It’s all relative: Genetic dyslipidaemia

It’s all relative: Genetic dyslipidaemia

CVD risk factors, and especially lipid metabolism, exemplify gene / environment interactions
Mainly genetic Co-dominant mutations: Either genetic allele affected Recessive mutations: Both genetic alleles affected Polymorphisms and SNPs: Alleles consistent with “normal” and markers in proximity to significant genetic effects. Genome-wide association studies (GWAS). Mainly environmental or secondary to other disorders.

A missing piece of the genetic puzzle
Uncommon genes with large effect Pathogenic mutations Autosomal recessive Autosomal dominant Clinical Effect Polymorphisms Common genes with small effect Gene Prevalence

Genetic hyper and hypo - cholesterolaemia
Dominant Monogenic Hypercholesterolaemia Familial hypercholesterolemia (FH) Familial defective apo-100 (FDB-100) PCSK9 gain-of-function (FH-3) Hyperalphalipoproteinaemia (CETP deficiency, non-atherogenic?) Recessive Monogenic Hypercholesterolaemia Autosomal recessive hypercholesterolemia (ARH) Lysosomal acid lipase deficiency: Wolman’s disease and Cholesterol ester storage disease Dominant Monogenic LDL deficiency PCSK9 loss-of-function Recessive Monogenic LDL deficiency Abeta and hypobeta – lipoproteinaemia Chyomicron retention disease

Familial Hypercholesterolaemia: What goes wrong?
NORMAL FH

Metabolic Defect in Familial Hypercholesterolemia
-100 INTESTINE VLDL LPL B -48 CIII B E LPL R LDL-R B Chylomicrons CII E LIVER CII IDL LRP E CIII SR-B1 FH AI E B HL AI HDL CE O . AII AI LDL LCAT B FC Macrophage OxLDL Other SR-B1 SR-A Tissues ABCA1 DAVIGNON 2006

Metabolic Defect in Familial Defective ApoB-100
INTESTINE VLDL LPL B -48  ApoB CIII B E LPL R LDL-R B Chylomicrons CII E LIVER CII IDL LRP E AI AII E SR-B1 HDL LCAT Other Tissues CE FC CIII FDB AI B HL O . LDL B Macrophage OxLDL SR-A ABCA1 DAVIGNON 2006

PCSK9 regulates the surface expression of LDLRs by targeting for lysosomal degradation
1. Qian YW, et al. J Lipid Res. 2007;48: 2. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 3. Zhang DW, et al. J Biol Chem. 2007;282:

Clinical Characteristics
Gain-of-Function Mutations in PCSK9 Cause Familial Hypercholesterolaemia (FH) PCSK9 Variant Population Clinical Characteristics D374Y British, Norwegian families, 1 Utah family Premature CHD Tendon xanthomas Severe hypercholesterolemia S127R French, South African, Norwegian families Tendon xanthomas; CHD, early MI, stroke R215H Norwegian family Brother died at 31 from MI; strong family history of CVD Associated with: High serum LDL-C2 Premature CHD and MI2 In vitro testing in many identified mutations show decreased levels of LDLRs3 1. Abifadel M, et al. Hum Gen. 2009;30: 2. Horton JD, et al. J Lipid Res. 2009;50:S172-S177. 3. Cameron J, et al. Hum Mol Genet. 2006;15: *For a full list of ADH mutations, please see refer to Abifadel reference.

What is “FH”? What does it cause?
FH is Co-dominant mutation of genes affecting formation or function of the LDL-receptor This causes metabolic and clinical consequences including precocious cardiovascular disease (CVD) Metabolic Increased LDL, Reduced clearance of remnants including LDL’s precursor, IDL. Increased Lp(a)? Reduced HDL? Clinical Dominant: 50% of each generation. Risk 50:50 Premature CHD, CVD and PVD Aortic stenosis Tendon xanthomas (11%) specific? Corneal arcus (27%) non-specific > 40y? Xanthelasmas (12%) nonspecific No signs highly sensitive FH IS NOT JUST HIGH CHOLESTEROL IN A PATIENT AND THEIR RELATIVE(S)

FH: Why is it important? Non-FH Women Non-FH Men FH Women
4/11/2017 FH: Why is it important? 1.0 Non-FH Women 0.9 Non-FH Men 0.8 FH Women 0.7 FH Men 0.6 Cumulative Probability of Clinical CAD 0.5 0.4 0.3 0.2 0.1 0.0 90+ 25-30 30-35 35-40 40-45 45-50 50-55 55-60 60-65 65-70 70-75 75-80 80-85 85-90 Age MED PED Registry 2001.

Why FH matters: Prevalence and Impact
5 – 10% of CHD events under age 60. J Lipid Res 34:269-77 CVD death in >80% of FH cases. Absolute CVD risk differs from general population models. High risk profile from birth, Interaction differs (smoking, gender) Circulation 97: Risk algorithms underestimate risk Eur Heart J 19:A2-11 Missed and misdiagnosed Prevalence: 0.2 – 0.5% (1 : ) Atherosclerosis 173:55-68 > 8 x 106 affected world-wide. Seminars in Vasc Med 4:87-92 > 40,000 Australians Equal numbers of unaffected relatives Up to 1:60 in local groups with “founder effect”. Detection rates 0 – 44% World’s best 20-40% World average (including Australia) < 5% Molecular Medicine meets Public Health

Case detection: Dutch Lipid Clinic Score?
Diagnostic criteria Process Case detection: Dutch Lipid Clinic Score? Detecting index cases Optimal components Diagnosis assessment Adults Clinical services Children, Adolescents Model of Care for FH Laboratory protocol Genetic testing Management Clinical protocol Adults Cascade Screening Children, Adolescents Process LDL-Apheresis

Severe triglyceride elevation due to recessive impairment of lipoprotein lipase
Chylomicrons persist after fasting, massive levels of TG. Homozygous deficiency of Lipoprotein Lipase (LPL) Homozygous deficiency of the cofactor for L PL, Apo CII Impaired transport of LPL to site of action (endothelium) due to homozygous defect in ANGPTL or GPIHBP Combined overproduction and undercatabolism of triglyceride-rich lipoproteins sufficient to saturate LPL, eg in Apo AV mutations.

. Metabolic Defects affecting Lipoprotein Lipase B B B E B E E E B B
Dietary fat INTESTINE B -100 LPL B -48 VLDL B -48 CII CIII E TG LDL-R CR B Chylomicrons E CII LPL LIVER IDL E LRP TG CIII FFA + MG TG SR-B1 AI E B HL AI HDL CE O . AII AI LDL LCAT ApoB-48 R B FC VLDLR Other OxLDL SR-B1 ABCA1 Macrophage Tissues SR-A DAVIGNON 2006

. Metabolic defects saturating in LPL, eg Apo AV mutations B E B B B E
ADIPOSE TISSUE Sugar fat calories Normal or reduced VLDL catabolism AV INTESTINE B -100 TG rich VLDL Overproduction of VLDL HSL CIII LPL E B -48 B -48 CII B CETP TG CE aGP + FFATG CR LPL E IDL E LDLR LRP CIII Chylomicrons SR-B1 CII HL AV E B AI AI HDL O . sdLDL AII AI CE SR-A B LCAT FC VLDLR CD-36 OxLDL Other Macrophage LOX-1 SR-B1 Tissues ABCA1 SR-PSOX DAVIGNON 2006

Algorithm for Diagnosis of Apo B Dyslipoproteinemias
HyperApo B > 1.2 g/L NormoApo B < 1.2 g/L NormoTG < 1.5 mmol/L HyperTG > 1.5 mmol/L NomoTG > 1.5 mmol/L Hyper TG > 1.5 mmol/L TG:Apo B > 0.12 TG:Apo B < 0.12 Apo B > 0.75 g/L Apo B < 0.75 g/L TC:Apo B > 6.2 TC:Apo B < 6.2 Lipoproteins Normal Chylo + VLDL Chylo Chylo + VLDL Remnants VLDL LDL VLDL + LDL Primary Causes ■ Normal ■ Hypoalpha- lipoproteinemia ■ Complete (FHC) or partial LPL deficiency associated with a secondary factor ■ Complete LPL deficiency (FHC) ■ Primary apoC-II ■ Familial dysbetalipoproteinemia (type III) ■ Hepatic lipase deficiency ■ (Primary cause associated with a secondary factor) ■ Familial hyperTG ■ Partial LPL deficiency ■ FH ■ Polygenic ■ FDB ■ PCSK9 deficiency ■ ARH deficiency ■ CYP7A1 ■ Hypoalphalipo-proteinemia ■ FCH ■ β-Sitosterolemia Abbreviations: apo, apolipoprotein; ARH, autosomal recessive hypercholesterolemia; CAPD, continuous ambulatory peritoneal dialysis; Chylo, chylomicrons; CP7A1, cytochrome P450 7A1; DM2, diabetes mellitus type 2; dysbeta; dysbetalipoproteinemia; FCH, familial combined hyperlipidemia; FDB, familial defective apoB; FH, familial hypercholesterolemia; FHC, familial hyperchylomicronemia; HAART, highly active antiretroviral therapy; LPL, lipoprotein lipase; PCOS, polycystic ovary syndrome; SLE, systemic lupus erythematosus; TC, total cholesterol; TG, triglyceride. de Graaf J et al. Nat Clin Pract Endocrinol Metab 2008;4:

Autosomal recessive disorders have revealed HDL metabolism
+ A-I Nascent HDL A-I CE LCAT FC FC ABCA1 Macrophage Rapid catabolism Homozygous (?heterozygous) Apo AI deficiency: Atherogenic Tangier’s Disease: ABC-AI deficiency: Atherogenic? LCAT Deficiency: Non-atherogenic? Apo A1 Milano: Anti-atherogenic?

Event Free Survival ( % )
Carriers of the ApoAI Leu178Pro Variant Are at Increased Risk of Developing CAD 100 50 family controls (n=147) Event Free Survival ( % ) apoAI (L178P) carriers (n=54) p = 0.008 ApoAI  50% HDL-C  63% 18.9 x  CAD risk Age ( years) Hovingh K et al. J Amer Coll Cardiol 44:1429, 2004 DAVIGNON 2006

Normal ApoAI and ApoAIMILANO Dimer
Lipid Binding In Vivo Catabolism AIm/AIm 1 243 173 s 35 143 187 99 AI 243 1 25 209 220 66 121 165 LCAT Activation Cholesterol Efflux “Receptor” Binding Franceschini G Eur J Clin Invest 26; 733, 1996 DAVIGNON 2006

Role of apolipoprotein E
Apo E: ligand for hepatic removal of remnants. Apo E knockout model is atherogenic Apo E2 has lower binding affinity (E4>E3>E2). E2:E2 only critical if lipids increase for other reasons. Other roles for Apo E (CNS lipid transport and neural repair). CR E AI CHYLOMICRON CIII TG OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO LPL LRP AII C CE HDL B 48 CII Lipolysis products FC

Metabolic Defects in Remnant Dyslipidaemia
Apo E2 homozygosity plus apo B overproduction or Hepatic Lipase deficiency Degradation (catabolic) Formation (anabolic) E B INTESTINE FC/PL FC LPL PLTP Chylo to ChyloRe AI LCAT FC CE CE LIVER HL Kidney SR-B1 ABCA1 LRP LDLR AI AII E CE AI E CE HDL3 HDL3 CELL B AI B LDL Ox LDL AI LCAT AI FC/PL HL HL E TG E CE B TG CE VLDL to Remn CETP HDL2 CE AII AII CE Modified from Deeb SS et al. J Lipid Res 44:1279, 2003

Unspecified hereditary Dyslipoproteinemia
Common: Lipoptotein (a) Rare: Familial Phytosterolemia Protease K V K IV Types 3-10 Apo(a) LDL Receptor Binding Site on apoB Type 2 ApoB LDL particle Type 1 C N -C -N DAVIGNON 2006 ABCG5 ABCG8 Micelles Phytosterols Cholesterol DAVIGNON 2006

Genetic dyslipidaemias have motivated treatment discovery
Clinical abnormalities represent real human problems Massive yield on research into genetic dyslipidaemia Familial Hypercholesterolaemia: Receptor mediated endocytosis Statins, PCSK9 antisense and Abs Familial Hyperchylomicronaemia: LPL gene therapy Apo A1 Milano: Synthetic HDL Deficiency of Apo B or MTP MTP inhibitors, Apo B antisense CETP Deficiency CETP inhibitors Apo E Valuable animal k/o model

IT’S ALL RELATIVE : MANAGEMENT OF GENETIC DYSLIPIDAEMIA
AAS MASTERCLASS 5-6TH OCTOBER 2012 IT’S ALL RELATIVE : MANAGEMENT OF GENETIC DYSLIPIDAEMIA CASE 1

HISTORY… Miss KM, 21year old Malay student nurse
Referred to Specialist Llipid Clinic Hypercholesterolaemia ‘Yellowish butterfly patterned lesion’ around her eyes On Pravastatin 20mg ON TC 13.7, LDL-c 11.6 mmol/L July 1997 Cycling accident at her home town Sustained minor soft tissue injury Noted to have xanthelasma around her eyes Lipid profile results: TC : 15.4 mmol/L LDL: 13.9 mmol/L Referred to Medical Clinic, home town Started Pravastatin 20 mg/ON 6th August 1998

HISTORY…… Noted ‘yellowish butterfly patterned lesion’ surrounding both eyes since primary school days Asymptomatic, well No h/o chest pain, palpitation, shortness of breath or poor effort tolerance No history of intermittent claudication or syncopal attacks. No h/o polyuria, polydipsia No h/o cold intolerance, lethargy or menstrual disturbance No past h/o jaundice, liver or renal diseases Not HT, DM No past surgical history

The youngest out of 11 siblings 3 siblings died :
HISTORY… The youngest out of 11 siblings 3 siblings died : eldest brother : 43 years – AMI (HC) elder brother (6th) : 23 years – AMI (HC, xanthelasma) eldest sister (2nd) : 33 years - ?MI, ? SCD Parents - consanguinous marriage (first cousins), not known to have DM/HPT/CAD

HISTORY… Student nurse at a Teaching Hospital, KL Single Non smoker No history of alcohol intake Exercise - once weekly (jogging), 30minutes Normal diet, low fiber intake

Physical Examination Anthropometry:
BMI : Waist circumference : cm (<80cm) Waist-to-hip ratio: (<0.85) PR: 72/min, regular, BP : 120/62 mmHg, DRNM Peripheral pulses – present, carotid & renal bruit : absent Other systems: Normal

Xanthelasma

Corneal Arcus Right Eye Left Eye Grade 4 Corneal Arcus grading:
0 – no arcus observable 1- upper or lower segments affected 2- both segments affected 3- both segments and just confluent 4- heavy confluent arcus (Wider et al, 1983) Corneal Arcus

Digital xanthomata Achilles tendon xanthomata

Test Result Reference range **FSL TC 15.4 13.7 < 5.7 mmol/L HDL-c
Baseline (1997) 1st visit to the SLC TC 15.4 13.7 < 5.7 mmol/L HDL-c 1.5 1.6 > 1.3 mmol/L LDL-c 13.9 11.6 <3.8 mmol/L Triglycerides 1.0 1.1 < 1.3 mmol/L On pravastatin 20 mg / ON x 1 year

Summary of KM’s risk factors
Negative risk factors: Positive risk factors: HDL > 1.3 mmol/L Non smoker Not hypertensive Not DM Not overweight/ obese Premenopausal female Markedly elevated TC & LDL levels Strong family history of premature CAD

Differential Diagnosis
1° Hypercholesterolaemia 2° Hypercholesterolaemia - TRO Familial hypercholesterolaemia (FH) Hypothyroidism Familial Defective ApoB100 Cholestasis PCSK9 gain-of-function mutation (FH-3) Nephrotic syndrome Polygenic hypercholesterolaemia

Tests Results Reference Ranges TFT fT4 11.78 9.10 – 23.8 nmol/L TSH 1.22 Iu/L FPG Glucose 4.4 <6.1 mmol/L Renal Profile Sodium 134 135 – 150 mmol/L Potassium 4.2 3.5 – 5.0 mmol/L Urea 3.2 2.5 – 6.4 mmol/L Creatinine 66 44-80 umol/L

Tests Results Reference Ranges LFT Albumin 42 35-50 g/L Total Protein 84 67-88 g/L Bilirubin Total 16 < 23 umol/L ALT 17 <44 U/L ALP 89 32 – 104 U/L Creatine Kinase U/L Lp(a) 0.58 <0.3g/L

Other Investigations….
ECG – normal Exercise stress test – normal ECHO – Normal, no evidence of aortic stenosis Carotid artery IMT – normal, no evidence of atheromatous plaques Other Investigations….

Question 1 What are the various criteria for the diagnosis of FH?
Dutch Lipid Clinic Network diagnostic scoring Simon Broome’s criteria MedPed criteria for FH NCEP ATPIII criteria

Question 2 According to the Dutch Lipid Clinic Network criteria, scoring for definite FH is: > 8 points 6 – 8 points 3 – 5 points

Question 3 In determining the Dutch Lipid Clinic diagnostic scoring for FH, the following are taken into account: Baseline LDL-c concentration: Yes/ No Clinical history of premature CAD: Yes/ No Clinical history of premature cerebral or PVD: Yes/ No Tendon xanthoma(ta) in the patient: Yes/ No Premature corneal arcus in the patient: Yes/ No Family history of hypercholesterolaemia: Yes/ No Family history of premature CAD/PVD in 1st degree relatives: Yes/ No Family history of tendon xanthomata and/or corneal arcus in 1st degree relatives: Yes/ No

Dutch Lipid Clinic – Diagnostic scoring for FH
Criteria: Family history: 1st degree relative with (a) premature CAD or vascular dis (men<55yrs, women<60yrs) OR (b) LDL > 95th percentile, in point and / or 1st degree relative with tendon xanthomata and/or corneal arcus OR childhood (<18yrs) LDL > 95th percentile points Clinical history Patient with premature CAD (men<55, women <60yrs) points Patient with premature cerebral or PVD (men<55, women <60yrs) -1 point Physical Examination - patient Tendon xanthomas - 6 points premature arcus points Lab Analysis – LDL-c >8.5mmol/> points LDL-c 6.5 – LDL-c LDL-c 4.0 – DNA analysis Functional DNA Mutation points Criteria: 8 points - DNA Mutation, or LDL-C > 8.5mmol/L 6 points - Tendon xanthomas 5 points - LDL-C 6.5 – 8.4mmol/L 4 points - premature corneal arcus < 45 yrs 3 points - LDL 5.0 – 6.4mmol/L 2 points - 1st degree relative with xanthomas or premature CA or childhood LDL > 95th percentile, or personal premature CAD 1 point - 1st deg relative with premature CAD/ vascular dis or LDL > 95th percentile, or personal history of premature cerebral or PVD, or LDL-c 4.0 – 4.9mmol/L Definite: > 8 points, Probable: 6 – 8 points, Possible FH: 3-5

US MedPed Criteria vs Simon Broome criteria for the dx of FH
Total cholesterol cutpoints (mmol/L) 1st-degree vs 2nd-degree vs 3rd-degree relatives with FH vs General population Age (years) 1st-degree 2nd degree 3rd degree General population < 20– 30– ≥ FH is diagnosed if TC levels exceed the cutpoint Key: FH = familial hypercholesterolaemia

Simon Broome Criteria - Diagnosis of FH
Criteria Description (A) TC > 7.5 mmol/L in adults or TC > 6.7 mmol/L in children <16 years, or LDL-c > 4.9 mmol/L in adults or > 4.0 mmol/L in children (B) Tendon xanthomas in the patient, or a first-degree or second-degree relative (C ) DNA-based evidence of mutation in the LDLR, or apo- B100 or PCSK9 gene (D) Family history of premature CHD (age <50 years in a second-degree relative or <60 years in a first-degree relative) (E) Family history of raised TC >7.5 mmol/L in a first- or second-degree relative, or >6.7mmol/L in child, brother or sister <16yrs of age Diagnosis Definite FH diagnosis requires either A + B or A + C Possible FH diagnosis requires either A +D or A + E Key: FH = familial hypercholesterolaemia

Summary Hypercholesterolaemia (LDL-c 13.9mmol/L)
Xanthomata, corneal arcus, xanthelasma No evidence of personal CAD Strong family history of premature CAD Positive family history of HC – 1st and 2nd degree relative Consanguity Ruled out secondary causes of HC

DEFINITE FAMILIAL HYPERCHOLESTROLEMIA
CLINICAL DIAGNOSIS DEFINITE FAMILIAL HYPERCHOLESTROLEMIA

Question 4: Should risk estimation tools eg Framingham Risk Scoring be used for her? YES NO

CHD Risk Stratification
CHD estimation tools such as those based on the Framingham risk scoring SHOULD NOT be used because people with FH are already at high risk of premature CHD. Heterozygous FH has >50% risk of CHD in men by the age of 50years, >30% in women by the age of 60years NICE Clinical Guideline Identification and Mx of FH 2.5.1 Individuals with FH are at high CHD risk. The 10-year CHD risk in the FH patient is not adequately predicted by any conventional risk assessment tools. Therefore, assessment of 10-year risk is not recommended.

Management Statin therapy - continued, titrated Add-on lipid lowering
Lifestyle modification Referred to dietician Referred to : - Cardiology - Plastic surgery Family tracing, cascade screening and counselling

Medication TC (mmol/L) TG HDL LDL Baseline, statin naive 15.4 1.0 1.5
13.9 Pravastatin 20 mg /ON 13.7 1.1 1.6 11.6 Pravastatin 20 mg/ON 11.9 10.4 Simvastatin 30 mg/ON 13.1 1.4 11.3 Atorvastatin 80 mg/ON 15.3 1.2 13.5 11.7 0.7 1.26 10.1 9.8 0.6 10.2 11.5 Simvastatin 80mg & Ezetimibe 10mg/ ON 8.9 7.3

Alternative or add-on lipid lowering therapy
Ezetimide Bile acid binding resin Fibric acid derivatives Nicotinic acid LDL-apharesis

LIFESTYLE MODIFICATION
Lifestyle modification - reduce LDL-c and other coronary RFs Modest, variable degree of LDL reduction ̴ 10% Diet – low fat <30% calories, saturated fat < 10% of calories, cholesterol < 200mg/day, fibre 10-25g/day, caloric deficit kcal/day Plant sterol esters or plant stanol esters 2g/day Physical activity – 30min/day, moderate intensity, at least 5days per week Ideal weight BMI < 23 (Asian) Smoking Alcohol intake HT, DM

Father Mother HC Xanthelasma HC Xanthoma 63 62 1 2 3 4 5 6 7 8 9 10 11 Xanthelasma CAD+ HC SD ?MI HC Xanthoma 33 39 23 33 32 26 23 21 38 40 HC Xanthoma 43 HC CAD+ Those found to have HC following family screening FAMILY TREE

Family Screening ( Lipid Screening) - 1999
Father Mother TC : 8.1 LDL : 5.7 TC : 8.7 LDL : 6.7 1 2 3 4 5 6 7 8 9 10 11 TC : 8.0 LDL : 6.4 TC : 16.2 LDL : 14.8 33 23 32 26 21 33 39 38 23 TC : 6.5 LDL : 4.6 TC : 7.0 LDL : 5.2 TC : 10.1 LDL : 6.7 TC : 4.9 LDL : 2.6 43 TC : 8.1 LDL : 5.7 40 TC : 15.3 LDL : 13.9 TC : 7.7 LDL : 4.6 TC : 8.7 LDL : 6.5 Affected with FH FH with known mutation TC : 8.7 LDL : 6.5 TC : 6.1 LDL : 4.4 Died Family Screening ( Lipid Screening)

Genetic testing was performed on KM and her family members
Question 5: What genes have been implicated in FH? LDL Receptor Lipoprotein lipase Apo B100 Apo CII PCSK9

Low Density Protein Receptor (LDLR) Gene
Cytogenetic Location: 19p13.2 Size: 44,469 bases Mosaic protein of ~840 amino acids (after removal of signal peptide); Molecular weight 95,376 Dalton

LDLR PROTEIN Made up of a number of functionally distinct domains that can function independently of each other. Ex 1 contains a signal sequence that localizes the receptor to the ER for transport to the cell surface Ex 2-6 code the ligand binding region Ex 7-14 code the EGFP domain Ex 15 codes the oligosaccharide rich region Ex 16 (and some of 17) code the membrane spanning region Ex 18 (with the rest of 17) code the cytosolic domain.

PCR PRODUCT ANALYSIS OF LDLR EXON 5 – DNA CHIP CAPILLARY GEL ELECTROPHORESIS
182bp Amplification of Exon 5 of LDLR gene by PCR. PCR products were analysed on an automated on-chip capillary gel electrophoresis (Agilent Bioanalyser). Lanes from left to right; 1-Kb ladder, Negative control sample, patients samples and normal control sample. The green band represent lower marker and purple band represent upper marker. The PCR products were detected at 182bp.

DHPLC RESULTS – LDLR Exon 5
Mutation Screening by DHPLC Heteroduplex peaks of FH patients with variant in exon 5 Wash peak g.763T>A Homoduplex peak of NC sample (wild type sample) Heteroduplex peaks of FH patients with variant in exon 5 g.763T>A The DHPLC chromatogram profiles of FH patients showed presence of heteroduplex peaks eluted at 5.2 mins and 5.8 mins at denaturing temperature of C. The presence of heteroduplex peaks were suggestive of disease-causing variants and subjected to DNA sequencing to confirm the variants.

DNA SEQUENCING RESULTS
FH Patient ID Affected LDLR regions DNA Sequence Description of variant and effect KM, SFM & WC (3 members of a Malay Family) Exon 5 Reference :GCCGGCAGTGTGACCG Variant :GCCGGCAGAGTGACCG g.763T>A; Cysteine (C) to Serine (S); Homozygous and Heterozygous mutation C234S NYK Exon 9 Reference : CTTCACCAACCGGCACG Variant : CTTCACCAACTGGCACG g.1216C>T; Arginine (R) to Tryptophan (W), Heterozygous mutation R385W

Description of variant and effect
DNA SEQUENCING RESULTS Reference sequence from normal control Homozygous: Substitution T>A at nucleotide position 763 (g.763T>A) DNA sequence of Homozygous FH patient Affected LDLR region DNA Sequence Description of variant and effect Exon 5 Reference: GCCGGCAGTGTGACCG Variant : GCCGGCAGAGTGACCG g.763T>A; Cysteine(C) to Serine (S) at codon 234. Identified as homozygous mutation C234S.

Heterozygous FH patient Description of variant and effect
DNA SEQUENCING RESULTS Reference sequence from normal control Heterozygous: Substitution T>A at nucleotide position 763 (g.763T>A) DNA sequence of Heterozygous FH patient Affected LDLR region DNA Sequence Description of variant and effect Exon 5 Reference: GCCGGCAGTGTGACCG Variant : GCCGGCAGAGTGACCG g.763T>A; Cysteine(C) to Serine (S) at codon 234. Identified as heterozygous mutation C234S.

KM is now married with 2 children
Questions 5: What is the probablity that her children may have FH with every pregnancy? 50% 25% Questions 6: When should diagnostic tests be done for her children? At birth By the age of 10 years or earliest opportunity thereafter

AD, one affected parent – 1:2 (50%) chance of heterozygous with every pregnancy
In children with one affected parent, the following Dx-tic tests should be done by the age of 10yrs or at the earliest opportunity thereafter: DNA test if the family mutation is known LDL-c if the family mutation is not known. To exclude the Dx of FH, LDL-c should be repeated after puberty - LDL-c concentration changes at puberty to almost similar to that of adult concentrations Cascade testing – combination of DNA testing and LDL-c to identify affected relatives In children at risk of homozygous FH (2 affected parents or presence of clinical signs eg cutaneous xanthomata), measure LDL-c before 5yrs of age or at the earliest opportunity thereafter. If LDL >11mmol/L, clinical Dx of homozygous FH should be considered NICE Clinical Guideline Identification and Mx of FH

Question 7: Who can be considered for LDL apheresis treatment?
Adults and children/young people with Homozygous FH Heterozygous FH

LDL lowering apheresis
Treatment of adults and children/ young adults with homozygous FH In children with 2 affected parents Presence of clinical signs eg cutaneous lipid deposits (xanthomata) LDL-c should be measured before 5 yrs of age, or earliest opportunity thereafter LDL-c > 13mmol/L (adults), > 11mmol/L (children/ young adults) – consider clinical diagnosis of homozygous FH Exceptional cases of heterozygous FH –progressive symptomatic CHD despite maximal tolerated lipid-modifying drug therapy, optimal medical and surgical therapy Specialist centre Cost implication

Question 8: What are the future lipid lowering therapy?
Antisense oligonucleotides (ASO) to inhibit Apolipoprotein B production –eg Mipomersen PCSK9 targeted therapy –eg PCSK9 inhibitor Microsomal TG Transfer Protein Inhibitors –eg lomitapide Thyroid Mimetics – eg eprotirome Cholesterol Ester Transfer Protein (CETP) Inhibitors - anacetrapib, dalcetrapib

(1) ANTISENSE OLIGONUCLEOTIDES (ASO) – MIPOMERSEN
Subcutaneous 200mg 1x/wk – phase 3 CT, LDL reduction lasted for 4/52 after last dose. No clinically relevant interactions wrt clearance of statins and ezetimide - impt as add-on therapy Yu et al Clin Pharmacokinet 2009;48:39-50 Homo FH (n=51) - Recent double blind CT, sc mipomersen 200mg/wk vs placebo x26wks – 25% vs 3% LDL reduction, 27% reduction apoB, 21% reduction TC Raal FJ et al. Lancet 2010;375: Hetero FH (n=124), CAD+ on max tolerated statins: 45% of these high risk subjects achieved target LDL of < 100mg/dL (2.6mmol/L) EAS Congress 2011

2. PCSK9 TARGETED THERAPY Animal studies – beneficial up to 80% LDL lowering, human studies not published yet Frank-Kamenetsky et al. Proc Natl Acad Sci USA 2008;105: Chan JC et al. Proc Natl Acad Sci USA 2009;106:9820-5 Statins and fibrates induce increase PCSK9 expression, thus PCSK9 inhibition could induce robust LDL reduction as add-on therapy in FH

3. MICROSOMAL TG TRANSFER PROTEIN (MTP) INHIBITORS
MTP inhibition with BMS in homozygous FH (n=6), dose 1mg/kg/day - 50% LDL reduction but noted to induce hepatic steatosis Cuchel et al. New Engl J Med 2007;356:148-56 MTP inhibition in homozygous FH (n=10) , dose 60mg/D – 44% LDL reduction; less steatosis Cuchel et al.Circulation 2009;120:S441 Ezetimibe 10mg vs MTP inhibition by lomitapide (5-10mg/ D x4/52) – 20% vs 20-30% in a dose-dependant manner Combined therapy (ezetimide + lomitapide) – similar but larger dose dependent LDL reduction (35-45%); SE: Mild ALT increase, diarrhoea Samaha et al. Nat Clin Pract Cardivasc Med 2009;2010:906-16 Potential attractive candidate for lipid lowering in FH patients if administered in lower doses

4. THYROID MIMETICS Thyroid hormone analogues – reduce LDL but associated with cardiac and bone related SEs More recently differential molecular mechanisms (cpds that act on TR-beta mainly expressed in the liver, do not affect TR-alpha expressed in brain and heart) – eg eprotirome, selective TR beta agonists (under Ix) Statin + placebo/ eprotirome for 12/52 – 20-30% additional reduction of LDL; no SE on heart or bone Ladenson et al. Use of thyroid hormone analoque eprotirome in statin-treated dyslipidaemia. New Engl J Med 2012;362:906-16

5. CETP (CHOLESTEROL ESTER TRANSFER PROTEIN) INHIBITORS
X2 approaches to inhibit CETP (a) Vaccine - CETi-1 synthetic CETP peptide vaccine, immune response anti CETP- Abs Animal studies of this vaccine – increase HDL, reduce aortic atherosclerosis but human studies poor response of autoAB production Rittershaus et al. Vaccine induced Abs inhibit CETP activity in vivo and reduce aortic lesions in a rabbit model of atherosclerosis, ATVB 2000;20: Davidson et al. The safety and immunogenicity of a CETP vaccine in healthy adults. Atherosclerosis 2003;169:113-20 (b) Small molecule CETP inhibitors – eg Torcetrapib, anacetrapib, dalcetrapib - antagonize CETP activity by binding to it. RADIANCE I – 800 FH pts, torce + atorva: No reduction in atherosclerosis (IMT) despite reduction in LDL (20%) and increase HDL (52%) Kasteleine JJ et al. Effect of torcetrapid on carotid atherosclerosis in FH. NEJM 2007;356:

Cont……. 5. CETP (CHOLESTEROL ESTER TRANSFER PROTEIN) INHIBITORS
ILLUMINATE – torce + atorva, prematurely terminated, unexpected increase in M&M – exact mechanisms unclear ?torce induced increase BP Barter et al. Effects of tercetrapid in patients at high risk of coronary event, NEJM 2007;357: Xie et al. Drug discovery using chemical systems biology:identidfication of the protein-ligand binding network to explain the side effects of CETP inhibitors. PLoS Comput Biol 2009;5:e 2 other CETP inhibitors have no effect on BP – molecule-specific off-target effect Anacetrapid and dalcetrapib – phase 3 clinical trials , mild HC and pts with CVD risk, effective lipid modifiers Pending CV outcome trials (DAL-outcomes I and II and HPS3/REVEAL) CETP inhibition likely to benefit patients with FH

Summary A 21year old Malay female student nurse who presented with incidental xanthelasma and severe hypercholesterolaemia associated with a strong family h/o premature CAD, xanthomata and grade 4 corneal arcus. There was consanguity between her parents. She is slim, non-smoker, euglycaemic, normotensive and has good HDL-c levels (>1.3mmol/L). Family tracing was performed. Molecular analyses confirmed the presence of homozygous FH with exon 5, position 763 T>A mutation of the LDLR, coding for the LDL binding domain. The updates on the present and future management, and the molecular pathogenesis of FH have been discussed.

Learning Issues FH criteria for diagnosis
High coronary risk category, should not use risk assessment tools LDL Receptor protein and gene Genetic testing – when to test for children? Lifestyle modification Current treatment, targets Plasma apheresis – when is it indicated? Future lipid lowering therapy

It’s all relative: Genetic dyslipidaemia

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It’s all relative: Genetic dyslipidaemia

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