Genetic and biochemical characteristics of patients with hyperlipidemia who require LDL apheresis

Transcription

1 Genetic and biochemical characteristics of patients with hyperlipidemia who require LDL apheresis Mato Nagel, Ioan Duma, Constantina Vlad, Mandy Benke, Sylvia Nagorka Zentrum für Nephrologie & Stoffwechsel, Weißwasser, 02943, Germany; Abstract. Hyperlipidemia is a well documented risk factor for premature atherosclerosis, cardiovascular disease and myocardial infarction. Lowering the blood lipid levels is therefore an important therapeutic approach involving changes in lifestyle, pharmacotherapy and in most serious cases also LDL apheresis. Among multiple causes of hyperlipidemia, mutations that involve at least one of the four genes: APOB, LDLR, LDLRAP1 and/or PCSK9 play a significant role. Other ones may also be involved. In current study we examined the genotype and phenotype of patients with hyperlipidemia that didn t respond well just to pharmacotherapy or developed adverse reactions and were therefore subjected to LDL apheresis. Eleven patients undergoing regular LDL apheresis were tested for their lipid levels before inception of this special treatment and then perpetually before and after apheresis treatment. Four of them had high LDL (M=3,73mmol/l SD±2,08) and Lp(a) (M=163,5mg/dl SD±14,47) levels, four had higher LDL (M=8,4mmol/l SD±3,81) but lower Lp(a) (M=45,6mg/dl SD±13,51) levels and three had lower LDL (M=2,05mmol/l SD±0,35) and higher Lp(a) (M=120,2mg/dl SD±29,41). All patients have been screened for mutations or SNPs in ABCA1, ABCB1, ABCG2, ANGPTL4, APOB, APOC3, APOE, CETP, GPIHBP1, HMGCR, LDLR, LDLRAP1, LIPA, LIPC, LMF1, LPA, LPL, PCSK9, SLC12A3 and SLCO1B1. Altogether we found 9 mutations and 110 noteworthy SNPs. Four mutations were located within the well established LDLR gene (67+2T>A, 662A>G, 798T>A, 2312-?_2389+?del) while other five were in ABCA1 (4760A>G), COQ2 (A117V), LIPC (1214C>T) and LPA ( T>C, 5673A>G). Among single nucleotide polymorphisms 47 were reported to significantly alter lipid levels, 37 have minor influence and 26 have a yet unknown effect. Conclusion. Although our sample was limited to a small group of patients we discovered a large number of mutations and relevant SNPs that allowed for an individual interpretation of lipid pathophysiology and should be considered for further analysis in larger studies. Key words: LDL apheresis, dyslipidemia, hypercholesterolemia, hypertriglyceridemia, lipoproteins

2 Objective. Our aim was to investigate biochemical lipid profile of patients undergoing LDL apheresis and to identify mutations and single nucleotide polymorphisms present in genes associated with abnormal lipid metabolism or with statine pharmacokinetics. Methods Participants. All participants in this study (n = 11), adults of European origin, were selected from patients that regularly undergo lipid apheresis at the Zentrum für Nephrologie & Stoffwechsel in Weißwasser, Germany. They all have risk factors and conditions that qualified them for apheresis as indicated by German law 1 which includes high lipid plasma concentrations in spite of changes made to their previous lifestyle and medication. Biochemical blood analyses were performed using standard laboratory procedures. The patients were tested for total cholesterol, high-density lipoproteins (HDL), low-density lipoprotein (LDL), triglyceride (TG), apolipoprotein A1 (ApoA), apolipporotein B (ApoB) and lipoprotein(a) (Lp(a)). Gene selection and genotyping. Genes: ABCA1, ABCB1, ABCG2, ANGPTL4, APOA5, APOB, APOC3, APOE, CETP, CLDN19, COQ2, EGF, GPIHBP1, HMGCR, KCNA1, LDLR, LDLRAP1, LIPA, LIPC, LMF1, LPA, LPL, PCSK9, SEIL1L, SLC12A3 and SLCO1B1 were selected based on literature reporting their association with lipid metabolism, or their influence on the pharmacokinetics of lipid lowering medication. Genotyping was performed using targeted next-generation sequencing. The vast majority of our patients were males (n=9). Average age of the subjects was SD±12.98 years (youngest: 32, oldest: 70) with majority (n=9) of them being over 50 years. Two patients were young: 32 and 34 years respectively. One patient needed apheresis twice per month while most of them required the procedure once weekly (n=10). Patients in our cohort presented as risk factors: weight problems (n=8), insulin dependent type 2 diabetes (present in 3 patients) and some were smokers or abused nicotine in the past (none was smoker at the time they started LDL apheresis). Patient Age Sex Lipid apheresis Risk factors From Frequency M 2016 Once weekly M 2009 Once weekly T2D, overweight F 2014 Twice monthly overweight M 2015 Once weekly T2D, overweight M 2016 Once weekly overweight M 2009 Once weekly T2D, overweight M 2014 Once weekly overweight, ex-smoker M 2016 Once weekly overweight M 2010 Once weekly ex-smoker M 2016 Once weekly overweight, ex-smoker F 1999 Once weekly ex-smoker 1. Kassenärztliche Bundesvereinigung: Zum Beschluss des Bundesausschusses der Ärzte und Krankenkassen vom zu den therapeutischen Apheresen. Deut Ärztebl. 2003; 100: A2033:

3 All patients had their cardiovascular system affected by high plasma lipid values suffering from: myocardial infarction (n=4), brain vessel sclerosis (n=1), carotid artery stenosis (n=2), coronary thrombosis (n=2), coronary artery disease (n=3), atherosclerosis (n=1) and, hypertension (n=10), apoplexy (n=1) According to their biochemical lipid profile measured before undergoing apheresis we distinguished three main groups of patients : - Group I (n=4) had high plasma concentrations of LDL(8,4 mmol/l SD±3,81), TG (2,85mmol/l SD±2,63), ApoA (1,56g/l SD±0,26), ApoB (1,86g/l SD±0,65) and low Lp(a) (45,6mg/dl SD±13,5). - Group II (n=3) had low plasma concentration of LDL (2,05mmol/l SD±0,35), TG (0,81mmol/l SD ±0,02), ApoA (1,54g/l SD ±1,14), ApoB (0,69g/l SD ±0,05) and high Lp(a) (120,2mg/dl SD ±29,41). - Group III (n=4) had high plasma concentrations of LDL (3,73mmol/l SD ±2,08), TG (1,12mmol/l SD ±0,35), ApoA (1,71g/l SD ±0,56), ApoB (1,003g/l SD ±0,62) and Lp(a) (163,5mg/dl SD ±14,47). Group I. Lp(a) < 60mg/dl LDL 2,6 mmol/l Group II. Lp(a) 60mg/dl LDL < 2,6 mmol/l Group III. Lp(a) 60mg/dl LDL 2,6 mmol/l Before apheresis After apheresis Before apheresis After apheresis Before apheresis After apheresis HDL 1,45 (SD ±0,14) 1,175 (SD ±0,377) 1,55 (SD ±0,21) 1,13 (SD ±0,23) 1,64 (SD ±0,88) 1,00 (SD ±0,2) LDL 8,4 (SD ±3,81) 1,55 (SD ±1,103) 2,05 (SD ±0,35) 0,56 (SD ±0,05) 3,73 (SD ±2,08) 1,4 (SD ±0,32) TG 2,85 (SD±2,63) 2,4 (SD ±2,011) 0,81 (SD ±0,02) 1,1 (SD ±0,95) 1,12 (SD ±0,35) 1,35 (SD ±0,77) ApoA 1,56 (SD ±0,26) 1,43 (SD ±0,229) 1,54 (SD ±1,14) 1,35 (SD ±0,09) 1,71 (SD ±0,56) 1,32 (SD ±0,25) ApoB 1,86 (SD ±0,65) 0,53 (SD ±0,22) 0,69 (SD ±0,05) 0,23 (SD ±0,01) 1,003 (SD ±0,62) 0,46 (SD ±0,11) Lp(a) 45,5 (SD ±13,5) 27,35 (SD ±30,49) 120,2 (SD ±29,41) 35,9 (SD ±9,93) 163,5 (SD ±14,4) 48,85 (SD ±28,27) Cholesterol 11,72 (SD ±2,29) 4,2 (SD ±1,66) 3,75 (SD ±0,21) 2,03 (SD ±0,15) 5,81 (SD ±1,46) 2,92 (SD ±0,76)

4 Genetical investigations performed on 26 genes within our study group revealed a number of 110 SNPs and 9 mutations. All SNPs and mutations were already known and reported ( Mutations found in our cohort were located in ABCA1, COQ2, LDLR, LIPC and LPA genes. Gene Exon/ Location Position Protein change cdna SNP No of patients Effects/Phenotype ABCA p.k1587r(aaa>aga) c.4760a>g rs /1 Low HDL COQ p.a117v(gcg>gtg) - - -/1 damaging LDLR 1 23 IVS1+2T>A c.67+2t>a rs /2 FH, Hypercholesterolemia LDLR p.d221g(gac>ggc) c.662a>g rs /1 FH, Hypercholesterolemia LDLR p.d266e(gat>gaa) c.798t>a rs /2 FH, Hypercholesterolemia LDLR _797 EX16del c.2312-?_2389+?del - -/1 FH LIPC p.t405m(acg>atg) c.1214c>t rs /1 FH LPA c t>c rs /1 High Lp(a), CAD LPA p.i1891m(ata>atg) c.5673a>g rs /3 High Lp(a), CAD Within the ABCA1 gene, mutation c.4760a>g (rs ) significantly associated with decreased HDL levels (Frikke-Schmidt et al. 2008, Boes et al. 2009, Clee et al. 2011) was found in three patients in hetero- and homozygous state. All three carriers of the mutation in our cohort had low HDL levels and in addition two of them carrying the mutation in homozygous state suffered from vascular diseases or myocardial infarction before their 30th birthday. Our data although deprived of statistical power seem to confirm that mutation is associated with decreased levels of HDL. Almost half of mutations (n=4) were identified in LDLR gene. These were present among four members of our group. The most affected patients with high lipid levels at a young age and with severely damaged cardiovascular system, had two mutations in LDLR: rs and rs (c.67+2t>a) associated with hypercholesterolemia and high risk of CAD (Amsellem et al. 2002). Mutation rs reported to meet at least one of three criteria considered pathogenic for familial hypercholesterolemia (Khera et al. 2016) was found in heterozygote state in a third patient. Mutation: c.2312-?_2389+?del considered pathogenic for familial hypercholesterolemia (Mardue et al. 2010) was also identified in a fourth patient. Mutations T>C (rs ) and 5673A>G (rs ) in LPA gene are associated with increased levels of Lp(a) and higher CAD risk. Rs was identified in one patient with low LDL and high Lp(a) levels suffering from carotid artery stenosis and undergoing multiple bypass and stent implants. Rs was present in heterozygous state in three patients with very high Lp(a) levels suffering from coronary artery disease and undergoing multiple bypass operations. Mutation 1214C>T (rs ) in LPIC was found in one patient suffering from brain vessel sclerosis who also had highest TG levels among all subjects of the study group.

5 We found in our cohort 110 SNPs within ABCA1, ABCB1, ABCG2, ANGPTL4, APOB, APOC3, APOE, CETP, GPIHBP1, HMGCR, LDLR, LDLRAP1, LIPA, LIPC, LMF1, LPA, LPL, PCSK9, SLC12A3 and SLCO1B1. genes. Among these 6 SNPs in ABCB1 (n=1), HMGCR (n=1) and SLCO1B1 (n=5 (table 1) are reported to significantly influence statine efficiency. Table 1. SNPs associated with statine pharmacokinetics Gene Exon Position Protein change cdna SNP No of Patients We also report in our group 13 SNPs that were previously found to be significantly associated with high LDL levels. These were found in in ABCB1 (n=1), APOB (n=7), APOC3 (n=1), APOE (n=1) LDLR (n=1) and PCSK9 (n=2) are significantly associated with high levels of LDL (table 2). Table 2. SNPs associated with high levels of Low Density Lipoprotein Gene Exon Positio Protein change cdna SNP No of n patients MAF (1000 Genome) MAF (1000 Genome) Expected/ Observed allele ABCB p.i1145i(att>atc) c.3435t>c rs / /1979 9/8 HMGCR p.i638v(ata>gta) c.1912a>g rs5908 -/ /25 0/1 SLCO1B p.n130d(aat>gat) c.388a>g rs / /1891 8/10 SLCO1B p.p155t(cct>act) c.463c>a rs / /325 1 / 2 SLCO1B p.v174a(gtg>gcg) c.521t>c rs / /439 2/3 SLCO1B p.l191l(ttg>ctg) c.571t>c rs / /1840 8/12 Expected/ Observed allele ABCB p.g412g(ggt>ggc) c.1236t>c rs / /2084 9/14 APOB 4 98 p.t98i(acc>atc) c.293c>t rs / /848 4/7 APOB p.t2515t(acc>act) c.7545c>t rs693 3/ /1257 6/11 APOB p.p2739l(cca>cta) c.8216c>t rs / /1834 8/2 APOB p.r3638q(cgg>cag) c.10913g>a rs / /193 1 / 2 APOB p.e4181k(gaa>aaa) c.12541g>a rs / /640 3 / 4 APOB p.r4270t(agg>acg) c.12809g>c rs / /314 1/1 APOB p.s4338n(agt>aat) c.13013g>a rs / /1855 8/20 APOC p.g34g(ggt>ggc) c.102t>c rs4520 5/ /2018 9/15 APOE p.c130r(tgc>cgc) c.388t>c rs / /754 3/ 4 LDLR p.r471r (AGG>AGA) c.1413g>a rs688 3/ /1381 6/11 PCSK p.v474i(gtc>atc) c.1420g>a rs / /656 3/19 PCSK p.g670e(ggg>gag) c.2009g>a rs / /506 2/22

6 16 SNPs in our group were significantly associated with high TG levels (table 3) in ABCB1 (n=1), APOA5 (n=1), APOB (n=2), GPIHBP1 (n=3), LDLR (n=1), LIPIC (n=1), LMF1 (n=4), LPL (n=2) and PCSK9 (n=1). Among all SNPs rs had highest correlation with TG levels in our cohort (r=0.97). Among SNPs associated with CAD we report in our group: rs in ABCG2, rs from APOB and rs5883 in CETP. Other 16 SNPs associated with low HDL levels, high Lp(a) or total cholesterol were also present in our group. Table 3. SNPs associated with high Triglycerid levels Gene Exon Position Protein change cdna SNP No of patients MAF (1000 Genome) Expected / Observed allele ABCB p.g412g(ggt>ggc) c.1236t>c rs / /2084 9/14 APOA p.v153m(gtg>atg) c.457g>a rs / /412 2/ 1 APOB p.r4270t(agg>acg) c.12809g>c rs / /314 1/ 1 APOB p.s4338n(agt>aat) c.13013g>a rs / /1855 8/20 GPIHBP1 1 4 p.l4l(ctc>ctt) c.12c>t rs / /707 3/6 GPIHBP p.c14f(tgc>ttc) c.41g>t rs / /795 3/1 GPIHBP p.t98t(acc>act) c.294c>t rs / /1 0/1 LDLR p.r744r (CGG>CGA) c.2232g>a rs5927 6/ /1101 5/16 LIPC p.t224t(acc>acg) c.672c>g rs6084 4/ /152 7/12 LMF p.t102t(acg>aca) c.306g>a rs / /1807 8/6 LMF p.t180t(acg>aca) c.540g>a rs / /1255 6/6 LMF p.g181g(ggg>gga) c.543g>a rs / /1913 8/8 LMF p.a252a(gcg>gca) c.756g>a rs / /887 4/2 LPL p.e145e(gag>gaa) c.435g>a rs248 -/ /194 1/ 2 LPL p.t388t(acc>aca) c.1164c>a rs316 -/ /764 3/3 PCSK p.v474i(gtc>atc) c.1420g>a rs / /656 3/19 Conclusion. In spite the small size of our study group we found a large number of mutations and SNPs. Putting aside the already well known mutations and SNPs we report a considerable number of SNPs that should be considered for further analysis in larger studies as possible involved in hyperlipidemia.