Clinical Expression of Familial Hypercholesterolemia inClusters of Mutations of the LDL Receptor Gene That

Cause a Receptor-Defective or Receptor-Negative PhenotypeS. Bertolini, A. Cantafora, M. Averna, C. Cortese, C. Motti, S. Martini, G. Pes, A. Postiglione,

C. Stefanutti, I. Blotta, L. Pisciotta, M. Rolleri, S. Langheim, M. Ghisellini, I. Rabbone, S. Calandra

Abstract—Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelatedItalian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases,we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutationson the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative,according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients’age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDLcholesterol was higher (118%) and high density lipoprotein cholesterol lower (25%) than the values found inreceptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higherin receptor-negative subjects. In patients.30 years of age in both groups, the presence of CAD was related to age,arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in thereceptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negativesubjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact thanin receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptor-defective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. ApolipoproteinE genotype analysis showed a lowering effect of thee2 allele and a raising effect of thee4 allele on the LDL cholesterollevel in both groups; however, the apolipoprotein E genotype accounted for only 4% of the variation in LDL cholesterol.Haplotype analysis showed that all families of the major clusters shared the same intragenic haplotype cosegregatingwith the mutation, thus suggesting the presence of common ancestors.(Arterioscler Thromb Vasc Biol.2000;20:e41-e52.)

Key Words: LDL receptor gene mutationsn familial hypercholesterolemian mutation clustersn receptor phenotype and clinical expression

Familial hypercholesterolemia (FH) is a common autoso-mal, codominant disease caused by mutations in the LDL

receptor (LDL-R) gene. It is characterized by elevated plasmaLDL levels, tendon xanthomas, premature coronary arterydisease (CAD), and a family history of 1 or more of these.1 Inmost populations, FH has proved to be extremely heteroge-neous at the DNA level:.600 mutations of the LDL-R genehave been reported so far1–3 (also see http://www.ucl.ac.uk/fh). Studies conducted in the rare patients with homozygousFH have shown that different types of mutations result inresidual functional LDL-R activity that varies from,2% to30%.2,4 This mutational heterogeneity explains, to someextent, the phenotypic variation found in FH homozygotes, in

whom there is a strong correlation between residual receptoractivity in cultured cells and the severity of the disease.1,2,4Asimilar genotype-phenotype correlation is less clear in FHheterozygotes (with documented mutations in the LDL-Rgene), because there is only a weak correlation betweenmaximally induced LDL-R activity in cultured cells and theplasma LDL cholesterol (LDL-C) concentration. In thesepatients, clinical expression of FH is highly variable in termsof the severity of hypercholesterolemia and the age of onsetand severity of CAD, as well as the response to diet andlipid-lowering therapy.5 A few studies on the genotype-phe-notype correlation in heterozygous FH have been carried outin isolated populations (such as the French-Canadian),

Received December 17, 1999; revision accepted March 17, 2000.From the Department of Internal Medicine (S.B., L.P., M.R., S.L.), University of Genoa; the National Institute of Health (A.C., I.B.), Rome; the

Institute of Internal Medicine (M.A.), University of Palermo, Palermo; the Department of Internal Medicine (C.C., C.M.), University of Rome “TorVergata,” Rome; the Department of Medical Sciences (S.M.), University of Padua, Padua; the Institute of Clinical Biochemistry (G.P.), University ofSassari, Sassari; the Department of Clinical and Experimental Medicine (A.P.), University of Naples “Federico II,” Naples; the Institute of MedicalTherapy (C.S.), University of Rome, Rome; the Department of Biomedical Sciences (M.G., S.C.), University of Modena and Reggio Emilia, Modena;and the Department of Pediatrics (I.R.), University of Turin, Turin, Italy.

Correspondence to Sebastiano Calandra, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi, 287, 41100Modena,-Italy. E-mail sebcal@unimo.it

© 2000 American Heart Association, Inc.

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wherein a founder effect had caused the accumulation ofmany patients with a limited number of mutant alleles of theLDL-R gene.6 Although these studies have provided someclue about the effect of LDL-R gene mutations on the clinicalexpression of FH, their results should be treated with caution,as it is likely that such patients might have other genes andenvironmental factors in common that might contribute totheir phenotype.

The marked heterogeneity of mutations of the LDL-R genecausing FH, which is present in most populations, makes itdifficult to collect a sufficiently large number of patients withthe same mutation and to compare the FH phenotype ingroups of patients (of the same population) carrying differentmutations at the LDL-R locus. Recently, Graham et al7 haveinvestigated genotype-phenotype correlations in heterozy-gous FH carrying different mutations of the LDL-R gene.They showed that frameshift (FS) mutations were associatedwith higher levels of LDL-C than those found in missensemutations.

In an attempt to determine to what extent the type ofgenetic defect is an important determinant of FH phenotype,we have investigated the clinical expression of the disease inseveral groups (clusters) of FH heterozygotes with definedmutations of the LDL-R gene that have been identified inItaly over the last few years.

MethodsPatientsThe study included a group of 725 unrelated patients (index cases)with the clinical diagnosis of heterozygous FH. Four hundredninety-five subjects had a diagnosis of “definite FH” according to thefollowing criteria: (1) untreated LDL-C levels.95th percentile ofthe distribution in the Italian population, stratified for sex and age;(2) vertical transmission of hypercholesterolemia and a bimodaldistribution of plasma LDL-C levels in the proband’s family; and(3) the presence of subjects with the FH homozygous phenotype inthe family (n572), tendon xanthomatosis in the proband or in at least1 first-degree relative (n5215), or severe hypercholesterolemia insome prepuberal children of the proband’s family (n5208). Twohundred thirty subjects were classified as “probable FH,” since eitherno xanthomas were detected in the probands or in their familymembers or no sufficient family data were available (Table I). Dataon smoking habits, arterial hypertension, diabetes, or other associ-ated diseases with relevant impact on cardiovascular riskwere collected.

FH patients were considered to have coronary artery disease(CAD1) if they (1) had history of a documented myocardialinfarction, coronary artery bypass graft, or percutaneous transluminalcoronary angioplasty; (2) suffered from angina pectoris with apositive exercise ECG and thallium test or an abnormal angiogram;and (3) had silent ischemia that was detected during bicycle exercisetesting and was confirmed by thallium test, stress echocardiography,

or angiography. FH subjects were considered free of CAD (CAD2)if they had a negative bicycle exercise test. CAD occurring before 60years of age was considered premature CAD. The families belongingto the FH clusters (see Results) were considered positive for thepresence of tendon xanthomas and/or premature CAD if at least 1adult member of the family was affected.

Informed consent was obtained from all subjects investigated, and,in the case of children, from their parents. The study protocol wasapproved by the human investigation committee of each participatinginstitution.

Biochemical AnalysisFasting plasma lipid concentrations were measured before anyhypolipidemic drug treatment. Total cholesterol, triglycerides, andHDL-C levels were measured enzymatically with commercial kits(Boehringer Mannheim GmbH) and an automated analyzer (Hitachimodel 704); LDL-C was calculated by the Friedewald formula.

Assay of LDL-R Activity in Skin FibroblastsThe assay of125I-labeled LDL binding, internalization, and degrada-tion in cultured skin fibroblasts was performed as described byGoldstein et al.8 Residual LDL-R activity (calculated as the maxi-mum rate of saturable125I-LDL degradation) was expressed as apercentage of the value obtained in control fibroblasts.

Analysis of the LDL-R GeneGenomic DNA was extracted from peripheral blood leukocytes by astandard procedure.9 All DNA samples were digested by using 5 to10 U/mg of several restriction enzymes, separated by agarose gelelectrophoresis, transferred to nylon membranes, and hybridizedwith various LDL-R cDNA probes.10 Polymerase chain reaction(PCR) amplifications of the promoter region and exons 1 to 18 of theLDL-R gene were carried out by using the primers reported byHobbs et al2 in a total volume of 50mL containing 100 ng ofgenomic DNA, 10 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/LMgCl2, 100 mmol of each of the 4 nucleotides, 10 pmol of eachprimer, and 2 U ofTaq DNA polymerase. Single-strand conforma-tion polymorphism analysis was performed by using a vertical gelunit; 5 mL of PCR product was mixed with an equal amount of 95%formamide, 20 mmol/L EDTA, 0.1% bromophenol blue, and 0.1%xylene cyanole; denatured at 96°C for 5 minutes; snap-cooled in 4°Cice water; and then loaded onto an 8% polyacrylamide gel containing5% glycerol. Electrophoresis was performed at room temperature ina standard Tris-borate-EDTA buffer, pH 8.0, at 150 V for 2 hours.After electrophoresis, the gels were stained with silver. The samplesshowing an abnormal single-strand conformation polymorphismpattern were sequenced by using an automated fluorescence ABIPrism 310 genetic analyzer (Applied Biosystems Inc) and followingthe manufacturer’s recommendations. Mutations identified by theautomated sequencer were confirmed by manual sequencing4 withthe use of appropriate primers. Several mutations reported in TableII have been characterized previously in our laboratory.4,10–23

The screening of some mutations in the probands’ family memberswas performed by using a variety of procedures: (1) Southernblotting (deletion of exons 2 to 12 and exons 13 to 14)12,16; (2) PCRamplification of fragments of the LDL-R gene with appropriatecanonic primers, followed by digestion with restriction enzymes(MspI for D200G,NlaIII for V502M, HinfI for D558Y, andPstI for

TABLE I. Selection Criteria for FH Families

Selection Criteria

No. ofFamiliesCollected

No. of Families WithIdentified Mutation

(% of Collected)

No. of HeterozygotesWith Identified

Mutation

No. ofHeterozygotes

per Family

Homozygous FH phenotype 72 69 (95.8%) 219 3.2

Tendon xanthomatosis 215* 120 (55.8%) 496 4.1

Hypercholesterolemic children 208* 58 (27.9%) 227 3.9

Probable FH 230* 35 (15.2%) 79 2.3

Total 725 282 (39.1%) 1021 3.6

*Screening in progress.

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TABLE II. Inventory of LDL-R Gene Mutations Causing FH in Italy

Mutation Fam Ho CHe He Receptor ProteinLDL-C, mmol/L

(n) Allele DesignationRef.No.

Exon (Ex) deletions (kb)

Promoter1Ex 1, 2 (20) 1 12 Not produced 7.2160.87 (8) FH Siracusa 18

Promoter1Ex 1–6 (.25) 2 4 Not produced 7.7060.89 (4) FH Bologna-1 10

Promoter1Ex 1–6 (24) 1 3 Not produced 8.9761.32 (3) FH Bari-3 New

Ex 2–12 (24) 10 2 61 Not produced 7.3661.40 (43) FH Pavia 12

Ex 3–10 (12) 1 6 (1) Truncated 6.9961.26 (6) FH Massa 16

(2) Inframe deletion

Ex 11, 12 2 6 Truncated 7.2161.24 (6) FH Genoa-1 16

Ex 13, 14 (3.7) 5 21 Truncated 8.5661.03 (16) FH Chieti-1 16

Ex 13, 14 (4.7) 1 2 Truncated 8.7661.65 (2) FH Rome 16

Ex 13–15 (4.5) 1 4 (1) Truncated 9.2861.73 (4) FH Potenza-1 13

(2) Inframe deletion

Ex 13–15 (5) 1 2 4 Truncated 8.7461.45 (4) FH Palermo-3 4

Ex 13–18 (.25) 2 1 5 Truncated 8.6661.60 (3) FH Catania 4

Ex 16 (2) 1 4 Inframe deletion 7.6061.39 (4) FH Padua-2 16

Exon (Ex) duplications (kb)

Ex 3–6 (8) 1 9 Inframe insertion 9.2361.41 (9) FH Caltanissetta 22

Ex 9–14 (10) 2 3 Inframe insertion 6.4561.66 (3) FH Salerno 15

Ex 13–15 (7) 1 3 Truncated 7.9360.40 (3) FH Bologna-2 11

Ex 16, 17 (5.5) 2 1 4 Inframe insertion 7.4160.46 (4) FH Viterbo 10

Nonsense (cDNA)

97C3T (Ex 2) 2 1 2 Q12X 7.5060.64 (2) FH Milan-4 4

103C3T (Ex 2) 1 1 2 Q14X 7.2261.08 (2) FH Milan-3 4

301G3T (Ex 3) 1 2 E80X 9.2960.78 (2) FH Imperia WS*

304C3T (Ex 3) 3 12 Q81X 7.8761.12 (8) FH Rapone WS*

373C3T (Ex 4) 3 1 5 Q104X 8.3461.03 (3) FH Foggia-2 4

418G3T (Ex 4) 2 6 E119X 8.2361.32 (4) FH Campobasso WS*

865G3T (Ex 6) 2 1 8 E267X 8.1561.01 (8) FH Milan-2 4

2311C3T (Ex 15) 2 8 Q749X 8.2960.99 (8) FH Mondovı New

Missense (cDNA)

28T3A (Ex 1) 1 1 W-12R 7.94 FH Enna 19

265T3C (Ex 3) 1 1 8 C68R 6.0060.93 (8) FH Catanzaro 4

346T3C (Ex 4) 1 6 C95R 8.6060.90 (6) FH Alghero 23

352G3T (Ex 4) 2 1 4 D97Y 4.2960.97 (4) FH Naples-3 4

401G3T (Ex 4) 1 1 C113F 8.45 FH Alessandria New

402C3G (Ex 4) 1 5 C113W 7.6861.41 (5) FH Lucca New

415G3A (Ex 4) 2 4 D118N 6.8161.01 (4) FH Sassari-2 23

662A3G (Ex 4) 48 4 135 D200G 6.6561.62 (95) FH Padua-1 4

665G3A (Ex 4) 4 10 C201Y 7.6660.88 (10) FH Genoa-2 New

682G3A (Ex 4) 7 16 E207K 8.4961.53 (14) FH Modena WS*

761A3C (Ex 5) 1 2 Q233P 7.6760.28 (2) FH Reggio Emilia-2 New

826T3C (Ex 6) 3 6 C255R 7.8961.66 (6) FH Ceva New

828C3G (Ex 6) 1 3 C255W 8.3660.56 (3) FH Sassari-3 23

829G3A (Ex 6) 1 1 E256K 6.34 FH Genoa-3 WS*

952T3C (Ex 7) 1 3 C297R 8.1761.23 (3) FH Perugia New

953G3T (Ex 7) 3 16 C297F 8.6661.03 (12) FH Trieste 14

1056C3G (Ex 7) 2 1 7 C331W 5.6060.47 (7) FH Avellino-1 4

1090T3C (Ex 8) 2 7 C343R 7.0061.70 (7) FH Palermo-2 WS*

1109A3C (Ex 8) 2 1 4 N349T 5.9360.47 (4) FH Reggio Calabria New

1118G3A (Ex 8) 3 2 13 G352D 6.5061.22 (8) FH Potenza-2 4

1135T3C (Ex 8) 7 5 1 12 C358R 6.7661.04 (8) FH Naples-1 4

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P664L); (3) PCR amplification of fragments of the LDL-R gene withappropriate mismatched primers, followed by digestion with restric-tion enzymes (NdeI for 31311 g3a, StyI for E207K, RsaI forFS453, andStuI for G571E); (4) PCR amplification of a specificallele (PASA) for G528D20; and (5) heteroduplex analysis forFS572.23 Details of the procedures adopted for screening thesemutations will be reported elsewhere.

Geographic Origin of MutationsThe putative geographic origin of each LDL-R gene mutation wasempirically established on the basis that individuals carrying aspecific mutation were proved to have stemmed from ancestors whohad been living in the same geographic area for at least 3generations.

Haplotype Analysis of the LDL-R LocusThe intragenic haplotypes cosegregating with some mutant alleles(D200G, FS453, and G528D) were constructed by using diallelicmarkers present in the LDL-R locus. Genotyping was performed by(1) Southern blotting (BstEII, 59ApaLI, PvuII, NcoI, and 39ApaLI);

(2) restriction enzyme digestion of PCR-amplified intron 7 (SmaI)and exons 8, 12, 13, and 15 (StuI,HincII, AvaII, and 59MspI); and(3) single-strand conformation polymorphism analysis (1413G/Ainexon 10).24–28

The polymorphic tetranucleotide microsatellite D19S394 that is250 kb telomeric to the LDL-R gene was genotyped by usingfluorescently tagged, PCR-generated fragments29 that were electro-phoresed in an Applied Biosystems automated DNA sequencer 373A Leon and sized by Genescan 672 software (Applied Biosystems).

Screening for Familial Defective Apo B-100All probands were screened for the presence of the R3500Q mutationin the apolipoprotein (apo) B gene causing familial defective apoB-100.30

Apo E GenotypingThe apo E genotype was determined by PCR amplification ofgenomic DNA according to the procedure of Hixson and Vernier.31

TABLE II. Continued

Mutation Fam Ho CHe He Receptor ProteinLDL-C, mmol/L

(n) Allele DesignationRef.No.

1195G3A (Ex 9) 2 1 3 A378T 4.2260.71 (3) FH Nuoro 23

1244A3G (Ex 9) 1 5 D394G 7.4660.89 (5) FH Padua-3 New

1301C3G (Ex 9) 1 1 T413R 10.33 FH Sassari-4 23

1454A3G (Ex 10) 1 1 2 H464R 5.5460.56 (2) FH Milan-1 4

1463T3A (Ex 10) 1 3 I467N 7.1960.77 (3) FH Piacenza WS*

1567G3A (Ex 10) 13 3 2 48 V502M 6.3261.21 (35) FH Bari-2 4

1646G3A (Ex 11) 29 3 2 127 G528D 7.5261.42 (66) FH Palermo-1 4, 20

1735G3T (Ex 12) 10 45 D558Y 8.1161.69 (31) FH Casale Monferrato New

1775G3A (Ex 12) 7 2 19 G571E 5.7560.92 (14) FH Foggia-1 4

2054C3T (Ex 14) 10 5 1 37 P664L 6.4861.39 (28) FH Frosinone-1 4

Frameshift (FS), deletion (del), insertion(ins) (cDNA)

623–644del (Ex 4) 1 2 FS187Term236 7.2660.50 (2) FH Asti New

682–684del, 681–685 ins 6 (Ex 4) 1 8 E207del, CK206–208ins 7.7661.01 (8) FH Chieti-3 21

681–682ins 18 (Ex 4) 1 9 CKDKSD206–207 ins 9.5361.28 (9) FH Padua-4 WS*

1067delA (Ex 8) 1 4 FS335Term348 9.0861.87 (2) FH Cuneo New

1068T3A, 1068–1069 ins 18 (Ex 8) 1 3 D335E, ECQDPD 335–336ins 8.9561.16 (3) FH Chieti-2 22

1122–1123insTGGC (Ex 8) 1 1 FS354Term360 8.27 FH Pisa 21

1374–1375delAG (Ex 10) 1 2 FS437Term444 8.6861.44 (2) FH Ancona WS*

1418–1419insACAT (Ex 10) 31 151 FS453Term515 7.4461.53 (101) FH Savona WS*

1439–1449del (Ex 10) 1 10 FS459Term510 8.4261.84 (10) FH Reggio Emilia-1 18

1478–1479delCT (Ex 10) 2 1 8 FS472Term513 6.3960.70 (4) FH Frosinone-2 4

1778delG (Ex 12) 8 40 FS572Term643 8.1461.86 (27) FH Sassari-1 23

Splicing

IVS3 31311 g3a 7 20 Inframe deletion 6.6561.40 (17) FH Olbia 23

IVS10 158611 g3a 2 1 5 (1) Inframe deletion 6.9561.75 (4) FH Agrigento 4

(2) Inframe insertion

IVS12 1846-1 g3a 2 1 3 (1) Truncated 7.2461.08 (3) FH Avellino-2 4

(2) Inframe deletion

IVS15 231111 g3a 2 1 4 (1) Truncated 5.4660.68 (3) FH Benevento 4

(2) Inframe insertion

IVS16 238911 g3a 1 1 1 Inframe deletion 7.33 FH Monfalcone 4

Fam indicates number of families; Ho, homozygotes; CHe, compound heterozygotes; He, heterozygotes; Term, termination; and WS, web site http://www.ucl.ac.uk/fh.LDL-C levels (adjusted for sex, age, and body mass index) are reported only for heterozygous patients whose untreated lipid values were available.*Not described before in Italy.

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Statistical AnalysisStatistical analysis was performed by using theSPSS9.0 (SPSS Inc)program. Statistically significant differences between groups oramong groups for continuous variables were evaluated by Student’st test for unpaired data or ANOVA, respectively. Triglyceride values,which were not normally distributed, were logarithmically trans-formed before analysis. Multiple comparisons among pairs of meansbased on unequal sample sizes were performed by the Dunnett Cmethod. Lipid values were adjusted for sex, age, and body massindex (BMI) by linear multiple regression analysis. When subjectswere grouped by apo E genotypes, their lipid values were alsoadjusted separately in the receptor-defective group and the receptor-negative group for the effect of each LDL-R gene mutation (ie, thelipid values for each mutation were adjusted to the grand mean of thewhole group). Differences in the distributions of categorical vari-ables were assessed by thex2 test with Yates’ continuity correctionwhen necessary. Genotype distribution for the apo E polymorphismwas determined by gene counting.x2 analysis was used to test forHardy-Weinberg equilibrium and to compare the observed genotypedistributions and allele numbers in CAD1and CAD2 subjects. Theproportion of the total phenotypic variability of adjusted LDL-C andHDL-C values attributable to the apo E gene polymorphism wascalculated according to Boerwinkle et al.32 The independent contri-bution of each variable to CAD was evaluated by logistic regressionanalysis, taking into account only those variables that were found tobe predictive in univariate analyses. The independent categoricalvariables were coded as follows: 1 for male and 0 for female sex; 0and 1 for the absence and presence, respectively, of arterial hyper-tension; 0, 1, 2, and 3 for nonsmoker, former smoker, current lightsmoker (,10 cigarettes per day), and current heavy smoker (.10cigarettes per day), respectively; 0 for mutations causing a receptor-defective phenotype and 1 for mutations causing a receptor-negativephenotype; 1 for genotypese2e2 ande2e3; 2 for genotypee3e3; and3 for genotypese2e4, e3e4, ande4e4. Age and lipid values enteredthe analysis as continuous variables. Likelihood ratio statistics wereused to compare models with different combinations of predictorswith an inclusion significance level of 0.05 and an exclusionsignificance level of 0.1. Odds ratios and 95% confidence intervalsare also reported.

ResultsMutations of the LDL-R Gene in FH PatientsTable I shows the number of families (unrelated index cases)and subjects with the clinical diagnosis of “definite” or“probable” FH, in whom an LDL-R gene mutation wasdetected during a national survey that is still in progress. Atthis time, analysis of the LDL-R gene mutation has beencompleted in all families of patients with homozygous FH, in42% of families with definite FH (presence of tendonxanthomas or hypercholesterolemic children), and in 15% offamilies with probable FH. By extending the genetic analysisto the relatives of index cases, we have been able to collect'1000 FH heterozygotes with identified mutations in theLDL-R gene.

Table II shows the mutations of the LDL-R gene found inour index patients to date, the number of apparently “unre-lated” families carrying the same mutation, and the number ofsubjects (heterozygotes, homozygotes, and compound het-erozygotes) belonging to these families. The allele designa-tion indicates the putative geographic origin of each mutationin our country. The table includes mutations that (1) werediscovered in Italian FH patients and were reported previ-ously by our group (see References 4 and 10 through 23 andthe web site http://www.ucl.ac.uk/fh); (2) were discovered inother countries and reported in the literature but also found tobe present in Italy (see the web site); and (3) are reported forthe first time in this article (new). During this survey, we

discovered that some mutations are overrepresented in spe-cific geographic districts of the country. For example, D200G(FH Padua-1), though detected in all districts of northernItaly, is found mostly in the Veneto region and has so far notbeen detected in central and southern Italy. On the other hand,G528D (FH Palermo-1) is found in southern Italy and Sicilybut not in northern Italy (Figure I).

The identification of several apparently unrelated individ-uals with the same mutation allowed us to define the presenceof clusters of mutations and to compare the FH phenotypeamong these clusters. In the present study, we arbitrarilydefined a “cluster” as any mutation group that included 5 ormore apparently unrelated families. At present, we haveidentified 12 such clusters (Table III).

Haplotype AnalysisHaplotype analysis was carried out in the largest clusters(D200G and FS453 found in northern Italy and G528D foundin southern Italy and Sicily). In each cluster, the haplotype ofintragenic markers cosegregating with the mutation was thesame in all families, thus suggesting a common ancestor.D200G and FS453 cosegregated with the following haplo-types:SmaI (2), StuI (1), 1413G/A (G),HincII (2), BstEII(2), AvaII (1), MspI 59 (1), ApaLI 59 (1), PvuII (2), NcoI(1), and ApaLI 39 (1). G528D cosegregated with thefollowing haplotype:StuI (1), HincII (1), BstEII (2), AvaII(2), ApaLI 59 (2), PvuII (2), NcoI (2), and ApaLI 39(2).Analysis of the microsatellite D19S394, located 250 kbupstream from the LDL-R locus, showed that the FS453mutation cosegregated in all families with a single allele (251bp); the D200G mutation cosegregated with 3 alleles (227,235, and 239 bp with a frequency of 0.18, 0.41, and 0.41,respectively); and the G528D mutation cosegregated with 4alleles (243, 255, 259, and 263 bp with a frequency of 0.07,0.41, 0.25, and 0.27, respectively). In 256 normal chromo-somes, the allele frequencies were as follows: 227 (0.05), 235(0.10), 239 (0.05), 243 (0.09), 251 (0.10), 255 (0.12), 259(0.12), and 263 (0.10).

FH Phenotype in Receptor-Defective andReceptor-Negative ClustersTable III shows the main clinical features of the clusters. Lipidvalues reported in the tables were adjusted for age, sex, and

Figure I. I Geographic location of the 3 major clusters ofLDL-R gene mutations found in Italy. The number beside theG528D symbol in Sicily indicates the number of families withthis mutation identified on the island.

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BMI. We separated the clusters into 2 groups designated“receptor-defective” and “receptor-negative,” respectively, onthe basis of (1) the residual LDL-R activity found by us and/orothers in cultured cells (fibroblasts) of homozygous subjects(.5% for receptor-defective,,5% for receptor-negative)1,2,4orheterozygous subjects (.55% for receptor-defective,,55% forreceptor-negative) carrying the same mutation and (2) thepresence of a mutation leading to a frameshift and a truncatedreceptor (null allele). One of the striking features emerging fromthe analysis of these clusters is the large variability of plasmaLDL-C levels observed in each cluster, regardless of the type ofmutation of the LDL-R gene. The mean difference between thelowest and the highest LDL-C values was 5.0461.61 mmol/L inthe receptor-defective group and 6.1361.58 mmol/L in thereceptor-negative group. Table III also shows that in thereceptor-negative group, the prevalence of tendon xanthomatosisin the families (defined as the number of families in which atleast 1 living member had tendon xanthomatosis) and theprevalence of premature CAD (defined as the number offamilies in which premature CAD had been detected in at least1 living member or had been reported as the cause of death in atleast 1 deceased family member) were respectively, 2.7- and2.1-fold those found in the receptor-defective group.

Table IV shows the comparison of the clinical features andlipid values between index cases (1 index case for eachunrelated family included in the major clusters shown inTable III) and between all heterozygous subjects. Index casesof the 2 groups were comparable in terms of sex distribution,mean age, BMI, smoking habits, and arterial hypertension.The unadjusted mean plasma levels of total cholesterol andLDL-C were significantly higher in the receptor-negativesubjects, whereas plasma triglyceride and HDL-C levels weresimilar in the 2 groups. When the data were adjusted for age,

sex, and BMI, plasma HDL-C levels were found to be slightlybut significantly lower in the receptor-negative subjects. Thepercentage of index patients with tendon xanthomas andCAD was higher in the receptor-negative subjects.

Considering all heterozygous subjects belonging to theclusters, the 2 groups were comparable in terms of mean age,sex distribution, arterial hypertension, and percentage ofcurrent and former smokers. BMI was found to be slightlyhigher in the receptor-defective group. Diabetic patients (1patient in the receptor-defective group and 2 patients in thereceptor-negative group) were not included.

The differences in the unadjusted and adjusted total cho-lesterol, LDL-C, and HDL-C levels observed in the indexpatients were clearly confirmed when we compared allsubjects belonging to the 2 groups (Table IV). In receptor-negative subjects, the adjusted LDL-C and HDL-C levelswere 18% higher and 5% lower, respectively, than thosefound in receptor-defective subjects. Figure II shows theage-related increase of LDL-C level in both groups; at eachage interval, the mean LDL-C value was higher in thereceptor-negative subjects. The differences in the prevalenceof tendon xanthomas and CAD in the 2 groups were similarto those found in index cases (Table IV).

In both receptor-defective and receptor-negative subjects,the genotype distributions of the apo E polymorphism were inHardy-Weinberg equilibrium (receptor-defective:e2e3n518, e2e4 n52, e3e3 n5137, e3e4 n-31, ande4e4 n51;receptor-negative:e2e2 n51, e2e3 n524, e2e4 n51, e3e3n5230, e3e4 n540, and e4e4 n52), and no significantdifferences in allele frequencies were observed between the 2groups (receptor-defective:e2 0.053,e3 0.854, ande4 0.093;receptor-negative:e2 0.045,e3 0.879, ande4 0.076). In bothgroups, plasma lipid values adjusted for sex, age, BMI, and

TABLE III. Clinical Features and Lipid Values (mmol/L) in FH Clusters

Families,(% Screened)

Families WithNo. of

Subjects

LDL-CHDL-C,

Mean6SDTG,

Mean6SDTX PCAD Mean6SD Range

Receptor-defective

G571E 7 (2.9%) 2 0 14 5.7560.92 4.28 –7.38 1.2560.25 1.4660.59

V502M 13 (4.8%) 0 5 35 6.3261.21 4.23 –8.52 1.1760.30 1.6861.10

P664L 10 (4.4%) 5 3 28 6.4861.39 4.89–10.56 1.3360.31 1.3460.54

31311 g3a 7 (8.7%) 0 1 17 6.6561.40 4.55 –9.28 1.1560.15 1.3460.71

D200G 48 (14.9%) 21 19 95 6.6561.62 4.08–11.49 1.4160.39 1.2960.58

Total 85 28 28 189 6.5061.46 4.08–11.49 1.3260.35 1.3960.72

Receptor-negative

D Ex 2–12 10 (3.0%) 7 5 43 7.3661.40 4.99–10.89 1.2260.31 1.4460.56

Fs453 31 (18.0%) 18 16 101 7.4461.53 4.07–12.27 1.3460.27 1.3260.63

G528D 29 (17.8%) 22 14 66 7.5261.42 4.74–11.41 1.1060.25 1.3760.51

D558Y 10 (6.7%) 7 7 31 8.1161.69 4.95–12.00 1.3560.35 1.2560.61

Fs572 8 (10.0%) 7 6 27 8.1461.86 4.86–11.37 1.1460.25 1.2160.52

E207K 7 (2.8%) 7 6 14 8.4961.53 5.22–10.68 1.3560.31 1.4660.52

D Ex 13, 14 5 (1.7%) 4 4 16 8.5661.03 6.99–10.11 1.2960.25 1.3560.43

Total 100 75* 58† 298 7.6961.55 4.07–12.27 1.2560.30 1.3460.57

TX indicates tendon xanthomas; PCAD, premature coronary artery disease; LDL-C, LDL cholesterol; HDL-C, HDL cholesterol; TG, triglycerides; Ex, exon; and FS,frameshift. The LDL receptor activity in fibroblasts of FH heterozygotes carrying Fs453, D558Y, and E207K were 44–48%, 42–46%, and 40–45%, respectively, ofthe values found in control fibroblasts. Lipid values were adjusted for sex, age, and body mass index.

*Receptor-negative vs receptor-defective x2 31.2, P,0.0001.†Receptor-negative vs receptor-defective x2 10.6, P50.001.

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the effect of each LDL-R gene mutation were found to beinfluenced by the apo E polymorphism. In comparison withsubjects carrying thee3e3 genotype, in the receptor-defectivegroup thee2e3 genotype was associated with lower HDL-Clevels (1.0960.24 versus 1.3260.30 mmol/L,P,0.05) andwith a tendency to higher triglyceride levels (1.7560.93versus 1.3960.65 mmol/L), whereas in the receptor-negativegroup, e2e3 was associated with lower LDL-C levels(6.9961.18 versus 7.6061.50 mmol/L,P,0.05). In bothgroups, subjects carrying thee4 allele (e2e4, e3e4, or e4e4genotypes) had higher LDL-C levels than did subjects withother genotypes (receptor-defective: 7.1361.72 versus

6.4961.36 mmol/L in thee3e3 group and 6.2361.09 mmol/Lin the e2e3 group, P,0.05; receptor-negative: 8.2561.33versus 7.6061.50 mmol/L in the e3e3 group and6.9961.18 mmol/L in thee2e21e2e3 group,P,0.05).

CAD in Receptor-Defective andReceptor-Negative ClustersBecause it is well established that the prevalence of tendonxanthomas and CAD is affected by age, we looked at thedistributions of these clinical features in subjects younger orolder than 30 years of age. In subjects#30 years of age (71in the receptor-defective group and 112 in the receptor-

Figure II. Age-related increase of mean plasmaLDL cholesterol in receptor-negative and receptor-defective clusters.

TABLE IV. Clinical and Biochemical Features of the Index Cases and All Heterozygotes in FH Clusters

Receptor-Defective Receptor-Negative P

M/F, n/n 39/46 45/55 NS

84/105 126/172 NS

Age, y 46.0615.3 47.0613.6 NS

38.5618.2 37.0618.7 NS

Body mass index, kg/m2 24.363.7 23.863.1 NS

23.264.0 22.463.6 0.02

Tendon xanthomas, % 24 (28.2%) 66 (66.0%) ,0.001

34 (18.0%) 122 (40.9%) ,0.001

Arterial hypertension, % 13 (15.3%) 17 (17.0%) NS

20 (10.6%) 40 (13.4%) NS

Current smokers, % 11 (13.0%) 15 (15.0%) NS

25 (13.2%) 29 (9.7%) NS

Former smokers, % 15 (17.6%) 17 (17.0%) NS

17 (9.0%) 22 (7.4%) NS

Coronary artery disease, % 17 (20.0%) 34 (34.0%) 0.04

21 (11.1%) 59 (19.8%) 0.01

Total cholesterol, mmol/L 9.1261.65 (8.7561.63) 10.5361.87 (10.2261.72) ,0.001 (,0.001)

8.4961.66 (8.4261.48) 9.4761.87 (9.5361.61) ,0.001 (,0.001)

LDL cholesterol, mmol/L 7.1361.60 (6.8161.59) 8.6661.77 (8.3761.66) ,0.001 (,0.001)

6.5761.61 (6.5061.46) 7.6561.76 (7.6961.55) ,0.001 (,0.001)

HDL cholesterol, mmol/L 1.3360.39 (1.3460.35) 1.2360.33 (1.2360.30) NS (,0.05)

1.3060.37 (1.3260.35) 1.2660.32 (1.2560.30) NS (,0.05)

Triglycerides, mmol/L 1.5060.73 (1.3560.70) 1.5060.65 (1.3960.62) NS (NS)

1.4360.79 (1.3960.72) 1.3160.64 (1.3460.57) NS (NS)

Values are mean6SD. Values in italics refer to all heterozygous subjects belonging to FH clusters. Lipid valuesshown in parentheses were adjusted for sex, age, and body mass index.

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negative group), no individual was detected with CAD ineither group, and only a few (2.8% in each group) were foundto have tendon xanthomas. When subjects.30 years werecompared (118 in the receptor-defective group and 186 in thereceptor-negative group), the prevalence of tendon xantho-mas and CAD in receptor-negative subjects was 2.4-fold(P,0.001) and 1.8-fold (P50.01), respectively, greater thanthat observed in receptor-defective subjects.

Table V compares the clinical features and lipid values inreceptor-defective and receptor-negative subjects.30 yearsof age who were CAD(1) or CAD(2). In both groups, age,the prevalence of arterial hypertension, previous smokinghabits, and total cholesterol and LDL-C levels were higher inCAD(1) patients. In the receptor-negative group, the male-to-female ratio and BMI were significantly higher inCAD(1) patients, whereas in the receptor-defective group,plasma triglycerides were higher in CAD(1) patients. Theprevalence of premature CAD (occurring before 60 years ofage in subjects.30) was significantly higher in receptor-negative patients (23.2% versus 10.6%;x254.76, P50.03),with a crude odds ratio (95% confidence interval) of 2.55(1.10 to 6.41).

The CAD(1) distribution according to quartiles of age wasexamined in subjects.30 years of age. In the first and secondquartile, the prevalence of CAD was similar in the receptor-defective and receptor-negative groups; in the fourth andespecially the third quartile, the prevalence of CAD wasmuch higher in the receptor-negative subjects (fourth quartile,56.3% versus 33.3%,P50.05; third quartile, 41.7% versus16.1%,P50.02).

Multiple logistic regression analysis, performed separately inthe 2 groups in subjects.30 years of age, showed thatindependent contributors to CAD were (1) male sex (P50.02),arterial hypertension (P50.02), and LDL-C level (P50.007) inthe receptor-defective group and (2) male sex (P50.001), age(P,0.0001), and arterial hypertension (P50.02) in the receptor-negative group. This analysis also showed that in the receptor-defective group, age had a marginal effect on CAD (P50.06),and in the receptor-negative group, the LDL-C level did notappear to be a good predictor for the development of CAD

(P50.08). In both groups, the apo E genotype did not signifi-cantly contribute to either premature CAD or total CAD.

Table VI shows the results of multiple logistic regressionanalysis performed in both groups taken together to comparethe risk of CAD associated with receptor-negative versusreceptor-defective mutations. This analysis showed that insubjects.30 years of age with receptor-negative mutations,the risk of CAD was'2.6-fold higher than that observed insubjects with receptor-defective mutations.

DiscussionMutation Spectrum and ClustersIn this study, we report partial characterization of the spec-trum of mutations of the LDL-R gene discovered in Italyduring a survey that started a few years ago and is still inprogress. So far, our inventory consists of 71 mutations andincludes 16 major rearrangements; 8 nonsense, 31 missense,and 11 frameshift mutations; and 5 splicing defects. Duringthis study, we found that'4% of subjects with clinicallysevere FH either do not carry detectable mutations in theLDL-R gene (proximal promoter, coding sequence, andexon/intron junctions) or show a phenotype that does not

TABLE V. Comparison Between Subjects >30 Years of Age With (1) or Without (2) Coronary ArteryDisease (CAD)

Receptor-Defective Receptor-Negative

CAD (2) CAD (1) P CAD (2) CAD (1) P

M/F, n/n 42/55 12/9 NS 45/82 33/26 0.01

Age, y 48.6612.5 55.4611.4 0.02 45.8611.3 55.669.7 ,0.001

Body mass index, kg/m2 24.763.5 25.463.1 NS 23.863.1 24.962.6 0.02

Tendon xanthomas, % 23 (23.7%) 9 (42.9%) NS 70 (55.1%) 49 (83.1%) ,0.001

Arterial hypertension, % 12 (12.4%) 8 (38.1%) 0.01 17 (13.4%) 23 (39.0%) ,0.001

Current smokers, % 18 (18.7%) 3 (14.3%) NS 20 (15.7%) 7 (11.9%) NS

Former smokers, % 8 (8.2%) 9 (42.9%) 0.001 6 (4.7%) 16 (27.1%) ,0.001

Total chol, mmol/L 8.8361.58 9.9261.56 0.005 9.9661.85* 10.5161.72 0.05

LDL chol, mmol/L 6.8561.57 7.8961.39 0.006 8.0661.76* 8.6661.61 0.02

HDL chol, mmol/L 1.3260.41 1.1860.24 NS 1.2860.33 1.2060.29 NS

Triglycerides, mmol/L 1.5560.86 1.9660.93 0.02 1.4260.66 1.5760.62 NS

Chol indicates cholesterol.*Significantly different from receptor-defective CAD (2) subjects.

TABLE VI. Parameters Associated With Coronary ArteryDisease in FH Patients >30 Years of Age WithReceptor-Defective and Receptor-Negative Mutations byMultiple Logistic Regression Analysis

Parameter b6SE (b) OR* 95% CI (OR) P

Male sex 1.10860.307 3.02 1.65–5.53 0.0003

Age 0.05860.013 1.06 1.03–1.09 0.0000

Arterial hypertension 1.20560.349 3.34 1.68–6.62 0.0006

Receptor-negative vsreceptor-defective

0.94860.320 2.58 1.37–4.83 0.003

b is the partial regression coefficient; SE(b), standard error of b; and CI,confidence interval.

*Odds ratio (OR), defined as eb, corresponds to changes in odds for malesvs females, for a 1-year increment in age, for patients with arterial hyperten-sion vs those without, and for patients with receptor-negative mutations vspatients with receptor-defective mutations, assuming that the other parametersremain constant at their mean values.

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segregate with the LDL-R gene haplotype within the family.This discovery is not surprising, as other studies havereported that a certain proportion of putative FH subjects donot have detectable mutations at the LDL-R locus.33–35Fromthe mutation list shown in Table II, the conclusion thatemerges is that Italy is like the United Kingdom,7,33 Germa-ny,36 and Japan,37 where many mutations have produced ahighly heterogeneous picture, in contrast to the situationfound in other countries (like Norway, Finland, and, to someextent, Denmark), where few mutations account for a largeproportion of FH patients.38–40For each index case, we triedto retrieve as much family data as possible to define thegeographic origin of the ancestor likely to carry the mutationidentified in the index case. This family history allowed us tolocate clusters of mutations in specific areas of the countryand to identify several clusters. To have a sufficient numberof subjects for the comparison of lipid values and clinicalfeatures in patients carrying different mutations, we decidedto pool the clusters according to the predicted effect of eachmutation on LDL-R function (receptor-defective versusreceptor-negative clusters; Table III). This analysis showedthat even in the setting of our large allelic heterogeneity,heterozygous FH patients who carry receptor-negative muta-tions have a more severe atherogenic lipid profile (higherLDL-C and lower HDL-C) than do patients with receptor-defective mutations. These results agree with previous obser-vations made in population samples living in other geo-graphic settings. In French-Canadian FH heterozygotescarrying a large deletion (.15 kb) in the promoter and thefirst exon of the LDL-R gene (receptor-negative phenotype),the mean serum cholesterol level was much higher than inheterozygous patients with the W66G mutation (receptor-defective phenotype).6 Afrikaner FH patients with a commonreceptor-defective mutation (D206E) have significantly lowercholesterol levels and milder disease than do Afrikanerscarrying a receptor-negative mutation (V408M).41 Vohl etal42 compared FH heterozygotes carrying null alleles, FHheterozygotes carrying defective alleles, and non-FH sub-jects. They found that carriers of null alleles had the highestplasma total cholesterol and LDL-C levels and the highesttotal cholesterol–to–HDL-C ratio, whereas the carriers ofdefective alleles had intermediate levels between null-allelecarriers and non-FH patients. Similar results, reported inother studies,7,43,44 suggest that in FH heterozygotes (aspreviously documented in FH homozygotes),1,4,6 the differ-ence in the magnitude of increase in plasma cholesterol andLDL-C levels largely derives from differences in the natureof the mutation in the LDL-R gene.

Prevalence of CADIn index cases as well as in all heterozygous FH patients, theprevalence of CAD in receptor-negative subjects (of bothsexes) was 1.7-fold higher than that found in subjects carry-ing receptor-defective mutations. Furthermore, when subjects.30 years old were further stratified by age, we found that inreceptor-negative patients, CAD occurred at a younger agethan in receptor-defective persons. This observation is inkeeping with that of Vohl et al,42 who showed that inFrench-Canadian FH heterozygotes, the development of CADas estimated from the age at first coronary angiography or atfirst revascularization occurred at an earlier age in FH

patients carrying a null mutation compared with carriers ofdefective alleles. Recently, Gaudet et al44 compared theexpression of CAD assessed by coronary angiography inyoung (25 to 49 years) versus middle-aged (50 to 64 years)male FH heterozygotes with receptor-negative mutations(.15-kb deletion, Y468X or R329X) and with missensemutations (W66G, E207K, or C646Y); in this latter group,92% of individuals carried the W66G mutation that producesa receptor-defective phenotype. In both age groups, carriersof receptor-negative mutations had higher plasma cholesteroland LDL-C levels than did carriers of missense mutations,and their relative odds of being affected by CAD before theage of 50 was'3-fold greater than that observed in patientswith missense mutations (in whom the receptor-defectivephenotype was prevalent).

Multiple logistic regression analysis performed in oursubjects who were.30 years of age showed that in bothreceptor-defective and receptor-negative groups, independentcontributors to CAD were male sex, age, and arterial hyper-tension (Table VI). The plasma level of LDL-C was a strongindependent contributor to CAD in the receptor-defectivegroup but was a weaker contributor in the receptor-negativegroup. This latter observation suggests that in our receptor-negative subjects, any increase in LDL-C above certainthreshold level (eg, 8.0 mmol/L LDL-C) contributes less tothe development of CAD than do age, male sex, and arterialhypertension. Taking into consideration all of these factors,the multiple logistic regression analysis showed that in bothsexes.30 years of age, the risk of CAD is'2.6-fold higherin patients carrying receptor-negative mutations.

In conclusion, compared with receptor-defective muta-tions, receptor-negative mutations are more strongly associ-ated with premature CAD, this relationship being indepen-dent of common risk factors such as sex, age, andhypertension, as well as the other risk factors such as smokinghabits, diabetes, and obesity,45 which were not contributors toCAD in our study.

Interindividual Variability of LDL-C LevelAs previously reported,6 we found wide variations in plasmalipid and lipoproteins, even in patients carrying the samemutation. Table III shows that the wide interindividualvariation in LDL-C level persisted even after adjustment forsex, age, and BMI and was present in all clusters regardlessof the type of mutation and its biological effect (receptor-negative and receptor-defective). These variations may bedue to genetic and/or environmental factors that affect ex-pression of the wild-type allele (in the case of receptor-negative mutations) or both alleles (in the case of receptor-defective mutations) or influence other pathways oflipoprotein metabolism. Several groups have investigated therole of common polymorphisms of several candidate genes(such as apo E, apo B, and the LDL-R) that are known to beassociated with variations in plasma LDL, HDL, and triglyc-erides in the general population. In light of the point dis-cussed above, the most reliable studies appear to be thoseconducted on FH patients with identified mutations of theLDL-R gene. Dallongeville et al46 were the first to show thatLDL-C levels were lower in the E3/2 subset than in the E3/3or E4/3 subset of heterozygous FH subjects with the.15-kbFrench-Canadian mutation. These observations were ex-

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panded by Ferrieres et al,47 who found that the contribution ofthe apo E polymorphism was different between the sexes.Women with thee2e3 genotype had lower total cholesterol,LDL-C, and apo B levels than did women with thee3e3genotype, whereas in men, thee2e2 genotype was associatedwith higher VLDL-C than was thee3e3 genotype. Overall,,20% of the variability for the various lipid traits amongthese FH patients was explained by apo E genotype. Surpris-ingly, these effects of apo E polymorphism were not observedin a large group of FH heterozygotes carrying the NorthKarelia mutation,48 although serum LDL-C levels were foundto be lower in subjects with the apoe4e2 genotype incomparison with subjects with other genotypes.

Betard et al49 examined whether the phenotypic variationin lipoprotein and apo B levels observed in a sample ofFrench-Canadians carrying the.15-kb deletion was associ-ated with genetic variability of the “wild allele” (ie, nonde-letion LDL-R gene). They found that some haplotypes,defined by using pairs of restriction fragment length poly-morphisms of the LDL-R gene, contributed to quantitativevariations in HDL-C and LDL-C levels in women but not inmen. However, in FH subjects carrying the North Kareliamutation, Vuorio et al48 failed to find a significant associationbetweenPvuII andAvaII polymorphisms of the LDL-R geneor XbaI polymorphism of the apo B gene and plasma lipidsand the prevalence of CAD.

In the present study, we found that in both receptor-negative and receptor-defective groups, apo E genotypeinfluences plasma LDL-C levels. Compared with thee3e3genotype, e2e2 and e2e3 genotypes have lower LDL-Clevels, whereas the opposite was true for genotypes contain-ing thee4 allele. Overall, apo E genotype accounted for 4.4%and 4.2% of the variation in plasma LDL-C level (adjustedfor sex, age, BMI, and the effect of each LDL-R genemutation) in receptor-defective and receptor-negative groups,respectively. Therefore, apo E polymorphism explains onlypart of the large interindividual variability of plasma lipidsand lipoproteins observed in each cluster of our series, thussuggesting the presence of other genetic and environmentalfactors. The important role played by environmental factorshas emerged from a recent study by Pimstone et al,50 whocompared lipid values and the prevalence of CAD in FHChinese subjects, with defined LDL-R gene mutations, livingin China and Canada. Higher LDL-C levels (and a higherprevalence of tendon xanthomas and CAD), not accounted forby an effect of the LDL-R gene mutation, were observed inFH heterozygotes living in Canada than in those living inChina. These differences could be ascribed, at least in part, tothe striking contrast in dietary fat consumption and level ofphysical exercise between subjects living in Canada and thoseliving in China. It is reasonable to assume that these factorshave a role to play in our series, too. A systematic study ofthese factors in the main clusters of our series is now inprogress. Our preliminary dietary survey, however, has failedto show striking differences in dietary habits (in terms of totaland saturated fat consumption) similar in magnitude to thatreported in the study of Pimstone et al.50 A recent comparison(1994 to 1996) of dietary habits between northern Italy(where, according to tradition, the diet is presumed to containmore meat and dairy products) and southern Italy (where aMediterranean-type diet is presumed to be consumed) has

revealed that the differences in total and saturated fat intakeare fairly small (35% versus 33% and 13% versus 12%,respectively; A. D’Amicis and A. Turrini, National Institutefor Food and Nutrition, personal communication, 2000). Inthis context, we need a very large number of genotyped FHsubjects to be able to ascertain the contribution of diet to theinterindividual variability of LDL-C level.

Common AncestorThe identification of clusters of families carrying the samemutation in specific areas of the country raises the question ofthe origin of these mutations (common ancestor versusrecurrent mutations). We examined the haplotype cosegregat-ing with each mutation in the largest clusters (D200G, FS453,and G528D). D200G has been reported in other Europeancountries in patients with different ethnic backgrounds, sug-gesting the hypothesis that D200G is a recurrent mutation.However, the observation that in our cluster, all patients sharethe same intragenic haplotype and the mutation is mostlyrestricted to the northeastern areas of Italy strongly suggeststhat D200G is identical by descent in all patients of the cluster(which also includes 4 identified homozygotes). The G528Dis a common mutation in southern Italy and Sicily (the clusterincludes 3 apparently unrelated homozygotes and 2 relatedcompound heterozygotes) but has been reported in otherEuropean countries, too. Interestingly, G528D is fairly com-mon in Greece, where it accounts for 23% of unrelated FHheterozygotes and shows 91% linkage disequilibrium with asingle D19S394 microsatellite allele (257 bp).51 In our series,all patients carrying the G528D mutation share the sameintragenic haplotype, but the mutation was found to be linkedwith 4 different alleles of the D19S394 microsatellite.

The finding that in our patients both D200G and G528Dare in linkage disequilibrium with several alleles of thepolymorphic microsatellite D19S394 telomeric to the LDL-Rgene may be explained in several ways: (1) a de novooccurrence of these mutations in some probands; (2) a seriesof recombination events between the mutations in the LDL-Rlocus and the D19S394 locus; or (3) replication slippage ofthe D19S394 variable number of tandem repeats duringmeiosis. If we assume that recombinations between the 2 locihave occurred over time, we may estimate that numerousgenerations have elapsed since the common ancestor, giventhat there is an expected 1/400 chance of recombinationbetween the D19S394 locus and LDL-R locus in a singlegeneration.29 Apparently, the D200G and G528D mutationsmight date back to thousands of years ago. The FS453mutation cosegregates in all 31 families not only with thesame intragenic haplotype but also with a single allele of theD19S394 microsatellite (251 bp); in this case, we can assumethat this mutation is considerably more recent, as it is limitedto a restricted geographic area of the country.

AcknowledgmentsThis work was supported by Telethon (project No. E. 0947), by agrant from the University of Genoa, and by a grant from the ItalianNational Institute of Health.

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12 Arterioscler Thromb Vasc Biol. September 2000

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CalandraC. Stefanutti, I. Blotta, L. Pisciotta, M. Rolleri, S. Langheim, M. Ghisellini, I. Rabbone and S. S. Bertolini, A. Cantafora, M. Averna, C. Cortese, C. Motti, S. Martini, G. Pes, A. Postiglione,

Receptor Gene That Cause a Receptor-Defective or Receptor-Negative PhenotypeClinical Expression of Familial Hypercholesterolemia in Clusters of Mutations of the LDL

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