Sian Ellard, Thalia Antoniadi and Emma Baple

Improving the interpretation of rare variants through inter-disciplinary working

▪ Guidelines▪ Training▪ Clinical practice

Sian Ellard, Thalia Antoniadi and Emma Baple

Improving the interpretation of rare variants through inter-disciplinary working

▪ Guidelines▪ Training▪ Clinical practice

Steve Abbs, NEQAS

Inconsistent variant classification

Next generation sequencing identifies more novel/rare variants

Single gene Small panel Large panel Exome Genome≤1 A few Many >200 >500,000

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Pathogenic

Likely pathogenic (>90%)

Uncertain significance

Likely benign (>90%)

Benign

2015: New guidelines from the USA

“This result does not confirm a diagnosis of disorder X and should not be used for clinical decision making”

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Pathogenic

Likely pathogenic (>90%)

Uncertain significance

Likely benign (>90%)

Benign

2015: New guidelines from the USA

Summary of evidence framework

Richards et al 2015 Genetics in Medicine; Jarvik and Browning 2016 Am J Hum Genet

Rules for combining evidence criteria

Assess the evidence for a variant across all patients for which information is available

Publications

Public/NHS databases

Laboratory database

Patient referredfor testing

Putting the ACMG guidelines into practice

Principles

▪ Use the guidelines as a framework to determine whether there is sufficient evidence to classify the variant as likely pathogenic or likely benign

▪ Use each data item only once (like Bayes’ theorem)

▪ Incorporate clinical data (functional and gene specific phenotype)

▪ Use your professional judgement

Patient Name: X

Gene: RYR1

Variant description: HGVS NM_000540.2: c.7306T>C (p.Cys2436Arg)

Variant location: GRCh37 (hg19): 19:38,990,639

Variant classification:

(Pathogenic/Likely pathogenic/ Uncertain

significance/Likely benign/Benign)

Likely Pathogenic

Evidence for variant classification (ACMG code – level)

▪ Cysteine is present at position 2436 of RYR1 in 150 orthologues, paralogues and other

homologous sequences. This residue is known to be important for redox-sensing. In silico

protein modelling suggests that a p.Cys2436Arg substitution would cause perturbation to the

secondary structure within that region and also to that of the neighbouring RYDR_ITPR

domain sequences. This variant maps to a known mutation hotspot for malignant

hyperthermia (PM1 - supporting).

▪ This variant has not been reported in the ExAC database (PM2 – moderate).

▪ The p.Cys2436Arg variant has been inherited in trans with a p. Glu3035Gln variant (PM3 –

moderate).

▪ The p.Cys2436 residue is conserved across 14 species from human to lamprey (Consurf score

9) and is predicted likely to be deleterious using SIFT, PolyPhen and AlignGVGD (PP3 –

supporting).

Appendix 1 - Evidence for variant classification using ACMG guidelines (Richards et al 2015 PMID 25741868)

Variant classification and interpretation workshop: November 4th 2016

Improving the quality of variant interpretation for clinical diagnosis

Train the trainers: February 28th 2017

Monthly WebEx for regional trainers

Cases

▪ Two cases per month circulated

▪ Submit classifications before meeting

▪ Review cases at meeting

▪ Present previous worked case examples

▪ Share ideas for local implementation

▪ Forum for questions/discussion

Draft UK guidelines for variant classification

Additional guidance for de novo variants

PS2 – (Strong) De novo (both maternity and paternity confirmed) in a patient with the

disease and no family history.

Note that the genotype must be consistent with the phenotype.

If a de novo variant was identified by trio exome or genome sequencing then maternity and

paternity have already both been confirmed . If a de novo variant is identified by trio exome

or genome sequencing and the patient’s phenotype is non-specific (eg intellectual disability),

this criterion would be used at a lower level.

Mutation hotspot and/or functional domain

PM1 – (Moderate) Located in a mutational hot spot and/or critical and well-established

functional domain (eg active site of an enzyme) without benign variation.

Plot functional domains, ExAC/gnomAD variants, HGMDPro/ClinVar/LOVD etc variants and

Consurf/conservation plots to show reported pathogenic variants, proxy population and

benign variants and amino acid conservation for a region of a gene.

PM1 Plot for SOX10 variant

Setting a threshold for constraint scores

PP2 – (Supporting) Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease.

ExAC constraint scores can be used as evidence for a low rate of benign variation (Lek et al 2016 Nature 536:285-291). Z scores ≥3 (marked amber in ExAC) are significant but it is important to consider constraint for the region encompassing the variant, not just across the entire gene (see Samocha et al 2017 bioRxiv).

Specificity of the phenotype

PP4 – (Supporting) Patient’s phenotype or family history is highly specific for a disease

with a single genetic aetiology.

This incorporates the prior probability that a patient will have a pathogenic variant in a

particular gene or genes. It does not have to be limited to diseases where there is a single

genetic aetiology.

▪ Test strategy – single gene, panel or trio exome/genome?

▪ Genetic heterogeneity of the disorder

▪ Specificity of the phenotype

Is the variant allele frequency too high?

Classifying benign variants

BS1 – (Strong) Allele frequency is greater than expected for disorder.

A very useful tool is available to determine whether the allele frequency of the variant is greater than expected for the disorder (Whiffin et al 2016 bioRxiv). In the absence of precise information about the disease prevalence and penetrance we recommend using conservative settings (by selecting the highest likely prevalence and the lowest likely penetrance) to see if the variant frequency on the ExAC database exceeds the maximum credible allele frequency. The tool can be accessed at http://cardiodb.org/allelefrequencyapp/.

NOTCH2 p.Arg1895His variant in patient with ?Alagille syndrome

http://cardiodb.org/allelefrequencyapp/

Improving the interpretation of rare variants through inter-disciplinary working

▪ Guidelines▪ Training: local implementation▪ Clinical practice

Sian Ellard, Thalia Antoniadi and Emma Baple

The Plan.

• CPD sessions

• ‘Pair’ scientists and clinicians

• Provide the training material and circulate the training cases for classification

• Collect the classification

• Organise a feedback session

Logistics…

• Identify the staff involved in variant interpretation: 80!!!

• Attempt pairing people according to their interests

• 1-2 clinical and 1-2 scientific staff per team: 34 teams!

Clinical Genetics: 44 Laboratory: 36

Consultants: 16 Senior scientists: 13

Counsellors: 23 Scientists: 23

Registrars: 5 STPs: 14

Organised the training in ‘rounds’

• Round 1: 29 teams were sent the cases for classification and were given little time to submit

• Feedback session – 24 April 2017

• Round 2: remaining teams and others that didn’t make it to the first round were given ample time to meet and complete the cases

• Feedback session – 15 June 2017

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Feedback session

• Presentation of the additional notes• Classification of the training cases and discussion

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Variant classifications during workshop

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pre- training classifications

Moving forwards

• Organising the WebEx participation

- a different team working up of the cases each time

- attending the WebEx

- feeding back in a CPD session – 5 July 2017

- one CPD session every 6-8 weeks

• Round 3: to take place in Autumn

• Working on updating the reporting templates (>600..)

What did the Guidelines ever do for us?

• Support the way towards consistency

• Provide the framework for dialogue

• Bringing the scientific and clinical expertise together to help with these variants that are sitting on the fence:

Can this variant explain this patient’s symptoms?

• Improve these communication networks between the scientists and the clinicians

• Essential infrastructure in view of 100,000 genomes and the wider application of WES and WGS

acknowledgments

• Helen Cox, Consultant Clinical Geneticist

• ACGS and Health Education England

• All the staff in WMRGL and CGU for their engagement and lively debate in the feedback sessions!

...we should be doing this with our non-genetics clinical colleagues, too

...shed light on some doubts and helped me see and interpret things differently

...appreciated the difficulties in interpretation from each other’s perspective

...I haven’t realised how variant classification was so prone to subjectivity

Sian Ellard, Thalia Antoniadi and Emma Baple

Improving the interpretation of rare variants through inter-disciplinary working

▪ Guidelines▪ Training▪ Clinical practice

Genome 4-5M

Exome 40,000

Panel 0->10

Disease-causing variant (or variant pair)

A cautionary tale…

• Move from:

“Guilty until proven Innocent”

• To:

“Innocent until proven Guilty”

NHS: A unique opportunity for MDT working

What is a Genomics MDT?Means different things to different people and in different places

▪ Who should attend?

▪ What format should the meeting take?

▪ What are the aims of the meeting and what questions need to be answered?

▪ Which cases should be discussed?

▪ Which stage(s) in the testing pathway should involve an MDT discussion?

▪ How and where should the discussion and outcomes be documented?

Selection of cases for testing

Phenotypedata

Primary Reviewby Clinical Scientists

EyeMDT

CardiacMDT

DDMDT

100kMDT

Sequencing

TumourMDT

Metabolic

What a Genomics MDT is not…..

▪ A competition

▪ An opportunity to show off

▪ A time trial

▪ An excuse to prevaricate

Clinical diagnostic exome service

Request form

▪ Contact details

▪ Clinical details

▪ Choice of strategy▪ Trio analysis▪ Couple analysis for lethal

disorders▪ Bespoke virtual panel etc..

▪ “Pre-analysis” MDT▪ Strategy▪ Virtual panel selection▪ Pipeline selection eg.

mosaicism, multiple disorders in the family, penetrance

Excel spreadsheet of filtered variants generated by a bioinformatician for review

by the clinical scientist

International cooperation

Fetus 2TOP 18/40

Rapidly progressing ventriculomegaly

No genetic diagnosis

2Fetus 1

TOP 22/40Rapidly progressing ventriculomegaly

No DNA

▪ Indian couple

▪ Same region, no known consanguinity

▪ Wanting to pursue prenatal diagnosis

▪ Limited DNA

▪ Parent only strategy

Fetus 2TOP 18/40

Rapidly progressing ventriculomegalyNo genetic diagnosis

2

POMGNT1 p.Arg497Gln/NPOMGNT1 p.Arg497Gln/N

POMGNT1 p.Arg497Gln/p.Arg497GlnNot homozygous

Fetus 1TOP 22/40

Rapidly progressing ventriculomegalyNo DNA

International cooperation

International cooperation

ACMG category

(Richards et al 2015 PMID 25741868 )

Evidence

PM1 Located in a mutational hot spot and/or

critical and well-established functional

domain without benign variation

p.Arg497 is located within the N-lobe

of the catalytic domain. Exon 17 of

POMGNT1 represents a mutation hot

spot (HGMD Pro).

PM2 Absent from controls (or at extremely low

frequency if recessive) in population

database(s)

1/121,334 alleles (European non-

Finnish).

PP1 Co-segregation with disease in multiple affected

family members

Variant identified in parents. Co-

segregates in one affected fetus and two

unaffected siblings (1/4 x ¾ x ¾ = 1/7)

but needs to be ≤1/8 for PP1 if only one

family.

PP3 Multiple lines of computational evidence

support a deleterious effect on the gene or

gene product

p.Arg497 is conserved across 23

species; Consurf score 9. SIFT and

Polyphen predict pathogenic, Align

GVGD class C35.

Additional evidence

ClinVar: Homozygous p.Arg497Gln variant identified in a patient with progressive muscle

weakness, cerebro-ocular dysgenesis, and septo-optic dysplasia (e-mail from Emory lab).

Criteria (*ACMG code) Level Evidence

Located in a mutational hot spot and/or critical

and well-established functional domain without

benign variation (PM1)

Moderate p.Arg497 is located within the N-lobe

of the catalytic domain. Exon 17 of

POMGNT1 represents a mutation hot

spot (HGMD Pro) with no

homozygous missense variants

reported in the ExAC database.

Absent from controls (or at extremely low

frequency if recessive) in population database(s)

(PM2)

Moderate One heterozygote (European non-

Finnish) in the ExAC database (60,667

individuals).

Co-segregation with disease in multiple affected

family members (PP1)

Supporting The p.Arg497Gln variant was

identified by exome sequence

analysis of the parents. Co-

segregation was demonstrated in one

affected fetus and two unaffected

siblings (1/4 x ¾ x ¾ = 1/7).

The Emory lab (USA)

identified this

homozygous variant in a

patient with Muscle-Eye-

Brain disease who

presented prenatally

with ventriculomegaly.

N ≤ 1 /4 if >1 FamilyMultiple lines of computational evidence

support a deleterious effect on the gene or gene

product (PP3)

Supporting p.Arg497 is conserved across 23

species; Consurf score 9. SIFT and

Polyphen predict pathogenic, Align

GVGD class C35.

International cooperation

• Non-consanguineous Caucasian parents

• One healthy older sibling

• Tonic clonic seizures from 6 months of age, controlled with antiepileptic medication

• CT and MRI normal

• Regression of skills at onset of seizures and ongoing poor developmental progress

• Walked independently at 6yrs, no meaningful vocalisations at 10yrs

• Normal growth parameters

Clinician and Clinical scientist cooperation

• Whole exome trio - DDG2P virtual gene panel analysis through the DDD project

• Patient is heterozygous for KMT2D variant in exon 48, NM_003482 c.15485T>G (p.Val5162Gly), confirmed de novo by Sanger sequencing

Testing undertaken and variant(s) found

Heterozygous pathogenic variants in KMT2D are associated with Kabuki

syndrome:

• Characteristic facial appearance• Minor skeletal anomalies• Persistence of fetal fingertip pads• Mild/moderate intellectual disability

and postnatal growth deficiency

PS2 – Maternity and paternity confirmed by exome, BUT the patient is not a good phenotypic fit for the disorder!

Conservation and variation in KMT2DAs previous slide, but showing data for residues encoded by exon 48 only (4882-5262); 11/21 previously reported DM mutations map within annotated functional domains or motifs

HGMD

ExA

C f

req

uen

cy

Co

nSu

rf g

rad

e

ZnF (PHD type)

5091 5137

FYRN

5175 5235

FYRC

PM1 – (Supporting) Exon 48 is a mutation hotspot, BUT there is benign variation within the same region of exon 48 as Val1562

Classification and summary of evidence

PP3 (Multiple lines of computational evidence support a deleterious effect on the gene or gene product conservation, evolutionary, splicing impact, etc.)

PM2 (Absent from controls) - Novel, not present in gnomAD

PP2 (Missense variant in a gene that has a low rate of benign missense variation and in which missense variants are a common mechanism of disease) – ExAC constraint score >3.0 and more constraint notable in exon 48

PM1 (Supporting) - Exon 48 is a mutation hotspot, BUT there is benign variation within the same region of exon 48 as Val1562

New DDD result!

KCHN1 c.1070G>A p.(Arg357Gln)

Temple-Baraitser syndrome and epilepsy

J Hum Genet. 2016 May;61(5):381-7. doi: 10.1038/jhg.2016.1. Epub 2016 Jan 28.De novo KCNH1 mutations in four patients with syndromic developmental delay, hypotonia and seizures

De novo KCHN1 p.(Arg357Gln) is reported as disease causing

Clinical summary

Clinical opinionCardiff

From OMIM:

So I asked Dr Sally Davies:Q1: What might this girl have?

•Quite hairy - Possibly Wiedemann-Steiner (KMT2A)•Large central incisors – Possibly KBG syndrome (ANKRD11) – quite a common findingin DDD project.

Q2: What about Kabuki syndrome?

No way!

▪ Born 2001▪ Polyglucosan disease, liver cirrhosis,

myopathy and mild dilated cardiomyopathy.

▪ Requested urgently as deteriorating clinically.

▪ GBE1 test and GSD panel negative

MDT: An opportunity for education

RBCK1 Small deletionp.Asn508fs (c.1522_1526del)/N

RBCK1 Nonsense variantp.Gln267Ter (c.799C>T)/N

RBCK1 Small deletion and nonsense variantp.Asn508fs (c.1522_1526del)/ p.Gln267Ter (c.799C>T)

▪ Polyglucosan body myopathy 1 (with or without immunodeficiency) is caused by biallelic mutations in the RBCK1 gene. Only 9 mutations described in the literature but these include nonsense, missense, small deletions and insertions

MDT: An opportunity for education

What is a Genomics MDT?▪ Who should attend? Depends on the case and the question(s)

▪ Clinical scientist, referring clinician, clinical geneticist, bioinformatician,

Genetic Counsellor, (other) experts.

▪ What format should the meeting take? Face to face versus virtual

▪ What are the aims of the meeting and what questions need to be answered? Preparation is key

▪ Which cases should be discussed? Not every result requires an MDT, focus is mainly on variant reporting, discussion of cases when no diagnosis has been made

▪ Which stage(s) in the testing pathway should involve an MDT discussion? Depends on the case and clinical question(s)

▪ How and where should the discussion and outcomes be documented? Genetics notes, hospital notes, electronic notes, appendix of the report