Tiered WES or Exome masking (icm...

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

http://en.wikipedia.org/wiki/Meckel_syndrome



http://en.wikipedia.org/wiki/Joubert_syndrome




• 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


• 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)


• 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


Details patient database Details

WES data


Details WES data

QC check


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


• 2y old girl • Unknown congenital disease • Myopathy

Genpanel: no mutation

Trio Analysis – results (S7)

• 2y old girl • Unknown congenital disease • Myopathy


Missense mutation in ACTA1 gene (c.282C>A)

Associated with congenital myopathy

Neuromuscul. Disord 21, 2011


• 4y old girl • Intellectual disability • Normal array profile

Not diagnosed with single patient WES analysis



• 4y old girl • Intellectual disability • Normal array profile


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

[email protected]

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.

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