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.
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
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
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