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The Laboratory Diagnosis Of Tuberous Sclerosis

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Presented at the 2007 ATSS Conference: Advances in Tuberous Sclerosis: From Pathway to Therapy. Michael Buckley presented outcomes from the SEALS laboratoty and described the basic processes being …

Presented at the 2007 ATSS Conference: Advances in Tuberous Sclerosis: From Pathway to Therapy. Michael Buckley presented outcomes from the SEALS laboratoty and described the basic processes being used at the lab.

Published in: Technology, Health & Medicine

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  • 1. The Laboratory Diagnosis of Tuberous Sclerosis Michael Buckley FHGSA FRCPA PhD Director, SEALS Genetics Laboratories Prince of Wales & Sydney Children’s Hospitals SEALS Genetics
  • 2. 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
  • 3.
    • 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)
  • 4. 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
  • 5. 3. Mutation Types SEALS Genetics GeneTests website
  • 6.
    • 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
  • 7.
    • 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
  • 8. 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
  • 9. 8. TSC mutation screen results SEALS Genetics
  • 10.
    • 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
  • 11. 9. Where are the missing mutations? SEALS Genetics
  • 12. 10. TSC1 Evolutionary conservation SEALS Genetics
  • 13. 11. TSC2 Evolutionary conservation SEALS Genetics
  • 14.
    • 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
  • 15. 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