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 Leading Conventional cause testing of Too slow Diagnosis of Death in To guide 8250 Genetic NICU & care Diseases in NICUs PICU Economics Work: Cost of care $4000 per day
Conventional Testing Too Slow For Optimal NICU Outcomes 1 0.9 Proportion Surviving 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 20 40 60 80 100 120 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 •
00:00 Baby CMH487: acute liver failure
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 Stereotyped Atypical presentations of 4,645 known genetic diseases presentations due to partially Genetic disease mimicking a non-genetic disease developed organ Genetic disease complicating a non-genetic disease systems and homeostatic Two concomitant genetic diseases responses 3,643 named, uncloned genetic diseases 20+ novel genetic diseases discovered per month
April 10, 2013 Hydrocele testis Infra-orbital crease Maternal diabetes Baby CMH487 Low-set, posteriorly rotated ears Feeding difficulties in infancy Ventilator dependence Single umbilical artery 00:00 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 HP:0009800 Maternal diabetes HP:0001539 Omphalocele HP:0008872 Feeding difficulties in infancy Baby CMH487 HP:0006533 Bronchodysplasia HP:0005946 Ventilator dependence HP:0006528 Chronic lung disease HP:0002020 Gastroesophageal reflux 00:00 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
00:00 Parents gave consent
00:00 Blood sample from mum, dad and baby
San Diego Synergy: Illumina + Edico + Rady Children’s
00:02 Transport to Institute
01:00 Robot isolates genomic DNA
06:00 Robot prepares DNA for sequencing
24:30 18 hour genome sequencing
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: Gene • One day faster • Samples 90% of genome • Reasonably good deletion structural variant detection Pros of Exomes: • Cost ¼ that of genomes Disadvantages of both: Whole • Limited phasing; requires trios Genome Sequence • Don’t detect insertion structural Whole variation or repeat expansions Exome Sequence = 2%
Co Cost an and T Time-to-Res esul ult o of Genomic S Sequenc uencing ng O Opt ptions ns 40X 4000 Trio Genome 48+ Hr 2x80nt 48+ 48+
Gap 4:
24:30 Infant CMH487
Compute & Storage 10 Gbps Remote Single backup monitoring dashboard 75 TB Simultaneous writes 500TB Compute cluster GPFS 9 TB/day 3 TB/day 6GB/sec 600 Mb/sec 10 Gbps 10 Gbps 100 cores High performance NAS In-house Genome sequencers researchers 5TB/day 1TB/day 24 x 7, 365 days/yr 1 GB/sec BAM FASTQ AWS FASTQ Google Public cloud Replication 1 Gbps http Azure 1 Gbps http 500TB Variant 25 yr archival External database Local object storage Collaborators Single global name space 2 yrs worth storage
24:45 Infant CMH487
25:00 Infant CMH487
Improved Analytic performance of WGS (nucleotide variants; not structural variants) Yeild Analytic Analytic Sample Site Pipeline Coverage (GB) A Sensitivity Specificity DRAGEN 99.93% 99.87% NA12878 143 45X Essex GSNAP/GATK-1.6/noVQSR 99.54% 98.57% DRAGEN 99.42% 99.46% a NA12878 65 20X CMH 97.29% 95.35% GSNAP/GATK-3.2/noVQSR
25:01 Infant CMH487
Does this DNA letter change cause a genetic disease? Hemoglobin- β DNA code Normal red Sickle cell blood cell
Variant pathogenicity categories Very Null variant (nonsense, frameshift, ±1 or 2 splice site position, initiation codon, exon Strong deletion) in gene where LOF known to cause disease Category CRITERIA • Strong Same amino acid change as previously established pathogenic variant • De novo in a patient with the disease and no family history Pathogenic 1VS + (1S or 2M/Supp) • Functional studies show damaging effect on the gene 2 Strong • Prevalence in affected individuals significantly greater than controls 1S + 3M or (2M+2Supp) • Moderate Located in mutational hot spot/functional domain without benign variation • Extremely low frequency in Exome Sequencing or 1000 Genomes Projects Likely 1 VS/S + 1 M • For recessive disorders, detected in trans with a pathogenic variant Pathogenic 1 S + (1 M or 2 Supp) • Protein length changed by in-frame indel in nonrepeat region or stop-loss 3 M • Novel missense at amino acid where different missense known to be pathogenic 2 M + 2 Supp • Assumed de novo, but without confirmation of paternity and maternity 1 M + 4 Supp 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 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.
25:41 Infant CMH487
25:42 Computer-Aided Comprehensive Differential Diagnosis 341 Possible Diagnoses
25:43 Infant CMH487 Diagnosis
26:00 • 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
• Confirmatory testing 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/mm 3 , 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 • Precision Medicine: – Meds. D/Cd – IV Ig and steroids
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 1 st tier test for genetic disease Dx): diagnostic rate ~15%
3. de novo Mutations: The most common mechanism of genetic disease diagnosis
35 NICU / PICU infants with likely genetic disease (Kansas City) 57% (20) 9% (3) Molecular Diagnosis By rapid WGS By standard methods Willig LK, et al. Science Trans. Med . April 2015
80 infants under care for likely genetic disease (Melbourne) 58% (46) 14% (11) Molecular Diagnosis By Exome Seq By standard methods Stark Z, et al. Genetics in Medicine 3/3/2016
Newborn Sequencing In Genomic medicine and public HealTh Explore use of genomic information for broadening understanding of diseases identified in the newborn period
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