2Bristol Genetics Laboratory What is FH?Bristol Genetics LaboratoryImage 2. Xanthelasma(Image from Pietroleonardo & Ruzicka, 2009)Image 1. Achilles tendonxanthomaAutosomal Dominant1/500 heterozygotes in UK(1/1,000,000 compound heterozygotes)Caused by LDLR, APOB & PCSK9 mutationsRaised cholesterol, Xanthomas/Xanthelasma,Risk of CVDSimon Broome Criteria‘definite’ or ‘possible’‘Normal’ life expectancyon statin treatmentNICE guidelinesDefiniteTotal cholesterol >7.5 mmol/LTendon xanthomasPossibleFamily history of myocardial infarction <age 60 in 1st degree relativeFH is an autosomal dominant disorder characterised by elevated serum cholesterol, tendon xanthoma and premature coronary artery disease. The prevalence is approximately 1 in 500 for the heterozygous form and 1 in 1,000,000 in the homozygous form. FH is a widely under-diagnosed treatable disorder, NICE guidelines in 2008 recommend that DNA testing is offered to all identified probands.Mutations in the low density lipoprotein, apolipoprotein B and the proprotein convertase subtilisin kexin genes all cause FH in the proportion shown. (The Low-Density Lipoprotein Receptor (LDLR) clears serum cholesterol (LDL-C) from the bloodstream. APOB mediates the binding of LDL to the LDL receptor. The exact function of PCSK9 remains unknown, but it is suspected to take part in liver regeneration, neuronal differentiation and cell death).The Simon Broome criteria are set out to aid diagnosis of FH. Patients with definite FH require to have a total cholesterol >7.5mmol/l and LDL-C >4.9 mmol/l in adults or, TC >6.7 mmol/l and LDL-C >4.0 mmol/l in children, plus the presence of tendon xanthoma in the patient or a 1st or 2nd degree relative (or DNA-based evidence of FH) for definite FH.Possible FH is diagnosed in patients with the same cut-off cholesterol values as dFH but have a family history of myocardial infarction below the age of 50 in a 2nd degree relative, below the age of 60 in 1st degree relative, or a family history of raised cholesterol levels.
3Why Perform Genetic Testing? Bristol Genetics LaboratoryPatient A, aged 8LDLR mutation confirmed in familyEquivocal cholesterol – 5.6mmol/L(FH >6.7mmol/L in children, ‘Normal’ <4.0mmol/L)Family history of extensive cardiovascular diseaseGreat uncle – Myocardial infarction at aged 31Patient A – mutation identifiedWill start statin treatment at ~ age 10Early treatment gives the maximum health benefitMore likely to adhere to treatmentThis patient has equivocal cholesterol at age 8. The patient has a poor family history with a great uncle that had an MI at age 31 with extensive cardiovascular disease. A mutation was identified in this patient which will enable the patient to commence treatment from the age of 10 and will therefore prevent the complications of FH in later life. Therefore, genetic testing provides a definitive, presymptomatic diagnosis of an under-diagnosed and under-treated disorder for which effective therapy exists.Also, a study in the Netherlands showed that the proportion of patients using cholesterol-lowering medication was significantly increased after FH diagnosis through genetic cascade screening (Kindt H et al, 2010).
4Why Perform Genetic Testing? Bristol Genetics LaboratoryPatient B, aged 55Pre-treatment cholesterol ~20mmol/L (FH >7.5mmol/L in adult)Unexpected compound heterozygote for two unclassified variantsc.[1766delA] + [932A>C]p.[Asp589fs] + [Lys311Thr]Pedigree:Instigated further cardiology investigationsExercise ECG positiveGenetic diagnosis allows consideration of LDL apheresisThis patient, age 55 had a pre-treatment cholesterol level of 20mmol/l, the patient was identified to be a compound heterozygote for 2 unclassified variants. This diagnosis led to extensive cardiology investigations, with a positive result for an exercise ECG and LDL apheresis to be considered. LDL apheresis (basically like dialysis to remove LDL) is expensive and can only be performed on patients with clinical need. In this case, as ‘homozygous’ FH is harder to treat as statins are not very effective, LDL apheresis may be indicated.Patient underwent partial ileal bypass – led to increased life expectancy for homozygote.(An exercise electrocardiogram helps to diagnose ischaemic heart disease, which is the common cause of angina and other heart problems. It can also help to assess the severity of ischaemic heart disease. An exercise ECG records the electrical activity of your heart whilst you exercise.)p.[Asp589fs] + [Lys311Thr]p.[Asp589fs] + [=]p.[=] + [=]
5Current Testing Method Bristol Genetics LaboratoryARMS –20 commonmutationsValidated on known positive control samplesTested 104 samples to dateMLPA (MRC-Holland: P062B) validated using 4 reported samples, obtained from a Norwegian lab+ve-veMutationconfirmationSequencingREPORT Offersequencing/MLPAREPORTSequencing+ve-veCASCADEREPORTProceed toMLPAPrimers designed for all 18 LDLR exons + promoterSequenced in 17 fragmentsValidated using 4 known positives+ve-veREPORTREPORT
6Simon Broome Audit Data Bristol Genetics LaboratorySimon Broome CriteriaNo. Patients TestedNo. +vedFH1515 (100%)pFH5323 (43%)Unclassified176 (35%)Criteria not met194 (21%)Total Diagnostic10448 (46%)Cascade2715 (56%)These are the results obtained so far, for over a years worth of testing. These results highlight that when the Simon Broome criteria is not strictly met, mutations are still identified. Based on this data, the Simon Broome criteria appears to be very effective as a diagnostic tool in predicting mutation positive cases.
7Bristol Genetics Laboratory Results to DateBristol Genetics Laboratory34 pathogenic variants detected to date+ 2 variants ‘likely non-pathogenic’c G>A and c.969C>T (p.[=])28 of these variants required UV studies(Nb. Most were previously reported to database but with no functional/family studies)cDNAProteinDiagnosticc.10580G>Ap.Arg3527Gln (APOB)9 (19%)c.1436T>Cp.Leu479Pro4 (8%)c.313+1G>AN/A3 (6%)c.1640T>Cp.Leu547Pro2 (4%)c.662A>Gp.Asp221Glyc.1049G>Cp.Arg350ProThese results are similar to that identified during the GOS Department of health pilot study, e.g. the APOB mutation was detected in 12% of patients in their study.Overall the current service would be unsuitable for a large scale approach due to the high cost of testing and the unsuitability of all these methods in combination for providing a large-scale service. This leads us to think of new technologies that could be used. A variety of methods are available and are already in use by other labs (e.g. Belfast uses an iplex system). During my project I have trialled the LIPOchip supplied by Progenika…this is the proposed replacement testing strategyCommonly detected mutations in SW diagnostic patients (n=48)
8Bristol Genetics Laboratory Assay SensitivityBristol Genetics LaboratoryTesting strategy not sustainable for disease frequencyAssaySensitivity (n=48)ARMS FH2052%Bi-directional sequencing of LDLR (Promoter + 18 exons)46%MLPA (P062B-C1)2%
9Bristol Genetics Laboratory LIPOchip® BackgroundBristol Genetics LaboratoryLIPOchip® has been in development since 2002 to detect themost prevalent Spanish mutationsCurrent Version (8) includes ‘European’ specific mutationsMore than 100 hospitals are using LIPOchip® throughout EuropeCopy number changes also detectedSpecific ‘BritChip’ due to be released June/July 2010Validation – 40 samples used (36 previously tested, 4 new cases)Blind testAll results concordantAs part of my project I have been trialling the LIPOchip assay produced by Progenika. This is a Spanish based company that currently produce slides that have a detection rate of 80% in the Spanish and other European populations. The current version of the LIPOchip has been shown to have a lower detection rate in the UK. A British version of the chip is expected to be released in July of this year.
10Bristol Genetics Laboratory LIPOchip® ProcessingBristol Genetics Laboratory5Resultsanalysis1Amplification2Fragmentation3Labelling4HybridizationPCR mixes1, 2, 3 and 4DNAse+Alkaline PhosphataseTdTBiotin-ddUTP2 hours45 minutes60 minutes3hours and 30 minutesOVERLAPPING PROCESSESTecan 4800 HS ProExtractionDAY 1DAY 2Overview of the process: DNA is amplified in four different multiplex PCR reactions. The PCR products are then fragmented and separately labelled with biotin. The labelled PCR products are applied to the surface of the slide for hybridisation (using a hyb station), mutant and normal oligos for each mutation are present on the slide in replicates of 10. A scanner detects the linked DNA as fluorescence emitted by the fluorophores using laser excitation.(Terminal transferase enzyme catalyses the addition of deoxi- and dideoxyNTPs labelled with biotin and fluorochromes at the 3’OH ends of double and single stranded DNA.)
11Bristol Genetics Laboratory Mutations DetectedBristol Genetics Laboratoryc.429C>A, p.Cys143Xc.1432G>A, p.Gly478ArgMutations not present in FH20 ARMSThis image gives the points of all samples included in the run (each point is a patient sample that is the mean value of all of the replicates for that specific mutation on the slide). The points plotted above give the ratio of the intensity of the normal oligo to the total intensity of both the normal and the mutant oligo.These are mutations that have been detected that are not present in the FH20 ARMS kit and would therefore not have been detected by the current initial screen. Mutation clearly seen on the right hand side – not called by the software as falls outside the box. The software defines the box based on the confidence that a mutant sample will fall within this region (based on processing thousands of samples – but obviously less samples processed for mutants, due to lack of available samples).
12Bristol Genetics Laboratory c.2093G>A (p.Cys698Tyr)Bristol Genetics LaboratoryPatient has c.2093G>T(p.Cys698Phe)Slight displacement fromthe ‘Normal’ groupThe mutation shown here is c.2093G>A, the patient circled has a G>T change identified by sequencing. A displacement from the normal group is detected, the software has correctly not called the mutation.
13Bristol Genetics Laboratory Del ex7-3’UTRBristol Genetics LaboratorySpecific controls are included in the chip for each PCR group. Chromosome 21 is used for normalisation of data and the X chromosome is used to detect changes in copy number.In each batch of hybridization, male and female controls are processed along with the samples. The ratio of the intensities of hybridization are used to detect whether a sample is duplicated or deleted.(E.g. For Chrs 21 ratio for male sample: female control =1, For chrs X ratio for male: female =0.5, For a normal dosage result sample:control = 1, deletion:control = 0.5, duplication:control = 1.5)
14Bristol Genetics Laboratory Duplication LDLR Exon 17Bristol Genetics LaboratoryMLPA resultLIPOchip resultLong-range PCR confirmationEx16_F Ex18_R3.5kb3.5kb~5kbN dup NEvidence from 3 separate methods. ?1.5kb. Work ongoing to map the breakpoints.
15LIPOchip® Trial Results Bristol Genetics LaboratoryPoint mutation analysis is robustCopy number detection results not always reportableMLPA will still be required in a significant proportion of casesAssaySensitivity (%) (n=48)Pick up (% of alldiagnostic cases) (n=104)ARMS (20 common mutations)5224Current LIPOchip (251 mutations)5827British LIPOchip (Personal Communication)7736Theoretical LIPOchip figures calculated based on if all cases tested to date had been screened by LIPOchip as first line test (based on whether the mutations identified by ARMS and seq were present on the current version of the chip).This only equates to 3 cases that would have been detected using the LIPOchip that would not have been picked up on the first line test using ARMS. Therefore, it would not be cost effective to use the chip as the first effective with the current version. If the sensitivity of the next version of the CHIP reaches 80% however, it is likely to be a cost effective solution, particularly if the copy number detection is improved as this will eliminate the need for MLPA in a large number of cases.British lipochip mutation list obtained from personal correspondence with Progenika. Based on our current cohort of patients the sensitivity is very close to the expected 80%.
16Proposed Method of Testing Bristol Genetics LaboratoryInitial screen using LIPOchip® platformCurrent European chip v.8 detects 251 mutations + copy number changesBritish LIPOchip predicted to detect 80% of UK mutationsFollowed by full bi-directional sequencing of LDLR (and MLPA where necessary)Negative patients meeting Simon Broome criteriaFull PCSK9 screen by bi-directional sequencing (Validation near completion, 12 fragments)Sequencing of APOB hotspot regions (exon 26 and 29)The detection rate of 80% will decrease the number of patients requiring a full LDLR screen by sequencing, thus reducing sequencing costs. If the LIPOchips can be purchased at a competitive cost, the initial ARMS screen will be replaced. It is also necessary to take into consideration the increased staff-time required for processing the LIPOchips.A further testing stage is due to be introduced for those patients that meet the Simon Broome criteria, this will be a full PCSK9 gene screen (Validation ongoing) and sequencing of the mutation hotspots in the APOB gene, validation of this is almost complete.
17Bristol Genetics Laboratory ConclusionBristol Genetics LaboratoryComprehensive testing service for FH implemented131 cases tested overall, 48% mutation positiveLIPOchip® evaluated – further work required to validate British version on releaseNetwork links with lipid and cardiac specialists have been established across the SW regionMechanism for robust funding is yet to be established
18Bristol Genetics Laboratory AcknowledgmentsBristol Genetics LaboratoryBristol Genetics LabMaggie WilliamsSarah Burton-JonesThalia AntoniadiGenetic Technologists – Teresa Tovey, Jenny Coles,Gemma Dennis, William Cross and Matthew GarnerExtraction Lab team and Array teamBiochemistry Department, BRIGraham BaylyMathangi BalasubramaniBath, Weston-super-Mare and Gloucester Biochemistry teamsGOS LabAlison TaylorProgenikaXabier AbadMaximilliano CrosettiGen-probe (Tepnel diagnostics)MRC-Holland