Genetic considerations in the treatment of familial hypercholesterolemia

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1 Clinical Lipidology ISSN: (Print) (Online) Journal homepage: Genetic considerations in the treatment of familial hypercholesterolemia Ann M Moyer & Linnea M Baudhuin To cite this article: Ann M Moyer & Linnea M Baudhuin (2015) Genetic considerations in the treatment of familial hypercholesterolemia, Clinical Lipidology, 10:5, To link to this article: Copyright 2015 Future Medicine Ltd Published online: 18 Jan Submit your article to this journal Article views: 487 View Crossmark data Full Terms & Conditions of access and use can be found at

2 Clinical Lipidology Genetic considerations in the treatment of familial hypercholesterolemia Familial hypercholesterolemia is an inherited disease characterized by a markedly increased concentration of LDL-bound cholesterol and can lead to premature cardiovascular disease. Most cases are due to autosomal dominant mutations in LDLR, APOB or PCSK9. Although most patients receive high-dose statin therapy, many are still unable to achieve desired lipid levels. For these patients, additional therapies, including LDL-apheresis are considered. Recently, there has been progress in the treatment of familial hypercholesterolemia with the development of PCSK9 inhibitors, a microsomal triglyceride transport protein inhibitor, and an antisense oligonucleotide against APOB. Addition of these new therapeutics to those in existence is likely to decrease morbidity and mortality associated with familial hypercholesterolemia. Ann M Moyer1 & Linnea M Baudhuin*,1 1 Department of Laboratory Medicine & Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA *Author for correspondence: Tel.: Fax: baudhuin.linnea@ mayo.edu Keywords: APOB familial hypercholesterolemia LDL LDLR LDLRAP1 LDL apheresis lomitapide mipomersen PCSK9 Background Familial hypercholesterolemia (FH; OMIM #143890) is an inherited disease characterized by defects in the LDL receptor (LDLR) pathway (Figure 1) that leads to a markedly increased concentration of LDL-bound cholesterol (LDL-C) in blood and increased risk of premature cardiovascular disease. Hypercholesterolemia is common, but most cases are polygenic and influenced by many factors, including obesity and diet. In contrast, FH is monogenic and LDL-C levels are often very high (>95th percentile for age and sex) from the time of birth and in the absence of additional risk factors. FH was first described by C Müller in the 1930s, and in the 1960s was classified into the heterozygous form (HeFH) and more severe homozygous form (HoFH). Although there is much overlap, LDL-C levels are typically >190 mg/dl (4.9 mmol/l) in untreated adults with HeFH (>160 mg/dl [4.1 mmol/l] in untreated children with HeFH) and >400 mg/dl (10.3 mmol/l) in patients with HoFH (Figure 2). In addition to markedly /clp Future Medicine Ltd increased LDL-C, increased lipoprotein (a) [Lp(a)] and decreased HDL-bound cholesterol (HDL-C) are often observed, further contributing to cardiovascular risk [1,2]. Often in children, the recognition of xanthomas is the first indication of a diagnosis of FH, especially HoFH [3,4]. Xanthomas typically develop by the age of 4 years in HoFH and by the end of the second decade in HeFH, ultimately affecting approximately 80% of heterozygotes [5]. Cholesterol deposition in the vasculature is the most serious concern because it can lead to premature coronary heart disease (CHD), which most often manifests as angina pectoris, myocardial infarction and peripheral vascular disease. Left untreated, CHD typically develops before the age of 55 years in men and before the age of 60 years in women with HeFH, and before the age of 20 years in HoFH, although it can occur in early childhood [3 4,6 7]. Early recognition and treatment initiation are essential since clinical outcomes are related directly to the degree and duration Clin. Lipidol. (2015) 10(5), part of ISSN

3 Upregulation of LDL receptors Moyer & Baudhuin Triglyceride Hydrolysis LDL apheresis Blood Legend LDL receptor synthesis MTP inhibitor MTP Degradation Antisense APOB LDL receptor recycling PCSK9 inhibitors Increase PCSK9 Statins LDL receptor LDL ApoB LDLR adaptor protein 1 PCSK9 ApoB mrna Cholesterol VLDL Hepatocyte Nucleus Cholesterol precursors Figure 1. LDL receptor and cholesterol pathway highlighting points of therapeutic intervention. After synthesis, the LDL receptor is transported to the cell surface where it can bind apob-containing LDL. After binding, the LDLR adaptor protein 1 interacts with cellular molecules in order for the complex to undergo endocytosis in a clathrin-coated vesicle. Next, the receptor and LDL dissociate and the receptor can either be recycled to the cell surface to bind another LDL molecule, or PCSK9 protein can direct LDL receptor degradation. Familial hypercholesterolemia results when mutations alter one or more of the following: the LDL receptor synthesis/ transport/function, LDL receptor-binding region of apob such that LDL cannot bind the receptor, LDLRAP1 and inhibit endocytosis, or PCSK9 (gain-of-function) to result in increased LDL receptor degradation. Therapeutics can act at several points in the pathway. By preventing apob synthesis and/or inhibiting MTP, less cholesterol can be incorporated with apob into VLDL and released into the bloodstream. PCSK9 inhibitors decrease the degradation of the LDL-receptor, increasing the available LDLR. Statins both inhibit cholesterol synthesis, as well as increasing the quantity of LDL receptors expressed on the cell surface. Finally, LDL apheresis works by removing LDL-cholesterol from circulation. of exposure to elevated LDL-C levels. However, even on treatment, the Simon Broome Register Group found that the relative risk of a fatal coronary event was increased 125-fold for women and 48-fold for men with HeFH aged years, though treatment modalities have improved since the time of this study [8]. In HoFH, atherosclerosis often affects the aortic root causing stenosis of the aortic valve, potentially compromising the coronary ostia. In addition, the carotid arteries, descending aorta, ileo-femoral arteries and renal arteries may be involved. Dyspnea, left ventricular cardiac failure and sudden cardiac death also may be observed in FH [9,10]. HeFH is a common genetic disorder, affecting approximately one in people in Europe and North America [7,11 12]. Due to founder effects, FH is more common in some populations, such as Afrikaners in South Africa (one in 70), and French Canadians (one in 150) [13,14]. While HoFH was previously thought to occur in about one in 1,000,000, it is now estimated to affect about one in 160, ,000 [15]. FH is thought to be both underdiagnosed and undertreated in most countries [7]. Genetic causes of FH FH is typically an autosomal dominant disorder associated with variants in three main genes: LDLR, APOB and PCSK9. Pathogenic variants in the LDLR gene, encoding for LDLR, are the most frequent genetic cause of FH and account for approximately 60 80% of all cases of FH [16]. The second most common genetic cause of FH is due to mutations in APOB, which encodes for apolipoprotein B-100, the protein component of LDL. Mutations in APOB contribute to about 1 5% of FH. FH that is caused by mutations in APOB is known as autosomal dominant type B hypercholesterolemia (OMIM #144010), sometimes also referred to as familial defective apob-100 (FDB). A third gene, PCSK9, is less frequently involved in the FH phenotype (autosomal dominant hypercholesterolemia-3, OMIM #603776), with a contribution of 1 3% [17]. Recently, rare autosomal dominant disease-causing mutations have also been identified in APOE and STAP1, which encode for apoe and signal transducing adaptor family member 1, respectively [18,19]. A rare autosomal recessive form of the FH phenotype, 388 Clin. Lipidol. (2015) 10(5) future science group

4 Genetic considerations in the treatment of familial hypercholesterolemia autosomal recessive hypercholesterolemia, is due to homozygous or compound heterozygous mutations in LDLRAP1. Sitosterolemia (OMIM #210250) is another rare autosomal recessive disorder that has phenotypic overlap with FH, but is characterized by elevated levels of plant sterols and is due to mutations in ABCG5 and/or ABCG8. Through landmark studies, M Brown and J Goldstein identified the LDLR and uncovered its role in cholesterol metabolism and FH [20,21], eventually receiving the Nobel Prize in Physiology and Medicine in 1985 for their work. LDLR facilitates the removal of LDL particles from the plasma via apob (Figure 1). When LDL binds to LDLR on the cell surface of hepatocytes, the LDL-ApoB-LDLR complex becomes internalized via uptake at clathrin coated pits into endosomes, mediated by the LDLRAP1 protein. Once inside the endosome, LDLR and LDL separate, Compound heterozygous LDLR and APOB or PCSK9 mutation Homozygous APOB or PCSK9 mutation Homozygous LDLR defective Homozygous LDLRAP1 (autosomal recessive FH) Homozygous LDL-receptor negative Polygenic hypercholesterolemia Heterozygous FH Homozygous FH 190 mg/dl (5 mmd/l) 300 mg/dl (7.8 mmoll) 500 mg/dl (13 mmol/l) Ezetimibe (10 20% reduction) Mipomersen (20 40% reduction) Lomitapide (40 50% reduction) Statin (15 30% reduction) PCSK9 inhibitor (35 75% reduction) LDL apheresis (60 70% reduction) Figure 2. Approximate cholesterol levels observed in patients by clinical diagnosis and by mutations identified (for homozygous familial hypercholesterolemia), and approximate decrease in cholesterol of a mock patient with a baseline LDL-cholesterol of 500 mg/dl. The red arrow shows increasing LDL-cholesterol level from left to right. LDL-cholesterol levels show considerable overlap between patients with varying clinical and mutational diagnoses. FH: Familial hypercholesterolemia. future science group 389

5 Moyer & Baudhuin where LDLR either undergoes lysosomal degradation (facilitated by PCSK9) or is recycled back to the cell surface. Another mechanism for LDLR degradation involves binding of exogenous PCSK9 to the LDLR receptor on the cell surface, which leads to LDLR internalization and lysosomal degradation. These pathways for LDL cellular uptake and LDLR recycling and degradation demonstrate the important interactions between the major known players in the FH phenotype: LDL, LDLR, APOB, PCSK9 and LDLRAP1. LDLR is comprised of six functional domains: the signal peptide (exon 1), the ligand-binding domain (exons 2 6), the EGF precursor homology domain (exons 7 14), the O-linked sugar domain (exon 15), the transmembrane domain (exons 16 17) and the cytoplasmic domain (exons 17 18) [22]. Exon 1 of LDLR encodes the signal sequence and is cleaved from the protein during translocation into the endoplasmic reticulum. While mutations have been observed throughout LDLR, the majority of mutations occur in the ligand binding and EGF precursor homology domains. Mutations occur most frequently in exon 4, which is also the largest exon in LDLR. Disease-causing mutations have also been observed in the promoter region of LDLR. Promoter region mutations have been detected in the R2 and R3 repeat elements, in a 280-bp region upstream of the transcription initiation site, and at FP1 and FP2 regulatory elements [23 25]. All described mutations have been functionally shown or theoretically suspected to impact transcriptional activity. There are five different classes of LDLR mutations, which are based on their impact on protein function [22,26 27]. Class 1 mutations, which are mainly null alleles, affect the synthesis of the receptor in the endoplasmic reticulum and involve the promoter and exon 1 regions. Class 2 mutations are transportdefective alleles involving the EGF-precursor homology domain. Class 2 mutations disrupt transport from the endoplasmic reticulum to the Golgi body, preventing addition of post-translational modifications necessary for receptor function. Class 3 mutations are the most common, involving the ligand binding domain, which prevents LDL binding to the receptor. Class 4 mutations are rare internalization-defective mutations that occur in exons Class 5 mutations prevent LDLR recycling, due to the inability of LDLR to release LDL particles in the endosomes, and are thought to mainly occur in the EGF-precursor homology domain. The majority of mutations in LDLR are missense mutations [28], but all types of mutations, including nonsense, insertion/deletion, splicing and major rearrangements, have been observed. Large deletion/duplication mutations, involving exon deletions or duplications, have also been observed in LDLR. These mutations are thought to occur due to recombination between Alu-type elements, which are common repetitive DNA sequences consisting of about 300 basepairs with two tandem repeats. Generally speaking, the majority of Alu repeats occur in intergenic regions. However, in the case of LDLR and some other genes (e.g., α- and β-globin), Alu repeats have been shown to be present in mature mrnas and can lead to exonic deletions and duplications. Mutations in LDLR can be divided into receptor negative (i.e., frameshift, truncating or splicing) that result in <2% residual activity and receptor defective (most missense), which typically retain 2 25% residual activity. Due to the residual activity of the defective LDLR allele, receptor defective mutations usually lead to a less severe phenotype. As the only lipoprotein component on the cell surface of LDL, apob-100 (apob) is the major LDLR ligand. ApoB was identified as a player in the FH phenotype due to observations that some patients with hypercholesterolemia and reduced clearance of LDL did not have defective LDLR, but rather had a defect in LDL itself, lending to a decreased affinity for LDLR [29,30]. Studies of the LDLR ligand binding domain of apob-100 have demonstrated that amino acid residues in APOB exon 26 are critical for binding [31]. Two mutations occur at the arginine residue at position p.3527 (legacy nomenclature is p.3500) in APOB (p.r3527q and p.r3527w), with the p.r3527q mutation frequency estimated at 1:500 1:700 Caucasians [32]. This residue is important for normal receptor binding due to its interactions with tryptophan at p.4369 [33]. Mutations in exon 22 and exon 29 have also been observed in association with the FH phenotype [34]. APOB truncating mutations and mutations in other areas of the gene have mostly been associated with hypocholesterolemia as a result of the decreased synthesis of apob-containing lipoproteins that transport cholesterol in plasma. PCSK9 has garnered tremendous interest lately for its role in hyper- and hypo-cholesterolemia and its potential as a promising drug target for patients with FH [35]. Its aspects as a drug target will be discussed elsewhere in this article. The role of PCSK9 in FH was uncovered in 2003 by Abifadel et al., based on studies done in a French family originally published in 1999 [36,37]. As a serine protease, PCSK9 mediates degradation of LDLR, by binding extracellular LDLR at the cell surface which prevents recycling of the internalized LDLR, leading to lysosomal degradation of LDLR [38,39]. Evidence also exists that PCSK9 acts 390 Clin. Lipidol. (2015) 10(5) future science group

6 Genetic considerations in the treatment of familial hypercholesterolemia intracellularly to degrade LDLR in a postendoplasmic reticulum compartment [40]. Similar to LDLR, there are several types of PCSK9 mutations, including mutations that affect PCSK9 stability or protein transport, null alleles and gain of function mutations. Gain of function mutations in PCSK9 cause increased PCSK9-mediated LDLR degradation, increased levels of LDL-C and the FH phenotype. Loss of function PCSK9 mutations are associated with low LDL-C and has a cardiovascular protective effect with a reduction in the risk of coronary heart disease [41]. Mutations in APOE have previously been associated with familial combined hyperlipidemia (FCHL, type III dyslipoproteinemia, OMIM #144250) which is characterized by high triglyceride levels in addition to high cholesterol. Recently, several APOE mutations have been identified in FH cohorts who lack mutations in LDLR, APOB and PCSK9. Specifically, within a French cohort, p.leu167del (c.500_502deltcc) and p.arg269gly (c.805c>g) mutations were identified, while a p.arg235trp (c.703c>t) mutation was identified in a Norwegian cohort [19]. The p.leu167del had also previously been reported in patients with seablue histiocytosis (OMIM #26960). The phenotypes observed among patients with the same APOE mutations appear to be variable and may be the result of additional genetic or environmental factors. STAP1 mutations were recently identified in several cohorts of patients with FH who did not have an LDLR, APOB or PCSK9 mutation [18,42]. The function of STAP1 is not well characterized and the mechanism by which mutations in this gene cause hypercholesterolemia also remains unknown at this time. Despite the discovery of additional genes associated with FH, the underlying genetic mutation(s) remain unknown for some cases and studies to uncover additional contributory genes continue. Genotype phenotype correlations Although there is phenotypic similarity in patients with mutations in LDLR, APOB and PCSK9, the severity and LDL-C levels are variable and can be associated with genotype (Figure 2). While heterozygous LDLR mutations are the most common genotypic occurrence in FH, patients can have homozygous or compound heterozygous mutations in LDLR, APOB or PCSK9, as well as digenic mutations (two different heterozygous mutations in separate genes, including involvement of LDLRAP1). Phenotypic presentation is accordingly more severe in patients with more than one mutation, with the exception of double mutations in APOB sometimes presenting similarly to heterozygous LDLR mutations. While homozygous/compound heterozygous FH is generally much more severe than HeFH, some patients with clinically-defined HeFH may have homozygous or compound heterozygous LDLR mutations, with only moderately higher LDL compared with true HeFH [43,44]. Genotype-related differences have been observed in patients with LDLR negative versus receptor defective mutations and LDL levels with LDL levels generally being moderately higher in receptor negative patients [43,45 46]. Furthermore, a higher occurrence of premature CHD has been observed in receptor negative compared with receptor defective patients [45,47 48]. The level of LDL-C is directly related to the residual LDLR function and correlates with phenotypic severity, regardless of the actual causative mutation. Overall, it is recognized that patients with the FH phenotype due to mutations in APOB tend to have a milder phenotype than patients with mutations in LDLR, including lower LDL cholesterol, better response to statins and lower risk of coronary heart disease [49]. It has been demonstrated that there is decreased production of apob-100 in patients with APOB defects, compared with patients with LDLR defects [50]. This is thought to be due to enhanced removal of apolipoprotein E-containing LDL precursors in patients with defective APOB. The phenotype resulting from mutations in PCSK9 is highly variable, with gain of function mutations ranging from mild to severe hypercholesterolemia [51]. The most severe mutation, p.d374y, is associated with total cholesterol levels reaching 506 mg/dl (13.1 mmol/l) and cardiovascular disease up to 10 years sooner than in patients heterozygous for severe LDLR mutations [52]. Patients with STAP1 mutations tend to have total cholesterol and LDL-C levels similar to patients with APOB mutations and lower than those with LDLR mutations [18]. Patients with homozygous LDLRAP1 mutations tend to have similar lipid levels and clinical features to those with homozygous receptor defective LDLR mutations [53]. Diagnosis of FH FH can be diagnosed clinically, genetically or through a combination of both. Historically, HoFH was diagnosed when the untreated LDL-C was >13 mmol/l (>500 mg/dl) or on treatment LDL-C was >8 mmol/l (>300 mg/dl), and cutaneous or tendon xanthomas were present by 10 years of age or both parents had untreated LDL-C concentrations elevated to a level consistent with HeFH. While presence of tendon xanthomas in the proband or a first-degree relative has traditionally been considered pathognomonic of FH and future science group 391

7 Moyer & Baudhuin enough to categorize a patient as having definite FH, they can also occur in sitosterolemia and cerebrotendinous xanthomatosis. These disorders are generally easily distinguished from FH in that they typically have normal or at most moderately elevated total cholesterol levels. In addition, sitosterolemia is characterized by markedly increased (>30-fold) plant sterol concentrations in sitosterolemia, while neurologic complications are seen in cerebrotendinous xanthomatosis. Although multiple diagnostic criteria have been developed, currently no one set of criteria is internationally agreed upon. The most commonly utilized diagnostic tools include the Simon Broome Register (SBR) criteria, the Dutch Lipid Clinic Network (DLCN) criteria and the Make Early Diagnosis to Prevent Early Death (MEDPED) criteria. The MEDPED criteria are based on age-specific thresholds for total cholesterol, taking into account family history of first-, second- and third-degree relatives [54]. Both the DLCN and SBR criteria are based on the WHO criteria and divide patients into categories of definite, probable and possible FH, and no diagnosis, based on LDL-C concentration, family history, clinical history and physical exam findings [55 57]. The DLCN and SBR criteria also include the results of molecular genetic testing, but differ in that genetic results are sufficient for a definite FH diagnosis in SBR, while in DLCN at least one other criteria is required for a definite diagnosis. Up to 40% of patients with a clinical diagnosis may not have a genetic diagnosis, potentially due to causal mutations and genes that have not yet been identified or a polygenic etiology that does not include the classical FH genes [58,59]. Conversely, family members of patients with FH have been identified through cascade testing who have a diagnostic mutation but fall below the cut-off for clinical diagnosis, possibly due to additional compensatory genetic variants and/or lifestyle factors [59]. Screening for FH In their 2013 consensus statement, the European Atherosclerosis Society (EAS) recommends cascade screening of family members for FH [7]. They indicate that probands should be identified based on the following clinical criteria: Plasma total cholesterol level 8 mmol/l ( 310 mg/dl) in an adult (or adult family member) or 6 mmol/l ( 230 mg/dl) in a child; or >95th percentile for age and sex; Premature CHD of the individual or a family member; Tendon xanthomas in the individual or a family member; Sudden premature cardiac death in a family member. In addition, a family pedigree should be drawn to assist with the evaluation. Cascade screening using LDL-C measurement should be performed including first-degree relatives, with consideration of screening second-degree relatives. If the proband has a known genetic mutation, genetic testing may also be incorporated into cascade screening. The NICE in the UK provide similar recommendations to EAS in their 2008 guidelines. However, they suggest that the possibility of HeFH should be considered in adults with total cholesterol >7.5 mmol/l (290 mg/dl) and endorse use of the Simon Broome criteria for phenotypic diagnosis [60]. The International FH Foundation also has published similar guidelines [61]. Of note, the International FH Foundation also discusses the ethical considerations for the notification of at-risk family members as well as logistical considerations in the presence (or potentially absence) of consent by the index case. The National Lipid Association (NLA) also has guidelines for screening in which they recommend universal screening for elevated serum cholesterol by age 20 (and as early as age 2 for children with a family history of premature CHD) [62]. According to the NLA, FH should be suspected when the LDL cholesterol is 190 mg/dl (4.9 mmol/l; or 160 mg/dl [4.1 mmol/l] in those <20 years of age) or the non-hdl cholesterol exceeds 220 mg/dl (5.7 mmol/l; or 190 mg/dl [4.9 mmol/l] in those <20 years) when in the untreated fasting state [62]. Presence of tendon xanthomas, arcus corneae (in patients under 45 years) and tuberous xanthomas or xanthelasma in those under age years should also be considered as signs of potential FH and prompt further testing. While cascade testing is mentioned, use of genetic testing is not covered in the NLA guidelines. Treatment options All patients with HeFH or HoFH should be encouraged to implement comprehensive lifestyle changes [63]. A referral to a dietician should be considered for assistance with dietary modification, consisting of reduction in intake of saturated fats, transfats and cholesterol. Initiation of an exercise routine and any additional risk factors, including hypertension, diabetes and smoking, should also be addressed. Despite the benefits of adopting a healthy lifestyle, additional therapy to lower lipids and reduce the risk of CHD will be required for most patients. 392 Clin. Lipidol. (2015) 10(5) future science group

8 Genetic considerations in the treatment of familial hypercholesterolemia Statins Statins lower cholesterol by inhibiting 3-hydroxy- 3-methylglutaryl-CoA (HMG-CoA) reductase, which slows the rate of mevalonate production and subsequent cholesterol production (Figure 1). In addition, the reduction in hepatic cholesterol production results in an increase in LDLR expression to increase cholesterol uptake from the serum. Since their introduction in 1987, statins have reduced the morbidity and mortality associated with CHD, and are currently the first-line therapy in FH, although there is wide variability in individual responses [57,64 65]. Maximal doses of potent statins, such as 80 mg/day of atorvastatin or 40 mg/day of rosuvastatin, should be initiated in patients diagnosed with FH. In HoFH, particularly with receptor-negative mutations, statin-mediated increases in LDLR expression are greatly diminished; however, high doses of potent statins have shown efficacy in both lipid lowering and reduction in CHD [66 68]. On average, patients with receptor-negative and receptor-defective mutations may experience 15 and 26% LDL-C reductions, respectively [10]. Several studies of HeFH have attempted to characterize the relationship between LDLR mutation class and response to statin therapy, but the results have been conflicting (reviewed in [69]). This has been in part due to small sample sizes, different types and doses of statins and different drug combinations, and use of different clinical end points. The NICE guidelines recommend a target of 50% reduction in LDL-C, while the American College of Cardiology/American Heart Association Guidelines simply recommend high-intensity statin treatment when LDL-C is >190 mg/dl (4.9 mmol/l) without a specific target [64,70]. Despite maximal statin therapy, some individuals with FH particularly those with HoFH will not achieve sufficient LDL-C reductions, in which case additional therapies are typically added. In the ENHANCE trial, addition of ezetimibe (10 mg/day) to simvastatin (80 mg/day) resulted in greater decreases in LDL-C (58 vs 41% reduction), but no significant difference in the change in intima-medial thickness was observed [71]. However, recently, results from IMPROVE-IT (ClinicalTrials.gov #NCT ) were presented at the 2014 American Heart Association Scientific Sessions, and showed a significant reduction in the primary end point (a composite of cardiovascular death, major coronary events, and stroke) by 6.4% in those on the combination of simvastatin and ezetimibe compared with statins alone [72]. In addition, a bile acid-biding resin, pure niacin, and/or fenofibrate may be recommended depending on the individual clinical scenario. Recent studies have identified an approximately 9 13% increased risk of Type 2 diabetes mellitus among patients taking statins, particularly in those taking high-dose statins [73 75]. While underlying biology for this observation is unclear, including whether the reduction in intracellular cholesterol levels or a nonlipid-related effect of statins is responsible, many mechanisms have been proposed [76]. Variants in HMGCR were found to be associated with both LDL-C levels and Type 2 diabetes risk, suggesting that the mechanism is related to cholesterol metabolism [77]. Despite the increased risk of diabetes, the benefits of reduction in atherosclerotic cardiovascular disease outweigh the risk, and statins remain a mainstay of treatment in patients with elevated cholesterol levels. Specifically, in one meta-analysis, for high-dose statins, the number needed to treat to prevent each cardiovascular event per year in patients with polygenic hypercholesterolemia is 155, compared with the number needed to harm of 498 to cause an additional case of diabetes [73]. The increased risk of diabetes in users of highdose statins is of particular concern for patients with FH who often begin therapy in childhood and will require lifetime treatment. Interestingly, a study by Besseling et al. found that the prevalence of Type 2 diabetes is significantly lower among patients with FH than among their unaffected relatives (after adjusting for potential confounders, OR: 0.49 [95% CI: ]) [78]. This finding is consistent with previous anecdotal observations and small studies [47]. This study also found an inverse relationship between mutation severity and diabetes prevalence in patients with FH. Patients with a LDLR mutation had a lower prevalence of diabetes than patients with APOB mutations (1.63 vs 2.42%, compared with 2.93% in unaffected relatives) [78]. Furthermore, patients with a LDLR negative mutation had a lower risk of diabetes than those with a LDLR defective mutation (1.41 vs 1.80%) [78]. In FH, because patients are at low risk for diabetes and high risk for CHD morbidity and mortality, the benefits of statin use far outweigh the potential increased risk for Type 2 diabetes. The relationship between Type 2 diabetes and cholesterol levels is fascinating, and further mechanistic work, perhaps involving patients with FH, may lead to better treatment of both diseases. Although statins remain a mainstay of treatment for FH, some patients are statin intolerant. Genetic testing of SLCO1B1 can be performed to identify individuals who are potentially at risk for statininduced myopathy [79]. In individuals predicted or demonstrated to be statin-intolerant, alternate treatment strategies, possibly including new anti-pcsk9 therapies, should be considered. future science group 393

9 Moyer & Baudhuin PCSK9 monoclonal antibodies As noted above, PCSK9 has gained great attention in recent years due to its promise in drug-development to reduce cholesterol levels. Pharmaceutical companies began to explore PCSK9 inhibitors after studies by Hobbs and Cohen revealed that patients with very low LDL cholesterol concentrations (as low as 14 mg/dl [0.36 mmol/l]) had PCSK9 null mutations and were surprisingly healthy [80]. Utilizing the Dallas Heart Study cohort, Hobbs and Cohen additionally demonstrated an association with PCSK9 null mutations and an 88% lower risk of developing cardiovascular disease (CVD), and an association with milder PCSK9 mutations and a 15% reduction in LDL-C and a 47% reduced risk of CVD [41]. Thus, a drug that could inhibit PCSK9 was theorized to lower LDL-C and risk of CVD. Additionally, since statins increase expression and secretion of PCSK9 (hence somewhat attenuating the impact of statins in LDL-C lowering), a drug that would inhibit PCSK9 would also potentially enhance the lipid-lowering effect of statins [81,82]. While the development of a small molecule that could inhibit PCSK9 and be packaged into a pill was not successful, the extracellular nature of PCSK9 lent itself to the development of a more costly monoclonal antibody approach. There are numerous PCSK9 monoclonal antibodies under investigation, including alirocumab (SAR236553/REGN727, Sanofi and Regeneron), evolocumab (AMG145, Amgen) and bococizumab (RN316, Pfizer). Phase I clinical trials demonstrated that PCSK9 inhibition by these molecules led to a 46 64% reduction in LDL-C for the two drugs given via subcutaneous injection (alirocumab and evolocumab) and up to approximately 75% for bococizumab, which is given intravenously, with few subjects discontinuing treatment due to adverse events [83 85]. An even further reduction in LDL-C was observed when alirocumab or evolocumab was given in the patients who were also on statin therapy [84,85]. Phase II, randomized, double-blind, placebocontrolled, week trials have also been completed on different cohorts such as hypercholesterolemic patients on a statin, hypercholesterolemic patients not on a statin, statin-intolerant patients, and patients with clinically defined FH (and on a statin, with or without ezetimibe; reviewed in [86]). In the case of evolocumab, biweekly administration resulted in 41 66% sustained LDL-C reduction, with no treatment-related serious adverse events reported [87]. Similarly, arilocumab treatment was associated with reductions in LDL-C of 50 70% [88,89]. Biweekly doses of bococizumab demonstrated placebo-adjusted LDL-C reductions of 35 53% at 12 weeks, with 2% of patients withdrawing from the study due to treatment-related adverse effects [90]. All three of these drugs have entered into placebocontrolled Phase III clinical trials. In the case of evolocumab, Phase III trials with dosing every 2 4 weeks in different cohorts (LAPLACE-2, GAUSS-2, MEN- DEL-2, RUTHERFORD-2 and DES-CARTES) have resulted in similar findings to their parallel Phase II trial results, with LDL reductions of up to 75% (reviewed in [86]). The OSLER-2 longer-term clinical trial of evolucumab, with median 11.1-month assessment of nearly 4500 patients who completed Phase II or Phase III trials, demonstrated reduced LDL-C of 61%, as well as a decreased rate cardiovascular events [91]. Overall, the clinical trials of PCSK9 inhibitors have been promising. Most trials investigating PCSK9 monoclonal antibodies have included patients diagnosed with FH through clinical criteria, rather than genetic analysis. Two exceptions to this were the TESLA Part B and RUTHERFORD 2 studies [43,92]. In the TESLA Part B study, 48 HoFH patients were genotyped and categorized into LDLR defective versus receptor negative. A difference was observed in receptor defective versus receptor negative response to evolocumab over the 12-week study. Specifically, there was a 46.9% decrease versus 24.5% decrease versus 10.3% increase in LDL in patients with two receptor defective mutations (n = 13), one receptor defective and one receptor negative mutation (n = 9), and two receptor negative mutations (n = 1), respectively. In the RUTHERFORD 2 study, 211 patients with clinically defined HeFH were genotyped, with 37% having a mutation classified as receptor defective, 32% receptor negative and 26% unclassified. Results from this 12-week study demonstrated LDL cholesterol-lowering response to evolocumab was not related to genotype in HeFH patients. Therefore, the utility of genotyping FH patients to determine LDL cholesterol-lowering response to anti-pcsk9 therapies may only be of benefit in suspected HoFH patients. Whether or not genotype is related to cardiovascular outcomes in heterozygous or homozygous FH patients receiving anti-pcsk9 therapies remains to be determined. LDL apheresis LDL apheresis has been demonstrated to be overall effective at reducing LDL and other atherogenic particles and reducing the risk for cardiovascular disease (reviewed in [93]). Some patients with severe HeFH and most patients with HoFH will not achieve sufficient LDL-C reduction despite maximum doses of statin or other therapies. In these patients, LDL apheresis, which directly and selectively removes circulating apob-con- 394 Clin. Lipidol. (2015) 10(5) future science group

10 Genetic considerations in the treatment of familial hypercholesterolemia taining lipoproteins by 60 75% [94], may be recommended. Furthermore, LDL apheresis is an important therapy to consider in the rare cases of pregnant HoFH patients and may be a consideration in HeFH patients who are pregnant and have significant atherosclerotic disease [95,96]. Criteria to warrant LDL apheresis has been proposed to include: functional HoFH patients with LDL-C 300 mg/dl (7.8 mmol/l; or non-hdl-c 330 mg/dl [8.5 mmol/l]); functional HeFH patients with LDL-C 300 mg/dl (7.8 mmol/l; or non-hdl-c 330 mg/dl [8.5 mmol/l]) and 0 1 risk factors; and functional HeFH patients with LDL-C 200 mg/dl (5.2 mmol/l; or non-hdl-c 230 mg/dl [5.9 mmol/l]) and high risk characteristics such as 2 risk factors or Lp(a) 50 mg/dl using an isoform insensitive mass assay [62,97]. Many patients receiving LDL-apheresis require repeat apheresis procedures every 2 4 weeks in order to maintain sufficiently lowered LDL-C levels. Oftentimes, apheresis and statins are provided in combination in order to further maximize LDL-C reduction. Although published genetic analyses of patients undergoing LDL apheresis are limited, we undertook a small study and observed that in patients undergoing LDL apheresis, those with an identifiable LDLR mutation had a more proatherogenic preapheresis profile and had significantly more small LDL particles removed during apheresis as compared with patients without an identifiable LDLR mutation [98]. This pilot cohort suggests that patients receiving the maximum lipid lowing therapy could be further stratified, based on genetic make-up, to optimize treatment. Additional studies to examine this and other effects in a larger cohort are underway. Antisense APOB Naturally occurring variants in APOB that disrupt or truncate the protein can cause familial hypobetalipoproteinemia (FHBL). Patients with heterozygous APOB-linked FHBL are typically asymptomatic and have a lower lifetime exposure to apob-containing lipoproteins, although many develop fatty liver [99]. Mipomersen, developed to mimic the reduction of APOB observed in heterozygous FHBL, is a 20-base pair single-stranded DNA oligonucleotide that is complementary to human apob-100 mrna and can cause selective degradation of the target mrna (Figure 1). It is US FDA-approved for the treatment of HoFH. The oligonucleotide has several modifications, including phosphorothioate ester linkages joining the nucleotides and 2 -methoxyethyl sugar residues incorporated into the first and last five positions, which enhance its stability, binding affinity and resistance to degradation [100]. Mipomersen is administered by weekly subcutaneous injection, achieves highest concentrations in the liver and kidneys, metabolized by nucleases, and its elimination half-life ranges from 1 2 months [100]. In a Phase II trial of 44 patients with HeFH, when mipomersen was added to conventional therapy over a 6-week treatment period, in the 200-mg treatment group LDL-C and apob were reduced by 21 and 23% [101]. Along with two additional Phase II trials by Akdim et al., this study demonstrated significant dosedependent reductions in apob and LDL-C in patients treated with mipomersen, alone or in combination [ ]. Based on the results of these studies and the side effect profile, the 200-mg dose was selected for Phase III trials. Multiple Phase III randomized, double-blind, placebo-controlled clinical trials were performed with patients with or without FH who were already receiving maximal medical therapy or maximally tolerated statin therapy [ ]. In these trials of patients, reductions of 25 37% were observed in LDL-C, 26 38% in apob and 21 33% in Lp(a) in the mipomersen arm. These results validated the efficacy of mipomersen and lead to the FDA approval of the 200 mg dose for the treatment of HoFH. However, the Committee for Medicinal Products for Human Use (CHMP) of the EMA decided not to approve mipomersen due to high discontinuation rates in trials and the potential hepatotoxicity. A recent study of long-term efficacy and safety in patients with FH demonstrated similar findings to the Phase III trials [108]. The most common side effects of mipomersen were injection site reactions, elevations in liver transaminases, flu-like symptoms and increased hepatic fat deposition. ALT elevations were typically >3, but occasionally >10 the upper limit of normal. Patients with the highest elevations in ALT tended to also achieve the largest reductions in apob [106]. Other studies have also found a potential correlation between drug efficacy and hepatic fat deposition in patients taking mipomersen and lomitapide (discussed below) [105,109]. Due to this potential hepatotoxicity risk, both mipomersen and lomitapide have both been FDA approved for restricted use, and monitoring of liver transaminases is recommended at least monthly in the first year of treatment and every 3 months during the second year of treatment. MTP inhibition MTP is a lipid-transfer protein located in the endoplasmic reticulum that is essential for the synthesis and secretion of apob-containing lipoproteins, including chylomicrons and VLDL (Figure 1). MTP inhibition results in reduced secretion of these lipoproteins into future science group 395

11 Moyer & Baudhuin the circulation. Lomitapide is an orally active small molecule inhibitor of MTP and is approved in both the US and in Europe for the treatment of HoFH. It is metabolized by CYP3A4 in the liver, necessitating caution when used with other drugs metabolized by CYP3A4, including several statins [110]. Few studies of lomitapide have been performed in patients with HoFH [109,111]. In a single-arm Phase III study that included 29 patients with HoFH, treatment with lomitapide in addition to other lipid-lowering therapies was shown to decrease LDL-C by 50% at week 26, and the reduction remained significant at week 56 (44% reduction) and week 78 (38% reduction). In addition, apob was decreased by 49% at week 26 and remained down by 43% at week 78, and Lp(a) was decreased by 15% at week 26, but not significantly decreased at week 78. HDL-C concentrations were also decreased by 12% at week 26, but this reduction did not persist. Although lomitapide appears promising, long term studies are required to verify that the reductions in LDL-C will persist and to evaluate the effect on cardiovascular morbidity and mortality. The main adverse reactions noted with lomitapide use include gastrointestinal symptoms, elevation of hepatic transaminases and increased hepatic fat [109]. Lomitapide is contraindicated in pregnancy as it has been found to be teratogenic in animal models. In addition, pediatric safety and effectiveness are unknown, and caution is advised in older patients who may be at increased risk for side effects due to decreased cardiac, renal and/or hepatic function. CETP inhibition CETP facilitates the transfer of cholesterol from HDL to LDL particles. Inhibition of this protein results in decreased concentration of atherogenic lipoproteins, including LDL-C, along with an increase in HDL-C. A recent Phase III study demonstrated a 39.7% decrease in mean LDL-C on anacetrapib compared with placebo in patients with HeFH [112]. Most of the patients in the study were on high dose statin, with over 70% additionally taking ezetimibe. Of note, the number of cardiovascular events was higher in patients on anacetrapib compared with placebo (four events in 203 patients vs zero events in 102 patients), but this study was not designed or powered to evaluate cardiovascular outcomes. However, these findings are reminiscent of another CETP inhibitor, torcetrapib, which compared with placebo resulted in an increased risk of cardiovascular events and mortality [113]. Surgical approaches In some patients with HoFH and inadequate response to medical management, surgical approaches have been taken, including liver transplant, portocaval shunt, and partial ileal bypass. Since the first transplant in 1984, at least 30 additional cases have been reported, including six combined heart and liver transplants [114]. Patients who underwent liver transplantation were generally young, ranging from 2 to 46 years of age, and in most cases experienced a rapid and marked improvement in their lipid profile to nearly normal levels, as well as regression of xanthomas. In addition to decreased LDL-C, in most cases HDL-C increased while Lp(a) decreased [114]. Because the group of patients who underwent liver transplant remains small, the long term cardiovascular benefits are unclear. Liver transplantation is not without disadvantages, including surgical complications and mortality, a limited donor pool, and the requirement of long-term immunosuppression. Portocaval shunts and partial ileal bypass procedures have also been used, but in a small number of patients with mixed results [ ]. Considerations in special populations Pediatric patients Studies have shown that atherosclerosis begins at an early age and initiation of early treatment can lead to beneficial cardiovascular outcomes [ ]. Furthermore, clinical trials with short and longer term followup have demonstrated the safety of statins in children [121,122]. The National Lipid Association Expert Panel on FH recommends universal cholesterol screening on all children aged 9 11 years old and screening at a younger age (starting at 2 years old) if the patient has a positive family history for hypercholesterolemia or premature coronary heart disease (CHD) [62]. One of the obvious goals of these recommendations is to maximize identification of children with FH at an optimal time point when treatment can be initiated. From a pharmacologic standpoint, statins are the preferred treatment option for children and consideration should be given to start treatment at the age of 8 years old or older [62]. In the case of HoFH, treatment may be started earlier. In pediatric FH patients, the treatment goal is to reduce LDL cholesterol by at least 50% or to <130 mg/dl (3.4 mmol/l). However, despite treatment from as early as 8 years of age, many children with FH do not achieve goal LDL-C levels [ ]. Furthermore, since increased carotid intimal medial thickness is already present in FH children before the age of 8 years, there is potential rationale for initiation of statins at a younger age than what is currently recommended [119]. Currently, pravastatin is approved for use in children ages 8 years and older, whereas other statins are approved for children ages 10 years and older. One study recently demonstrated that treatment of children with FH as young as 6 years old with rosu- 396 Clin. Lipidol. (2015) 10(5) future science group

12 Genetic considerations in the treatment of familial hypercholesterolemia vastatin, one of the most potent statins, was both safe and efficacious [118]. Recently, ezetimibe monotherapy was demonstrated to result in 27% reduction in LDL-C after adjustment for placebo and was well-tolerated in a group of 6 10-year-old children, suggesting that it may provide an additional treatment option [126]. Pregnancy Management of patients with FH who are or may become pregnant is a particular challenge. Contraceptive options must be carefully considered as hormonal products further elevate lipids and are associated with an increased risk of thrombosis. Prior to pregnancy, an assessment of cardiovascular risk, particularly in homozygotes, is recommended to evaluate the risk of pregnancy to the mother. In a typical pregnancy, cholesterol levels and triglycerides increase, and while the relative increases are similar between women with FH and the general population, the absolute concentration changes are much larger in women with FH [127]. Although it has been hypothesized that increased cholesterol during pregnancy may lead to accelerated atherosclerosis for the mother and/or predispose the fetus to increased cardiovascular risk, the full effects of hypercholesterolemia in pregnancy are not well characterized [128]. Routine cholesterol measurement is not recommended during pregnancy since no therapy is indicated. Statins are contraindicated during pregnancy and lactation because of concerns of teratogenicity; however, the studies supporting this concern are limited [128]. Nevertheless, according to the NICE guidelines, women should stop statins for 3 months prior to attempting to conceive and to not resume until after lactation [129]. Pregnant women should be encouraged to follow a cholesterol free (<50 mg/day) or restricted (<300 mg/day) diet, which has been shown to decrease LDL-C [130]. Bile acid sequestrants may be considered in some women. LDL apheresis may also be considered and has been successfully used in controlling lipid levels in several case reports of patients with FH during pregnancy [131,132]. Newer agents, such as PCSK9 inhibitors, mipomersen and lomitapide, may be good options in the future, but will require significant additional study prior to use in this population. Conclusion FH is an inherited disorder that causes significant morbidity and mortality, primarily due to the premature development of cardiovascular disease. While most cases are caused by mutations in LDLR, several additional genes have also been identified, including APOB, PCSK9, LDLRAP1, APOE, and STAP1; however, the underlying genetic etiology for some FH cases remains unknown. Currently, no one set of criteria has been internationally agreed upon for the diagnosis of FH, resulting in variability across countries and across studies. Although statins remain a mainstay of therapy, new therapies, including PCSK9 inhibitors, have recently emerged. As new genes contributing to FH are uncovered, additional mechanisms of disease and therapeutic targets may also be identified, potentially improving both the diagnosis and treatment of FH, as well as polygenic hypercholesterolemia. Future perspective FH continues to be an underdiagnosed disorder with significant morbidity and mortality. Continued efforts Executive summary Background Familial hypercholesterolemia (FH) is an inherited disorder characterized by increased LDL-bound cholesterol and high risk for premature cardiovascular disease. Genetic causes of FH Most cases are caused by mutations in LDLR (60 80%), APOB (1 5%), PCSK9 (1 3%), with rare cases related to LDLRAP1. The disease phenotype varies based on which mutation(s) is present, with the phenotype being more severe in cases with less residual receptor activity. Diagnosis of FH & screening FH can be diagnosed by clinical criteria, genetic testing or a combination; after diagnosis, it is important to screen family members. Treatment options Statins are the mainstay of therapy, and although they may increase the risk for Type 2 diabetes, the risk in patients with FH is lower than the general population, such that the benefits of statin therapy far outweigh the risks of diabetes. Several promising new drug classes have recently been developed, including monoclonal antibodies that inhibit PCSK9, an antisense oligonucleotide against APOB, and an inhibitor of MTP; while the PCSK9 inhibitors are well-tolerated, both the antisense APOB and MTP inhibitor can cause elevated hepatic fat. future science group 397