Multiplex PCR assay for screening deletions in the low density lipoprotein receptor gene

Clinica Chimica Acta 309 Ž2001. 7–12 www.elsevier.comrlocaterclinchim

Multiplex PCR assay for screening deletions in the low density lipoprotein receptor gene ´ ´ a,) Zsuzsa Pocsai a , Gyorgy ¨ Paragh b, Roza ´ Adany a

Department of PreÕentiÕe Medicine, School of Public Health, Medical and Health Science Centre, UniÕersity of Debrecen, P.O. Box 9, Debrecen H-4012, Hungary b First Department of Medicine, Medical and Health Science Centre, Faculty of Medicine, UniÕersity of Debrecen, Debrecen, Hungary Received 5 September 2000; received in revised form 5 February 2001; accepted 14 February 2001

Abstract Background: In different populations of the world, more than 150 genetic alterations of the LDL receptor gene have been identified; each of which can result in hypercholesterolaemia, but no hot spots in the gene were detected so far. Because of the existence of very variable genetic alterations in different ethnic communities, none of the assays developed for screening mutationsrdeletions in a population defined can be adapted to study the possible genetic defects. The present study was designed to develop a new, multiplex PCR-based, molecular biological method to screen the whole coding region of the LDL receptor gene. Methods: Using primer pairs completely flanking the promoter and the entire exonal region, in the PCR reactions 83–386-bp long, DNA sequences were synthesised in seven different reaction mixtures. The reaction conditions of the multiplex PCR system were optimised in order to synthesise all exons and the promoter region of the gene using only two annealing temperatures. The products could be visualised separately by agarose gel electrophoresisrethidium bromide staining. Results: A rapid, effective test enabling the screening of DNA alterations in the entire LDL receptor gene was developed. Using this simple multiplex PCR assay, deletions affecting more than 10 bp in any part of the gene can be easily detected by a single agarose gel electrophoresis. Conclusions: The simplicity, specificity and versatility of the assay make it suitable system for routine screening of LDL receptor gene mutations in large population samples. This PCR assay can be recommended for screening of LDL-RG deletions in populations or groups at high risk for cardiovascular diseases. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Multiplex PCR assay ŽM-PCR.; LDL receptor gene ŽLDL RG.; Familiar hypercholesterolaemia ŽFH.; Deletions; Screening

1. Introduction Cardiovascular diseases with multifactorial origins resulting from the interplay of genetic and ) Corresponding author. Tel.: q36-52-417-267; fax: q36-52417-267. ´ ´ .. E-mail address: [email protected] ŽR. Adany

environmental factors present a particular challenge to different medical disciplines and are frequently associated with acquired hypercholesterolaemia. Recently, a number of experimental studies were carried out to identify and evaluate both genetic and nongenetic determinants of atherosclerosis and hypercholesterolaemia. It was clearly shown that the increase in plasma LDL Žlow density lipoprotein.

0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 9 6 - X

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cholesterol is frequently due to genetic alterations at the gene locus specifying the formation of the LDL receptors, leading to defective catabolism of LDL w1x. The monogenic disease resulting from the structural and functional alterations of low density receptor gene ŽLDL-RG. can be characterized by lifelong elevation of serum LDL cholesterol level, and its frequency in the populations of Europe and North America averages about 0.2% w2x. However, in some regions of the world, it is much higher due to effects consequent on emigration or due to local customs, which encourage consanguineous marriages. Studies carried out in the last decades identified a large number of genetic defects, which strongly alter the synthesis and consecutively the function of the LDL receptor and clinically result in the manifestation of hypercholesterolaemia in human w3,4x. Recently identified aberrations in different FH Žfamiliar hypercholesterolaemic. families have proved to vary extensively, ranging from large and small deletions and insertions to nonsense and missense types of single nucleotide alterations predisposing to the premature onset of atherosclerotic vascular disease w5–9x. In most populations of the world, including the population of North America and Europe, more than 150 different genetic variants of the gene involving major gene rearrangements or single nucleotide alterations have been identified so far, each of which can give rise to the disease w3x. So far, no hot spots characteristic for the LDL receptor gene were identified among the different ethnic communities. Because of the very variable genetic alterations in different populations, none of the assays developed for the detection of different mutationsrdeletions in the regions of the gene can be adapted to study the possible DNA damages. Thus, we decided to develop a new molecular biological assay, which allows to screen genetic abnormalities in the whole gene in most routine diagnostic and risk assessment laboratories w10x. The multiplex polymerase chain reaction ŽPCR. assay seems to be the most appropriate among the routinely available molecular biological methods that can fulfill these requirements. In this paper, we now publish a newly developed multiplex PCR-based molecular biological method that can give the possibility to screen the whole coded region of the LDL receptor gene composed of 17 introns and 18 exons,

most of which correlate with functional domains previously defined at the protein level. 2. Materials and methods 2.1. Patients inÕolÕed in the study Twenty consecutive unrelated patients with heterozygous FH Ž8 males and 12 females. were invited to participate in this study, and none of them refused it. Cases were admitted to the health care program of the Eastern-Hungarian Regional Lipid Centre at the University School of Medicine, Debrecen, Hungary between January 2, 1995 and December 30, 1996. They agreed to provide peripheral blood samples for LDL receptor analysis simultaneously with samples for a Alipid-panelB Žcholesterol, HDL-C, LDL-C, apoA-1  apolipoprotein A-14 , apoB  apolipoprotein B4 , LpŽa.  lipoprotein Ža.4 , triglyceride and lipid electrophoresis. before starting with any dietary andror therapeutic intervention. Patients with chronic diseases, such as diabetes, hypertension or hyperlipidaemia, or receiving anticoagulant drugs were excluded from the study. None of the study group had heart failure, or renal, liver or thyroid disease. The diagnostic criteria of FH were Ž1. serum total cholesterol level greater than 7.8 mmolrl, Ž2. serum LDL cholesterol level greater than 4.9 mmolrl, Ž3. normal triglyceride level, Ž4. the presence of tendon xanthomas, Ž5. the presence of hypercholesterolaemia andror tendon xanthomas in at least one first-degree relative, and Ž6. positive family history for early myocardial infarction. Data obtained on this population were analyzed and are presented in this paper. The study had been approved by the Ethical Committee of the University Medical School, Debrecen, Hungary. 2.2. DNA samples Genomic DNA isolated with salt extraction from peripheral white blood cells from 20 EasternHungarian heterozygous FH patients served as samples of the study. FH status of the patients was characterized previously by standard criteria. In addition, test DNA samples were a generous gift of Professor K. Kontula ŽSecond Department of Medicine, University of Helsinki., and the presence

Z. Pocsai et al.r Clinica Chimica Acta 309 (2001) 7–12

of this genetic aberration in these samples was confirmed by his laboratory. DNA samples were quantified by GeneQuant II. RNArDNA calculator ŽPharmacia Biotech. using spectrophotometric method. 2.3. Genetic analysis The whole coded region of the LDL receptor gene Žpromoter and 18 exons. was amplified by PCR. For

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the amplification of exons 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, and 16, we have used self-designed primer pairs, and for synthesis of promoter region and exons 1, 7, 8, 9, 17 and 18, oligonucleotides designed by Hobbs et al. w3x were applied. Primer pairs were synthesized by Pharmacia Biotech and sequences of them are given in Table 1. The primer sets of different reaction mixtures are listed in Table 2. Each 50-ml reaction mixture consists of 0.3 mg of genomic DNA, 10 mmolrl Tris–HCl ŽpH 9.0., 50

Table 1 Sequences of oligonucleotides used in multiplex PCR assay for amplifying all exons and the promoter region of the LDL receptor gene Primer

Sequence

PP.1 PP.2 P1.1 P1.2 P2.1 P2.2 P3.1 P3.2 P4.1 P4.2 P5.1 P5.2 P6.1 P6.2 P7.1 P7.2 P8.1 P8.2 P9.1 P9.2 P10.1 P10.2 P11.1 P11.2 P12.1 P12.2 P13.1 P13.2 P14.1 P14.2 P15.1 P15.2 P16.1 P16.2 P17.1 P17.2 P18.1 P18.2

5 -gag tgg gaa tca gag ctt cac ggg t-3 X X 5 -cca cgt cat tta cag cat ttc aat g-3 X X 5 -act cct ccc cct gct aga aac ctc a-3 X X 5 -ttc tgg cgc ctg gag caa gcc tta c-3 X X 5 -tgg gcg aca gat gtg aaa gaa ac-3 X X 5 -agc acg tct cct ggg gac tca t-3 X X 5 -tgt ctg tca cct gca aat cc-3 X X 5 -act tac gac agc ctt gct cgt c-3 X X 5 -tgc agc ccc caa gac gtg ct-3 X X 5 -cgc agt ttt cct cgt cag at-3 X X 5 -tcc tgt ttt cca gct gtg gcc-3 X X 5 -cat taa cgc agc caa ctt c-3 X X 5 -tga cac tct gcg agg gac cca aca a-3 X X 5 -cgc act ctt tga tgg gtt ca-3 X X 5 -agt ctg cat ccc tgg ccc tgc gca g-3 X X 5 -agg gct cag tcc acc ggg gaa tca c-3 X X 5 -cca agc ctc ttt ctc tct ctt cca g-3 X X 5 -cca ccc gcc gcc ttc ccg tgc tca c-3 X X 5 -tcc atc gac ggg tcc cct ctg acc c-3 X X 5 -agc cct cat ctc acc tgc ggg cca a-3 X X 5 -cac cca gct tga cag agc cca-3 X X 5 -cca tga aca gga tcc acc a-3 X X 5 -ctt cat gta ctg gac tga ctg g-3 X X 5 -cta ggg tga tgc cat tgg g-3 X X 5 -atc tcc tca gtg gcc gcc tct act-3 X X 5 -ctc aaa gac ggc caa gga gaa-3 X X 5 -tgc ctg ttt agg aca aag ta-3 X X 5 -ctc ttg gct ggg tga ggt tgt-3 X X 5 -gag tga act ggt gtg aga gga-3 X X 5 -ctg tga ggc agc tcc tca tgt-3 X X 5 -tct ttc aga ggc tga ggc tg-3 X X 5 -ctt ggt gag aca ttg tca cta tct cc-3 X X 5 -tgc agc tct ggg cga cgt tg-3 X X 5 -cga tgg gga gga caa tgg aca ga-3 X X 5 -tga cag agc gtg cct ctc cct aca g-3 X X 5 -gct ttc tag aga ggg tca cac tca c-3 X X 5 -tcc gct gtt tac cat ttg ttg gca g-3 X X 5 -aat aaa aca agg ccg gcg agg tct c-3

X

X

Product size Žbp.

Amplified fragment of LDL-R gene

155

promoter region

234

exon 1

123

exon 2

131

exon 3

386

exon 4

136

exon 5

123

exon 6

169

exon 7

175

exon 8

271

exon 9

228

exon 10

119

exon 11

140

exon 12

153

exon 13

153

exon 14

179

exon 15

83

exon 16

207

exon 17

135

exon 18

Z. Pocsai et al.r Clinica Chimica Acta 309 (2001) 7–12

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Table 2 Composition of reaction mixes used in multiplex PCR assay Reaction mixtures

Amplified region

Used primers

Size of amplicons Žbp.

Annealing temperature Ž8C.

Mg 2q concentration ŽmM.

1

exon 17 exon 18 exon 1 exon 7 exon 9 promoter exon 8 exon 3 exon 4 exon 13 exon 5 exon 6 exon 14 exon 10 exon 11 exon 12 exon 2 exon 15 exon 16

P17.1–P17.2 P18.1–P18.2 P1.1–P1.2 P7.1–P7.2 P9.1–P9.2 PP.1–PP.2 P8.1–P8.2 P3.1–P3.2 P4.1–P4.2 P13.1–P13.2 P5.1–P5.2 P6.1–P6.2 P14.1–P14.2 P10.1–P10.2 P11.1–P11.2 P12.1–P12.2 P2.1–P2.2 P15.1–P15.2 P16.1–P16.2

207 135 234 169 271 155 175 131 386 153 136 123 153 228 119 140 123 179 83

51

6.0

51

4.7

51

4.5

47

5.6

47

4.4

47

4.5

47

4.3

2

3 4

5

6

7

mmolrl KCl, 1.5 mmolrl each of dATP, dTTP, dCTP, and dGTP ŽPharmacia Biotech., 1–1 mmolrl of both primers ŽPharmacia Biotech., 2.5 U Taq polymerase ŽPharmacia Biotech. and 10% dimethylsulfoxide ŽSigma Aldrich.. Reaction mixes number 1, 2, 3, 4, 5, 6, and 7 contain 6.0, 4.7, 4.5, 5.6, 4.4, 4.5, and 4.3 mmolrl MgCl 2 ŽPromega., respectively. After initial denaturation, 35 PCR cycles were performed in a programmable DNA thermal cycler ŽGene Machine Junior, UNITEK. using the temperature profile of 1 min at 948C, 1 min at annealing temperature and 2 min at 728C. The primer extension of the 35th cycle was extended to 12 min. In case of reaction mixes 1, 2, and 3, the 518C annealing temperature was applied, while in case of the other mixes, the temperature was 478C. After amplification, the post-PCR samples were concentrated under vacuum by Automatic Environment SpeedVac ŽAES 1000 Savant. for 30 min. The products were electrophoresed on a 3.5% agarose gel ŽA9539, Sigma Aldrich. in a buffer containing 90 mmolrl Tris– borate ŽpH 8.4. and 4 mmolrl EDTA for 2 h at a voltage of 70 V. The fragments were dyed with ethidium bromide Ž10 mgrml; Sigma Aldrich. and were visualised by ultraviolet illumination. Large

quantities of the pre-made reaction mixes were prepared in advance and stored at y868C. For analysis, a tube was thawed, DNA and Taq polymerase were added, and the reaction was performed in an automatic thermocycler.

3. Results and discussion We have developed a rapid, effective screening test enabling the simultaneous analysis of DNA alterations in the entire LDL receptor gene. In a relatively simple multiplex PCR system, all the 18 exons and the promoter region of the gene can be amplified and analyzed using primer pairs flanking completely the coding region of the gene. Sizes of amplified fragments and reaction conditions were taken into consideration to evolve the PCR assay. The principle of the method is summarized in Fig. 1a. Primer compositions were positioned to have efficient amplification yield of all relevant DNA regions under approximately similar conditions in the same test tube, and to synthesize DNA fragments that could be size-fractionated in a fully informative way by a single electrophoretic run. Using the system 83–386-

Z. Pocsai et al.r Clinica Chimica Acta 309 (2001) 7–12

bp long, DNA sequences were synthesised at not more than two annealing temperatures in seven different reaction mixtures ŽFig. 1a,b.. Reaction conditions, including magnesium chloride concentration, annealing temperature, the effect of different addi-

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tives were set up in each reaction mix separately to optimise the assay. For the synthesis of exons 1, 7, 8, 9, 17, 18 and promoter region, 478C annealing temperature was applied, while for others it was 518C. The assay requires only 2 mg genomic DNA isolated from peripheral lymphocytes by salt extraction for analysing the whole gene. Using this simple assay, deletions affecting more than 10 bp in any part of the LDL receptor gene can be easily detected by a single agarose gel electrophoresis. For preliminary experiments, we used a collection of DNA samples with a known carrier status of the FH-Helsinki gene, where a large deletion from intron 16 to exon 18 exist ŽFig. 1c.. In lanes 1, 3, and 5, results are shown using the same type of reaction mixtures containing primer pairs for the 17, 18 exons of the gene Žreaction mixture 1., while lanes 2, 4, 6 show the results of the reaction mixture 7, which was suitable to amplify 2, 15, 16 exons. Applying these two reaction mixtures is suitable to detect FHHelsinki. In presence of the FH-Helsinki mutation, only 123- and 179-bp fragments were amplified in the reaction mixture 7 using multiplex PCR assay Žlane 4., and no DNA fragment became visible in reaction mixture 1 containing oligonucleotides for synthesis of exons 17 and 18 Žlane 3.. In the case of 20 Hungarian hypercholesterolaemic patients, all these regions of the gene were amplified, presenting that no FH-Helsinki DNA mutation can be found in the LDL receptor gene Žlanes 5, 6.. Further analysis of the entire coding region of the gene indicated that

Fig. 1. Ža. Principle of the multiplex PCR assay carried out on the LDL receptor gene. Electrophoretic pattern of the entire LDL receptor gene in the multiplex PCR assay. Exons are indicated above the lines. Annealing temperature ŽTa . of 518C was applied for reaction mixes number 1, 2, 3; while for mixes number 4, 5, 6, 7, it was 478C. Žb. Visualization of the multiplex PCR fragments carried out on LDL receptor gene after agarose gel electrophoresis and ethidium bromide staining. Lane 1: reaction mix 1; lane 2: reaction mix 2; lane 3: reaction mix 3; lane 4: reaction mix 4; lane 5: reaction mix 5; lane 6: reaction mix 6; lane 7: reaction mix 7; lane 8: pBR 322 DNA marker. Žc. Confirming the diagnostic value of the newly developed multiplex PCR assay. Reaction mixes in lanes 1, 3, 5 contain primer pairs for exons 17, 18 of the LDL receptor gene, while lanes 2, 4, 6, primer pairs for exons 2, 15, 16 were used. Lanes 1, 2: DNA sample of control subject; lanes 3, 4: DNA sample of known carrier status of the FH-Helsinki gene; lanes 5, 6: DNA samples of an Eastern-Hungarian hypercholesterolaemic subject; lane 7: pBR 322 DNA marker.

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Z. Pocsai et al.r Clinica Chimica Acta 309 (2001) 7–12

no large deletions or other large rearrangements in the LDL receptor gene were present in any of the Eastern-Hungarian hypercholesterolaemic patients. As genetic alterations of the LDL receptor gene are very variable among ethnic communities, the identification of these deletions characteristic for different ethnic groups is needed for developing special screening tests, which can be used to test patients being at high risk for genetic abnormalities. The availability of new methods that speed up the process of finding genetic aberrations in genes has allowed the identification of an ever-increasing number of LDL receptor gene variants that cause hypercholesterolaemia. The aim of these studies is to identify disease-associated genes and to examine the structure and function of the encoded protein w11x. Through these studies, the functional consequences of this alteration and the molecular mechanism can also be defined. Human molecular genetics offers the possibility of improved disease diagnosis prevention and treatment through genetic testing and mechanism-based therapy w12x. The fact that relatively efficient therapeutic measures can be offered today for both heterozygous and homozygous FH patients emphasises the importance of early diagnosis and intervention of FH. Our new multiplex PCR assay is designed so that hypercholesterolaemic patients subsequently can be screened for larger than 10-bp deletions in the LDL receptor gene. The simplicity, specificity, and versatility of the assay make it an ideal system for routine screening of LDL receptor gene mutations in large population samples. This PCR assay can be recommended for screening the gene for genetic aberrations even in groups at high risk and individuals of cardiovascular diseases. Further analysis of small deletions, insertion mutation and other genetic aberrations in the gene can be studied by using the same PCR products by singlestrand conformation polymorphism ŽSSCP. analysis.

Acknowledgements The authors thank Professor Kimmo Kontula of the Second Department of Medicine, University of

Helsinki, Finland for the generous gift of a known carrier status FH-Helsinki DNA samples. This study way supported by the Hungarian Ministry of Education ŽContract no. FKFP 0498r1999..

References w1x Kontula K, Koivisto UM, Koivisto P, Turtola H. Molecular genetics of familiar hypercholesterolaemia: common and rare mutations of the low density lipoprotein receptor gene. Ann Med 1992;24:363–7. w2x Soutar A. Familiar hypercholesterolaemia: mutations in the gene for the low density lipoprotein receptor. Mol Med Today 1995;1:90–7. w3x Hobbs H, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familiar hypercholesterolaemia. Hum Mutat 1992;1:445–66. w4x Kontula K. Molecular genetic diagnosis of hypercholesterolaemia. Klin Labor 1993;10:34–6. w5x Sun XM, Webb JC, Gudnason V, Humphries S, Seed M, Thompson GR, et al. Characterization of deletions in the LDL receptor gene in patients with familial hypercholesterolaemia in the United Kingdom. Arterioscler, Thromb, Vasc Biol 1992;12:762–70. w6x Meiner V, Landsberger D, Berkman N, Reshef A, Segal P, Seftel HC, et al. A common Lithuanian mutation causing familiar hypercholesterolaemia in Ashkenazi Jews. Am J Hum Genet 1991;49:443–9. w7x Leitersdorf E, Tobin EJ, Davignon J, Hobbs HH. Common low density lipoprotein receptor mutations in the French Canadian population. J Clin Invest 1990;85:1014–23. w8x Koivisto UM, Turtola K, Aalto-Setala K, Top B, Frants RR, Kovanen PT, et al. The Familiar Hypercholesterolaemia ŽFH.-North Karelia mutation of the low density lipoprotein receptor gene deletes seven nucleotides of exon 6 and is a common cause of FH in Finland. J Clin Invest 1992;90:219– 28. w9x Aalto-Setala K, Helve E, Kovanen P, Kontula K. Finnish type of low density lipoprotein receptor gene mutation ŽFH Helsinki. deletes exons encoding the carboxy terminal part of the receptor and creates an internalization-defective phenotype. J Clin Invest 1989;84:499–505. w10x Kontula K. The use of PCR in diagnosing lipoprotein disorders. Ann Med 1992;24:195–9. w11x Lashley FR. Genetic testing, screening and counseling issues in cardiovascular disease. J Cardiovasc Nurs 1999;13:110–26. w12x Ellsworth DL, Sholinsky P, Jaquish C, Fabsitz RR, Manolio TA. Coronary heart disease. At the interface of molecular genetics and preventive medicine. Am J Prev Med 1999; 16:122–33.