Rapid screening for monogenic diseases in severely ill...
Transcript of Rapid screening for monogenic diseases in severely ill...
Rapid screening for monogenic diseases in severely ill newborns
Cleo van Diemen
Department of Genetics
GOAL: rapid genetic diagnostics for severely ill newborns in the NICU and IC
Why?
Need for speed in clinic in severely ill patients
Routine molecular testing is time consuming
Rapid cytogenetic testing is limited
Results too late to aid in decision making
Relevance:
Early diagnosis can prevent/limit unnecessary (invasive) diagnostics
Facilitates complementary diagnostics/treatment
Early diagnosis is important for the parents
Pilot project UMCG Pediatrics and Genetics
Inclusion of newborns and severely ill young children (<1 year) in ICU with suspected genetic disease
Unexplained and severe neurological manifestations; intractable seizures, severe neonatal onset movement disorder
Suspected metabolic disorder
Unexplained syndromal manifestations with multiple congenital anomalies
Acute organ failure (liver, kidney, lungs, heart)
Prerequisites
No clear acquired cause/explanation (high a-priori risk for monogenetic disorder)
Blood/DNA is available from both parents
Aim pilot project: provisional 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”
Use HPO terms for filtering
Expected 25-30 patients per year
Consultants related to clinical genetics, child neurology/cardiology,
metabolic diseases, etc.
MRI, EEG, echocardiogram, etc.
Biochemical testing
SNP-array
(when appropriate) targeted gene panel tests, single gene analysis
Standard procedures continue
Selection patient in multidisciplinary consultation
Phenotype
Consent and DNA isolation
Genome sequencing of patient
Data analysis In house pipeline/Cartagenia
Filtering CGD genes/phenotype
Multidisciplinary consultations
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 4 weeks; 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
All procedures are validated and described in SOPs
Exclusion late onset disease gene
Multidisciplinary meetings
First multidisciplinary meeting:
Technician, lab specialist, clinical geneticist (not patient consultant)
Evaluate each variant based on OMIM annotation/disease, check inheritance match and phenotypes
Evaluate incidental findings
Second multidisciplinary meeting:
At least present: technician, lab specialist, consulting clinical geneticist, bioinformatician and paediatrician
Evaluate all matched variants and incidental findings: if necessary consult review board installed for this purpose
Discuss follow-up
Coverage results
Main characteristics: - Flexion contracture (HPO 0001371) - Myopathy (HPO 0003198) Additional characteristics: - Cryptorchidism (HPO 0008689) - Supernumerary nipple (HPO 0002558) - Congenital bilateral hip dislocation (HPO 0008780) - Slender long bones (HPO 0003100) - Narrow mouth (HPO 0011337) Additional diagnostics: - MRI brain: probably normal brain. Suggestion of very thin or absent muscles in back. No polyhydramnion. Muscle biopsy: "congenital myopathy“, possibly nemalin myopathy Additional info: Parents consanguineous
Example Patient
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
Primary Consultation N =35
Variant Filtering Cartagenia
Inheritance match N = 31
Phenotype match N = 7
HGMD N = 0
Other N = 7
Second disciplinary consultation N = 7
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 Nemaline myopathy 9 AR
Variant Filtering
Number of variants after filtering N = 35
Number of variants after filtering N = 35
Inheritance match N = 31
phenotype match N = 7
HGMD N = 0
Other N = 7
Interdisciplinary consultation N = 7
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 Nemaline myopathy 9 AR
Variant Filtering
Human Phenotype Ontology terms Mean coverage
Nr of candidates after filtering*
Provisional diagnosis
1 Abnormality of the nervous system, Abnormality of movement 51x 5 no
2 Dilated cardiomyopathy (Myopathy) 29x 5 no
3 Cleft palate, Abnormality of finger, Abnormality of the cerebral ventricles 40x 0 no
4 Epileptic encephalopathy, myoclonus, hypotonia 33x 4 no
5 Status epilepticus, myoclonus 36x 3 no
6 Abnormality of the nervous system, microcephaly 38x 5 Yes, Vici syndrome EPG5
7 Acute liver failure 30x 1 no
8 Hydrocephaly, hypotonia, macrocephaly 32x 2 no
9 Cardiomyopathy 39x 3 Yes, 1p36 microdeletion syndrome#
10 hydrops fetalis, chylothorax, Pulmonary lymphangiectasia, Delayed CNS myelination
36x 5 no
11 Abnormality of the nervous system, Abnormality of movement 40x 4 Yes, RNMD1
12 Malformation of the heart and great vessels, Abnormality of the upper limb 34x 6 no
13 Flexion contracture, Myopathy 25x 4 Yes, nemalin myopathy 9 KLHL41
14 Interstitial pulmonary disease 38x 3 no
15 Myopathy, Fatigable weakness 27x 4 no
16 hyperinsulinism, cholestasis 43x 3 no
*number of variants followed up for sanger sequencing/biochemical testing etc #identified by SNP array
Project with additional patients: deceased children
Retrospective inclusion of additional patients (not rapid screening): newborns that died with unknown cause
- Trio design using Agilent SSID kit (clinical exome) - No result? -> WGS
Inclusion of 8 patient-parent trios thus far
Gender: M Partus 36+4 (spontaneous) Death: 1 day Main features: • Hypoplastic left heart • Pulmonary artery atresia • Absent/hypoplastic toes • Absent toenail • Hypoplastic fingernail Additional features: • Cupped ear (HP:0000378) li • Thickened nuchal skin fold (HP:0000474) • SUA (HP:0001195) • Wide intermamillary distance (HP:0006610)
Example Patients
Gender: M Partus 36+4 (spontaneous) Death: 1 day old Main features: • Hypoplastic left heart • Pulmonary artery atresia • Absent/hypoplastic toes • Absent toenail • Hypoplastic fingernail Additional features: • Cupped ear • Thickened nuchal skin fold • SUA • Wide intermamillary distance
Example Patients
Gender: V Age: TOP 23 weeks Main features: • Hypoplastic ostium mitralisvalve • Bicuspide aortaklep • Tubular hypoplastic aortic arch • Persistent VCS to sinus coronarius • Postaxial polysyndactyly left foot Additional features: • Abnormality of the gingiva • broad nose
Variants after filtering tree
N = 135
Inheritance match N = 23
Phenotype match N = 2
Filtering with data parents
N = 18
Variants after filtering tree
N = 162
Inheritance match N = 49
Phenotype match N = 2
Filtering with data parents
N = 41
Results
Variants after filtering tree
N = 135
Inheritance match N = 23
Phenotype match N = 2
Filtering with data parents
N = 18
DDX11 c.2052+4A>G Warsaw breakage syndrome
KMT2D c.4418+4A>G Kabuki syndrome 1
Variants after filtering tree
N = 162
Inheritance match N = 49
Phenotype match N = 2
Filtering with data parents
N = 41
KMT2D p.P3794S Kabuki syndrome 1
KMT2D p.R2645* Kabuki syndrome 1
Results
Variants after filtering tree N = 135
Inheritance match N = 23
Phenotype match N = 2
Filtering with data parents N = 18
DDX11 c.2052+4A>G Warsaw breakage syndrome
KMT2D c.4418+4A>G Kabuki syndrome 1
Variants after filtering tree N = 162
Inheritance match N = 49
Phenotype match N = 2
Filtering with data parents N = 41
KMT2D p.P3794S Kabuki syndrome 1
KMT2D p.R2645* Kabuki syndrome 1
LB Also in father
De novo De novo
Results
Variants after filtering tree N = 135
Inheritance match N = 23
Phenotype match N = 2
Filtering with data parents N = 18
DDX11 c.2052+4A>G Warsaw breakage syndrome
KMT2D c.4418+4A>G Kabuki syndrome 1
Variants after filtering tree N = 162
Inheritance match N = 49
Phenotype match N = 2
Filtering with data parents N = 41
KMT2D p.P3794S Kabuki syndrome 1
KMT2D p.R2645* Kabuki syndrome 1
LB Also in father
De novo De novo
Results
Similar features and some differences, same syndrome
Difficult to phenotype in these very young patients,
underlining the importance for molecular genetic testing
Age of death diagnosis 2800 genes SSID
1 IUVD 31 wks twin;hydrops foetalis;consanguineous parents;recurrence no diagnosis
2 15 months interstitial pneumonitis, PMR no diagnosis
3 6 weeks epileptic encephalopathy, bilateral hip luxation; IUGR RMFSL, compound BRAT1
4 1 day sudden death no diagnosis
5 perinatal 33 wks atresia of trachea+ oesophagus, BAV, BPV; "CHAOS" no diagnosis
6 26+1 wk hydrops Joubert, compound ZNF423
7 TOP 23 weeks HLHS, postaxial polydactyly one foot Kabuki, de novo KMT2D
8 1 day hypoplastic left heart, pulmonary artery atresia, absent toes, absent toenails, hypoplastic fingernail Kabuki, de novo KMT2D
Summary
• In a pilot study of 15 severely ill newborns and infants using rapid whole genome sequencing we found mutations with clinical relevance in 3 patients (3/15)
• In the same cohort one microdeletion was detected with SNP-array (1/15)
• 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 4 more patients (4/8)
First conclusions
Technically feasible within one week, good coverage
Interdisciplinary approach works very good: highlighted strong and weaker aspects of whole procedure. Begin to speak and understand each other’s language
Also better procedures/faster for routine diagnostics
BUT…. Why so many unsolved cases?
• Gene is yet unknown (outside CGD panel)
• Causal gene/variant is detected, but not recognized (very unlikely)
• Disease is not the result of a genetic defect
• Variant is not detected by current pipeline
Currently ongoing
Whole exome/genome analysis in research setting of unsolved cases (when consent is obtained)
Validation of CNV detection in the pipeline (Convading, Delly)
Additional filtering and annotation tools (i.e. CADD scores, gene network analysis to prioritize new candidate genes)
Parallel transcriptome sequencing next to WGS in research
setting
Whole exome/genome follow-up
CoNVADING: Detection of deletions/duplications/insertions (10kB resolution)
• 1 additional potential diagnosis (HetZ deletion of 30 kB including part of candidate gene, with compound splice site variant)
Gene network analysis based on co-expression • 1 additional potential diagnosis (suspected metabolic disease,
mitochondrial respiratory chain protein)
Re-evaluation of variants in splice-sites and intronic regions for splice-enhancers using RNA analysis
• 2 potential diagnoses
Future perspectives
Reduction in run-through time From Hiseq2500 to NextSeq500
New bioinformatics pipeline (i.e. Genalice trial)
Fast (clinical) exome sequencing in trio design or WGS?
Parallel transcriptome sequencing next to NGS
Clinical Genetics: Mieke Kerstjens Anne Herkert Katharina Löhner Patrick Rump Conny van Ravenswaaij-Arts Irene van Langen
Genomics Coordination Center: Mark de Haan Gerben van der Vries Roan Kanninga Joeri van der Velde Freerk van Dijk Patrick Deelen Pieter Neerincx Morris Swertz Genome diagnostics:
Martine Meems-Veldhuis Martijn Viel Arjen Schepers Jos Dijkhuis Renée Niessen Birgit Raddatz Jan Jongbloed Kristin Abbott Richard Sinke
Research: Cleo van Diemen Kim de Lange Desirée Weening Lennart Johansson Juha Karjalainen Lude Franke Pieter van der Vlies Tom de Koning Rolf Sijmons Cisca Wijmenga
Pediatrics: Tom de Koning Klasien Bergman
Many thanks to: