICU

Rationale

Economics Work: Cost of care

$4000 per day

Leading cause of Death in NICU & PICU

Conventional testing

Too slow To guide

care

Diagnosis of 8250 Genetic Diseases in

NICUs

Conventional Testing Too Slow For Optimal NICU Outcomes

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Days of Life

Why single gene diseases?

• Simple, deterministic genetics: – 1 or 2 pathogenic variants in/near 1 gene that causes

disease with symptoms similar to that in the infant = necessary and sufficient

• 50yr medical genetic infrastructure – Medical geneticists, genetic counselors, public health

services • Orphan drug companies, gene therapies

Baby CMH487:

acute liver failure

00:00

Gap 2: Making a differential diagnosis: Complexity of Genetic Diseases in Symptomatic Infants

Typical presentations of 4,645 known genetic diseases

Partial presentations of 4,645 known genetic diseases

Atypical presentations of 4,645 known genetic diseases

20+ novel genetic diseases discovered per month

Genetic disease mimicking a non-genetic disease

Genetic disease complicating a non-genetic disease

Two concomitant genetic diseases

3,643 named, uncloned genetic diseases

Stereotyped presentations due

to partially developed organ

systems and homeostatic

responses

April 10, 2013 Baby CMH487

00:00

Hydrocele testis Infra-orbital crease Maternal diabetes Low-set, posteriorly rotated ears Feeding difficulties in infancy Ventilator dependence Single umbilical artery Cholestasis Thrombocytopenia Prolonged partial thromboplastin time Prolonged prothrombin time Chronic lung disease Hypertelorism Thoracolumbar scoliosis Bronchodysplasia Omphalocele Chin dimple Duplicated collecting system Ventricular hypertrophy Nevus flammeus Gastroesophageal reflux

12,000 HPO terms x

5,500 genetic diseases x

3,500 disease genes

April 10, 2013 Baby CMH487

00:00

HP:0009800 Maternal diabetes HP:0001539 Omphalocele HP:0008872 Feeding difficulties in infancy HP:0006533 Bronchodysplasia HP:0005946 Ventilator dependence HP:0006528 Chronic lung disease HP:0002020 Gastroesophageal reflux HP:0002944 Thoracolumbar scoliosis HP:0000081 Duplicated collecting system HP:0000034 Hydrocele testis HP:0001195 Single umbilical artery HP:0100876 Infra-orbital crease HP:0000368 Low-set, posteriorly rotated ears HP:0000316 Hypertelorism HP:0010751 Chin dimple HP:0001052 Nevus flammeus HP:0001396 Cholestasis HP:0001873 Thrombocytopenia HP:0003645 Prolonged partial thromboplastin time HP:0008151 Prolonged prothrombin time HP:0001714 Ventricular hypertrophy

Parents gave consent

00:00

Blood sample from

mum, dad and baby

00:00

San Diego Synergy: Illumina + Edico + Rady Children’s

Transport to Institute

00:02

Robot isolates genomic DNA

01:00

Robot prepares DNA for sequencing

06:00

18 hour genome sequencing

24:30

Each of my 37 trillion cells contains 2 genomes of 3.2 billion DNA letters

We are fearfully and wonderfully made. Psalm 139

Scope: Comparison of Short Read Genome and Exome Sequencing Pros of Genomes:

• One day faster • Samples 90% of genome • Reasonably good deletion

structural variant detection

Pros of Exomes: • Cost ¼ that of genomes

Disadvantages of both: • Limited phasing; requires trios • Don’t detect insertion structural

variation or repeat expansions

Gene

Whole Genome Sequence Whole

Exome Sequence

= 2%

Cost and Time-to-Result of Genomic Sequencing Options

48+ 48+

40X 4000 Trio Genome 48+ Hr 2x80nt

Gap 4:

24:30 Infant CMH487

9 TB/day 6GB/sec

3 TB/day 600 Mb/sec

5TB/day 1 GB/sec

Remote backup

Genome sequencers 24 x 7, 365 days/yr

High performance NAS

Compute cluster

In-house researchers

Single monitoring dashboard 75 TB

10 Gbps

10 Gbps 10 Gbps

Sim

ulta

neou

s writ

es

500TB

100 cores

1TB/day

External Collaborators

500TB Public cloud

AWS Google Azure

25 yr archival 1 Gbps http

Single global name space

Replication

2 yrs worth storage Local object storage

1 Gbps http

Compute & Storage

FASTQ

BAM FASTQ

Variant database

GPFS

24:45 Infant CMH487

25:00 Infant CMH487

Improved Analytic performance of WGS (nucleotide variants; not structural variants)

SampleYeild (GB)

Site Pipeline

A

DRAGENGSNAP/GATK-1.6/noVQSRDRAGENGSNAP/GATK-3.2/noVQSR

Essex

CMH

NA12878

NA12878 65a

143

Analytic Sensitivity

Analytic Specificity

99.93% 99.87%99.54% 98.57%99.42% 99.46%97.29% 95.35%

45X

20X

Coverage

25:01 Infant CMH487

Hemoglobin-β DNA code

Normal red blood cell

Does this DNA letter change cause a genetic disease?

Sickle cell

Variant pathogenicity categories

Genet Med. 2015 Mar 5. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of ACMG and AMP. Richards S, et al.

Very Strong

Null variant (nonsense, frameshift, ±1 or 2 splice site position, initiation codon, exon deletion) in gene where LOF known to cause disease

Strong • Same amino acid change as previously established pathogenic variant • De novo in a patient with the disease and no family history • Functional studies show damaging effect on the gene • Prevalence in affected individuals significantly greater than controls

Moderate • Located in mutational hot spot/functional domain without benign variation • Extremely low frequency in Exome Sequencing or 1000 Genomes Projects • For recessive disorders, detected in trans with a pathogenic variant • Protein length changed by in-frame indel in nonrepeat region or stop-loss • Novel missense at amino acid where different missense known to be pathogenic • Assumed de novo, but without confirmation of paternity and maternity

Supporting • Cosegregation with disease in multiple affected family members in gene known to cause disease

• Missense variant in gene with low rate of benign missense variants and where missense variants commonly cause disease

• Multiple computational tools call deleterious • Phenotype highly specific for disease with single genetic etiology • Reputable source reports as pathogenic, but unpublished

Category CRITERIA

Pathogenic 1VS + (1S or 2M/Supp) 2 Strong 1S + 3M or (2M+2Supp)

Likely Pathogenic

1 VS/S + 1 M 1 S + (1 M or 2 Supp) 3 M 2 M + 2 Supp 1 M + 4 Supp

25:41 Infant CMH487

341 Possible Diagnoses

25:42 Computer-Aided Comprehensive Differential Diagnosis

Infant CMH487

Diagnosis

25:43

• Perforin 1 heterozygous c.272C>T [p.Ala91Val] P, supported by functional studies

• Perforin 1 heterozygous c.1310C>T [p.Ala437Val] LP, supported by case-control studies

• Diagnosis: Hemophagocytic lymphohistiocytosis type 2

26:00

• Confirmatory testing

• Precision Medicine: – Meds. D/Cd – IV Ig and steroids

Hemophagocytic Lymphohistiocytosis Diagnostic Criteria (need 5) Present in CMH487 Fever No Hepatomegaly or splenomegaly Modest (1) 2 cytopenias: hemoglobin<9 g/dL, platelets <100,000/mm3, ANC <1000 Yes (2) Serum ferritin >500 ng/mL Yes (3) Serum triglyceride >265 mg/dL or fibrinogen <150 mg/mL Yes (4) Absent/decreased natural killer cell assay Yes, after Dx (5) Soluble IL2 receptor (CD25) >2,400 units/mL Not done Hemophagocytosis without malignancy Not done

Outcome • Coagulopathy resolved on d. 7 • 7 surgeries for correction of congenital anomalies • He is now 32 months old, normal liver function • 72 quality adjusted life years saved

Diagnostic Utility: Meta-Analysis of 9 Studies of Short Read Exome & Genome Sequencing in Children

1. Overall Diagnosis Rate = 28% = most effective method for making molecular diagnosis of

childhood genetic disease

Chromosomal microarray (current 1st tier test for genetic disease Dx): diagnostic rate ~15%

3. de novo Mutations: The most common mechanism of genetic disease diagnosis

Willig LK, et al. Science Trans. Med. April 2015

35 NICU / PICU infants with likely genetic disease (Kansas City)

57% (20) By rapid WGS

9% (3) By standard methods

Molecular Diagnosis

Stark Z, et al. Genetics in Medicine 3/3/2016

80 infants under care for likely genetic disease (Melbourne)

14% (11) By standard methods

58% (46) By Exome Seq

Molecular Diagnosis

Explore use of genomic information for broadening understanding of diseases identified in the newborn period

Newborn Sequencing In Genomic medicine and public HealTh

65 Randomized

NICU infants of age < 4 months with clinical features suggesting a genetic disease

33 Infants with Standard Tests

32 Standard Tests and Trio Rapid WGS

NSIGHT Randomized Controlled Study of rapid WGS in NICU infants

5 Cross-overs

Molecular Diagnosis 5 (15%) 13 (41%) p<0.05

Study terminated early due to loss of equipoise of neonatologists

Time to Diagnosis 65 days 16 days

2.9 QALYs per family tested

YES: 2.9 QALYs per newborn genome Cost per QALY: $3500

Kansas City Melbourne Change in care 13 (37%) 16 (20%) Life-saving treatment 1 (3%) 2 (3%) Major morbidity avoided 3 (9%) 1 (1%) Major Procedure Change 3 (9%) 4 (5%) Palliative Care Guidance 6 (17%) 0 (0%) Medication Change 4 (11%) 8 (10%) NICU stay decreased by >1 month 1 (3%) 0 (0%) Parent or sibling diagnosed 1 (3%) 10 (13%) Diet Change 2 (6%) 2 (2%) Complication monitoring 1 (3%) 11 (14%)

CMH5033, a 3-week-old male with isolated, symptomatic atrial flutter, and

his parents

CMH5033: 3 week old male with atrial tachydysrhythmias

DOL2: Local NICU; Atrial tachycardia; Premature atrial contractions; Some non-conducted P waves; Heart rate normal; Discharged DOL7. 2 weeks old: Outpatient; tachypneic, gained 1 oz. EKG: Atrial tach 190-230. Some non-conducted P waves. Admitted CMH NICU. Episodes atrial flutter 217 bpm with pallor, thready pulse. Cardioverted x 2. ECG: 6/2: Vent.Rate:164BPM, Atrial Rate:178BPM, Normal P-R Interval, QRS Interval and QTc. Rx: Amiodarone, esmolol, flecainide. ECHO: Structurally normal. Patent foramen ovale. Ectopic beats. During sinus beats, LV ejection fraction 52%. During ectopic beats, abnormal septal motion. Amiodarone and esmolol D/Cd after flecainide therapeutic. 3 weeks old: Enrolled for WGS; Hemodynamically stable; Eating well; Discharged.

Trio Whole Genome Sequencing

• Day 1: Enrolled • Day 2: Blood samples from trio • Day 4: Finished 2 x 100 nt, HiSeq 2500, proband, 108GB &

mother 109GB • Day 4: Proband VCF analyzed • Day 5: Finished father WGS (89GB), re-analysis of trio • Day 9: Laboratory director completed analysis/interpretation

Automated Differential Diagnosis • HPO terms: atrial flutter (mandatory), AFib (present),

multifocal atrial tachycardia (present), 1 gene (SCN5A) • Phenomizer: terms present: P<0.05; 259 genes • 17 OMIM AFib loci • These genes harbor 1 possibly pathogenic variant in the

proband with minor allele frequency<0.5% Coordinates (GRCh37) Locus Locus name MIM# Phenotype Phenotype# 12:21950322-22094796 ABCC9, ATFB12 ATP-binding cassette, subfamily C, member 9 (sulfonylurea receptor 2) 601439 Atrial fibrillation, familial, 12 614050 10:70600000-105800000 ATFB1 Atrial fibrillation, familial, 1 608583 Atrial fibrillation, familial, 1 608583 6:75900000-105500000 ATFB2 Atrial fibrillation, familial, 2 608988 Atrial fibrillation, familial, 2 608988 4:107700000-114100000 ATFB5 Atrial fibrillation, familial, 5 611494 {Atrial fibrillation, familial, 5} 611494 16:66700000-74100000 ATFB8 Atrial fibrillation, familial, 8 613055 Atrial fibrillation, familial, 8 613055 1:147228331-147253164 GJA5, ATFB11 Gap junction protein, alpha-5, 40kD (connexin 40) 121013 Atrial standstill, digenic (GJA5/SCN5A) 108770 1:147228331-147253164 GJA5, ATFB11 Gap junction protein, alpha-5, 40kD (connexin 40) 121013 Atrial fibrillation, familial, 11 614049 12:5153084-5155953 KCNA5, ATFB7 Potassium voltage-gated channel, shaker-related subfamily, member 5 176267 Atrial fibrillation, familial, 7 612240 21:35736322-35743439 KCNE2, ATFB4 Potassium voltage-gated channel, Isk-related family, member 2 603796 Atrial fibrillation, familial, 4 611493 17:68164756-68176188 KCNJ2, ATFB9 Potassium channel, inwardly rectifying, subfamily J, member 2 600681 Atrial fibrillation, familial, 9 613980 11:2466220-2870339 KCNQ1, ATFB3 Potassium voltage-gated channel, KQT-like subfamily, member 1 607542 Atrial fibrillation, familial, 3 607554 1:11905765-11907839 NPPA, ATFB6 Natriuretic peptide precursor A 108780 Atrial standstill 2 615745 1:11905765-11907839 NPPA, ATFB6 Natriuretic peptide precursor A 108780 Atrial fibrillation, familial, 6 612201 5:37291734-37371227 NUP155, ATFB15 Nucleoporin, 155kD 606694 ?Atrial fibrillation 15 615770 19:35521554-35531352 SCN1B, ATFB13 Sodium channel, voltage-gated, type I, beta polypeptide 600235 Atrial fibrillation, familial, 13 615377 11:118033518-118047336 SCN2B, ATFB14 Sodium channel, voltage-gated, type II, beta polypeptide 601327 Atrial fibrillation, familial, 14 615378 11:123499894-123525314 SCN3B, ATFB16 Sodium channel, voltage-gated, type III, beta subunit 608214 Atrial fibrillation, familial, 16 613120 11:118004091-118023629 SCN4B, ATFB17 Sodium channel, voltage-gated, type IV, beta subunit 608256 Atrial fibrillation, familial, 17 611819 3:38589552-38691163 SCN5A, ATFB10 Sodium channel, voltage-gated, type V, alpha polypeptide 600163 Atrial fibrillation, familial, 10 614022

IGV: Proband, 3:38591853-38591853 A > G Good coverage, bidirectional reads call variant, clean alignments

Filters: Potentially pathogenic variant(s) with allele frequency <0.5% in genes assoc. with atrial fibrillation: 1

heterozygous variant

Inherited from father (As far as we know he is unaffected)

Proband

Mother

Father

Complication: There are 9 OMIM SCN5A Phenotypes

1. Non-synonymous hgvs_c: NM_000335.4:c.6007T>C, hgvs_p: NP_000326.2:p.Phe2003Leu hgvs_c: NM_001099404.1:c.6010T>C, hgvs_p: NP_001092874.1:p.Phe2004Leu hgvs_c: NM_001099405.1:c.5956T>C, hgvs_p: NP_001092875.1:p.Phe1986Leu hgvs_c: NM_001160160.1:c.5911T>C, hgvs_p: NP_001153632.1:p.Phe1971Leu hgvs_c: NM_001160161.1:c.5848T>C, hgvs_p: NP_001153633.1:p.Phe1950Leu hgvs_c: NM_198056.2:c.6010T>C, hgvs_p: NP_932173.1:p.Phe2004Leu In silico tools largely predict to be benign SIFT: tolerated (0.65) MutPred: Medium risk Grantham score: 22 (range 5 – 215) LRT: Neutral FATHMM: Damaging (-3.6) 2. Occurs in a functional domain of the protein: Unstructured COOH terminus is involved in inactivation gating 5 variants in this region have physiologic phenotypes 3. Occurs in a 20 AA region that is well conserved; Albeit PolyPhen2 = benign (0.001), PhyloP 0.04 And, Dog and Chinchilla have leucine at this position

Support for Pathogenicity of 3:38,591,853-38,591,853A>G

4. Allele frequency – rare, but not extremely low CMH MAF 0.24%, 11 het., 0 hom., / 4666 total alleles EVS MAF European American 0.31% ExAC MAF 0.20% dbSNP: rs41311117 MAF 0.14% 5. Reported in patients with sudden infant death syndrome, sudden cardiac death, Brugada syndrome, and in apparently healthy individuals…Oleson et al. Circ Cardiovasc Genet 2012 5:450-459 – F2004L father and proband had early onset AF 6. Altered electrophysiologic effects in 2 manuscripts Wang: “F2004L alone is not sufficient to evoke arrhythmia susceptibility, and other factors are probably needed for full pathophysiological expression” Bebarova: “We believe that the F2004L loss-of-function variant is a disease-associated mutant”. “…the question arises why the pathogenic potential of this SCN5A variant is so variable. Undoubtedly, modifier genes could play a role.”

Support for Pathogenicity of 3:38,591,853-38,591,853A>G

Wang et al. Circulation. 2007;115:368-376

Bebarova et al. Am J Physiol Heart Circ Physiol. 2008 Jul; 295(1): H48–H58.

Cells transfected CHO cells tsA201 cells Fast inactivation Impaired Recovery from inactivation Faster Delayed Slow inactivation Faster Persistent Na current Increased Decreased Steady-state inactivation Depolarizing voltage shift Peak Na current Unaltered Decreased

Genet Med. 2015 Mar 5. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of ACMG and AMP. Richards S, et al.

Very Strong Null variant (nonsense, frameshift, ±1 or 2 splice site position, initiation codon, exon deletion) in gene where LOF known to cause disease

Strong • Same amino acid change as previously established pathogenic variant • De novo in a patient with the disease and no family history • Functional studies show damaging effect on the gene • Prevalence in affected individuals significantly greater than controls

Moderate • Located in mutational hot spot/functional domain without benign variation • Extremely low frequency in Exome Sequencing or 1000 Genomes Projects • For recessive disorders, detected in trans with a pathogenic variant • Protein length changed by in-frame indel in nonrepeat region or stop-loss • Novel missense at amino acid where different missense known to be pathogenic • Assumed de novo, but without confirmation of paternity and maternity

Supporting • Cosegregation with disease in multiple affected family members in gene known to cause disease • Missense variant in gene with low rate of benign missense variants and where missense variants commonly

cause disease • Multiple computational tools call deleterious • Phenotype highly specific for disease with single genetic etiology • Reputable source reports as pathogenic, but unpublished

Category NEW CRITERIA

Pathogenic 1 VS + (1 S or 2 M/Supp) 2S 1S + (3M or 2M+2Supp)

Likely Pathogenic

1 VS/S + 1 M 1 S + (1 M or 2 Supp) 3 M 2 M + 2 Supp 1 M + 4 Supp

Interpretation 1: Likely benign (1 strong + 2 supporting) • BS1: Allele frequency greater than expected • BP6: LMM assertion is likely benign in Clinvar • BP4: In silico predictions Interpretation 2: Pathogenic (2 strong) • Functional studies show damaging effect on the gene • Prevalence in affected individuals significantly greater than controls

Questions to be answered at the end of the presentation:

Based on the aggregate evidence, do you believe SCN5A p.Phe2004Leu is:

Pathogenic Likely Pathogenic Likely Benign Benign

1 2% 22 61% 13 36% 0 0%

Questions to be answered at the end of the presentation:

Based on the aggregate evidence, do you believe that, in this case, SCN5A p.Phe2004Leu is:

Causative Likely causative A risk factor Unrelated

1 3% 10 37% 15 55% 1 3%

Questions to be answered at the end of the presentation:

Based on these two answers, in this research study, would you report SCN5A p.Phe2004Leu as:

Diagnostic No report, since non-diagnostic Report, clinical correlation warranted

1 2% 8 19% 32 78%

Summary

1. 15% of the 15% of babies admitted to ICUs may benefit from rapid genome sequencing

2. Case studies show WGS saved 2.9 QALYs per test in ICU/ICU infants 3. Clinical utility is multi-dimensional and requires novel multidisciplinary teams e.g.

for precision palliative care 4. Additional clinical trials are needed to define optimal methods and delineate

infants who will benefit 5. Systematic healthcare changes needed for broad adoption of genomic medicine in

ICU infants

Proposal for the SADS Foundation • (Blame Jim Perry) • A randomized, controlled study of rapid WGS

as a first tier test in Code Blue children

Support: Children’s Mercy Hospital

Rady Children’s Hosptial NICHD NHGRI NIDDK

The LORD is my creativity, Psalm 27

Josh Petrikin Laurel Willig Carol Saunders John Lantos Neil Miller Emily Farrow

Lance Prince MD PhD Gail Knight MD Farhad Imam MD PhD Nathaly Sweeney MD Nicole Coufman MD PhD Mallory Boutin Julie Ryu MD Cynthia Kuelbs MD George Chiang MD Lynn Byrd MD James Perry MD Amber Hildreth DO Jenni Friedman MD Jonathan Sebat PhD Tina Chambers PhD Albert Oriol Jeff Neul MD PhD Cyndi Kuelbs MD Lauge Farnaes MD PhD Bruce Barshop MD

Kevin Hall PhD James Richardson Kyle Farh MD Susan Tousi Severine Catreux Mike Ruehle John Reynders

Todd Laird Yan Ding MD Joe Gleeson MD PhD Michelle Clark PhD Julie Cakici RN Wendy Benson Ray Veeraraghavan PhD Matt Bainbrindge PhD Jennifer Azares Sergei Batalov PhD Vanessa Wertheim RN PhD 4 new folk Shareef Nahas PhD FACMG Shimul Chowdhury PhD FACMG David Dimmock MD FACMG Julie Reinke

Zornita Stark MD Sue White MD Tiong Tan MD

The children are waiting….