Department of Genetics,
University Medical Center Groningen
The Netherlands
Rapid whole genome sequencing in
severely ill newborns and infants
Mieke Kerstjens
Department of Genetics
-No disclosures
-patient data: not to be communicated with
others
-research data, confidential, paper submitted
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Outline
Rapid WGS in newborns and infants, pilot study
• Introduction: Why?
• Design
• Results
• Not just a succes story: struggles and mistakes
• Conclusions
• Future Perspectives
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GOAL: Rapid genetic diagnostics
for severely ill newborns and infants
Why?
• Routine molecular testing is time consuming
• Results too late to aid in decision making
• Need for speed in clinic in severely ill patients
Relevance:
• Early diagnosis can prevent/limit unnecessary
(invasive) diagnostics
• Facilitates complementary diagnostics/treatment
• Early diagnosis is important for the parents
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The start… Science Translational Medicine 2012
Rapid Whole-Genome Sequencing for
Genetic Disease Diagnosis in Neonatal
Intensive Care Units Carol Jean Saunders,1,2,3,4,5* Neil Andrew Miller,1,2,4* Sarah Elizabeth Soden,1,2,4*
Darrell Lee Dinwiddie,1,2,3,4,5* Aaron Noll,1 Noor Abu Alnadi,4 Nevene Andraws,3
Melanie LeAnn Patterson,1,3 Lisa Ann Krivohlavek,1,3 Joel Fellis,6 Sean Humphray,6
Peter Saffrey,6
Zoya Kingsbury,6 Jacqueline Claire Weir,6 Jason Betley,6 Russell James Grocock,6
Elliott Harrison Margulies,6 Emily Gwendolyn Farrow,1 Michael Artman,2,4 Nicole
Pauline Safina,1,4
Joshua Erin Petrikin,2,3 Kevin Peter Hall,6 Stephen Francis Kingsmore1,2,3,4,5†
6Illumina Inc., Chesterford Research Park,
Little Chesterford, CB10 1XL Essex, UK.
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Aim : genetic diagnosis within 72 hours
• Start set-up of project: September 2013
• Start inclusion of patients: May 2014
• Analysis of ~2800 genes from the Clinical Genomics
Database (CGD) which are “clinically actionable”
• All procedures validated and in “SOP’s”
• 30 patients included
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• Unexplained and severe neurological manifestations
• Suspected metabolic disorder
• Unexplained multiple congenital anomalies
• Unexplained acute organ failure (heart, lungs, liver,
kidney)
• Age < 12 months at disease onset
Prerequisites
• No clear acquired cause/explanation (high a-priori risk for
monogenetic disorder)
Inclusion
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• SNP-array
• Consultants related to clinical genetics, child neurology/cardiology, metabolic diseases, etc.
• MRI, EEG, echocardiogram, etc.
• Biochemical testing
• (when appropriate) targeted gene panel tests
Standard procedures continue
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Selection patient Phenotype
Consent and DNA isolation
Genome sequencing
Data analysis
In house pipeline/Cartagenia
Filtering CGD genes/phenotype
Multidisciplinary consultation
Preliminary results:
Candidate gene/genes
Initiate complementary tests
(Sanger confirmation; examine parents; etc)
Pre-test counseling
Consult clinical geneticist
Post-test counseling
Yes
No Follow-up
now max 1 month;
Goal several days
Logistics
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Logistics lab
Sequencing:
Illumina HiSeq 2500
4 lanes 100 bp paired-end
Rapid run
DNA isolation: QIAamp DNA Blood Mini Kit
Data Processing:
In-house pipeline
GCC
Variant filtering/
interpretation:
Cartagenia
DNA library preparation:
Nextera/NEB
hour 0-1 hour 1-9 hour 9-44 hour 44 -68 hour 68 -72
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Exclusion late onset disease genes
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Interdisciplinary meetings
1. Weekly meeting of large team to evaluate
results and follow up
2. Adhoc meeting of small team (technician, lab
specialist, bioinformatician clinical geneticist
and paediatrician) after results
-Evaluate each variant based on OMIM
annotation/disease, check inheritance match and
phenotypes
-Evaluate incidental findings: if necessary consult
review board installed for this purpose
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Example Patient
Male, 9 month, non-consanguineous
parents, first child, slow motor
development
Sudden relapse after fever, respiratory
arrest, hypotonia
Additional:
Abnormal regulation of temperature
Additional diagnostics:
MRI: white matter abnormalities
white matter
grey matter
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Considered Diagnoses
- Leukoencephalopathy with vanishing white matter (EIF2B1-B5)
-Megalencephalic leukoencephalopathy with subcortical cysts (MLC1)
- Mitochondrial complex I deficiency (NUBPL)
- Leukoencephalopathy with brain stem and spinal cord involvement and lactate
elevation (DARS2)
- Progressieve leukoencephalopathy (LKENP)
- Brain small vessel disease with or without ocular anomalies (COL4A1)
-Acute infection-induced encephalopathy (RANBP2, CPT2)
-MRCD (mitochondrial resp chain disorders)
-Zellweger (PBD1A,PBD1B,PBD2A, PBD3A,PBD4A, PBD5A,
PBD7A,PBD8A,PBD10A, PBD11A, PBD12A,PBD13A, PEX1)
-X-ALD (ABCD1)
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Selection patient in multidisciplinary
consultation
Consent parents
Pre-test counseling
Consult clinical geneticist
Phenotype in HPO terms
Example Patient
Leukoencephalopathy (HPO:0002352) Abnormal CNS myelination (HPO:0004335)
accepted
yes
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Human Phenotype Ontology (HPO)
Phenomizer:
http://compbio.charite.de/phenomizer/
HPO browser:
http://compbio.charite.de/hpoweb/show
term?id=HP:0000118
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Filtering Cartagenia Pop.Fr <2%
Pop.Fr <0,5%
Phenotype (HPO)
Homozygous
Dominant (de novo)
Splice site effect
Transcriptional
effect
Candidate genes
Multidisciplinary consultation
Dominant
Compound heterozygous
Recessive
HPO Phenotype: HPO:0002352, HPO:0004335
leukoencephalopathy, abnormal CNS myelination
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multidisciplinary consultation
Preliminary results: Candidate gene/genes
Initiate complementary tests (Sanger confirmation; examine parents; etc)
AF Gene cDNA Protein Prediction OMIM Inheritance
0.5 EIF2B5 c.1015C>T p.R339W P Leukoencephalopathy with vanishing white matter AR
0.5 EIF2B5 c.1208C>T p.A403V LP Leukoencephalopathy with vanishing white matter, AR
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Diagnosis in 5 days
Clinical impact:
• Patient was not transported to tertiary hospital with specific
expertise
• Further treatment was withdrawn, comfort care
• Parents were counseled, prenatal diagnostic test offered for
future pregnancies
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ID phenotype diagnosis
1401 myoclonic seizures, hypotonia, movement disorder no diagnosis
1402 DCM no diagnosis
1404 cleft palate, absent thumbs, hydrocephaly no diagnosis
1405 perinatal asphyxia e.c.i (resuscitated), encephalopathy no diagnosis
1406 status epilepticus; intracerebral haemorrhage R frontal no diagnosis
1407 microcephaly, epilepsy, PMR,VSD, poor vision EPG5 c.7475T>G (p.Phe2492Cys)
and c.5869+1G>A
1408 liver failure e.c.i.; liver biopsy severe fibrosis; 24 wks/475 g no diagnosis
1501 hypotonia, hypoventilation; hydrocephaly no diagnosis
1502 NCCM, VSD's, PDA,AV-block del 1p36.33p36.32
1503 hydrops, chylothorax no diagnosis
Results
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ID phenotype diagnosis
1504 neonatal seizures, PMR, movement disorder no diagnosis
1505 COA, postaxial polydactyly fingers, diaphragmatic hernia,
hypospadias
no diagnosis
1506 prenatally contractures, congenital myopathy; consanguineous
parents
homozygous KLHL41 c.1213G>A
p.(Gly405Ser) ;nemalin myopathy
1508 microcephaly, PMR, failure to thrive, hyponatremia, deafness,
hypertension
RMND1c.1262_1275del;p.Asn421T
hrfs*18 and c.713A>G p.Asn238Ser
combined OXPHOS type 11
1509 interstitial pulmonary disease; consanguineous parents no diagnosis
1511 myopathy, fatigable weakness no diagnosis
1513 hyperinsulinism, cholestasis no diagnosis
1514 bulbar weakness/palsy, abnormal muscle tone, central
hypotonia
no diagnosis
1515 cardiomyopathy no diagnosis
1516 congenital microcephaly, cleft palate, short stature, Fallot’s
tetralogy
no diagnosis
Results
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ID phenotype diagnosis
1518 abnormal gyration, nephrotic syndrome no diagnosis
1519 lactic acidosis, dysmorphic features Homozygous GFER; c.580C>T
(p.R194C) progr. mit.myopathy
1601 leukoencephalopathy, abnormal CNS myelination compound EIF2B5; vanishing
white matter; c.1015C>T
(p.R339W), c.1208C>T (p.A403V)
1602 growth delay, cholestasis, ventricular septal defects no diagnosis
1603 IUGR, microcephaly, epilepsy, encephalopathy, contractures no diagnosis
1604 abnormal muscle tone, feeding problems compound heterozyg. RAPSN;
congenital myastenic syndrome
1605 cardiomyopathy, hepatomegaly, parents first cousins homozygous GLB1 c.176G>A
p.R59H GM1-Gangliosidosis
1606 osteopetrosis/hypomineralisation of the skull Heterozygous ANKH; c.1015T>C
(p.Cys339Arg);
Craniometaphyseal dysplasia
1607 dysmorphic features, anal atresia, COA, pulm.hypertension no diagnosis
1608 cholestase, hypospadie, leverfalen, IUGR, hypothyroidism no diagnosis
Results
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Results
Diagnostic yield 30%
30 patients included
9 diagnoses (one microdeletion by SNP)
1 incidental finding (PALB2 LP paternal)
Mean turnaround time 12 days
Serious clinical impact
• retrieval of intensive treatment in 5
• therapy in 1
• counseling parents, choices for future pregnancies in all
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Additional Project deceased children
Retrospective inclusion:
infants that died from undiagnosed disease with
onset <12 mo
• Trio design using Agilent SSID kit (clinical exome)
- not rapid screening
• Inclusion of 8 patients thus far
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Results project deceased patients
n=8
ID phenotype diagnosis
1 hydrops foetalis;consanguineous parents;recurrence no diagnosis
2 interstitial pneumonitis, PMR homozygous NSMCE3*
3 epileptic encephalopathy, bilateral hip luxation; IUGR BRAT1 c.1313_1314delAG
(p.Gln438Argfs*51) and
c.638dupA (p.Val214Glyfs*189)
4 sudden death no diagnosis
5 atresia of trachea+ oesophag, BAV, BPV; "CHAOS" no diagnosis
6 hydrops no diagnosis
7 HLHS, postaxial polydactyly one foot Kabuki KMT2D c.7933C>T
p.R2645* de novo
8 hypoplastic left heart, pulmonary artery atresia, absent toes,
absent toenails, hypolplastic fingernail
Kabuki KMT2D c.4418+4A>G de
novo,effect on splicing
*WES Radboud UMC
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Summary
• In a pilot study of 30 severely ill newborns and
infants, using rapid whole genome sequencing,
we found mutations with clinical relevance in
patients (9/30) (30%)
• In a second cohort of children who had died in
infancy without a diagnosis, we performed trio-
analysis, using the same pipeline, and found
mutations in one more patient (4/8)
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Lancet Respir.Medicine 2015
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Comparison with previous studies
(Kingsmore group)
• Willig Lancet Resp Med 2015 (57%) 35 retrospective patients<4 months; research set-up
20 diagnoses
• This study (30%) 30 prospective patients <12 months from NICU/PICU; diagnostic set-up
9 diagnoses (30%)
8 retrospective patients
4 diagnoses
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not just a succes story
struggles and mistakes
KLHL41 mismatch HPO
GFER not yet in Cartagenia (old version HPO)
RMND1 deafness later added to phenotype
RAPSN no inheritance match, second mutation was a deletion
NSMCE3 new gene, not recognized
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Example patient
Main characteristics:
• Flexion contractures
(HPO 0001371)
• Myopathy
(HPO 0003198)
Additional info:
Parents consanguineous
Additional diagnostics:
Muscle biopsy: "congenital myopathy“, possibly nemalin myopathy
SNP-array: 4 large homozygous regions
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AF<2%
N =3204
AF<0,5%
N =2649
Phenotype
N =479
Phenotype
N =404
Homozygous / compound heterozygous
N =29
Dominant
N =6
Transcriptional effect
N =21
Splice site effect
N =8
Transcriptional effect
N =6
Splice site effect
N =0
Homozygous-compound
Dominant
Consultation
N =35
Variant Filtering
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Team meeting, no diagnosis
Strategy:
“loosen up” HPO terms:
Abnormality of the musculature (higher up in the tree)
Focus on homozygous regions
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Variant Filtering
AF Gene cDNA Protein Prediction OMIM Inheritance
0,5 MYH3 c.1411T>C p.Y471H LP
Arthrogryposis, distal,
type 2A, AD
1 NEB
c.20467-
4_20467-3insT LB Nemaline myopathy 2 AR
1 NEB c.16544A>C p.K5515T LB Nemaline myopathy 2 AR
0,5 TTN c.36202+1G>A LP
Myopathy, early-onset,
with fatal
cardiomyopathy AD
0,5 COL6A2 c.2182G>A p.V728M LP
Ullrich congenital
muscular dystrophy AD/AR
0,5 SCN5A c.5938G>T p.V1980F LP Cardiomyopathy AD/AR/Digenic
1 KLHL41 c.1213G>A p.G405S LP Nemalin myopathy 9 AR
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HPO id HPO label
HP:0002093 Respiratory insufficiency
HP:0001270 Motor delay
HP:0000218 High palate
HP:0001989 Fetal akinesia sequence
HP:0002804 Arthrogryposis multiplex congenita
HP:0003828 Variable expressivity
HP:0000774 Narrow chest
HP:0001324 Muscle weakness
HP:0000007 Autosomal recessive inheritance
HP:0000347 Micrognathia
HP:0001623 Breech presentation
HP:0002650 Scoliosis
HP:0000175 Cleft palate
Why did we miss it at first?
KLHL41 matched HPO terms Flexion contractures (HPO 0001371) Myopathy (HPO 0003198) HPO is not perfect…
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What did we learn?
• HPO is not perfect, loosen up ( in tree)
• Clinical diagnosis is leading, follow up is important, add genelist to
HPO
• Multidisciplinary approach is the key, communicate!
• Don’t exclude heterozygotes of recessive diseases
• Check literature for new genes
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Why so many unsolved cases?
• Gene is yet unknown (outside CGD panel)
• Causal gene/variant is detected, but not recognized
• Disease is not the result of a genetic defect
• Variant is not detected by current pipeline
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Conclusion
• A multidisciplinary approach using rapid
NGS is a good strategy in clinical
diagnostics of severely ill newborns and
infants. It is technically feasible within one
week, with good coverage.
• Future studies are needed to judge cost-
effectiveness, however, 9 relevant
diagnoses in 30 patients is a promising
result.
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Future perspectives
• Additional CNV detection in the pipeline
and additional filtering and annotation tools
must be developed
• Follow-up of unsolved patients: whole
exome sequencing/whole genome
sequencing/transcriptome analysis
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Thanks to:
Clinical Genetics:
Anne Herkert
Patrick Rump
Conny van Ravenswaaij-Arts
Irene van Langen
Mieke Kerstjens
Genome diagnostics:
Martine Meems-Veldhuis
Martijn Viel
Arjen Schepers
Jos Dijkhuis
Birgit Raddatz
Jan Jongbloed
Kristin Abbott
Richard Sinke
Pediatrics:
Tom de Koning
Klasien Bergman
Genomics Coordination Center:
Mark de Haan
Gerben van der Vries
Roan Kanninga
Joeri van der Velde
Freerk van Dijk
Pieter Neerincx
Elisa Hoekstra
Morris Swertz
Research:
Cleo van Diemen
Kim de Lange
Krista van Dijk-Bos
Jelkje de Boer-Bergsma
Desirée Weening
Lennart Johansson
Pieter van der Vlies
Rolf Sijmons
Cisca Wijmenga
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