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Tiered WES or Exome masking (icm LOVD+)
Claudia Ruivenkamp
Clinical Genetics
21/09/2016
Diagnostic workflow for patients with intellectual disability (ID)
~1000 patients/yr
Genomic microarray
Single gene test
Whole Exome Sequencing
Majority of ID patients has NO diagnosis
~1-5%
~15-20%
Why exome sequencing? versus targeted and genome
• ID is genetic very heterogeneous (>500 genes involved), Sanger sequencing is time consuming and too expensive
• Diagnostic yield will increase compared to Sanger sequencing (less covered regions are no problem)
• Simple to add genes to genepanel
• Can analyze the whole exome (not all responsible genes are known)
• Whole Genome Sequencing (WGS) too expensive
• WGS data analysis and interpretation too complex
WES Pilot study - question
27-Sep-16 4
• Trio-sequencing: successful, but expensive… • Sequencing only the index: reduction in costs • but is it sufficient?
Pilot study WES – single patient approach
• 32 patients with unexplained ID and/or MCA • No etiological or syndromic diagnosis • Negative family history
• Patients reached end stage of conventional strategies
• Target gene tests are normal • Genomic array profile is normal
Start beginning of 2013
Pilot study WES – Method
• WES is outsourced to a commercial company • Agilent Sureselect XT Human All Exon V5 kit • Illumina Hiseq2000 system (pools of 6-8 samples/lane)
• Sequencing criteria: • Q30>80% • >90% of targeted exome 10X • >80% of targeted exome 30X
~60 million reads
WES data analysis pipeline
Base calling
Read mapping
Annotation
Identify variants
Re-align (indels)
Variant recalibration
Interpretation
Filtering
FASTQ file
BWA
GATK
Seattle seq / VEP
> 5% in the population; intergenic regions
MAGPIE Modular Automated GATK Pipeline
Data analysis and interpretation is done at the LDGA
Pilot study WES – Variant interpretation
Pilot study WES – Results
a well-known gene, but the syndrome was not recognized
a known gene, but different phenotype
a gene that is not yet linked to a phenotype
Pilot study WES – Results (S32)
Prenatal case • hygroma colli • intracerebral cysts • ? abnormal facial anatomy
a ciliopathy?
Well known gene: Mutations in the CC2D2A gene are associated with Meckel syndrome (12) as well as Joubert syndrome (23)
Array Analysis WES Analysis (BAM file) Sanger confirmation
Father
Mother
Patient S32
Control
NKX3-2 CC2D2A PROM1
Chr 4
Pilot study WES – Results (S19)
• Boy • 3y 3 m • IQ 70 • severe sleep disorder • behavioral problems
(aggression) • no epilepsy
Smith-Magenis syndrome? no deletion 17p11.2 no mutation RAI1 no mutation GRIN2B
@ 6y: Autism Spectrum Disorder (ASD)
de novo missense mutation in SCN2A known cause for epilepsy!?
Rauch et al. Lancet 2012 : Recurrently mutated gene in ID series (3/51) Baasch et al., Epilepsia 2014: De novo mutations in 5/38 ID/ASD patients with no epilepsy
a known gene, but different phenotype
Pilot study WES – Results (S18)
• Boy • 7y • severe ID • no speech • non-ambulant • healthy
de novo missense mutation in GRIN1 no known syndrome, causative? c.1858G>A
Pathogenicity clues Highly conserved nucleotide (phyloP: 4.73 [-14.1;6.4]) Highly conserved amino acid, up to Fruitfly (considering 12 species) Moderate physicochemical difference between Gly and Arg (Grantham dist.: 125 [0-215]) This variation is in protein domains:
Ionotropic glutamate receptor Extracellular solute-binding protein, family 3 NMDA receptor
Align GVGD: C15 (GV: 206.04 - GD: 124.98) SIFT: Deleterious (score: 0, median: 3.64) MutationTaster: disease causing (p-value: 1)
Pilot study WES – Results (S18)
• Boy • 7y • severe ID • no speech • non-ambulant • healthy
de novo missense mutation in GRIN1 no known syndrome, causative? c.1858G>A
Hamdan et al. AJHG 2011 • 2 de novo mutations in GRIN1: - missense mutation in 10y-old girl with
moderate ID, no epilepsy - in frame-duplication in 7y-old boy with severe
ID, partial complex epilepsy and hypotonia Nature 2013, Epi4K consortium • 1 de novo missense mutation in GRIN1 18 y-old woman with severe epilepisy and ID, no speech mouse studies: • GRIN1 expression behavioral problems (cf. schizophrenia) and learning problems
GRIN1 forms complexes with GRIN2A&B
Enough evidence for pathogenicity
Pilot study WES – Results
• 7 of 32 patients were diagnosed (22% versus ~30%-55%)
• Relatively high amount of potentially pathogenic variants remain
(average of 13 variants/patient; range 2 to 27) • All these variants require clinical interpretation which is very
time-consuming
• All these variants require Sanger validation
• Majority of these variants were likely benign because they are inherited from one of the parents
Pilot study WES – Conclusions
• At this moment trio-analysis (affected child and parents) is more efficient in identifying the causative variant
• In the future when databases are filled with more and more exome data and consequently with more rare benign variants, exome sequencing single
patients will become a more realistic diagnostic approach.
Is exome sequencing of single patients with intellectual disability an effective diagnostic strategy?
Trio-analysis in undiagnosed cases
Adjustments: - Automated the WES process for implementation in diagnostics - MAGPIE for single patients and trios (affected child and parents) - Controlled by Collibri automation framework (web interface) - Filtering and interpretation : Custom made program (LOVD+)
25 of 32 were not diagnosed with single patient analysis Advantages: • Filter out inherited variants (ID mostly de novo) • Recessive filtering; inheritance directly visible • Open the exome (not limited to genpanel) • Trio-genotype check (QC) to exclude sample swaps
WES workflow
Primerdesign ‘Prinia’
Colibri: automation framework (web interface)
Custom made program
Filtering the remaining variants
Filter on • Gene panel of interest • Quality of the variant • Present in ANY frequency in normal controls (2%) • Present in the parents • Coding sequence (UTR, intronic)
Ivo Fokkema
WES data analysis - LOVDplus
WES data analysis - LOVDplus
Details patient database Details
WES data
WES data analysis - LOVDplus
Details WES data
QC check
WES data analysis - LOVDplus
Details WES data
coverage
Example gene panel analysis
Gene panel analysis 711 ID genes • 3 standard analysis (de novo, X-linked, recessive) • Stringent variant filtering
Example whole exome analysis (open the exome)
Whole exome analysis Only! For trios and informed consent (de novo, X-linked, recessive)
Variant interpretation 1
gene
BAM file
Variant classification
5 different classes
Variant interpretation is complex
• Variant present in in-house database? • Variant known in dbSNP/normal population? • Coding – Non-coding? • Mutation type? Truncating, splice-site, missense, synonomous…. • Which gene? Genpanel or exome? • What is known about the gene? • Does it fit the phenotype of the patient? • ……
Classification and interpretation is complex
• Known variants in known genes, that are linked to the phenotype, are pathogenic • However: - Novel variants in known genes, with a different phenotype - Variants in genes that are not linked to a phenotype; OMIM, literature study…
Trio analysis - Results
• 25 of 32 were not diagnosed with single patient analysis
• 22 trio analysis was performed (WES on parents) (for 3 no parental DNA available) • 9 (potentially) pathogenic variants are detected
Id gene type Genomic Change (GRC37) Coding Protein change
L1 WAC frameshift g.28872407_28872408insA NM_016628.3:c.356dup p.Asn119Lysfs*2 exome de novo
S2 SNX14 nonsense g.86217761delT NM_153816.3:c.2670delT p.Cys890* exome homozygous
S3 PCDH8 missense g.237048462T>G NM_000254.2:c.2718T>G p.Phe906Leu exome de novo
S5 ADNP nonsense g.49509038G>C NM_015339.2:c.2213C>G p.Ser738* exome de novo
S7 ACTA1 missense g.229568475G>T NM_001100.3:c.282C>A p.(Asn94Lys) exome de novo
S21 SLC9A6 missense g.135080689G>A NM_001042537.1:c.652G>A p.(Gly218Arg) X linked genepanel de novo
FGD1 missense g.54492214A>G NM_004463.2:c.1412T>C p.(Val471Ala) X linked genepanel maternal
S25 KMT2A frameshift g.118376268del NM_001197104.1:c.9661del p.Leu3221Serfs*35 exome de novo
S29 CACNA1A missense g.13446642G>A NM_023035.2:c.1060C>T p.Leu354Phe exome de novo
S31 ECEL1 missense g.233348907C>G NM_004826.2:c.1211G>C p.Arg404Pro homozygous exome maternal/paternal
Trio Analysis – Results (S7)
• 2y old girl • Unknown congenital disease • Myopathy • Normal array profile
Trio Analysis – Results (S7)
• 2y old girl • Unknown congenital disease • Myopathy
Genpanel: no mutation
Trio Analysis – results (S7)
• 2y old girl • Unknown congenital disease • Myopathy
Genpanel: no mutation
Missense mutation in ACTA1 gene (c.282C>A)
Associated with congenital myopathy
Neuromuscul. Disord 21, 2011
Trio Analysis – Results (S5)
• 4y old girl • Intellectual disability • Normal array profile
Not diagnosed with single patient WES analysis
Genpanel: no mutation
Trio Analysis – Results (S5)
• 4y old girl • Intellectual disability • Normal array profile
Genpanel: no mutation
Truncating mutation in ADNP gene (c.2213C>G(p.Ser738*))
• Associated with autosomal dominant mental retardation nr 28
• 3 cases with de novo truncating mutations, same exon
Nat Genet;46, 2014
Why does WES not always work?
No pathogenic variant detected?? • Lack of sequence coverage of a variant • Bio-informatic variant calling issues • Variant filtering issues (mosaic/imprinting) • Miss-interpretation of variants • The cause of the disease is located outside the coding sequences • The cause of the disease is a large indel or structural genomic variant
missed by exome sequencing
Other errors… • Sample swaps • Father is not the father • Clinical heterogeneity or incorrect diagnosis
Summary & Conclusion
• WES detected (potentially) pathogenic variants in: • 7 of 32 patients with single patient strategy (22%) • 9 of the 22 patients with trio approach (41%)
In total, (potentially) pathogenic variants were detected in 16 of 32 patients (50%) • Majority ‘solved’ by opening the exome (up to date gene panel needed
for single analysis)
• Trio approach: variant filtering is easier
• WES increases the diagnostic yield for ID patients
27-Sep-16 37 Insert > Header & footer
Bio informatics Gijs Santen (KG) Wibowo Arindrarto (SASC) Sander Bollen (LDGA) Adri Krol (LDGA) Patrick van Bunderen (LDGA) Ivo Fokkema (LOVD+) Jeroen Laros (HG) Martijn Vermaat (HG) Other SASC members
LDGA Claudia Ruivenkamp Mariëtte Hoffer Antoinet Gijsbers Esther Nibbeling Monique Losekoot Esther Nibbeling Marlies Laurense-Bik Saskia Smith Ivonne Minderhout Michiel van der Wielen Johan den Dunnen Bert Bakker Lies Hoefsloot
Clinical Genetics Emilia Bijlsma Arie van Haeringen Emmelien Aten Nicolette den Hollander Martijn Breuning Sarina Kant Marjolein Kriek Christi van Asperen et al.