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


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



Filtering with data parents

N = 18


N = 162




N = 41

Results


N = 135




N = 18

DDX11 c.2052+4A>G Warsaw breakage syndrome

KMT2D c.4418+4A>G Kabuki syndrome 1


N = 162




N = 41

KMT2D p.P3794S Kabuki syndrome 1

KMT2D p.R2645* Kabuki syndrome 1

Results

Variants after filtering tree N = 135



Filtering with data parents N = 18









LB Also in father

De novo De novo

Results













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:

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