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Characterisation of the breakpoints of Low-Density Lipoprotein (LDL) Receptor gene deletions in families with familial h

Liverpool John Moores University and Liverpool Women’s NHS Foundation Trust. Characterisation of the breakpoints of Low-Density Lipoprotein (LDL) Receptor gene deletions in families with familial hypercholesterolemia. Frances White – 435387 BSc Honours Project – BMLGN3005

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Characterisation of the breakpoints of Low-Density Lipoprotein (LDL) Receptor gene deletions in families with familial h

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  1. Liverpool John MooresUniversity and Liverpool Women’s NHS Foundation Trust Characterisation of the breakpoints of Low-Density Lipoprotein (LDL) Receptor gene deletions in families with familial hypercholesterolemia. Frances White – 435387 BSc Honours Project – BMLGN3005 Tutors:Dr Janice Harland School of Pharmacy and Biomolecular Sciences Roger MountfordLiverpool Women’s NHS Foundation Trust

  2. What is Familial Hypercholesteraemia (FH)? • Autosomal Dominant condition • Incidence of 1/500 in UK. • 3 genes have been identified: LDLR – Low Density Lipoprotein Receptor (Chr.19) PCSK9 - Proproteinconvertasesubtilisin/kexin type 9 gene(Chr.1) APOB1 – Apolipoprotein-B (Chr.2)

  3. Clinical Presentation and Treatment • High Total Cholesterol Level (>7.5) • Strong Family History • Xanthomas, Xanthelasmas, Corneal arcus • Lead to Increase risk of Heart Disease • Treatment: Statin Drugs to lower cholesterol– Simvastatin Figure 2. www.medicalobserver.com.au Figure 3. Figure 1. www.medic.cardiff.ac.uk www.emedicine.medscape.com

  4. Role of Low Density Lipoprotein (LDL) • Small package that carries cholesterol around the body. • LDL carries cholesterol for ~2.5days until disposal. • At disposal, LDL cholesterol binds to the LDL receptor on liver cells. • The LDL cholesterol undergoes endocytosis and digested. • Cholesterol is used immediately or stored until later use. • The LDL receptors return to cell surface. • FH patients • Some FH patients have less amount of LDL receptors. • LDL cholesterol lingers for ~4.5 days until disposal. • Body believes it is not taking in and storing enough cholesterol. • So...it produces even more cholesterol which is left to linger! Figure 5. Figure 4. Normal LDL Receptors www.faculty.clintoncc.suny.edu FH LDL Receptors

  5. LDLR gene • Chromosome 19 (19p13.2) • 44,359bp • 18 exons • 2,583 base mRNA transcript • 860 amino acids • 1051 mutations described on mutation database (www.ucl.ac.uk/ldlr)

  6. LDLR mutation classes • Class 1 mutations affect the synthesis of the receptor • Class 2 mutations prevent proper transport of recpetor within the cell, not localised on cell surface • Class 3 mutations stop the binding of LDL to the receptor. • Class 4 mutations inhibit the internalisation of the receptor-ligand complex. • Class 5 mutations give rise to receptors that cannot recycle properly. This leads to a relatively mild phenotype as receptors are still present on the cell surface (but all must be newly synthesised)

  7. LDL Receptor Figure 6.

  8. NICE Guidelines 2008 • Aim: to offer cascade screening to those of FH families, identify at risk children and treat with statins as preventative measure by age 10yrs. • This has shown to reduce risk of heart disease to below that of the normal population. • In heterozygote FH families, high chance of heart attack in 30’s/40’s. • In homozygote children (rare), heart disease can exist in their teens.

  9. Aim of Project • Genetics Service at LWH found 5 families with a deletion of exons 2-6 in LDLR gene. • Aim: Identify the breakpoint of the deletion • Why?To allow simple and quick testing for at risk family members

  10. Methods • DNA was stored on 12 patients (9 families) • Multiplex Ligation-dependent Probe Amplification-MLPA • Quick and accurate technique that identifies copy number variations within known genes. • Long-Range Polymerase Chain Reaction (LR-PCR) • PCR used to amplify very large fragments of DNA. • Sanger Sequencing • Fluorescent sequencing is used to identify each base within the DNA sequence of interest.

  11. Results - MLPA MLPA Results show two copies of LDLR gene. Normal Figure 7. MLPA Results show one copy of exon 2-6. Patient Figure 8.

  12. Results – Long-Range PCR 1Kb Ext 1 2 H2O • Initial PCR product of >10Kb – Deleted allele Lane 1- Patient and Lane 2 - Normal • A ‘walk-in’ process from intron 1 and intron 7 with various primers produced a PCR product of ~2.5Kb. • More precise ‘walk-in’ process produced a 300bp PCR product – Small enough to sequence. PCR product from target DNA. Figure 9. 1Kb 1 2 3 4 5 6 7 8 9 10 11 12 13 H2O Figure 10. 1 2 3 4 5 6 7 8 9 10 11 12 H2O50bp Figure 11.

  13. Results – Sequencing 300bp PCR product was purified and sequenced. The sequence clearly read through intron 1 and continued in intron 7 after the breakpoint. The change was mapped to c.68-2467C>A in intron 1 and resulted in a deletion of ~11Kb of DNA. breakpoint INTRON 1 INTRON 7 Figure 12.

  14. Discussion and Conclusion • MLPA results confirmed the deletion in all 12 patients. • >10Kb PCR product produced by Long-Range PCR is likely to be the deleted allele as it is of the correct size. • The breakpoint identified is the same in all 12 patients. • The results pose two questions: • Is this a common deletion in the North-West region or within the UK? • If so, how common is it? Literature suggests there are populations where this deletion is common. • Could these patients actually be distantly related?

  15. Further Work • Design a simple and more cost effective assay • Allow at risk family members cascade screening. • Microsatellite Analysis • Identify a possible common haplotype amongst the patients that may prove they are related. • Search for more patients with LDLR del ex2-6 • Do they have the same breakpoint? • Approach Elucigene to incorporate this deletion into the FH20 kit. • Currently the kit is UK mutation specific and NOT region specific

  16. Key References • Brown M. S., Goldstein J. L., A Receptor-mediated pathway for cholesterol homeostasis (1986), Science, 232(4746); 34-47 • Mullis, K. B. and Faloona, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol, 155, 335-350. • Sanger F., Nicklen S., Coulson A. R.., DNA Sequencing with Chain-terminating inhibitors (1977), Proceedings of the National Academy of Sciences of the United States of America, 74(12); 5463-5467 • Schouten J. P. et al., Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification (2002), Nucleic Acids Research, 30(12); e57.

  17. Acknowledgments I would like to thank the following people: • Dr Janice Harland, Project Tutor, School of Pharmacy and Biomolecular Sciences, John Moores University • Roger Mountford, Project Tutor, Head of Laboratory, Merseyside and Cheshire Regional Molecular Genetics Laboratory • Everyone within the Merseyside and Cheshire Regional Molecular Genetics Laboratory with special thanks to Julie Sibbring, Kimberley Spencer, Emma McCarthy, Diane Cairns and Abigail Rousseau. • Regional Molecular Genetics Laboratory, Great Ormond Street Hospital, London. • Finally a huge thanks to the patients.

  18. Thank you.Questions?

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