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A New PCSK9 Gene Promoter Variant Affects Gene Expression and Causes Autosomal Dominant Hypercholesterolemia

Laboratorio de Estudios Genéticos (S.B., A.-B.G.-G., S.M.-H., C.I., V.G.-A., F.J.C.), Fundación Investigación Hospital Clínico Universitario de Valencia, and Instituto de Biomedicina de Valencia (S.V., M.C.), E-46010 Valencia, Spain; Servicio de Endocrinología (S.M.-H., J.F.A., J.T.R., R.C.), Hospital Clínico Universitario de Valencia, Universidad de Valencia, E-46022 Valencia, Spain; Servicio de Medicina Interna (J.C.M.-E.), Hospital Rio Hortega, E-47005 Valladolid, Spain; and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (S.B., A.-B.G.-G., S.M.-H., V.G.-A., J.F.A., J.T.R., R.C., F.J.C.)

Address all correspondence and requests for reprints to: F. Javier Chaves, Fundación de Investigación Hospital Clínico, Universitario de Valencia, Avda. Blasco Ibáñez 17, E-46010 Valencia, Spain. E-mail: felipe.chaves{at}uv.es.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Autosomal dominant hypercholesterolemia (ADH) is a genetic disorder characterized by increased low-density lipoprotein (LDL)-cholesterol levels, leading to high risk of premature cardiovascular disease. More than 900 mutations in LDL receptor, six in APOB and 10 in PCSK9 have been identified as a cause of the disease in different populations. All known mutations in PCSK9 causing hypercholesterolemia produce an increase in the enzymatic activity of this protease. Up to now, there are data about the implication of PCSK9 in ADH in a low number of populations, not including a Spanish population.

Objective: The objective of the study was to study the prevalence of PCSK9 mutations in ADH Spanish population.

Participants: We screened PCSK9 gene in 42 independent ADH patients in whom mutations in LDL receptor and APOB genes had been excluded.

Results: None of the known mutations causing ADH was detected in our sample, but we found two variations in the promoter region that could cause ADH, c.-288G>A and c.-332C>A (each in one proband). The analysis of the effect of these two variations on the transcription activity of the PCSK9 promoter showed that c.-288G>A did not modify the transcription, whereas c.-332C>A variant caused a 2.5-fold increase when compared with the wild-type sequence, either with or without lovastatin.

Conclusions: PCSK9 is a rare cause of ADH in Spanish population and, up to what we know, none of the previously described mutations has been detected. We have identified a new mutation that could cause ADH by increasing the transcription of PCSK9.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
Autosomal dominant hypercholesterolemia (ADH) (OMIM 143890) is a frequent genetic disorder being present in about one in 500 people in open Caucasian populations. It is characterized by increased levels of low-density lipoprotein cholesterol (LDL-C) in plasma, which can produce skin and tendon xanthomas and, overall, premature cardiovascular disease. Up to now, ADH is known to result from mutations at three main loci, although other loci could also be involved. Two loci are well characterized: LDLR (encoding the low density lipoprotein receptor) and APOB (encoding apolipoprotein B100); mutations in these genes cause familial hypercholesterolemia (OMIM 143890) and familial defective apolipoprotein B (FDB) (OMIM 144010), respectively. More than 900 mutations in LDLR gene have been described, whereas only six in APOB gene causing FDB have been identified (1, 2, 3, 4). Other mutations in the ApoB gene can cause hypobetalipoproteinemia (OMIM 107730). Recently it has been demonstrated that specific mutations increasing activity in proprotein convertase subtilisin/kexin type 9 precursor (PCSK9; OMIM 607786) are responsible for ADH, whereas those reducing PCSK9 activity are associated with lower LDL-C levels (5, 6, 7).

Human PCSK9 gene is approximately 22 kb long, comprising the promoter region and 12 exons, and it is located on chromosome 1p32. The gene produces an mRNA of 3636 bp encoding a 692-amino acid protein. This protein, also called neural apoptosis regulated convertase, is a serine protease belonging to the protease K subfamily of subtilases. It is a subfamily of proteases largely involved in the processing of inactive precursor proteins to the active product and seems to be involved in the inactivation and degradation of LDLR (8, 9, 10). Several studies have found that mutations increasing the activity of PCSK9 result in an increase of LDL-C levels whereas those reducing PCSK9 activity decrease LDL-C levels (5, 7, 11, 12, 13). The regulation of LDL-C levels has been related to LDLR-dependent and -independent pathways (14, 15, 16, 17, 18).

Several studies have aimed to identify mutations in PCSK9 causing ADH in a limited number of populations, but no data are available on the prevalence of these mutations in Spanish population (11, 12, 19, 20, 21, 22). Previous reports by our group have described mutations in LDLR and APOB genes causing ADH in our population (23, 24, 25, 26). Thus, the aim of this study was to establish the importance of PCSK9 as a cause of ADH in Spanish population and to characterize the mutations responsible for it.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients and controls

The study was conducted in 42 ADH probands attending our Lipid Clinic at the Hospital Clínico Universitario (Valencia, Spain) in whom mutations in LDLR or APOB genes had been previously excluded. The initial group was composed by approximately 150 ADH patients. ADH was diagnosed following the Med Ped criteria, summarized as plasma levels of total cholesterol (TC) and LDL-C higher than the 95th percentile corrected for both age and sex, together with presence of tendon xanthomas, coronary artery disease in the proband or in a first degree relative, and bimodal distribution of TC and LDL-C plasma levels in the family (autosomal dominant pattern of lipid IIa phenotype). Participants were not under cholesterol-lowering therapy at the time of sample extraction. All subjects were Caucasian and lived in the Valencian community (Spain).

A control group of 450 normocholesterolemic subjects with LDL-C and TC levels lower than percentile 70 and without treatments capable of influencing cholesterol levels was also studied. Individuals with glucose alterations were excluded. Patients and controls were not matched.

The protocol was approved by the institutional ethics committee and all subjects gave their written consent.

Genetic analysis

DNA was extracted as described or using Chemagic system (Chemagen, Baesweiler, Germany) from whole blood (23). RNA was extracted from lymphocytes after separation with Ficoll-Hypaque.

In all patients the presence of mutations in LDLR or APOB genes was excluded by previous analysis of these genes as previously described (25). In brief, LDLR promoter region and exons (including intronic junctions) were sequenced to detect small mutations and analyzed by Southern blot and semiquantitative procedure to detect large rearrangement (23, 27). Regions of exons 26 and 29 of APOB gene, in which all mutations causing FDB have been found, were sequenced (3).

For PCSK9 analysis, we sequenced the promoter region (–1000 bp upstream from ATG), all exons and intronic boundaries (a minimum of 50 bp in each intron sequence was analyzed). Primers were designed by Primer3 software and synthesized by Sigma (St. Louis, MO) (28).

Database for LDLR mutations is accessible at http://www.hgmd.cf.ac.uk/ac/index.php. Single-nucleotide polymorphism (SNP) databases can be consulted (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=255738&chooseRs=all).

Genetic sequences used as reference for PCSK9 are the following: for mRNA, accession no. NM_174936.2; for genomic sequence, accession no. NC_000001.1.

pGL3-PCSK9 promoter-reporter gene constructs

A fragment of the human PCSK9 promoter from nucleotides –1028 to –1 relative to the translation initiation site was amplified with the following primers: 5-CATCTGCAAGGGAGGATCATAAATTC-3 (forward) and 5-GGAGCTGACGGTGCCCATGGGGGCCAGGGGAGA GG-3 (reverse) from heterozygote DNA samples for mutations c.-332C>A and c.-288G>A. PCR products (wild type and variants) were cloned into pGEMTEasy vector (Promega, Madison, WI) and then subcloned into SacI/NcoI sites of pGL3basic vector (Promega). The sequence and integrity of each construct were verified by sequencing in both directions.

Cell culture and luciferase assay

For promoter assays, NIH3T3 or HepG2 cells were grown in DMEM containing 25 mM glucose, supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) at 37 C in a humidified atmosphere of 5% CO₂. Plasmid transfections were performed on attached cells at 70% confluence. Cells were transfected by Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA) with 750 ng of pGL3-PCSK9 reporter constructs (wild type or mutated) and 40 ng of a promotorless Renilla luciferase construct (pRL-0). When indicated, 10 µM lovastatin (Merck Sharp & Dohme, Madrid, Spain) was included. Transactivation activities were measured 24 h after transfection in a Wallac 1420 VICTOR luminometer using a dual-luciferase reporter assay system (Promega), following the manufacturer’s instructions. Relative light units were determined by quantification of the signal from the Firefly luciferase, normalized with cotransfected Renilla luciferase activity in the same sample. Finally, these relative values were normalized against mock transfection. Each expression construct was transfected in triplicate wells. The experiments were repeated three times.

PCSK9 expression and plasma measurement

To measure the expression of PCSK9 in plasma from patients carrying c.-332C>A mutation, RNA was extracted from lymphocytes and cDNA was obtained using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA). Real-time PCR was performed using SYBGreen (Applied Biosystems) and primers amplifying exons 3–4 of the gene. A fragment of the major histocompatibility complex, class I, G (b2 microglobulin) gene (HLA-G) was used as housekeeping. Amplification plots and dissociation curves were obtained. Ratio [PCSK9 threshold cycle (Ct)/HLA-G Ct] was calculated for each sample.

To quantitate PCSK9 from plasma, ProteoPrep Blue albumin and IgG depletion kit (Sigma; PROTBA) was used to remove albumin and IgG from eight samples of human plasma. HepG2 extract (40 µg) was the positive control. Twenty-five microliters of human depleted plasma were loaded onto SDS-PAGE gels. Proteins were separated and transferred to polyvinyl difluoride membrane (Roche, Stockholm, Sweden). For immunoblotting the primary goat anti-PCSK9 (ab28770; Abcam, Cambridge, UK), used at 1:250, was incubated overnight at 4 C. Membrane was next incubated with peroxidase-labeled rabbit antigoat IgG (Pierce, Rockford, IL) at 1:5000 for 60 min at room temperature. Proteins were visualized using enhanced chemiluminescence and exposure to x-ray film.

Statistical analysis

Values are given as mean ± SD. One-way ANOVA with Bonferroni correction was used to analyze differences between groups. P <0.05 was considered significant.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Clinical data

Clinical characteristics of 42 ADH subjects without LDLR and APOB gene mutations and controls are shown in Table 1. As expected, their LDL-C and TC levels are higher in patients than in controls.

We sequenced promoter, exons, and its corresponding intron-exon boundaries and found several known polymorphisms, one not previously described intronic polymorphism and two new variations (c.-332C>A and c.-288G>A) in the promoter region (Table 2).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Schematic representation of human PCSK9 promoter. A, The Mat Inspector 7.4 software was used to detect consensus motifs. Position –1 was fixed to the nucleotide preceding the initial ATG codon. The position of studied genetic variations and nuclear transcription factor Y (NF-Y) and SP1 binding sites are indicated. B, Nucleotide sequences corresponding to the wild-type (WT) (c.-332C) and mutated (c.-332A) probes used in the EMSA. The core SREBP binding site sequences and c.-332C>A variation are indicated with opened boxes. Putative Sp1 site suggested by Jeong et al. (29 ) is indicated.

Mutation effect

Analysis using MatInspector software and previous publications (29, 30) suggest that putative binding sites for sterol regulatory binding proteins (SREBP), SP1 and nuclear transcription factor Y might regulate the activity of PCSK9 human promoter (Fig. 1). The regulation of PCSK9 expression by depletion of sterol levels and statins has been previously reported, which indicates that SREBP transcription factors could be responsible for this regulation (30).

Both variants are located close to the core SRE in PCSK9 promoter. Furthermore, c.-332C>A is located inside a Sp1 site recently described (29). Therefore, we analyzed their ability to modify its promoter transcription activity.

Luciferase reporter analysis indicated that c.-288G>A variant had no significant effect, whereas c.-332C>A variant caused a 2.5-fold increase in PCSK9 promoter activity relative to wild-type construction activity when transfected in HepG2 cells (Fig. 2). Similar results were obtained in 3T3 cells (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Transcription activity analysis of the –1028/–1 region of PCSK9 human promoter. NIH3T3 and HepG2 cells were transiently transfected with 750 ng of PCSK9 luciferase reporter constructs plus 40 ng of promotorless-renilla plasmid. Luciferase activities were normalized against the internal control Renilla values. The data represent mean ± SEM (n = 3) and are expressed as fold induction respect empty vector (pGL3basic). *, P < 0.005; **, P < 0.05.

Mutant PCSK9 expression and plasma measurements

Once the effect of c.-332C>A mutations in vitro was shown, the question whether this mutation was able to modify PCSK9 gene expression and protein levels in carrying patients arose. Although it has been shown that lymphocytes do not express PCSK9, we decided to try to quantify lymphocytes expression to check this possibility. RNA was extracted from lymphocytes from the mutant PCSK9 proband and her relative: two familial hypercholesterolemia probands with mutations in LDLR gene (p.C660X and p.V408M) and two controls without any mutation in LDLR nor PCSK9 genes. The mutant PCSK9 proband’s relative was taking plant stanol esters with a lipid-lowering diet at the moment of the extraction (Benecol; Kaiku Corporation Alimentaira, Urnieta, Spain). Therefore, controls used in the experiment took the same supplement for 3 wk, and samples before and after this treatment were collected.

Real-time PCR was performed as explained in Patients and Methods. PCSK9 expression was very low in all the individuals and almost undetectable in controls and patients with LDLR gene mutations (in the level of 0–1 copy). However, PCSK9 expression in both patients with c.-332C>A was detectable in a range from 1 to 10 copies. PCSK9 expression was calculated as the ratio of PCSK9 to HLA-G Cts. Results obtained were the following: 1.958 ± 0.223 for controls (with and without Benecol supplementation), 1.650 ± 0.014 for c.-332C>A patients and 1.153 + 0.081 for LDLR gene mutation carriers. Patients with LDLR or PCSK9 mutations are under the same condition of hypercholesterolemia and when comparing their PCSK9 expression differences are significant (P 0.05), whereas no differences were found between controls and PCSK9 gene mutants. However, due to the low PCSK9 expression in lymphocytes, real-time PCR measurements are very close to the detection limits, and these results have to be interpreted with caution. Figure 3 shows the amplification plot from patients with LDLR or c.-332C>A PCSK9 mutation.

View larger version (13K): [in this window] [in a new window]   FIG. 3. Amplification plot of PCSK9 expression in lymphocytes. PCSK9 from lymphocytes extracted from both c.-332C>A carriers and ADH patients with mutations in LDLR gene (p.C660X and p.V408M) was measured by real-time PCR. LDLR mutation carriers present a lower PCSK9 expression than c.-332C>A PCSK9 mutants, and this lower expression is maintained when correcting with the housekeeping gene. The figure also shows the low expression levels in lymphocytes.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In our previous work, we were able to identify a mutation in LDLR or ApoB genes in about 75% of the probands diagnosed as ADHs (24, 25, 26). We identified a group of 42 ADH patients in whom mutations in LDLR or APOB genes were not found, using the procedures for mutation detection described by our group (24, 26, 27).

In this group of ADH subjects, we found polymorphisms previously described by other groups or by public SNP databases (see Patients and Methods) and only one new polymorphism with low frequency downstream exon 6 (c.996 + 44G>A) (5, 6, 12). Although some of these polymorphisms have been identified as modifying LDL-C and TC in population studies, they have not been identified as causing ADH (6, 7). In addition, we have found two novel variations located in the promoter region, c.-332C>A and c.-288G>A, close to an SRE site. Variation –332C>A was inside the SP1 site described recently (29) in the promoter area and could be thought as causing ADH.

We tested the presence of these two new variations in a control population with normal LDL-C and TC levels. Mutation c.-288G>A was present in the control population but at a very low frequency (three of 450 controls). In addition, we failed to find a cosegregation of mutation and disease in the proband’s family. Unfortunately, we could not test the c.-332C>A segregation with the disease because the proband’s family was unavailable and only one relative, a cousin, could be studied. His TC and LDL-C were close to dyslipidemic levels (TC and LDL-C, 257 and 190 mg/dl, respectively), but he was taking a lipid-lowering diet and supplements. He also carried mutation c.-332C>A. The proband with c.-332C>A was a woman and had TC 292 and LDL-C 208 mg/dl at baseline. She had neither xanthomas nor xanthelasmas, and the same was found in her relative. Her family history showed several relatives were dyslipidemic and mortality history from cardiovascular disease: her father and her two paternal uncles died from myocardial infarction at 57, 48, and 66 yr, respectively. On the other hand, she was unresponsive to statin treatment (Table 3). Differences in lipid levels between proband and relative could be due to the proband’s unresponsiveness to statin treatment and an slight cholesterol-lowering effect of control diet and supplement (Benecol) (33).

Up to now, only mutations increasing PCSK9 protein activity have been identified as causing hypercholesterolemia, and no one has been found in the promoter region of this gene (5, 11, 12, 13, 19, 21). To confirm the possible implication of these two new mutations in the development of ADH, we tested their ability to modify the PCSK9 promoter activity. Our results indicated that mutation c.-288G>A does not modify the promoter activity, whereas mutation c.-332C>A causes a 2.5-fold increase in the transcriptional activity of this promoter, compared with the wild-type sequence. Recently a paper regarding regulation of PCSK9 expression by SREBP2 has been published (29). The authors suggested a new Sp1 site in PCSK9 promoter, which involves position c.-332. Furthermore, specific mutation of this Sp1 site increases promoter transcription and maintains regulation by sterols. Our results agree with those from this work about the implication of this position in promoter regulation, supporting the fact that c.-332C>A mutation affects PCSK9 expression and can be responsible for ADH.

The statin used in our experiments increases the activity of PCSK9 promoter in all sequences tested, as has been previously described (30). Our results show that wild-type sequence and c.-288G>A mutation have similar promoter activity, whereas c.-332C>A produces an increase in promoter activity, maintaining the 2.5 times of overexpression, compared with wild sequence.

Activation of PCSK9 expression by statins has been suggested as one of the mechanisms involved in loss of statins treatment efficiency. It is known that inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activates SREBPs, which in turn increases LDLR levels but also PCSK9 and 3-hydroxy-3-methylglutaryl coenzyme A reductase levels. Genetic polymorphisms affecting PCSK9 expression or activity could therefore contribute to interindividual differences in response to pharmacological treatment with statins. Preservation of the increased activity of c.-332C>A mutation, compared with the wild-type sequence, was demonstrated in transfected cells with and without addition of statins (Fig. 2). This result could explain the poor response of this patient to statins, shown in Table 3. The proband’s clinical history confirms a poor response to statins, with plasma lipid levels kept elevated along the years despite different statin treatments. The increased PCSK9 expression found in cells treated with statins supports the clinical findings.

We have also tried to demonstrate an effect of this c.-332C>A PCSK9 mutation on PCSK9 gene expression and plasma protein levels. PCSK9 is a protein made by the liver, which exerts its function mainly in this organ. Only RNA extracted from lymphocytes was available to quantification. We watched that both patients with c.-332C>A mutation have a slightly higher expression when compared with samples with similar plasma LDL-C levels but not with samples from normocholesterolemic individuals. However, expression is very low, close to detection limits, and data could be considered not conclusive. RNA from other tissues could confirm these results. Analysis in a sample with more patients would be necessary to confirm these results. The same problem was found when measuring PCSK9 protein levels on plasma, as other authors have also seen. Serum PCSK9 levels have been measured by immunoprecipitation and a newly developed ELISA method (31, 32), showing a wide range of values. Our difficulty in detecting PCSK9 protein levels can be due to the method used. On the other hand, a correlation between serum PCSK9 and LDL-C levels has been described in men but not women (32). PCSK9 mutation carriers are a woman (proband) and a man (his cousin); therefore, the quantification of PCSK9 in plasma will not be conclusive.

Unfortunately, the size of the family carrying this new PCSK9 mutation and the available samples do not allow demonstrating the effect of the alteration in blood but previous data (29), the absence of this mutation in control population and our in vitro studies argues in favor to the effect of this mutation in PCSK9 gene overexpression and involvement in hypercholesterolemia.

In conclusion, we have screened the complete PCSK9 gene and found only two mutations, c.-332C>A and c.-288G>A, which can be related to hypercholesterolemia. Our data indicate that mutation c.-322C>A increases the transcription of PCSK9 and can cause hypercholesterolemia, whereas c.-288G>A does not. We have identified only one patient with a mutation in PCSK9 of 42 ADH subjects without mutations in APOB or LDLR genes. This seems to indicate that in our population ADH caused by PCSK9 is uncommon, as has also been reported in other populations (11, 12, 19, 20, 22).

Thus, we have identified and characterized a new point mutation, c.-332C>A, in the promoter sequence of PCSK9 capable of causing hypercholesterolemia, which reduces statin treatment efficiency. Its frequency in our ADH population seems to be low, in accordance with previous reports from other European populations.

	Footnotes

 
This work was supported by grants from the Spanish Ministry of Health, Instituto de Salud Carlos III (Madrid) (to F.J.C., S.M.-H., and C.I.) in the programs for incorporating researcher to the National Health Service (Reference FIS01/3047) and postspecialized formation (CM06/0060), respectively; "Juan de la Cierva" Program (Spanish Ministry of Science and Education, starting in 2004) (to A.-B.G.-G.); Red de Hiperlipemias Primarias (C03/181 and PI05/0174); Spanish Ministry of Science and Education Grants SAF05/02883 and SAF2006-06760; Generalidad Valenciana Grants CTIDIA 2002/65, ACOMP2007--075, and GV04/255; and Spanish Ministry of Industry, Bargain, and Transportation Grants FIT-010000--2007--69. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is an Instituto de Salud Carlos III initiative.

Disclosure Summary: S.B., S.V., A.-B.G.-G., S.M.-H., C.I., V.G.-A., J.F.A., J.C.M.-E., J.T.R., M.C., and F.J.C. have nothing to declare. R.C. consults for Pfizer and Merck Sharp & Dohme and received lecture fees from Pfizer, Merck Sharp & Dohme, and Astra Zeneca.

First Published Online June 17, 2008

Abbreviations: ADH, Autosomal dominant hypercholesterolemia; APOB, ApoB, apolipoprotein B100; Ct, threshold cycle; FDB, familial defective apolipoprotein B; HLA-G, histocompatibility complex, class I, G gene; LDL-C, low-density lipoprotein cholesterol; LDLR, LDL receptor; PCSK9, proprotein convertase subtilisin/kexin type 9 precursor; SNP, single-nucleotide polymorphism; SP1, specificity protein-1; SRE, sterol regulatory element; SREBP, sterol regulatory binding protein; TC, total cholesterol.

Received February 5, 2008.

Accepted June 10, 2008.

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