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Institute of Cytological Research (F.J.C., A.B.G.-G., M.E.A.) and Service of Endocrinology and Nutrition (J.T.R., M.C., J.F.A., R.C.), Hospital Clínico Universitario, Department of Medicine, University of Valencia, Valencia, Spain E-46010
Address all correspondence and requests for reprints to: Dr. Rafael Carmena, Department of Medicine, Hospital Clínico Universitario, Avda. Blasco Ibáñez 17, E-46010 Valencia, Spain. E-mail: carmena{at}uv.es
Abstract
The aims of this study were to examine the presence of mutations in the low-density lipoprotein receptor gene among subjects clinically diagnosed with familial hypercholesterolemia and to analyze whether the molecular diagnosis helps to predict the response to simvastatin treatment in our familial hypercholesterolemia population. Fifty-five probands and 128 related subjects with familial hypercholesterolemia were studied. Genetic diagnosis was carried out following a three-step protocol based on Southern blot and PCR-single strand conformational polymorphism analysis. A randomized clinical trial with simvastatin was conducted in 42 genetically diagnosed subjects with familial hypercholesterolemia classified as carriers of null mutations (n = 22) and of defective mutations (n = 20). A mutation-causing familial hypercholesterolemia was identified in 46 probands (84%). In 41 of them (89%), a total of 28 point mutations were detected, 13 of which have not been previously described. The remaining five probands (11%) were carriers of large rearrangements. Familial hypercholesterolemia with null mutations showed a poor response to simvastatin treatment. The mean percentage reduction of plasma total and low-density lipoprotein cholesterol levels in these subjects were significantly lower (24.8 ± 10.3 vs. 34.8 ± 10.9, P = 0.04 and 30.0 ± 39.8 vs. 46.1 ± 18.2, P = 0.02, respectively) than in subjects with defective mutations. Baseline and posttreatment high-density lipoprotein cholesterol plasma values were significantly lower in subjects with familial hypercholesterolemia with null mutations (P < 0.001). In an outbreed Caucasian population, a three-step protocol for genetic screening detected a mutation in the low-density lipoprotein receptor gene in a high percentage (84%) of subjects with familial hypercholesterolemia. Subjects with familial hypercholesterolemia with null mutations (class I) showed lower plasma high-density lipoprotein cholesterol values and a poor low-density lipoprotein cholesterol response to simvastatin treatment.
FAMILIAL HYPERCHOLESTEROLEMIA (FH) is an autosomal dominant disease defined at the molecular level by the presence of mutations in the low-density lipoprotein receptor (LDLR) gene and characterized by markedly elevated LDL cholesterol (LDLc) levels, tendon xanthomata, and increased risk of coronary artery disease (1, 2, 3, 4). Despite the hereditary nature of the disease, FH shows great variability in phenotypic expression. The expression of this disease may be influenced by factors such as age, gender, diet, type of LDLR mutations, or other genes (5, 6, 7, 8).
Correct genetic diagnosis and early treatment of subjects with FH could prevent premature coronary heart disease (CHD). Compared with Northern European subjects with FH, Spanish subjects present lower levels of plasma LDLc and lower prevalence of tendon xanthomata (9), making genetic diagnosis even more important. So far, a limited number of FH mutations have been described in our population. Identification of LDLR gene mutations in family groups facilitates follow-up of affected individuals and eliminates the problems associated with equivocal lipid profiles, especially in younger patients (10, 11). Even in adults, the use of total cholesterol (TC) as a marker of the disease produces an error of up to 40% (11).
In addition, the type of LDLR gene mutation has been associated with different phenotype expression (6, 8, 12), response to statins (13, 14), and risk of premature CHD (5). Thus, DNA diagnosis could allow the study of genotype-phenotype correlation in terms of estimating the clinical severity and prognosis.
The present study was undertaken to: 1) assess the efficacy of a three step protocol in the genetic diagnosis of FH in an outbreed South European population and 2) to evaluate the influence of LDLR gene type of mutation on the response to treatment with simvastatin.
Subjects and Methods
Subjects
The study population consisted of a total of 183 subjects with FH: 55 probands and 128 related subjects from 30 FH families that had been referred to our lipid clinic. All subjects were Caucasian and lived in the Valencia region. The institutional ethics committee approved the protocol, and all subjects gave written informed consent to enter the study.
Diagnostic criteria for FH included plasma levels of TC and LDLc higher than the 95th percentile corrected for both age and sex (15), presence of tendon xanthomata, coronary artery disease in the proband or in a first-degree relative, and bimodal distribution of TC and LDLc levels in the family, indicating an autosomal dominant pattern of phenotype IIa.
A complete medical history and physical examination were obtained in all participants. Body mass index (BMI) was calculated as weight divided by height squared (kg/m2). Blood pressure was measured with a von Recklinghausen sphygmomanometer in the sitting position and after a 5-min rest. The mean value of three measurements was considered. CHD was diagnosed if there was a documented history of previous myocardial infarction, coronary artery bypass surgery or angioplasty, angina pectoris with positive electrocardiogram, and thallium test or abnormal coronary angiogram (stenosis of >70% in a major vessel).
Study design
A genetic diagnosis of FH could be established in 155 subjects, and 82 of them were randomly selected to enter the study intervention trial. Twenty-six molecularly defined subjects with FH could not be phenotypically classified as null or non-null mutation or presented exclusion criteria and were excluded, and 12 molecularly defined subjects with FH declined to participate. The remaining 44 subjects (22 with null mutations and 22 with defective mutations) were randomized to enter the clinical trial with simvastatin. Two subjects with defective mutations did not complete the study protocol and were excluded.
The study was conducted on an outpatient basis under the supervision of a physician and a clinical dietician at the lipid clinic. All potentially molecularly defined subjects with FH were individually screened 6 wk (visit 1) before the study to verify inclusion and exclusion criteria, explain different study phases, and obtain the informed consent. This prestudy visit included a complete medical history, physical examination, and laboratory test. All lipid-lowering medications were discontinued in this visit, at least 6 wk before the start of the study. Inclusion criteria were heterozygous FH with detected mutation on the LDLR gene, age > 18 < 65 yr, and male or female (postmenopausal or adequate anticonceptive method and a negative pregnancy test). Exclusion criteria were homozygous FH; presence of metabolic, hepatic, renal, or endocrine disease; acute coronary events in the preceding 3 months; treatment with drugs that could affect lipid metabolism (corticosteroids, E, androgens, theophyline, coumarin derivates, barbiturates, antiacids, fish oil preparations, thiazides, or ß blockers), and ethanol consumption more than 30 g/d.
All eligible participants received simvastatin 20 mg once daily for 6 wk (visits 0–2) at visit 0. This visit included physical examination, dietary advice on a standard cholesterol-lowering diet (National Cholesterol Education Program type 1), and blood sampling. Caloric intake was regulated to maintain constant weight throughout the trial. Subjects were instructed to maintain their regular level of physical activity and lifestyle. Laboratory tests included plasma lipids and lipoproteins and routine hematology and blood chemistry determinations. Patient compliance was verified by tablet counts at 3 and 6 wk (visits 1–2). No adverse side effects were reported. Plasma lipid and lipoproteins were determined at visit 0 (baseline) and 2 (after 6 wk of simvastatin treatment). During treatment, patients and physicians were unaware of the specific molecular defect in the LDLR gene.
Laboratory methods
Measurement of lipids and lipoproteins. After fasting for 12–14 h, blood samples were obtained from an antecubital vein into containing EDTA (Vacutainer) and were centrifuged within 4 h. Plasma was stored at 4 C for a maximum of 3 d. Cholesterol and triglyceride levels were measured by enzymatic techniques (16, 17). High-density lipoprotein cholesterol (HDLc) was measured after precipitation of apoB-containing lipoproteins with polyanions (18) and very low-density lipoprotein (VLDL) cholesterol (VLDLc) after separation of VLDL (d < 1.006 g/ml) by ultracentrifugation (19). The LDLc was calculated by subtraction of VLDLc and HDLc from TC. Total plasma apoB was measured by immunoturbimetry (20). The coefficients of variation for lipids and lipoproteins were less than 5%.
Genetic methods
DNA extraction was performed by standard procedure (21). Genetic diagnosis of FH was established by identification of different mutations of LDLR gene by Shouthern blot analysis of BglII and KpnI+XbaI digested genomic DNA probed with a mix of different exons (22) and with exon 2, respectively, to detect major rearrangements. Other digestions and probes and standard and long PCR were used to confirm and delimit the rearrangements (Chaves, F. J., personal communication). mRNA from internal rearrangements was analyzed by RT-PCR and DNA sequencing was used to characterize them (23).
The fragment containing the apoB mutations responsible for familial ligand-defective apoB-100 (FDB) was amplified using the primers described by Schuster et al. (24) and analyzed by single strand conformational polymorphisms (SSCP) using 10 x 10 x 0.5 cm gels (12% acrilamide [29:1], 5% glycerol, 1x TEB, 8 C, and under 400 V). DNA samples carrying the mutations R3480P, R3500Q, R3500W, and R3531C, responsible for FDB, were used as controls.
The promoter, 18 exons and the intronic regions close to them (about 50–70 bp) were amplified in duplex or triplex PCRs and analyzed by SSCP (22, 25) using different conditions ranking from 10–15% of acrilamide (29:1), 0–15% of glycerol, 2–25 C, 400–500 V, and 0.5 or 1 x Tris-borate/EDTA during 2–4 h. After the electrophoresis, DNA was stained with silver (26). When an abnormal band was identified in a subject, the corresponding exon was amplified and automatically sequenced to characterize the mutation.
Genotyping for the apoE polymorphism was performed following the method described by Hixon and Vernier (27) with minor modifications.
Statistical analysis
Data were analyzed with the Statistical Package for the Social
Sciences (6.1.3 for Windows; SPSS, Inc., Chicago, IL) and
expressed as mean ± SD. The reduction in plasma
lipids and lipoprotein levels was expressed as a percentage of
untreated baseline levels. Mean values of quantitative variables were
compared with one-way ANOVA. Proportions were compared with contingency
tables and the 
2 test or the Fisher’s exact
test (n < 5). Changes in plasma lipids and lipoproteins values
were tested using the paired t test.
Multiple regression analysis was used to estimate the independent contributions of the different LDLR gene mutations, age, sex, BMI, and mean baseline lipid values to the TC and LDLc responses to simvastatin.
The general lineal model for repeated measures was used to test the significance of lipid response to simvastatin intervention as well as gene-simvastatin interactions between LDLR gene mutations (null vs. defective) and simvastatin response for each lipid variable examined. Age and BMI were also used as covariates in these analyses.
Results
Genetic diagnosis of FH
The clinical and biochemical characteristics of the 55 FH probands
studied are presented in Table 1
. No
significant differences in age, clinical characteristics, BMI, lipids
and apoB values were found between men and women. The genetic diagnosis
of FH was established using a three-step protocol that screened
mutations at the apoB gene, responsible for FDB, and major
rearrangements and minor or point mutations in the LDLR gene,
responsible for FH.
|
In a second step, we screened the sample for major rearrangements
localized in the LDLR gene using Southern blot analysis. This screening
procedure allowed us to detect and characterize 5 not previously
described major rearrangements (Table 2);
3 of them were deletions (2 affecting the promoter region) and 2
partial duplications of the receptor (23). The 2 deletions
affecting the promoter region were null mutations, while the other
major rearrangements were defective mutations. The mRNA analysis of
these alleles has shown a production of in frame mRNA in all of them
(23).
|
shows
the 28 point mutations causing FH that were identified; 13 of them have
not been previously described in other populations: M-21L, 112insA,
C68W, P160R, 790delATGA, 920insTCAG, Y354C, C358Y, ITL545MPN, G642E and
2312-3 C
T, V779M and N804K (3, 4). These 28 point or
minor mutations produce different alterations of the LDLR gene: one
altered the translation initiation codon, 3 were stop mutations, 3 were
frame shift mutations, 3 altered the splicing and 18 were missense
mutations.
|
The response to simvastatin treatment was studied in a subgroup of
42 molecularly defined FH subjects (see study design). No differences
were observed between men and women with respect to age, BMI, or
baseline concentrations of plasma lipids and apoB (Table 4). After treatment with simvastatin 20
mg once daily for 6 wk, there was a significant percent reduction
(P < 0.001) in both sexes in TC (28 ± 12 in males
vs. 27 ± 10% in females), LDLc (35 ± 14 vs. 33
± 13) and apoB (20 ± 29 vs. 25 ± 25, respectively), with
no significant changes in VLDLc, triglycerides (TG), and HDLc.
|
Effect of LDLR gene mutation on plasma lipids and treatment response to simvastatin
Subjects were classified according to the presence of null
mutations (class I; n = 22) or defective mutations (n = 20).
Plasma lipids and apoB values at baseline (wk 0) and after 6 wk of
treatment with simvastatin 20 mg once daily are shown in Table 5 separated according to the type of LDLR
gene mutation (null mutations vs. defective mutations).
|
and 3 for molecular
characteristics). A defective mutation was considered by changes of
aminoacids at different position on LDLR: S516L (n = 1), R395W
(n = 2), C358Y (n = 5), C68W (n = 2), C95R (n = 1),
T413R (n = 7), G642E (n = 1), and P160R (n = 1). No differences were found between the two molecularly defined FH groups with respect to BMI (26.2 ± 4.7 in null mutations vs. 25.6 ± 3.4 in defective mutations group), gender (11 males vs. 8 males), presence of CHD (n = 5 vs. n = 4), and APOE genotype distribution (E3/E3 17 vs. 15, E2/E3 0 vs. 2, E2/4 0 vs. 1, E3/E4 5 vs. 2). As expected, compared with baseline values, simvastatin significantly reduced TC, LDLc, and apoB levels (P < 0.001) in both groups. No statistically significant differences were observed between men and women in plasma lipid and lipoprotein levels at baseline and after treatment and all analysis were carried out in the entire group.
Significant differences with respect to the percentage decreases in TC and LDLc depending on LDLR type of mutations were observed. The mean percentage reduction of plasma TC and LDLc levels in subjects with null mutations were significantly smaller (24.8 ± 10.3 vs. 34.8 ± 10.9, P = 0.04 and 30.0 ± 39.8 vs. 46.1 ± 18.2, P = 0.02, respectively) than in subjects with defective mutations. The multiple regression analysis of percentage LDLc changes with simvastatin treatment showed that only the type of LDLR mutation, but not age, BMI, or gender, was an independent variable.
Compared with the 12% decrease in plasma TG levels observed with
simvastatin treatment in the defective receptor group, subjects with
null mutations showed a 8% increase. These differences, however, were
not significant because of the great variability of the response to
simvastatin. No statistical difference was observed in the mean
percentage changes in HDLc between the two groups. However, as shown in
Table 5, at baseline and after simvastatin, the null mutation group
showed HDLc values significantly lower than defective mutation
subjects: baseline 1.02 ± 0.21 mmol/liter vs.
1.45 ± 0.42 mmol/liter, respectively (P <
0.001), and posttreatment 1.10 ± 0.38 vs. 1.52 ±
0.35, respectively (P = 0.001). This between-group
statistical difference was also observed when age and BMI
(P = 0.017 and P = 0.022, respectively)
were used as covariates. No statistical differences at baseline and
after treatment with simvastatin were observed in TC, TG, VLDLc, and
apo B values between both groups, whereas LDLc did not show differences
before treatment, but the LDLc levels were higher in the null mutations
group (5.09 ± 1.24 vs. 4.18 ± 0.85;
P = 0.033).
Using the general lineal model for repeated measures and FH genotype
(presence or absence of null mutations in the LDLR gene) as
between-subject factors and lipid measurements as dependent variables,
we found a significant within-group effect in the response to
simvastatin treatment on TC (P < 0.001), LDLc
(P < 0.001), and apo B (P < 0.001),
but not in TG, VLDLc, and HDLc. We also found significant within-group
gene-treatment interactions on TC (P = 0.008) and LDLc
(P = 0.032) (Table 5). When age and BMI were used as
covariates in the analysis, a significant effect of gene-treatment
interaction was found in TC (P = 0.037) and apo B
(P = 0.039) plasma values.
Compliance was assessed by tablet counting; no differences were found between groups. There was no significant variation in body weight at baseline and after treatment period.
Effect of APOE genotype on plasma lipids and apo B and response to simvastatin
In the 42 molecularly defined subjects with FH, the APOE genotype distribution was 2/3 (n = 2), 2/4 (n = 1), 3/3 (n = 32), and 3/4 (n = 7). No significant differences between genders, nor between null/defective mutations were observed.
Because of the small sample size, baseline lipids and apo B, and response to statins were compared only between APOE E3/E3 and E3/E4 carriers. We found no significant differences in sex distribution and BMI between both groups, or in baseline and after treatment response of lipids and apo B plasma concentrations. Mean percentage reduction on TC (27 ± 11 in APOE E3/E3 group vs. 25 ± 5 in APOE E3/E4 group), LDLc (33 ± 13 vs. 32 ± 8), and apo B (40 ± 44 vs. 44 ± 43) were similar in both groups.
Discussion
This is the first genetic screening searching for large and point
mutations of the LDLR gene carried out in subjects with FH in Spain. As
can be expected in an open population, the LDLR gene mutations found
(Tables 2 and 3) were very heterogeneous. This is in contrast with
findings in closed populations in which the founder effect is evident,
such as French-Canadians in Québec, Christian-Lebanese in
Lebanon, or European descendents in South Africa. In all of them, the
number of mutations giving rise to FH is limited, facilitating a rapid
genetic diagnosis (28, 29). By contrast, in the type of FH
population reported here, this diagnosis can be more difficult;
nevertheless, analyzing with PCR-SSCP exons 2, 4, and 6 in which most
of the LDLR gene mutations are located, we were able to establish a
genetic diagnosis in 41% of our FH population.
Our diagnostic protocol, besides demonstrating the absence of carriers of apo B mutations, has shown a high sensibility to detect mutations in the LDLR gene, allowing the identification of a mutation in a high percentage of patients with FH. About 84% of our patients with FH have been diagnosed as carriers of LDLR gene mutations (5 subjects with large rearrangements and 41 with point mutations). We were unable to identify mutations in 15% of our subjects with FH; this could be due to the existence of mutations in regions of the LDLR gene (intronic regions) that were not analyzed or in other genetic regions (30, 31, 32). In a previous study (22), we analyzed the segregation of polymorphisms of the LDLR gene in 15 FH families (the index cases of these families have been included in the present study), and we did not find segregation with the LDLR or apo B genes in three of them. These data support the notion that in some FH families, the hypercholesterolemia could be explained by genes different from those mentioned above. In another study, screening for the R3500Q mutation in 110 FH probands, we detected the first Spanish family with FDB (33).
Although we have identified a total of 33 LDLR gene mutations (28 point
and minor mutations and 5 large rearrangements), only 11% of the
subjects with FH studied shared the same one (mutation 112insA), an
indication of the heterogeneity of our population. Eighteen of the
minor and point mutations identified are missense mutations, three are
stop, three frameshift, one alters the translation initiation codon
(34), three alter splicing regions (35), two
delete the promoter region, one deletes internal exon,s and two
duplicate an internal region of the gene. Thirteen of the minor and
point mutations identified have not been described in other FH
populations (Tables 2 and 3), including one in another Spanish region
(3, 4, 36) geographically close to Valencia.
Interestingly, some of the most common mutations found in our
population (112insA, C95R, and C358Y) have not been found in the other
Spanish sample, while we have found only one case of mutation E10X and
none of 518delG that were the more common mutations in the other study.
In our population, mutations W-18X and E256K segregated in the same
allele in two of the three patients in whom E256K was present,
mutations Q71E and 313+1GC segregated together in one allele and
313+1GC alone in another patient. Interestingly, other mutations
described to segregate together in the other population do not do so in
ours (36). These data again indicate the great genetic
heterogeneity among populations form different regions of Spain,
including those geographically close. The mutations we described were
distributed along all the gene sequence and, only in some exons and
intron-exon regions, we failed to find any mutation. This is in
accordance with other studies in outbreed populations and with data
reported by Varret et al. (3) and Wilson
et al. (4) and the corresponding databases
accessible by the Internet (http//www.umd.necker.fr and
http//www.ucl.ac.uk/fh).
Another goal of the study was to consider the impact of LDLR gene mutations on the plasma lipids response to simvastatin. The null mutations included impair the production of LDLR protein because of complete deletions of the promoter region (total absence of mRNA production) or to early stop codons; the defective mutations include missense mutations. We have shown that, in our population, the type of LDLR mutation had a profound effect on total and LDLc response to simvastatin. Therefore, genetic diagnosis of FH could be a useful tool in predicting response to treatment with statin drugs.
In previous studies in heterozygous FH, differences in treatment response to statins have been associated to age, BMI, and type of LDLR mutations (13, 14). Because of the small number of molecularly characterized subjects with FH included in our and other studies, the power to detect statistical differences is limited. This could explain the different results reported. In different Caucasian populations, LDLR mutations with no protein expression (null mutations) have been associated with higher baseline TC and LDLc levels, poor response to statins, and higher risk of CHD (5, 11, 12, 13, 14). Thus, carriers of LDLR mutations predicted to be severe (like null mutations or mutations that affect exon 4 repeat 5) will not decrease lipid levels with treatment to the same extent than carriers of mild mutations. Our study confirms this hypothesis in a South European FH population, in which no data were so far available. This is in agreement with the results of the FH regression study (37), in which baseline and posttreatment LDLc levels were highest in individuals with severe mutations, intermediate in those with mild mutations, and lowest in the group with no identified mutation.
By contrast, Leitersdorf et al. (38) investigated the lipid response to fluvastatin in patients with FH with three different types of distinct LDLR mutations that are retained and degraded in the endoplasmic reticulum. Carriers of the Lebanese mutation, which functionally behaves like a null allele, had greater cholesterol-lowering responses than carriers of the Sephardic and the Lithuanian mutations, functioning as defective. In addition, these authors showed that carriers of the same mutation displayed a wide range in responses to fluvastatin, implicating the participation of additional genetic or environmental factors.
In another study, Sijbrands et al. (39) observed similar responses to simvastatin treatment in genetically defined heterozygous subjects with FH carrying two functionally different classes of LDLR gene mutations: mRNA negative vs. mRNA positive mutations. As expected, patients with mRNA-negative mutations had higher TC and LDLc levels, lower HDLc, and higher prevalence of tendon xanthomata.
The observed differences in LDLc responses to statins among the various LDLR gene mutations are not yet completely understood. One possible explanation could be that upregulation of the wild-type LDLR allele is affected by the nature of the mutant allele or that some defective mutations show additional LDLR activities (40). Moreover, genetic variability in DNA polymorphism in the wild-type allele could quantitatively influence LDLc concentrations, as has been reported by Bétard et al. (41) in French Canadian women with FH with carrying the 15-kb deletion.
Another possible explanation, advanced by Jeenah et al. (42) is that mutant LDLR protein, when upregulated, interacts with the normal LDLR protein along its intracellular processing, inhibiting its normal function.
We have also investigated the effect of simvastatin treatment on plasma triglyceride and HDLc levels. No significant differences in triglyceride were observed. On the other hand and in agreement with Sijbrands et al. (39), our subjects with FH with null mutations (no mRNA or protein) showed plasma HDLc values significantly lower at baseline and postsimvastatin treatment than those carrying defective mutations. In our null mutation carriers, baseline total and LDLc values were not significantly higher than in those with defective mutations. The fact that HDLc levels were significantly lower in the former could help explain these findings.
Our observation on HDLc values suggests that the LDLR could be important in HDL metabolism in patients with FH. The effect of the LDLR on the clearance of remnant chylomicrons and VLDL particles could explain in part the HDLc plasma levels in our subjects with FH. Bowler et al. (43) have demonstrated a 50% reduction in the clearance of chylomicron remnants in Watanabe hereditable hyperlipidemic rabbits. In recent human studies, there was a markedly delayed clearance of retinol labeled triglyceride rich lipoproteins following an oral fat load in six Japanese homozygous subjects with FH. In addition, the binding and clearance of chylomicron remnants by fibroblasts was substantially decreased in the homozygous FH group (44). Along the same lines, Castro-Cabezas et al. (45) reported, in heterozygous subjects with FH, a 2-fold delay in the area under the curve for clearance of remnant particles. Thus, it is possible that subjects with FH with "null mutations" would have a lower clearance of remnant particles (chylomicron, VLDL, and intermediate density lipoprotein) and, in consequence, lower HDLc concentrations. In addition, this subgroup of FH presented the highest risk of CHD in the study of Vohl et al. (5).
Several studies have suggested that APOE genotype does not alter the response to statins (46, 47), although a lower response in E4 carriers treated with lovastatin has been reported when compared with E3 carriers (48). In the present study with a small sample of only seven E4 carriers, we found no significant differences in response to simvastatin treatment.
In conclusion, genetic diagnosis of FH is important for early detection of affected family members, particularly among young subjects in whom TC values may overlap between affected and nonaffected subjects. In our study, we have demonstrated that a protocol including a three-step genetic diagnosis could detect mutations in the LDLR in about 84% of subjects diagnosed as FH in a South European outbreed population. Baseline plasma lipid values of patients with null mutations show a lower level of HDLc. In addition, the degree of response to simvastatin treatment was related to the type of mutation present in the patient. Subjects carrying null mutations showed a more limited response, including a lower percentage of reduction in total and LDLc without significantly increasing their levels of HDLc. However, variations of this response within carriers of the same mutation have been observed, suggesting the influence of additional factors.
As it is well known, TC, LDLc, and HDLc plasma levels are related to cardiovascular risk. Hence, the relation shown in this study between the mutation type and plasma HDLc levels, and the response of TC and LDLc levels to simvastatin treatment may be important in the prediction of the cardiovascular risk in this population. More prospective studies should be carried out to confirm this hypothesis.
Acknowledgments
We thank Dr. Clive R. Pullinger of the Cardiovascular Research Institute (San Francisco, CA) for providing DNA samples of each mutation of the apo B gene.
Footnotes
This work was supported by grants from Fondo de Investigaciones Sanitarias: FIS 96/2063 and 99/008.
Abbreviations: BMI, Body mass index; CHD, coronary heart disease; FDB, familial ligand-defective apoB-100; FH, familial hypercholesterolemia; HDLc, high-density lipoprotein cholesterol; LDLc, low-density lipoprotein cholesterol; LDLR, LDL receptor; SSCP, single strand conformational polymorphism; TC, total cholesterol; TG, triglycerides; VLDL, very low-density lipoprotein; VLDLc, VLDL cholesterol.
Received March 8, 2001.
Accepted June 4, 2001.
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