Journal of Clinical Lipidology (2013) 7, 174–177

Duplication of exon 7–12 in the low-density lipoproteinreceptor gene in three Danish patients with familialhypercholesterolemia

Søren Feddersen, MSc, PhD*, Martin Overgaard, MSc, PhD, Mads Nybo, MD, PhD

Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Sdr. Boulevard 29, DK–5000Odense, Denmark

KEYWORDS:Cholesterol;Duplication;Familialhypercholesterolemia;Genomic rearrangements;LDL receptor

* Corresponding author.

E-mail address: soeren.feddersen@o

Submitted August 14, 2012. Accep

2012.

1933-2874/$ - see front matter � 2013

http://dx.doi.org/10.1016/j.jacl.2012.1

Abstract: Familial hypercholesterolemia (FH) is one of the most frequent single-gene disorders;nevertheless, it is commonly underdiagnosed and undertreated. To increase the number of individualsdiagnosed and treated for FH, an ongoing discovery of novel FH mutations is necessary as a prereq-uisite to implement good nationwide genetic FH screening strategies. Here we report on the findingof a seldom exon 7–12 duplication in the low-density lipoprotein receptor gene of three Danish patientswith FH.� 2013 National Lipid Association. All rights reserved.

Familial hypercholesterolemia (FH) is an autosomal-dominant disease causing severe elevations of bloodcholesterol levels as the result of defects in the genesencoding the low-density lipoprotein receptor (LDLR), theapolipoprotein B (APOB), and the proprotein convertasesubtilisin/kexin type 9 (PCSK9).1–4 FH causes hypercho-lesterolemia from childhood, and patients with FH whoare untreated may have up to 20-fold increased risk of pre-mature coronary heart disease.5 Although no cure existsfor FH it can be efficiently treated by lifestyle changesand lipid-lowering drugs.6 Nevertheless, FH is underdiag-nosed and undertreated,5 and it is therefore important toreport on novel FH mutations as well as mutations appear-ing for the first time in certain populations as a prerequi-site to implement good nationwide genetic FH screeningstrategies. The number and kind of mutations in LDLR,

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ted for publication November 19,

National Lipid Association. All right

1.003

APOB, and PCSK9 vary considerably between differentpopulations, and among these a few duplications hasbeen described.7–9 Here we describe three unrelated Dan-ish FH patients with a seldom duplication of exon 7–12 inthe LDLR.

Methods

Subjects

Proband A was a 52-year-old woman who had noxanthomas or cardiovascular disease (CVD) but withhypercholesterolemia and acute myocardial infarction infirst-degree relatives (lipid profile; see Table 1). ProbandB was a 27-year-old man without xanthomas or CVD butwith hypercholesterolemia in first-degree relatives. Pro-band C was a 54-year-old woman with hypertension, an-gina pectoris, and hypercholesterolemia and a familyhistory with male persons dying at early age, includingher father, who died of acute myocardial infarction at

s reserved.

Table 1 Fasting plasma lipid concentrations without medication

Plasma total cholesterol Plasma LDL cholesterol Plasma HDL cholesterol Plasma Triglycerides

Proband A 8.0 6.0 1.8 0.6Proband B 8.9 6.3 0.8 4.9Proband C 9.4 7.0 2.0 0.9

HDL, high-density lipoprotein; LDL, low-density lipoprotein.

All concentrations are in mmol/L.

Feddersen et al Duplication in the LDL receptor gene 175

the age of 53 years. The probands were unrelated. Allprobands had two blood samples taken separately to al-low result confirmation by analysis of a second sample.Samples were taken after the patients fasted overnight.The most common causes of secondary hypercholesterol-emia were ruled out by investigating for diabetes, hypo-thyroidism, and renal or liver disease. All plasma lipidmeasurements were performed on an Architect c16000Instrument (Abbott, Chicago, IL) with dedicatedreagents.

FH analysis

Genomic DNA from peripheral leukocytes was ex-tracted with a Maxwell 16 Blood DNA purification kit(Promega Corporation Madison, WI). Samples werescreened for small deletions, insertions, or point mutationsin all 18 exons; the promoter; and the flanking introns ofthe LDLR gene with temperature-gradient capillary elec-trophoresis (TGCE).10 On the basis of TGCE results, rel-evant regions were selected for sequencing on an ABI3730xl DNA Analyzer (Applied Biosystems, Foster City,CA) using BigDye Terminator v3.1 Cycle sequencing kit(Applied Biosystems). For all samples, exon 7, 12, 15,and 18 of LDLR and exon 26 of APOB were sequencedwithout previous TGCE analysis. The multiplex ligation-dependent probe amplification (MLPA) Salsa P062LDLR Kit (MRC-Holland) was used to identify largegenomic rearrangements in LDLR.11 The MLPA productswere run on an ABI 3730xl DNA Analyzer in accordancewith the manufacturer’s protocol, and data were analyzedwith the use of Genemarker software v1.50 (SoftGenetics,LLC). MLPA analysis was performed in duplicatesor triplicates on two independent samples from eachindividual.

Copy number variation analysis

To validate MLPA results, copy number variation (CNV)was determined for selected introns of LDLR by the use ofquantitative real-time polymerase chain reaction (PCR).TaqMan CNV assays for intron 8 and the intron 10-exon11 boundary targeted the duplicated region of LDLR,whereas the assay for intron 5 targeted a region notsupposed to be duplicated according to MLPA results.TaqMan CNV assays (Hs7151140_cn, Hs07136751, andHs07156441_cn) consisting of probes and primers targeting

intron 5, intron 8, and the intron 10-exon 11 boundary ofLDLR were used together with the TaqMan RNase Pcopy number reference assay. LDLR assays were run in du-plex together with the reference assay on a StepOnePlusSystem (Applied Biosystems) in accordance with the man-ufacturer’s protocol, and copy numbers were determinedusing CopyCaller software v2.0 (Applied Biosystems).All samples were run in triplicate.

Results

Investigation by TGCE and sequencing of selectedregions of LDLR did not reveal any mutations. However,MLPA analysis revealed duplication of exon 7–12 in theLDLR: Fig. 1A shows the MLPA pattern for one of theFH patients with duplication of exon 7–12 (the other FHpatients had similar MLPA patterns). Peak heights ofexon 7–12 relative to the mean peak height of each in sixcontrol samples were above the 1.3-fold line, indicatingthat these exons are duplicated. To validate MLPA results,DNA samples were subjected to exon CNV analysis by theuse of quantitative real-time PCR (Fig. 1B). This clearly il-lustrated an increased copy number of intron 8 and the in-tron 10-exon 11 boundary compatible with duplication,whereas intron 5 had a copy number comparable with thecontrols.

Discussion

During routine molecular genetic testing in Danishpatients with FH we identified three unrelated patientsheterozygous for a duplication of exon 7–12 in LDLR. Inmany populations, large rearrangements account forw10% of the mutations in the LDLR gene,12 which con-firms the importance of applying tests that detect large re-arrangements in routine genetic screening. We use theMLPA analysis for this purpose because it is faster thanlabor-intensive techniques such as Southern blotting andmore sensitive than long-range PCR.11 Of note, this dupli-cation was not revealed by use of TGCE or standard DNAsequence analysis.

The three individuals tested in this study all had clinicalindications of FH, which implies that part of the LDLRepidermal growth factor precursor homology domain hasbeen duplicated in these FH patients because this domain isencoded by exon 7–14.13 Breakpoint sequences of

Figure 1 MLPA and exon CNV analysis of LDLR in FH pa-tients. (A) Representative MLPA pattern from FH patient with du-plication of exon 7–12. Exons present at more than 1.3-fold wereconsidered to be duplicated (upper region), and those at less than0.75-fold to be half the normal copy number (lower region). (,)peaks obtained with probes to exon 7–12 of LDLR; (B) peaks ob-tained with control probes; (C) peaks obtained with probes to thepromoter, exon 1–6 and exon 13–18 of LDLR and probes to thecontrol genes SMARCA4 and ANKRD25. (B) CNV analysis ofLDLR. For validation of MLPA results, TaqMan CNV assayswere performed. The intron 5 assay served as a control. Openbars, hatched bars, and solid bars represent results obtained withintron 5, intron 8, and intron 10-exon 11 boundary assays, respec-tively. Pt, patient; Control, genomic DNA from a healthy individ-ual; Mix control, a mix of genomic DNA from several healthyindividuals.

176 Journal of Clinical Lipidology, Vol 7, No 2, April 2013

duplications identified in our FH patients were not deter-mined and reading frames are therefore unknown. If thereading frame is disrupted, we would expect a defect trun-cated protein consisting of mainly the LDLR ligand-binding domain due to a premature stop codon. On theother hand, if the reading frame is unaltered, an abnormallylarge LDLR may be produced. Interestingly, a duplicationof exon 9–14 of LDLR that does not produce a shift inthe reading frame was previously reported.14 Althoughnot disrupting the reading frame, this duplication was

shown to result in an abnormally large receptor proteinthat is degraded faster than normal LDLR. In agreementwith the lipid profiles of our patients, we therefore concludethat a duplication of exon 7–12 of LDLR is a disease-causing rearrangement that leads to reduced levels ofLDLR and a corresponding increase in plasma cholesterol.

Alu elements are short (w300) repetitive mobile DNAelements that are extensively interspersed throughout thehuman genome. They contribute to nonallelic homologousrecombination events causing CNV. Rearrangement ofLDLR involving Alu elements in intron 6 and intron 12has previously been observed,12 suggesting that theherein-described duplication of exon 7–12 may resultfrom Alu-mediated recombination.

Duplication of exon 7–12 in LDLR was first reported byChiou and Charng, who found the duplication in TaiwaneseFH patients.15,16 However, this report is the first docu-mented report on the finding of the exon 7–12 duplicationin a European population such as the Danish population.We speculate whether the fact that three Danish individualsturn out to have the same (with the current knowledge) un-common duplication could be due to a local founder. Asmentioned, we have not been able to establish any kinshipbetween the probands, but hopefully we can unravel thequestion through a continuous investigation for this dupli-cation in our Danish FH patients in the future.

A diagnosis of FH is primarily suspected on the basis ofclinical criteria; however, to confirm this genetic screeningis required. Advantages of genetic screening include iden-tification of patients at a younger age and thereby improve-ment of treatment and treatment compliance.5 Geneticscreening thus enables early intervention, which may pre-vent atherosclerotic damage and lower the risk of CVD inFH patients.17 Interestingly, Chiou and Charng found thatthe effect of lipid-lowering drugs on serum LDL cholesterollevels in patients with FH depends on the type of mutation.More specifically, the serum LDL cholesterol-lowering ef-fect of treatment with rosuvastatin and/or ezetimibe was re-duced in subgroups with abnormal MLPA patterns,nonsense mutations, or frameshift mutations comparedwith subgroups with undetected mutation or missense mu-tations.15 Clinically, FH patients with large rearrangementsas the duplication described here would presumably benefitfrom a higher starting dose of lipid-lowering drugs com-pared to missense mutation carriers.

We believe that the most frequently appearing FHmutations have been identified, whereas identification ofthe more rare mutations such as the exon 7–12 duplicationhas just started. Although rare these mutations may collec-tively constitute a considerable part of FH-causing muta-tions. Precaution must therefore be taken if genetic FH testsbased on DNA array technology are implemented in futuregenetic FH screening strategies, because most of these testsonly detect a limited number of genetic mutations. On theother hand, FH tests based on targeted next-generationsequencing technologies seems promising, since pointmutations and CNVs can be detected with a specificity,

Feddersen et al Duplication in the LDL receptor gene 177

accuracy and sensitivity comparable to that of Sangersequencing and MLPA.

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