The Laboratory Diagnosis of Tuberous Sclerosis Michael Buckley FHGSA FRCPA PhD Director, SEALS Genetics Laboratories Prince of Wales & Sydney Children’s Hospitals SEALS Genetics

Advances in Tuberous Sclerosis: From Pathway to Therapy Medical and Family Conference 3 – 4 November 2007 Sydney Children’s Hospital Randwick For more information: www.atss.org.au

Two TSC genes have been identified; TSC1 (Hamartin) at 9q34 and TSC2 (Tuberin) at 16p13.3 Normal function is the regulation of cellular proliferation Pathological function as tumour suppressor genes, with an inherited mutation plus a second mutation in hamartomas TSC1 has 21 coding exons including 3.4kb of sequence TSC2 has 41 coding exons including 5.4kb of sequence 1. History SEALS Genetics TSC1 : Science 277:805-8 (1997); TSC2 : Cell 75:1305-15 (1993)

Two TSC genes have been identified; TSC1 (Hamartin) at 9q34 and TSC2 (Tuberin) at 16p13.3

Normal function is the regulation of cellular proliferation

Pathological function as tumour suppressor genes, with an inherited mutation plus a second mutation in hamartomas

TSC1 has 21 coding exons including 3.4kb of sequence

TSC2 has 41 coding exons including 5.4kb of sequence

2. Sensitivity of Mutation Detection SEALS Genetics Overall, there is a mutation detection sensitivity of approximately 75% MLPA on Previously Screened Mutation Negative Samples Mutation Screening Results for Small Mutations in TSC

3. Mutation Types SEALS Genetics GeneTests website

Standard approach is to screen for large scale mutations using a commercially provided Multiplex Ligation-dependant Probe Amplification assay for TSC1 and TSC2. Subsequent screening is via semi-automated DNA sequencing using M13-tagged primers for each exon. Sequence is analysed by Seqscape software, requires average quality score >40 ( p <0.05 of error) Didirection sequencing with repeat analyses to clarify any data ambiguities All mutations confirmed by bidirectional sequencing of two samples 5. TSC mutation screening at SEALS SEALS Genetics Kwiatkowski D (2005) Eur J Hum Genet 13;695

Standard approach is to screen for large scale mutations using a commercially provided Multiplex Ligation-dependant Probe Amplification assay for TSC1 and TSC2.

Subsequent screening is via semi-automated DNA sequencing using M13-tagged primers for each exon.

Sequence is analysed by Seqscape software, requires average quality score >40 ( p <0.05 of error)

Didirection sequencing with repeat analyses to clarify any data ambiguities

All mutations confirmed by bidirectional sequencing of two samples

A total of 50 cases have been received for analysis Referred principally from the major Clinical Genetics Units in NSW. Time for analysis is approximately 1 week/patient Involves the analysis of 64 amplicons, and a total of 8,500 base pairs of DNA MLPA analyses are essentially complete DNA sequence analyses - 20 have been completely analysed, with 5 in progress and 25 due for analysis in the next 7 months Expectation is that the validation project will be finished mid-2007 Subsequent testing will be provided on a fee for service basis to cover reagents and labour costs only – approximately $1750 for sequencing and MLPA 6. TSC mutation screening at SEALS SEALS Genetics Kwiatkowski D (2005) Eur J Hum Genet 13;695

A total of 50 cases have been received for analysis

Referred principally from the major Clinical Genetics Units in NSW.

Time for analysis is approximately 1 week/patient

Involves the analysis of 64 amplicons, and a total of 8,500 base pairs of DNA

MLPA analyses are essentially complete

DNA sequence analyses - 20 have been completely analysed, with 5 in progress and 25 due for analysis in the next 7 months

Expectation is that the validation project will be finished mid-2007

Subsequent testing will be provided on a fee for service basis to cover reagents and labour costs only – approximately $1750 for sequencing and MLPA

7. TSC MLPA results SEALS Genetics Three MLPA identified mutations in a cohort of 50 patients Contrary to published data the NSW TSC2 deletion frequency is lower than that of TSC1

8. TSC mutation screen results SEALS Genetics

Comprehensive mutation detection fails to identify a mutation in 100% of patients suggests That there may be other genes that cause TSC That there are other mutational mechanisms that are not well covered by current diagnostic techniques (eg inversions) That unclassified variants may truly be mutations That mosaicism plays a significant role There is currently no evidence for additional TSC loci Possibly we are ignoring important regulatory regions of TSC1 and TSC2? 4. TSC1, TSC2, TSC3? or? SEALS Genetics Kwiatkowski D (2005) Eur J Hum Genet 13;695

Comprehensive mutation detection fails to identify a mutation in 100% of patients suggests

That there may be other genes that cause TSC

That there are other mutational mechanisms that are not well covered by current diagnostic techniques (eg inversions)

That unclassified variants may truly be mutations

That mosaicism plays a significant role

There is currently no evidence for additional TSC loci

Possibly we are ignoring important regulatory regions of TSC1 and TSC2?

9. Where are the missing mutations? SEALS Genetics

10. TSC1 Evolutionary conservation SEALS Genetics

11. TSC2 Evolutionary conservation SEALS Genetics

The ATSS and its donors for funding the project Jacinta Dzarir, Glenda Mullan, Peter Taylor in the SEALS Genetics Laboratories for putting the program together SEALS for infrastructure 12. Thanks! SEALS Genetics

The ATSS and its donors for funding the project

Jacinta Dzarir, Glenda Mullan, Peter Taylor in the SEALS Genetics Laboratories for putting the program together

SEALS for infrastructure

Advances in Tuberous Sclerosis: From Pathway to Therapy Medical and Family Conference 3 – 4 November 2007 Sydney Children’s Hospital Randwick For more information: www.atss.org.au