rapid whole genome sequencing in severely ill newborns and...

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


Outline

Rapid WGS in newborns and infants, pilot study

• Introduction: Why?

• Design

• Results

• Not just a succes story: struggles and mistakes

• Conclusions

• Future Perspectives


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


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.


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


• 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


• 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


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


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


Exclusion late onset disease genes


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


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


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)


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


Human Phenotype Ontology (HPO)

Phenomizer:

http://compbio.charite.de/phenomizer/

HPO browser:

http://compbio.charite.de/hpoweb/show

term?id=HP:0000118

http://compbio.charite.de/phenomizer/

http://compbio.charite.de/hpoweb/showterm?id=HP:0000118

http://compbio.charite.de/hpoweb/showterm?id=HP:0000118


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


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


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


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



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



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


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


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


Results project deceased patients

n=8


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


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)


Lancet Respir.Medicine 2015


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


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


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


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


Team meeting, no diagnosis

Strategy:

“loosen up” HPO terms:

Abnormality of the musculature (higher up in the tree)

Focus on homozygous regions


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


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…


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


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


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.


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


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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