Journal of Clinical Apheresis 00:00–00 (2014)

Genetic and Biochemical Analyses in Dyslipidemic Patients Undergoing LDL Apheresis Leslie J. Donato,1 Amy K. Saenger,1 Laura J. Train,1 Katrina E. Kotzer,1 Susan A. Lagerstedt,1 Jean M. Hornseth,1 Ananda Basu,2 Jeffrey L. Winters,1 and Linnea M. Baudhuin1* 1

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 2 Division of Endocrinology, Mayo Clinic, Rochester, Minnesota

Objective: Familial hypercholesterolemia (FH) can be due to mutations in LDLR, PCSK9, and APOB. In phenotypically defined patients, a subset remains unresponsive to lipid-lowering therapies and requires low densitylipoprotein (LDL) apheresis treatment. In this pilot study, we examined the genotype/phenotype relationship in patients with dyslipidemia undergoing routine LDL apheresis. Patients and Research Design: LDLR, APOB, and PCKS9 were analyzed for disease-causing mutations in seven patients undergoing routine LDL apheresis. Plasma and serum specimens were collected pre- and post-apheresis and analyzed for lipid concentrations, Lp(a) cholesterol, and lipoprotein particle concentrations (via NMR). Results: We found that four patients harbored LDLR mutations and of these, three presented with xanthomas. While similar reductions in LDL-cholesterol (LDL-C), apolipoprotein B, and LDL particles (LDL-P) were observed following apheresis in all patients, lipid profile analysis revealed the LDLR mutation-positive cohort had a more pro-atherogenic profile (higher LDL-C, apolipoprotein B, LDL-P, and small LDL-P) pre-apheresis. Conclusion: Our data show that not all clinically diagnosed FH patients who require routine apheresis have genetically defined disease. In our small cohort, those with LDLR mutations had a more proatherogenic phenotype than those without identifiable mutations. This pilot cohort suggests that patients receiving the maximum lipid lowering therapy could be further stratified, based on genetic make-up, to optimize treatment. J. Clin. C 2014 Wiley Periodicals, Inc. Apheresis 00:000–000, 2014. V Key words: familial hypercholesterolemia; LDL apheresis; dyslipidemia; lipoproteins; LDLR

INTRODUCTION

Familial hypercholesterolemia (FH) is an autosomal dominant disorder which is characterized by elevated concentrations of low density-lipoprotein cholesterol (LDL-C) and is strongly associated with premature cardiovascular disease and myocardial infarction. Heterozygous FH has a worldwide incidence of 1 in 500 individuals, but may have increased prevalence in certain populations, as high as 1 in 67. Patients with heterozygous FH have two-fold elevations in plasma cholesterol and premature development of coronary atherosclerosis [1]. Homozygous (or compound heterozygous) FH is a rare condition, occurring at a frequency of approximately 1 in 1,000,000 individuals and is characterized by severely elevated LDL-C (usually >650 mg/ dL) [2,3]. The mortality rate in homozygous FH is extremely high and, if left untreated, individuals do not usually survive beyond the age of 30. The recently expanded definition of FH as proposed by Goldberg et al. includes disease-causing mutations in the LDLR, APOB, and PCSK9 genes, and has also been previously described as autosomal dominant hypercholesterolemia (ADH) [2,3]. Individuals who are mutation-positive most frequently have deleterious mutations in the LDLR gene (85–90%), which encodes C 2014 Wiley Periodicals, Inc. V

the LDL receptor. Approximately, 5–10% of individuals have deleterious mutations in the APOB gene which is associated with familial defective apolipoprotein B100 (FDB). Less than 5% of genetic mutations causative of a FH phenotype are due to gain-of-function mutations in the PCSK9 (encoding proprotein convertase subtilisin/kexin type 9) gene. Therapeutic treatment strategies for FH patients focus on reduction of LDL-C by >50%, with the statin therapies being the first line of treatment. Statin pharmacotherapy is frequently combined with other lipidlowering agents, although some heterozygous FH and polygenic hypercholesterolemia individuals are refractory or show intolerance to statins or combination pharmacotherapies. A last resort for treatment therapy in Additional Supporting Information may be found in the online version of this article. *Correspondence to: Linnea M. Baudhuin, Ph.D., DABMG, Department of Laboratory Medicine and Pathology, 400 Hilton Building, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905, USA. E-mail: [email protected] Received 24 April 2013; Accepted 27 December 2013 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jca.21317

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patients unable to meet their LDL-C goal is therapeutic LDL apheresis (also called lipid apheresis or lipoprotein apheresis) which directly and selectively removes circulating apo-B containing lipoproteins by 60–75% [4,5]. The purpose of this study was to characterize the underlying genetic cause for the dyslipidemia in patients undergoing routine therapeutic LDL apheresis, inclusive of mutations in LDLR, APOB, and/or PCSK9. We further examined the biochemical phenotype of these patients in order to determine the effectiveness of the apheresis, using traditional lipid and NMR lipoprotein particle analyses on specimens collected pre- and post-apheresis. MATERIALS AND METHODS Patient Population

Patients with hypercholesterolemia who were undergoing routine LDL apheresis by the dextran sulfate cellulose absorption system (Liposorber LA-15 System, Kaneka, Osaka Japan) were recruited from the Mayo Clinic Therapeutic Apheresis Treatment Unit (Rochester, MN). Three plasma volumes were treated at each apheresis procedure. Criteria used at our institution to warrant biweekly LDL apheresis are synonymous with those defined by the Food and Drug Administration: either (1) LDL-C level >200 mg/dL and established vascular disease or (2) LDL-C level >300 mg/dL with no vascular disease. In addition, there must be demonstrated failure of pharmacological and lifestyle therapies to lower cholesterol to desirable levels. This study was approved by the Institutional Review Board at the Mayo Clinic and written informed consent was obtained from all study participants.

tems, Foster City, CA). Sequence analysis was done using Mutation SurveyorTM software (SoftGenetics, State College, PA) and visual inspection. PCSK9 Sequencing

The entire coding region (all 12 exons plus flanking regions) of PCSK9 (GenBank NM_174936) were sequenced. First, each exon and a minimum of 20 bp of intronic DNA flanking each exon were amplified via PCR, with amplicons ranging in size from 342 to 536 bp. Amplification, sequencing, and subsequent analysis were performed as detailed for LDLR. APOB Genotyping

APOB was genotyped for the two most common mutations associated with ADH: p.R3500W and p.R3500Q. Primers flanking APOB codon 3500 were used in a 30 cycle PCR reaction (30 sec at 94 C, 30 sec at 55 C, 1 min at 72 C), with a final 10 min extension at 72 C. Amplicons were subjected to allele-specific primer extension (40 cycles: 30 sec at 94 C, 30 sec at 55 C, and 2 min at 72 C) incorporating biotinylated dCTP (Roche) and products were hybridized to fluorescent Flexmap beads (Luminex Corp, Austin, TX). The biotin was conjugated to a Streptavidin, R-Phycoerythrin reporter solution (Molecular Probes, Eugene, OR). Excess reporter solution was washed away from the beads with 31 Hybridization Buffer (0.2M NaCl, 0.1M Tris, 0.08% Triton XR 100, pH 8.0) and samples were loaded into a LuminexV 100 for detection. Median fluorescence intensity signals were analyzed to determine allelic ratios (mutant signal/ total signal) and genotype calls.

LDLR Sequencing

The entire coding region (all 18 exons plus flanking regions) and promoter of LDLR (GenBank NT_011295.10) were sequenced. First, the promoter region and each exon, with a minimum of 20 basepairs (bp) of intronic DNA flanking each exon, were amplified by multiplexed polymerase chain reactions (PCR), with amplicons ranging in size from 278–755 bp. Amplification was performed using a common master mix containing Platinum Taq DNA Polymerase, 310 PCRx Enhancer System, 310 PCR Buffer (-MgCl), MgSO4 (all Invitrogen, Carlsbad, CA), and a 10 mM dNTP mixture (Roche, Indianapolis, IN). Master mix, forward and reverse primers were combined with genomic DNA and amplified by 35 cycles of PCR (30 sec at 95 C; 30 sec initially at 68 C then decreased by 0.5 C each cycle, with the last 20 cycles performed at 60 C; and 1 min extension at 72 C, with a final 10 min extension at 72 C). Amplicons were bidirectionally sequenced using Big Dye Terminator technology on an ABI 3730 system (Applied BiosysJournal of Clinical Apheresis DOI 10.1002/jca

APOE Genotyping

APOE was genotyped for three common alleles designated e2, e3, and e4. Primers flanking the region encompassing codons 112 and 158 were used in a 35 cycle PCR reaction (30 sec at 94 C, 30 sec at 65 C, 30 sec at 72 C), with a final 10 min extension at 72 C. Restriction enzyme digestion was then performed utilizing Hha I (New England Biolabs, Ipswich, MA) for 4 h at 37 C. Digested product was loaded on an 8% polyacrylamide gel and run for 26 min at 200 V. Following electrophoresis, the gel was stained with ethidium bromide and then visualized with the AlphaImager system (Protein Simple, Santa Clara, CA). The combination of alleles present was determined by the pattern of banding visualized on the gel. LDLR Large Genomic Rearrangement Analysis

Testing for large rearrangements of the LDLR gene was done by use of a multiplex ligation-dependent

Analysis of Dyslipidemic LDL Apheresis Patients

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TABLE I. Clinical and Family History of LDL Apheresis Patients Patient 1 2 3 4 5 6 7

Age 58 49 57 71 60 81 71

Gender

Lipid Deposits

Events

Family History

F M M M M M M

— Xanthelasmas Xanthomas Xanthomas — — —

CAD, stent, angioplasty Angioplasty, stent, bypass CAD, MI, stent, angioplasty CAD CAD LVD, bypass, heart transplant CAD, heart transplant

Daughter, son Mother, sister, grandfather, son Father, 3 siblings, son Brother, mother, 2 children Father, mother, sister, 2 sons, grandparents Father, 6 siblings Father, 2/6 siblings

probe amplification (MLPA) kit (MRC Holland, Amsterdam, The Netherlands), according to the manufacturer’s instructions. Briefly, genomic DNA was hybridized to probe pairs targeted to the 18 LDLR exons and the proximal promoter. After a 16-h hybridization, probes were ligated at 54 C for 15 min, and amplified by PCR (35 cycles of 30 sec at 95 C, 30 sec at 60 C, 1 min at 72 C; with a final 20 min extension at 72 C). Detection was performed using capillary electrophoresis on an ABI 3130xl (Applied Biosystems). LDLR target peak heights were normalized to internal control targets and assessed for exon loss (e.g., a relative gene ratio of 0.5 for a single copy deletion) or exon gain (e.g., a gene ratio of 1.5 for a single copy gain) using GeneMarker software v1.41 (SoftGenetics).

Statin Y Y Y Y N N Y

dL 5 0.01129 mmol/L, apoA—1 mg/dL 5 0.01 g/L; apoB—1 mg/dL 5 0.01 g/L. NMR Analysis

Lipoprotein particle concentrations (HDL-P, LDL-P, VLDL-P, IDL-P, total and subclasses) were analyzed using an automated NMR instrument (Bruker-Biospin, 400 MHz). Lipoprotein particle concentrations were quantitated utilizing the LipoScience NMR LipoProfiler R software (LipoScience, method. The NMR LipoProfileV Raleigh, NC) evaluates the signal produced by the terminal methyl groups of lipoproteins and separates the total lipid signal into component signals attributed to the various lipoproteins and their subclasses. RESULTS

Lipoprotein Metabolism Profile Analysis

Patient History and Results of Genetic Analysis

A single apheresis treatment was chosen to assess lipid profiles before and after treatment. EDTA plasma and serum samples were collected pre- and postapheresis, subjected to lipid fractionation and analyzed in a CDC-certified lipid laboratory. Lipid analysis included total cholesterol (TC; Roche Diagnostics, Indianapolis, IN), total triglycerides (TG, non-glycerol blanked; Roche Diagnostics, Indianapolis, IN), highdensity lipoprotein cholesterol (HDL-C) by MgCl2/dextran sulfate precipitation, and beta-quantification LDL cholesterol (LDL-C). Lipoproteins were fractionated by overnight ultracentrifugation. Apolipoprotein A (apoA; Roche Diagnostics, Indianapolis, IN), apolipoprotein B (apoB; Roche Diagnostics, Indianapolis, IN), total cholesterol, and triglyceride concentrations were quantitated on a Roche Cobas c501 (Indianapolis, IN). Glycerol blanked triglycerides (Roche Diagnostics, Indianapolis, IN) were run on any sample found to contain elevated triglycerides in the HDL fraction (glycerol, Patients 3 and 7 in Supporting Information Table 1). Lp(a) cholesterol was analyzed using cellulose acetate electrophoresis (Helena Laboratories, Beaumont, TX) with reference interval < 3 mg/dL. All results are shown in conventional units. Conversions to SI units are as follows: TC, HDL-C, LDL-C, Lp(a) cholesterol—1 mg/dL 5 0.02586 mmol/L; TG—1 mg/

A total of nine patients undergoing LDL apheresis were eligible for inclusion of the study. Seven of these patients consented to participate in the study (Table I). Four were found to harbor a heterozygous mutation in LDLR (Patients 1–4 below). None of the seven patients had mutations in either APOB or PCSK9. All gene polymorphisms are displayed in Supporting Information Table 2. Patient 1 was a 58-year-old Caucasian female with a history of Graves’ disease and severe hyperlipidemia who, upon referral for hypertriglyceridemia at age 45, presented with a plasma total cholesterol of 476 mg/ dL, triglycerides of 832 mg/dL, and an HDL-C of 21 mg/dL. LDL-C could not be calculated using the Friedwald equation due to elevated triglycerides and a betaquantification LDL-C was not performed. She did not have a family history of coronary artery disease, lipid disorders, or premature familial heart disease or vascular disease; however, her daughter and son have both been diagnosed with FH. The patient’s APOE genotype was found to be E2E3, indicating she did not have familial dysbetalipoproteinemia. She did not have skin xanthomas and was diagnosed with unexplained mixed hyperlipidemia. With medication and diet modifications she was effectively able to lower her triglycerides which ranged from 88 to 300 mg/dL within the last Journal of Clinical Apheresis DOI 10.1002/jca

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Fig. 1. Analysis of Exon 12 duplication. A: Schematic representation of primer hybridization to template with either one or two copies of exon 12. A PCR product will only be generated if exon 12 is duplicated. B: Results of PCR analysis. Lane 1—patient sample with primer set to detect exon 12 duplication (dup primer set). Lane 2—control sample with dup primer set. Lane 3—patient sample with housekeeping primers. Lane 4—control sample with housekeeping primers. Lane 5—No template control with dup primer set. Lane 6—No template control with housekeeping primers. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

five years, and she was ultimately diagnosed with FH. However, she was ultimately intolerant to simvastatin, pravastatin, fenofibrate, colesevelam, and atorvastatin, all which caused myalgias, but was able to tolerate rosuvastatin. The patient developed significant coronary calcification and was initiated on LDL apheresis eight years ago. Genetic analysis revealed a duplication of exon 12 in LDLR, a mutation which is predicted to lead to protein truncation and receptor inactivity. To verify that the identified duplication of exon 12 was the result of tandem duplication, an alternative fragment analysis technique was used. Primers were designed to hybridize just outside of exon 12 and to synthesize away from the exon, confirming the presence of a tandem duplication of exon 12 (Fig. 1). Patient 2 was a 49-year-old Caucasian male with hypercholesterolemia that was first diagnosed at 18 years of age. He has a significant history of coronary artery disease and suffered a myocardial infarction (MI) at the age of 40. He has undergone coronary angiography, cardiac stenting, and a single vessel coronary artery bypass graft. He had a history of claudication and peripheral vascular disease with involvement in both of his legs and was noted to have cholesterol deposits around his eyes (xanthelasmas). He has a strong family history of FH in his maternal lineage. Journal of Clinical Apheresis DOI 10.1002/jca

His mother had elevated cholesterol, lower extremity deep vein thromboses (DVTs) on three separate occasions, and died at age 66 of a stroke. His maternal grandfather died from an MI at the age of 44. His sister had a stroke and coronary artery disease in her 40s, recurrent DVTs, and has cardiac stents. The patient has one son with reportedly high cholesterol. The patient first presented for a lipid consultation at age 43 following an abnormal lipid screen (LDL-C of 378 mg/dL, triglycerides of 215 mg/dL, HDL cholesterol of 35 mg/ dL, and total cholesterol of 456 mg/dL) which failed to substantially improve with combination pharmacotherapy. He began LDL apheresis and has undergone biweekly apheresis treatments for the past four years. Genetic evaluation of Patient 2 revealed a deletion of exons 17 and 18 in LDLR. This deletion would be predicted to lead to protein truncation and receptor inactivity. Patient 3 was a 57-year-old Caucasian male with a personal history of mixed hypercholesterolemia first diagnosed at age 38. He first presented upon referral at age 42 with a total cholesterol of 293 mg/dL, triglycerides of 83 mg/dL, HDL-C of 49 mg/dL, and LDL-C of 227 mg/dL. He was a competitive marathon runner but was advised to refrain from competitive running due to his history of significant coronary artery disease. He suffered an MI at the age of 40, which resulted in percutaneous transluminal coronary angioplasty of the distal right coronary artery and stent placement. He continued to have chest discomfort following placement of the stent and underwent coronary angiography which revealed severe coronary artery disease and 90% lesions in his first diagonal and second diagonal. He had periodic appearance of xanthomas in the palmar areas and the left tendo-Achilles. He has a significant family history of hyperlipidemia that includes his father who had coronary disease and died at age 67, three siblings with hypercholesterolemia and an adult son with high cholesterol. He had previous intolerance to cholesterol-lowering medications and developed myopathy and elevated liver enzymes which caused him to discontinue the medications. He has undergone bi-weekly LDL apheresis for the past decade. Genetic analysis of Patient 3 revealed a mutation in LDLR exon 7, nucleotide c.1012T>G, amino acid p.Cys338Gly (p.C338G). The cysteine (Cys) to glycine (Gly) substitution is predicted to lead to receptor misfolding. Patient 4 was a 71-year-old Caucasian male with poorly controlled FH with inadequate diet and exercise compliance. He had a documented history of elevated cholesterol since he was 20 years old. He had a strong family history of coronary disease. His brother had hypercholesterolemia and suffered an MI at age 49 and underwent coronary artery bypass at age 59. His mother had hypercholesterolemia, xanthelasmas, and

Analysis of Dyslipidemic LDL Apheresis Patients

suffered an MI at the age of 71. His two adult children were diagnosed as teenagers with borderline hypercholesterolemia. At age 55, his lipid screen revealed a total cholesterol of 298 mg/dL, HDL-C of 39 mg/dL, LDL-C of 200 mg/dL, and triglycerides of 294 mg/dL. He developed coronary artery disease as documented by the presence of diffuse coronary calcification in the setting of severe hypercholesterolemia which was resistant to multiple drug interventions including combination therapy. He had Achilles tendon xanthomas and xanthomas on the extensor tendons of both hands. After six months of LDL apheresis, he had a dramatic response in lowering his LDL-C and has continued apheresis therapy bi-weekly for the past eleven years. Genetic analysis revealed a mutation in LDLR intron 3 (IVS3), c.31311G>A, which is predicted to abolish splicing at the splice donor site of intron 3. The other three patients (Patients 5–7) did not have observable mutations in any of the three genes associated with FH. Patient 5 was a 60-year-old Caucasian male with a history of severe hypercholesterolemia with coronary artery disease who was intolerant to all statins, ezetimibe, colesevelam, as well as niacin. His father was a smoker, had an MI at the age of 49, and subsequently died of cardiac disease at age 61. His mother, also a smoker, died at age 87 of coronary artery disease. His sister had hyperlipidemia requiring statin use. His two sons had high cholesterol, and his grandparents also had high cholesterol. He was diagnosed with type 2 diabetes and dyslipidemia and his lipid profile prior to LDL apheresis revealed a total cholesterol of 398 mg/dL, triglycerides of 247 mg/dL, HDL-C of 61 mg/dL, and LDL-C of 288 mg/dL. Apheresis was initiated and bi-weekly treatments have been ongoing for the past 6 years. Patient 6 was an 81-year-old Caucasian male with a significant family history of hyperlipidemia and coronary artery disease in several family members who were diagnosed in their 50s. His father had a history of heart disease and stroke and he had six siblings who all had heart disease, high cholesterol, high triglycerides, and high blood pressure. The patient’s own cardiac history included coronary artery disease with marked left ventricular dysfunction with a left ventricular ejection fraction (LVEF) of 25%, coronary bypass graft surgery, and underwent an orthotopic heart transplant for his ischemic cardiomyopathy. He was diagnosed with mixed hyperlipidemia and was previously on simvastatin and pravastatin but discontinued therapy following development of rhabdomyolysis. His lipid profile while on 20 mg/day of pravastatin remained abnormal (total cholesterol of 293 mg/dL, triglycerides of 334 mg/dL, HDL-C of 44 mg/dL, LDL-C of 182 mg/dL). His APOE genotype was E3/E3 indicating he did not have type 3 hyperlipoproteinemia. He has undergone biweekly LDL apheresis for the past eleven years.

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Patient 7 was a 71-year-old Caucasian male who was suspected to have FH and diagnosed with severe coronary artery disease prior to an orthotopic heart transplant. None of his five siblings had a history of premature vascular events, although two had hyperlipidemia. His father died at age 77 and had heart disease in his early 50s while his mother had no history of heart disease. He was diagnosed with hypercholesterolemia and four years prior to initiation of apheresis therapy his lipid profile revealed hypertriglyceridemia (total cholesterol 270 mg/dL, triglycerides 555 mg/dL, HDL-C 35 mg/dL) which only modestly improved with dietary and pharmacological treatment. He continued to develop progressive atherosclerosis post cardiac transplant necessitating apheresis every 3 weeks for the last 6 years to treat his dyslipidemia. Lipid Evaluation

To determine whether the patient’s genetic test results correlate to their lipid profile phenotype, an indepth lipoprotein metabolism profile was performed on all seven patients. Results of pre- and post-apheresis concentrations of LDL-C, apolipoprotein B (apoB), and HDL-C are shown in Figure 2. There was a significant reduction in LDL-C following apheresis in all patients, but no difference noted between patients without identifiable LDLR mutations and those with LDLR mutations (average 265% and 260%, respectively). Similar overall reductions in apolipoprotein B concentrations were noted between the group without and with LDLR mutations (average 64% and 61%, respectively) postapheresis (Figs. 2a and 2b). Two patients (Patients 4 and 5) had elevated Lp(a) cholesterol pre-apheresis and apheresis treatment was successful in removing a significant portion of Lp(a) from circulation (Supporting Information Table 1). In contrast, apheresis did not significantly affect HDL-C or apolipoprotein A (Fig. 2c and Supporting Information Table 1). Individuals who possessed LDLR mutations had, on average, greater LDL-C (238 mg/dL) and apoB (191 mg/dL) concentrations pre-apheresis compared to those without mutations (138 mg/dL and 120 mg/dL, respectively). Comparison of pre-apheresis HDL-C concentrations in those with and without identifiable mutations revealed, on average, less HDL-C in those with LDLR mutations (36 mg/dL versus 45 mg/dL, respectively). A majority (n 5 6) of apheresis patients had elevated triglycerides of >150 mg/dL (mean 5 282 mg/dL) preapheresis, however mean triglyceride levels between the two groups did not differ (273 mg/dL in LDLR mutation-positive and 293 mg/dL in LDLR mutationnegative). Five of the patients (Patients 1–5) met the criteria for the familial combined hyperlipidemia (FCH) phenotype (triglycerides >133 mg/dL and ApoB > 120 mg/dL) [6]. Journal of Clinical Apheresis DOI 10.1002/jca

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centrations, we plotted our results for pre-apheresis total LDL particles on a population study comparing total LDL-P to LDL-C within the MESA population (Fig. 4) [7]. This comparison showed the total LDL particles measured pre-apheresis in those with mutations were farther from the population mean than those without identifiable mutations. However, a similar relationship was not observed in the large LDL particles (Fig. 3c). Interestingly, those without mutations had higher pre-apheresis levels of small HDL-P (28.1 vs. 17.8 mmol/L, respectively). In the post-apheresis samples, those with identifiable mutations had, on average, higher total LDL particle numbers and small LDL particle numbers as compared to those patients without mutations, whereas the large LDL particles did not show the same pattern. LDL apheresis caused a similar reduction in total LDL-P in those with and without

Fig. 2. Laboratory analysis of lipid markers pre- and postapheresis. Patients with identified LDLR mutations are shown in gray. Patients without identified mutations are shown in black. A: LDL-c, B: Apo-B, C: HDL-C.

Lipoprotein Particle Analysis

The apheresis patients lipoprotein particle profiles were analyzed by NMR spectroscopy because lipoprotein particles are heterogeneous in both size and cholesterol content (Fig. 3 and Supporting Information Table 1). In the pre-apheresis samples, those with identifiable mutations had higher total LDL particle concentrations and small LDL particle concentrations compared to those patients without mutations (3,268 nmol/L vs. 1,739 nmol/L and 2,806 nmol/L vs. 1,303 nmol/L, respectively). Because these patients have a unique starting threshold and varying total LDL-C conJournal of Clinical Apheresis DOI 10.1002/jca

Fig. 3. Laboratory analysis of lipoprotein particles from pre- and post-apheresis samples. Patients with identified LDLR mutations are shown in gray. Patients without identified mutations are shown in black. A: Total LDL-P, B: small LDL-P, C: large LDL-P.

Analysis of Dyslipidemic LDL Apheresis Patients

Fig. 4. Distribution of LDL-P in the seven study patients compared to the MESA population (gray). Patients with identified LDLR mutations are shown in black. Patients without identified mutations are shown in white. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

mutations (64% vs. 58%, respectively). However, apheresis did not significantly alter HDL-C, HDL-P, or apolipoprotein A concentrations. A genotype–phenotype relationship was not observed with total HDL-P, triglyceride-rich very low density lipoprotein (VLDL), or chylomicron particles (Supporting Information Table 1). DISCUSSION

In our cohort of seven LDL apheresis patients, four individuals were found to harbor a mutation in one of the known FH-causing genes, specifically LDLR (Table II). Interestingly, all of the patients with xanthomas or xanthelasmas noted on physical examination also harbored a disease-causing LDLR mutation. This correlation of genotype and clinical presentation in these patients is consistent with the current understanding about the role of the LDL receptor and the impact LDLR mutations can have on clinical presentation [8]. However, xanthomas/xanthelasmas do not present in all FH patients nor do they usually start to present until the end of the second decade of life, and only half of heterozygotes have xanthomas by the third decade (80% by the time of death) [1]. Thus, the presence or TABLE II.

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absence of lipid deposits may not be a reliable indicator of which suspected FH patients may or may not have a disease-causing mutation, and is dependent on the age of the patient. In our cohort, other than the presence of the xanthomas or xanthelasmas, there were no other distinguishing clinical features that were helpful in predicting which patients were likely to harbor a disease-causing mutation. Two patients (Patients 1 and 2) were found to have gene dosage mutations in LDLR. The theoretical consequence of these mutations is the synthesis of nonfunctional protein. Patient 1 was shown to have a duplication of exon 12, a region where a similar genetic defect has been previously reported to be associated with FH [9]. Patient 2 was shown to have a deletion of exons 17 and 18, also a genetic defect previously reported to associate with FH [10,11]. Other such gene dosage mutations in LDLR have been documented in reported FH patients and are assumed to result in non-functional LDL receptor protein. The mechanism of these kinds of large gene alterations is predicted to be homologous recombination between Alu sequences that have been identified in LDLR [12]. Two of the patients (Patients 3 and 4) were found to harbor point mutations in LDLR that had been previously reported in FH cases. Patient 3 had a nucleotide substitution in exon 7 (c.1012T>G), which resulted in the amino acid change p.Cys338Gly. This mutation is expected to negatively affect overall folding of the protein. Multiple reports of variation at LDLR residue p.Cys338 have been reported including nonsynonymous changes of this residue to glycine (Gly), serine (Ser), arginine (Arg), and tyrosine (Tyr), as well as a nonsense change (p.Cys338X) [13–17]. The p.Cys338Tyr variant occurs in the epidermal growth factor (EGR) precursor homology domain and functional studies have shown that binding, uptake, and degradation of iodinated LDL in skin fibroblasts from a patient homozygous for this variant was A) that abolished the splice donor site. The c.31311G>A mutation has an increased frequency in individuals of Norwegian descent and has been termed FH Elverum [15,18,19]. Different mutations at the same nucleotide, namely c.31311G>C and c.31311G>T, have also been previously reported [20,21]. Functional studies of c.31311G>A have been performed and demonstrated

LDLR Mutations Identified in Cohort

Patient

Gene

Nucleotide change

1 2 3 4

LDLR LDLR LDLR LDLR

Duplication of exon 12 Deletion of exons 17–18 c.1012T>G c.31311G>A

Amino acid change n/a n/a p.Cys338Gly n/a (intronic)

Significance Non-functional protein Non-functional protein Predicted misfolding Abolish splice donor site

Journal of Clinical Apheresis DOI 10.1002/jca

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that this mutation causes skipping of exon 3 and inclusion of intron 3, resulting in the absence of functional LDL receptor produced from this transcript [19]. In Patients 5–7 there were no disease-causing mutations observed in LDLR, APOB, or PCSK9. Overall, the mutation detection rate observed in our LDL apheresis cohort was 57%. Reported frequencies of causal mutations in FH patients vary widely with the method of detection and the selection of population included in the analysis. Not surprisingly, studies using targeted detection of a limited set of known mutations report lower frequencies compared to those using whole gene sequencing. However, even in those studies that employ whole gene sequencing technology there is a wide range of reported frequencies. In a study of 68 patients who were diagnosed with definite familial hypercholesterolemia (DFH, a patient population with the highest likelihood of genetically caused FH as defined by the presence of tendon xanthoma in the proband or a relative) the frequency of mutation identification was reported as 87% [22]. However, another study of 190 DFH patients found a mutation frequency in the LDL receptor of 56% [23]. While we observed a mutation detection rate of 57% in our cohort, the true frequency is likely limited by our small cohort size. The patient population in our study was diagnosed with FH but required LDL apheresis to maintain adequate lipid control. Also, all of our apheresis patients have been clinically diagnosed with therapyresistant FH but not all have a personal or family history of xanthoma. Therefore, our data show that it should not be assumed that the cohort of FH patients who require LDL apheresis have disease-causing mutations. The absolute cause of hypercholesterolemia in the three patients without identified mutations is unknown, but unlikely to be caused only by lifestyle given the complete lack of response to any lipid lowering agents. It is more probable that there are other genetic factors that place patients at risk for uncontrollable hypercholesterolemia either by causing an elevated LDL cholesterol level or causing the resistance to lipid lowering agents. It is possible that these patients are multi-gene or single-gene carriers for hyperlipidemia-causing mutations that have yet to be identified. As with all genetic tests, there are limitations to the testing in that mutations could occur in regions of the genes that were not analyzed, such as deep intronic or regulatory regions. Furthermore, APOB mutation analysis in this study covered only two common APOB mutations: p.R3500W and p.R3500Q. Thus, rare APOB variants could be present in these individuals in other areas of the gene. Of note, most of the apheresis patients at the time of our analysis displayed elevated triglycerides (mean5 282 mg/dL). FCH is characterized by elevated cholesterol and triglycerides although it is not genetically Journal of Clinical Apheresis DOI 10.1002/jca

defined. The mean plasma triglyceride concentration in patients with heterozygous FH is not significantly different from that of the general population, but it is not uncommon for FH patients to present with elevated plasma triglycerides [1,24]. One study in a Japanese cohort found greater than 20% of heterozygous FH patients presented with an FCH phenotype [25]. In addition, a study in Spain found the prevalence of FHcausing mutations in 143 unrelated patients that present with clinically diagnosed FCH was nearly 20% [26]. Here, in our small cohort, 3 out of 4 (75%) of LDLRmutation positive patients and 6 out of 7 (86%) overall presented pre-apheresis with hypertriglyceridemia. Patients with FH have been shown to have two-fold higher LDL particles compared to controls [27]. Our study indicates that it may be possible to further stratify FH patients who require LDL apheresis by their physical presentation and analysis of their phenotypic lipid profiles. Upon physical examination, all of the patients who had documented lipid deposits in the form of xanthomas or xanthelasmas were in the cohort of FH-patients with disease-causing mutations. After analysis of the patients’ lipid profiles, on average, those with disease causing mutations had a higher proatherogenic profile as compared to those without disease causing mutations. Specifically, those with mutations had, on average, lower HDL-cholesterol and HDL particles, and higher LDL-cholesterol, apolipoprotein B, total LDL particles, and small LDL particles in the samples collected immediately prior to their apheresis procedure. In addition, it is important to note that the increase in total LDL particles is not simply a result of increases in all subfractions of LDL particles since the large LDL particles were not, on average, higher in those with mutations as compared to those without. The comparison of LDL-P concentration to the MESA population study reveals the differences in patients with and without identifiable disease-causing mutations; namely that those with mutations fall farther from the mean LDL particle concentration within their LDL-C cohort of the population as compared to those without mutations (Fig. 4). Two patients (4 and 5) had elevated Lp(a) cholesterol pre-apheresis (reference range for healthy individuals by this method is < 3 mg/dL). Studies have demonstrated a synergistic effect of Lp(a) and elevated LDL-cholesterol on the development of coronary artery disease in the general population [28,29]. Therefore, it could be expected that elevated Lp(a) may have increased the risk of cardiovascular disease in patients with FH. However, conflicting results from studies have variably supported or not observed this association in FH heterozygotes (reviewed in Ref. 30). In our study, most of the patients had a significant history or personal and/or familial coronary artery disease, thus the elevated Lp(a) cholesterol in Patients 4 and 5 did

Analysis of Dyslipidemic LDL Apheresis Patients

not appear to put these patients at a higher risk for cardiovascular disease compared to the others in our small cohort. CONCLUSIONS

In this study, we show that not all FH patients that require routine LDL apheresis possess identifiable disease-causing mutations. However, those with identifiable mutations tended to have more proatherogenic lipid profiles as compared to those that did not have identifiable mutations. Traditional lipid profile and LDL particle distribution could potentially assist in stratifying patients who are more or less likely to have disease-causing mutations. A similar genotype–phenotype relationship was not observed with the patient’s VLDL and chylomicron particles indicating the genetic effect is specific to LDL particles. In addition to the small cohort size in this study, another caveat to this analysis is that many of the patients were still receiving statin therapy at the time of lipid profile analysis (Table I). Because lipoprotein subclasses may be affected post-treatment [31], particle analysis of a larger cohort of FH patients would be required to more rigorously analyze this genotype–phenotype particle relationship. Identifying genotype–phenotype correlations could potentially lead to a pharmacogenetic approach to FH where discovery of certain genotypes might prompt the implementation of more aggressive LDL-lowering therapies, such as LDL apheresis, at an earlier stage. REFERENCES 1. Kwiterovich PO Jr., Fredrickson DS, Levy RI. Familial hypercholesterolemia (one form of familial type II hyperlipoproteinemia). A study of its biochemical, genetic and clinical presentation in childhood. J Clin Invest 1974;53:1237–1249. 2. Goldberg AC, Hopkins PN, Toth PP, Ballantyne CM, Rader DJ, Robinson JG, Daniels SR, Gidding SS, de Ferranti SD, Ito MK, McGowan MP, Moriarty PM, Cromwell WC, Ross JL, Ziajka PE. Familial hypercholesterolemia: screening, diagnosis and management of pediatric and adult patients: clinical guidance from the National Lipid Association Expert Panel on Familial Hypercholesterolemia. J Clin Lipidol 2011;5(3 Suppl):S1–S8. 3. Goldberg AC, Hopkins PN, Toth PP, Ballantyne CM, Rader DJ, Robinson JG, Daniels SR, Gidding SS, de Ferranti SD, Ito MK, McGowan MP, Moriarty PM, Cromwell WC, Ross JL, Ziajka PE. Familial hypercholesterolemia: screening, diagnosis and management of pediatric and adult patients: clinical guidance from the National Lipid Association Expert Panel on Familial Hypercholesterolemia. J Clin Lipidol 2011;5:133–140. 4. Winters JL. Low-density lipoprotein apheresis: principles and indications. Semin Dial 2012;25:145–151. 5. Winters JL. Lipid apheresis, indications, and principles. J Clin Apher 2011;26:269–275. 6. Veerkamp MJ, de Graaf J, Hendriks JC, Demacker PN, Stalenhoef AF. Nomogram to diagnose familial combined hyperlipidemia on the basis of results of a 5-year follow-up study. Circulation 2004;109:2980–2985.

9

7. Otvos JD, Mora S, Shalaurova I, Greenland P, Mackey RH, Goff DC Jr. Clinical implications of discordance between lowdensity lipoprotein cholesterol and particle number. J Clin Lipidol 2011;5:105–113. 8. Bertolini S, Cantafora A, Averna M, Cortese C, Motti C, Martini S, Pes G, Postiglione A, Stefanutti C, Blotta I, Pisciotta L, Rolleri M, Langheim S, Ghisellini M, Rabbone I, Calandra S. Clinical expression of familial hypercholesterolemia in clusters of mutations of the LDL receptor gene that cause a receptor-defective or receptor-negative phenotype. Arterioscler Thromb Vasc Biol 2000;20:E41–E52. 9. Marduel M, Carrie A, Sassolas A, Devillers M, Carreau V, Di Filippo M, Erlich D, Abifadel M, Marques-Pinheiro A, Munnich A, Junien C, Boileau C, Varret M, Rabes JP, Farnier M, Luc G, Lecerf JM, Bruckert E, Bonnefont-Rousselot D, Giral P, Kalopissis A, Girardet JP, Polak M, Chanu B, Moulin P, Perrot L, Krempf M, Zair Y, Bonnet J, Ferrieres J, Ferrieres D, Bongard V, Cournot M, Reznick Y, Schlienger JL, Fredenrich A, Durlach V. Molecular spectrum of autosomal dominant hypercholesterolemia in France. Hum Mutat 2010;31:E1811– E1824. 10. Hobbs HH, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum Mutat 1992;1:445–466. 11. Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Annu Rev Genet 1990;24:133–170. 12. Lehrman MA, Russell DW, Goldstein JL, Brown MS. Alu-Alu recombination deletes splice acceptor sites and produces secreted low density lipoprotein receptor in a subject with familial hypercholesterolemia. J Biol Chem 1987;262:3354–3361. 13. Maruyama T, Miyake Y, Tajima S, Harada-Shiba M, Yamamura T, Tsushima M, Kishino B, Horiguchi Y, Funahashi T, Matsuzawa Y, Yamamoto A. Common mutations in the lowdensity-lipoprotein-receptor gene causing familial hypercholesterolemia in the Japanese population. Arterioscler Thromb Vasc Biol 1995;15:1713–1718. 14. Nauck MS, Scharnagl H, Nissen H, Schurmann C, Matern D, Nauck MA, Wieland H, Marz W. FH-Freiburg: a novel missense mutation (C317Y) in growth factor repeat A of the low density lipoprotein receptor gene in a German patient with homozygous familial hypercholesterolemia. Atherosclerosis 2000;151:525–534. 15. Lombardi MP, Redeker EJ, Defesche JC, Kamerling SW, Trip MD, Mannens MM, Havekes LM, Kastelein JJ. Molecular genetic testing for familial hypercholesterolemia: spectrum of LDL receptor gene mutations in The Netherlands. Clin Genet 2000;57:116–124. 16. Kotzer MS, Baudhuin L. Novel human pathological mutations. Gene symbol: LDLR. Disease: hypercholesterolemia. Hum Genet 2009;126:352. 17. Varret M, Rabes JP, Thiart R, Kotze MJ, Baron H, Cenarro A, Descamps O, Ebhardt M, Hondelijn JC, Kostner GM, Miyake Y, Pocovi M, Schmidt H, Schuster H, Stuhrmann M, Yamamura T, Junien C, Beroud C, Boileau C. LDLR Database (second edition): new additions to the database and the software, and results of the first molecular analysis. Nucleic Acids Res 1998;26:248–252. 18. Leren TP, Solberg K, Rodningen OK, Tonstad S, Ose L. Two founder mutations in the LDL receptor gene in Norwegian familial hypercholesterolemia subjects. Atherosclerosis 1994;111: 175–182. 19. Cameron J, Holla OL, Kulseth MA, Leren TP, Berge KE. Splice-site mutation c.31311, G>A in intron 3 of the LDL receptor gene results in transcripts with skipping of exon 3 and inclusion of intron 3. Clin Chim Acta 2009;403:131–135. 20. Lombardi P, Sijbrands EJ, van de Giessen K, Smelt AH, Kastelein JJ, Frants RR, Havekes LM. Mutations in the low Journal of Clinical Apheresis DOI 10.1002/jca

10

21.

22.

23.

24.

25.

Donato et al. density lipoprotein receptor gene of familial hypercholesterolemic patients detected by denaturing gradient gel electrophoresis and direct sequencing. J Lipid Res 1995;36:860–867. Jensen HK, Jensen LG, Hansen PS, Faergeman O, Gregersen N. Two point mutations (313 1 1G –> A and 313 1 1G –> T) in the splice donor site of intron 3 of the low-density lipoprotein receptor gene are associated with familial hypercholesterolemia. Hum Mutat 1996;7:269–271. Graham CA, McIlhatton BP, Kirk CW, Beattie ED, Lyttle K, Hart P, Neely RD, Young IS, Nicholls DP. Genetic screening protocol for familial hypercholesterolemia which includes splicing defects gives an improved mutation detection rate. Atherosclerosis 2005;182:331–340. Taylor A, Wang D, Patel K, Whittall R, Wood G, Farrer M, Neely RD, Fairgrieve S, Nair D, Barbir M, Jones JL, Egan S, Everdale R, Lolin Y, Hughes E, Cooper JA, Hadfield SG, Norbury G, Humphries SE. Mutation detection rate and spectrum in familial hypercholesterolaemia patients in the UK pilot cascade project. Clin Genet 2010;77:572–580. Schrott HG, Goldstein JL, Hazzard WR, McGoodwin MM, Motulsky AG. Familial hypercholesterolemia in a large indred. Evidence for a monogenic mechanism. Ann Intern Med 1972; 76:711–720. Bujo H, Takahashi K, Saito Y, Maruyama T, Yamashita S, Matsuzawa Y, Ishibashi S, Shionoiri F, Yamada N, Kita T. Clinical features of familial hypercholesterolemia in Japan in a database from 1996–1998 by the research committee of the ministry of health, labour and welfare of Japan. J Atheroscler Thromb 2004;11:146–151.

Journal of Clinical Apheresis DOI 10.1002/jca

26. Civeira F, Jarauta E, Cenarro A, Garcia-Otin AL, Tejedor D, Zambon D, Mallen M, Ros E, Pocovi M. Frequency of lowdensity lipoprotein receptor gene mutations in patients with a clinical diagnosis of familial combined hyperlipidemia in a clinical setting. J Am Coll Cardiol 2008;52:1546–1553. 27. Jarauta E, Mateo-Gallego R, Gilabert R, Plana N, Junyent M, de Groot E, Cenarro A, Masana L, Ros E, Civeira F. Carotid atherosclerosis and lipoprotein particle subclasses in familial hypercholesterolaemia and familial combined hyperlipidaemia. Nutr Metab Cardiovasc Dis 2012;22:591–597. 28. Armstrong VW, Cremer P, Eberle E, Manke A, Schulze F, Wieland H, Kreuzer H, Seidel D. The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis. Dependence on serum LDL levels. Atherosclerosis 1986;62:249–257. 29. Cremer P, Nagel D, Mann H, Labrot B, Muller-Berninger R, Elster H, Seidel D. Ten-year follow-up results from the Goettingen Risk, Incidence and Prevalence Study (GRIPS). I. Risk factors for myocardial infarction in a cohort of 5790 men. Atherosclerosis 1997;129:221–230. 30. Goldstein JL Hobbs HH, Brown MS, Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis SE, Ballabio A, editors. The Online Metabolic and Molecular Bases of Inherited Disease. Chapter 120: Familial Hypercholesterolemia: 1-138. New York: McGraw-Hill; 2013. 31. Bays H, Conard S, Leiter LA, Bird S, Jensen E, Hanson ME, Shah A, Tershakovec AM. Are post-treatment low-density lipoprotein subclass pattern analyses potentially misleading? Lipids Health Dis 2010;9:136.