Inactivating mutations in TSC1 and TSC2 cause tuberous sclerosis complex (TSC). This disease is variable in severity and classically causes seizures, intellectual disability, behavioural difficulties, brain tumours, heart tumours, renal tumours and a facial rash. It occurs worldwide and is found in approximately one in 10,000 live births. It can be inherited as an autosomal dominant disorder, but about 70% of cases are new in the family and there is a significant risk of recurrence in a second child because of gonadal mosaicism in one of the parents. In some cases, mildly affected individuals are not recognised till the birth of a severely affected child. Genetic testing is needed both for diagnosis and for reproductive decisions, but in at least 10% of tests the causative change is not found. There are also many TSC variants of uncertain significance which are often unique. The TSC1 and TSC2 databases (www.lovd.nl/TSC1 and www.lovd.nl/TSC2) attempt to record all TSC variants which have been reported, both in the contest of genetic testing for TSC and in other clinical conditions. Classification of the pathogenicity of these TSC variants in the TSC1 and TSC2 databases refers to their ability to cause TSC. Data from August 2015 show 889 different small variants for TSC1 of which 66% were pathological and 9% were not. For TSC2, there were 2522 different small variants of which 50% were pathological and 9% were not. All others were to some extent uncertain. Recent advances in the quantity and quality in NGS, and the access to enormous amounts of population data have produced many challenging opportunities to improve variant classification. We have now formalised our decision-making on the pathogenicity of variants and this uses type and position, likely effect of variant, confidence in diagnosis, frequency of variant in different patients, family details including de novo reports, co-occurrence with known harmful variant, and where appropriate, an in vitro function assay. We now also screen our database automatically for duplicates in very large cohort datasets, most of which do not have extensive phenotype data. We will present our current practice for interpretation and would welcome comments. The TSC variation databases are funded by the TSA and the TS Alliance.

1. Pathogenicity Decision Pathway
in Tuberous Sclerosis Complex
Rosemary Ekong, Yoan Diekmann, Sue Povey

2. Special Features of Tuberous sclerosis complex (TSC)
Cause: Inactivating variants in TSC1 and TSC2
Clinical presentation: Variable in severity even within families
Features: Seizures, intellectual disability, tumours in the brain, heart, kidney and eye, a
facial rash and other skin signs
Frequency: Approximately one in 10,000 live births
Inheritance: Autosomal dominant, but ~70% of cases are sporadic
Risks:
Recurrence in a second child due to gonadal mosaicism in one parent
Mildly affected individuals only diagnosed after the birth of a severely affected child
Problem:
70% of cases have new variants
Several unique TSC variants of uncertain significance
No causative change found in 10-15% of genetic tests

3. What We Consider in Pathology Assessment, i.e. Cause of TSC
Type of variant and its predicted effect on the protein
Type of base change and position in exon
Confirmed homozygotes with the variant = variant does not cause TSC
Co-occurrence with well-established or a potentially pathological variant
Number 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 family
Clinical diagnosis of having TSC or of not having TSC in fully examined relatives
Genetic 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

4. Our Classification: Five Categories
Pathogenic (+)
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 (?) = VUS
Not enough data to classify

5. 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 conclusion
This is the same for all examples of the same variant
May 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

6. Pathogenicity Assessment Pathway

7. Reclassification Using Large Population Datasets
These have limited or no access to clinical data
We have classified 1845 different small variants as definitely pathogenic
 588 TSC1 + 1257 TSC2 - Aug 2015 data
 None occur in data from ~70,000 individuals (EVS and ExAC)
Only 5 single examples of variants classified as probably pathogenic occur in these datasets
 1x TSC1 and 4x TSC2 missense variants
 Population frequencies = 1:33,045 - 1:3712 individuals
 MAF = 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

8. Reclassification of Variants

9. 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?

10. 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 LOVD
Co-occurrence with very rare TSC-causing variants (privacy)
Privacy outweighs any gain from ethnicity information
Definition 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
TSC
oversight
committee

11. 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 TSC
Report of ‘de novo’ from an experienced TSC centre
With or without additional data
Reclassification from (+?) to (+) requires at least 2 different de novo cases
Situation
Policies for testing for biological parentage vary in different settings
Many diagnostic labs take care not to discover the family structure
De novo reports without confirmation of biological parentage
Where paternity was confirmed, should this be indicated?

12. 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