The Laboratory Diagnosis of Tuberous Sclerosis Michael Buckley  FHGSA FRCPA PhD Director, SEALS Genetics Laboratories Prin...
Advances in Tuberous Sclerosis: From Pathway to Therapy Medical and Family Conference 3 – 4 November 2007 Sydney Children’...
<ul><li>Two TSC genes have been identified; TSC1 (Hamartin) at 9q34 and TSC2 (Tuberin) at 16p13.3 </li></ul><ul><li>Normal...
2. Sensitivity of Mutation Detection SEALS Genetics Overall, there is a mutation detection sensitivity of approximately 75...
3. Mutation Types SEALS Genetics GeneTests website
<ul><li>Standard approach is to screen for large scale mutations using a commercially provided Multiplex Ligation-dependan...
<ul><li>A total of 50 cases have been received for analysis </li></ul><ul><li>Referred principally from the major Clinical...
7.  TSC MLPA results SEALS Genetics Three MLPA  identified mutations in a cohort of 50 patients Contrary to published data...
8.  TSC mutation screen results SEALS Genetics
<ul><li>Comprehensive mutation detection fails to identify a mutation in 100% of patients suggests </li></ul><ul><ul><li>T...
9.  Where are the missing mutations? SEALS Genetics
10.  TSC1 Evolutionary conservation SEALS Genetics
11.  TSC2 Evolutionary conservation SEALS Genetics
<ul><li>The ATSS and its donors for funding the project </li></ul><ul><li>Jacinta Dzarir, Glenda Mullan, Peter Taylor in t...
Advances in Tuberous Sclerosis: From Pathway to Therapy Medical and Family Conference 3 – 4 November 2007 Sydney Children’...
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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 used at the lab.

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The Laboratory Diagnosis Of Tuberous Sclerosis

  1. 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. 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. 3. <ul><li>Two TSC genes have been identified; TSC1 (Hamartin) at 9q34 and TSC2 (Tuberin) at 16p13.3 </li></ul><ul><li>Normal function is the regulation of cellular proliferation </li></ul><ul><li>Pathological function as tumour suppressor genes, with an inherited mutation plus a second mutation in hamartomas </li></ul><ul><li>TSC1 has 21 coding exons including 3.4kb of sequence </li></ul><ul><li>TSC2 has 41 coding exons including 5.4kb of sequence </li></ul>1. History SEALS Genetics TSC1 : Science 277:805-8 (1997); TSC2 : Cell 75:1305-15 (1993)
  4. 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. 5. 3. Mutation Types SEALS Genetics GeneTests website
  6. 6. <ul><li>Standard approach is to screen for large scale mutations using a commercially provided Multiplex Ligation-dependant Probe Amplification assay for TSC1 and TSC2. </li></ul><ul><li>Subsequent screening is via semi-automated DNA sequencing using M13-tagged primers for each exon. </li></ul><ul><li>Sequence is analysed by Seqscape software, requires average quality score >40 ( p <0.05 of error) </li></ul><ul><li>Didirection sequencing with repeat analyses to clarify any data ambiguities </li></ul><ul><li>All mutations confirmed by bidirectional sequencing of two samples </li></ul>5. TSC mutation screening at SEALS SEALS Genetics Kwiatkowski D (2005) Eur J Hum Genet 13;695
  7. 7. <ul><li>A total of 50 cases have been received for analysis </li></ul><ul><li>Referred principally from the major Clinical Genetics Units in NSW. </li></ul><ul><li>Time for analysis is approximately 1 week/patient </li></ul><ul><li>Involves the analysis of 64 amplicons, and a total of 8,500 base pairs of DNA </li></ul><ul><li>MLPA analyses are essentially complete </li></ul><ul><li>DNA sequence analyses - 20 have been completely analysed, with 5 in progress and 25 due for analysis in the next 7 months </li></ul><ul><li>Expectation is that the validation project will be finished mid-2007 </li></ul><ul><li>Subsequent testing will be provided on a fee for service basis to cover reagents and labour costs only – approximately $1750 for sequencing and MLPA </li></ul>6. TSC mutation screening at SEALS SEALS Genetics Kwiatkowski D (2005) Eur J Hum Genet 13;695
  8. 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. 9. 8. TSC mutation screen results SEALS Genetics
  10. 10. <ul><li>Comprehensive mutation detection fails to identify a mutation in 100% of patients suggests </li></ul><ul><ul><li>That there may be other genes that cause TSC </li></ul></ul><ul><ul><li>That there are other mutational mechanisms that are not well covered by current diagnostic techniques (eg inversions) </li></ul></ul><ul><ul><li>That unclassified variants may truly be mutations </li></ul></ul><ul><ul><li>That mosaicism plays a significant role </li></ul></ul><ul><li>There is currently no evidence for additional TSC loci </li></ul><ul><li>Possibly we are ignoring important regulatory regions of TSC1 and TSC2? </li></ul>4. TSC1, TSC2, TSC3? or? SEALS Genetics Kwiatkowski D (2005) Eur J Hum Genet 13;695
  11. 11. 9. Where are the missing mutations? SEALS Genetics
  12. 12. 10. TSC1 Evolutionary conservation SEALS Genetics
  13. 13. 11. TSC2 Evolutionary conservation SEALS Genetics
  14. 14. <ul><li>The ATSS and its donors for funding the project </li></ul><ul><li>Jacinta Dzarir, Glenda Mullan, Peter Taylor in the SEALS Genetics Laboratories for putting the program together </li></ul><ul><li>SEALS for infrastructure </li></ul>12. Thanks! SEALS Genetics
  15. 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

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