Pathogenicity Decision Pathway in Tuberous

Sclerosis ComplexRosemary Ekong, Yoan Diekmann, Sue Povey

Special Features of Tuberous sclerosis complex (TSC)

Cause: Inactivating variants in TSC1 and TSC2Clinical presentation: Variable in severity even within familiesFeatures: Seizures, intellectual disability, tumours in the brain, heart, kidney and eye, a

facial rash and other skin signsFrequency: Approximately one in 10,000 live birthsInheritance: Autosomal dominant, but ~70% of cases are sporadicRisks:

Recurrence in a second child due to gonadal mosaicism in one parentMildly affected individuals only diagnosed after the birth of a severely affected child

Problem:70% of cases have new variantsSeveral unique TSC variants of uncertain significance

No causative change found in 10-15% of genetic tests

What We Consider in Pathology Assessment, i.e. Cause of TSC

Type of variant and its predicted effect on the proteinType of base change and position in exonConfirmed homozygotes with the variant = variant does not cause TSCCo-occurrence with well-established or a potentially pathological variantNumber of times variant already reported in TSC LOVD

Excludes, as far as possible, multiple reports from the same case or relative

Frequency of variant in large population datasets (e.g. Exome Variant Server or ExAC)Segregation of variant independently from the disease in the familyClinical diagnosis of having TSC or of not having TSC in fully examined relativesGenetic test results in parents and/or other relatives Is anything in the family description inconsistent with a single variant causing TSC?

Example: 2 different variants in the same family – 1 TSC1 and 1 TSC2 – 1 familial, 1 new

Reported variants checked for correct HGVS nomenclature DNA and predicted effect on protein - Mutalyzer

Results from in vitro functional assay on missense and in-frame variants

Our Classification: Five CategoriesPathogenic (+)

Established as a cause of TSC; expected to be at least 99% correct

Probably pathogenic (+?)Probably causes TSC; expected to be at least 90% correct

Probably not pathogenic (-?)Probably does not cause TSC; expected to be at least 90%

correct

No known pathogenicity (-)Established as not causing TSC; expected to be at least 99%

correct

Unknown (?) = VUSNot enough data to classify

Our Classification: Variant Pathogenicity

In the format: Reported/Concluded

Reported = That of the submitter Several submissions of the same variant may be

different We do not change this

Concluded = Our current conclusionThis is the same for all examples of the same variantMay be different from that of the submitter

• We are more cautious• New evidence obtained since the submission

Examples:+/+ +/+? +/? ?/- ?/+? +/-

TSC2 c.856A>G; 7 reports

Co-occurrence: 2/7 reports (truncating & splicing) Homozygous in a grandparent MAF in 6 populations = 0.36%

3.6% (2 homozygotes) in one population

Pathogenicity Assessment Pathway

Reclassification Using Large Population DatasetsThese have limited or no access to clinical data

We have classified 1845 different small variants as definitely pathogenic 588 TSC1 + 1257 TSC2 - Aug 2015 dataNone occur in data from ~70,000 individuals (EVS and ExAC)

Only 5 single examples of variants classified as probably pathogenic occur in these datasets1x TSC1 and 4x TSC2 missense variantsPopulation frequencies = 1:33,045 - 1:3712 individualsMAF = 0.0015% - 0.0135%

243 TSC variants that we have classified as VUS appear in these datasets TSC1 = 52 (49 reclassified); TSC2 = 191 (105 reclassified)

Reclassification from (-?) to (-)At least 3 or more individuals in a population with at least 1 for every 4000 individuals (MAF of 0.0125%)

Reclassification from (?) to (-)At least 10 individuals in a population with at least 1 for every 2000 individuals (MAF of 0.025%)

Occurrence of homozygotes (preferably >1) useful

Reclassification of Variants

Discussion 1: Does the distribution of observed variants in

populations matter?

For example, if the variant has been seen at very low levels in 3 different populations? TSC2 c.2545+10C>T (exon 21)1 report; pathogenicity = -/?MAF = EU 1/64988 or EA 1/8599, Latino 1/11450, South Asian 1/16420

What does this mean?

Discussion 2: Should ethnic groups be mentioned?

We note ethnicity was discussed at the GDSDAC meeting (March)Suggestion that ethnicity field be excluded - “political and ethical issue”

Currently, ethnicities given in ExAC are not displayed in TSC LOVDCo-occurrence with very rare TSC-causing variants (privacy)Privacy outweighs any gain from ethnicity informationDefinition of ethnicity – is it social, cultural, geographical, racial? Ethnicity disclosure covered in consent?

In some entries, MAFs in TSC LOVD fall below 0.01% because all the populations with the variant are reflected in this figure

But in assessment, we consider the population with the highest frequency if the variant is very low in the others

TSCoversight committee

Discussion 3: How much weight to give to reports that a

variant is de novo?To classify a VUS as TSC-causing we currently consider:Certain diagnosis of TSCReport of ‘de novo’ from an experienced TSC centre With or without additional dataReclassification from (+?) to (+) requires at least 2 different de novo cases

SituationPolicies for testing for biological parentage vary in different settingsMany diagnostic labs take care not to discover the family structureDe novo reports without confirmation of biological parentageWhere paternity was confirmed, should this be indicated?

Acknowledgement

We thank the patients, clinicians and scientists for sharing their data

Funding for the TSC variation databases: TSA (UK) and TS Alliance (USA)

www.lovd.nl/TSC1 www.lovd.nl/TSC2