1. Mutation screening in patients affected with autosomal dominant hypercholesterolemia – results from the Department of Health Pilot Project Alison Taylor Molecular Genetics GOSH

2. Autosomal Dominant Hypercholesterolaemia  Characterised by: Increased plasma levels of total cholesterol and low density lipoprotein Increased plasma levels of total cholesterol and low density lipoprotein Tendon xanthoma Tendon xanthoma Premature symptoms of coronary heart disease Premature symptoms of coronary heart disease  Heterozygous form 1/500  Homozygous form 1/1,000,000

3. Autosomal Dominant Hypercholesterolaemia  Mutations in: LDLR LDLR Apolipoprotein B-100 gene (APOB) Apolipoprotein B-100 gene (APOB) Proprotein convertase subtilisin/kexin type 9 gene (PCSK9) Proprotein convertase subtilisin/kexin type 9 gene (PCSK9)  Majority of mutations in the LDLR gene – >1200 identified  Cascade testing is cost effective way to identify new patients

4. Simon Broome Criteria  Simon Broome FH register criteria: A) TC > 7.5mmol/l or LDL cholesterol >4.9mmol/l A) TC > 7.5mmol/l or LDL cholesterol >4.9mmol/l B) Tendon xanthoma in patient or first degree relative B) Tendon xanthoma in patient or first degree relative C) Family history of MI before age 50yrs in 2nd degree relative or 60yrs in 1st degree relative C) Family history of MI before age 50yrs in 2nd degree relative or 60yrs in 1st degree relative D) Family history of TC >7.5mmol/l in first/second degree relative D) Family history of TC >7.5mmol/l in first/second degree relative

5. Dept of Health Project  Present data from a cohort of 635 patients with clinical diagnosis of heterozygous FH  Samples referred from six adult lipid clinics in UK  Patients classified using Simon Broome criteria 190 definite FH (DFH) 190 definite FH (DFH) 394 possible FH (PFH) 394 possible FH (PFH) 51 unclassified (UFH) 51 unclassified (UFH)

6. FH20 ARMS Kit   FH20 kit from Tepnel has 3 mixes (A, B and C)   Efficient and cost effective screen for FH testing   Doesn’t require specialised equipment, completed in 1-2 days   Used as initial screen   Multiplex ARMS analysis to detect 13 common mutations in familial hypercholesterolaemia Taylor et al Clinical Genetics 2007: 71: 561-568 p.Arg3527Gln p.Asp482His p.Asp374Tyr

7. Point mutation screen  18 exons and promoter region of LDLR gene  Initially done by SSCP, then dHPLC  Now – direct sequence analysis Beckmann Coulter Robots Beckmann Coulter Robots 20 fragments 20 fragments 6 patients, 1 normal control and blank 6 patients, 1 normal control and blank 2 PCR plates – 4 sequence plates 2 PCR plates – 4 sequence plates

8. MLPA analysis  GeneMarker software (SoftGenetics)

9. Results  Mutations found in 232 patients (36.5%) DFH – 106 (55.8%) DFH – 106 (55.8%) PFH – 113 (28.7%) PFH – 113 (28.7%) UFH – 13 (25.5%) UFH – 13 (25.5%)  Of mutations detected ARMs – 43% ARMs – 43% Point mutation screen – 52.3% Point mutation screen – 52.3% MLPA – 4.7% MLPA – 4.7%

10. Results  107 different mutations 12% APOB p.Arg3527Gln 12% APOB p.Arg3527Gln 5% c.654_656delTGG 5% c.654_656delTGG 5% c.313+1G>A 5% c.313+1G>A 5% p.Pro685Leu 5% p.Pro685Leu 1.7% p.Asp374Tyr PCSK9 1.7% p.Asp374Tyr PCSK9 6.9% novel – all predicted to impact on receptor function 6.9% novel – all predicted to impact on receptor function

11. Cascade testing  All but one of the sites carried out cascade testing where a mutation found in proband  Cascade testing in 100 families 290 relatives tested 290 relatives tested Mutation found in 166 relatives (56.1%) Mutation found in 166 relatives (56.1%)

12. Conclusion  NICE guidelines recommend that DNA testing is offered to all identified probands  Data presented strongly supports the clinical utility of DNA testing in patients with ADH  Overall detection rate in 635 patients was 36.5%  FH20 ARMs kit is an extremely efficient test in UK patients 49% DFH 49% DFH 38% PFH 38% PFH

13. Conclusion  Mutation screening still required as 6.9% mutations novel  Even in subjects with low clinical suspicion of FH mutation testing will be useful

14. Acknowledgements  RMG GOSH Gail Norbury Gail Norbury Darrell Wang Darrell Wang Brendan Martin Brendan Martin Kunjan Patel Kunjan Patel  London IDEAS Steve Humphries Steve Humphries Ros Whittall Ros Whittall Gretta Wood Gretta Wood Mabella Farrer Mabella Farrer JE Cooper JE Cooper Gaye Hadfield Gaye Hadfield Sarah Leigh Sarah Leigh  Referring Centres RDG Neely S Fairgrieve D Nair M Barbir J Jones S Egan Y Lolin E Hughes  Tepnel Diagnostics  Dept of Health

15. I am running the London Marathon in April for Great Ormond Street Children's Charity www.justgiving.com/aetaylor