Heterozygous familial hypercholesterolemia in children: low-density lipoprotein receptor mutational analysis and variation in the expression of plasma lipoprotein-lipid concentrations

atherosclerosis ELSEVIER

Atherosclerosis126(1996)163-171

Heterozygous familial hypercholesterolemia in children: low-density lipoprotein receptor mutational analysis and variation in the expression of plasma lipoprotein-lipid concentrations Ana L. ‘T’orres, Sital Moorjani’, Marie-Claude Vohl, Claude GagnC, Benoit Lamarche, Louis-Daniel Brun, Paul-J Lupien, Jean-Pierre DesprCs* Lipid Research Center, Lava1 University Medical Research Center, CHUL,

2705 Laurier Blvd., Qutbec GI V 4G2, Canudo

Received27 February 1996;revised7 May 1996;accepted16 May 1996

Abstract

The phenotypic expression 01‘heterozygous familial hypercholesterolemia (FH) is variable from biochemical and clinical standpoints and several genetic and environmental factors could contribute to explain this variability. We have compared, in a cohort of 266 heterozygous FH children and adolescents (l-19 years), the variation in plasma lipoprotein-lipid levels among patients defined by three mutations in the low density lipoprotein receptor (LDLR) gene. Comparison of the plasma total and LDL-cholesterol (LDL-C) levels among the three mutation groups revealed significant differences. Plasma total and LDL-C levels were significantly higher (P < 0.05) in the group bearing the French-Canadian A > 15 kb null allele mutation (8.17 rt 1.45 and 6.58 + 1.42 mmol/l) and in the group with the defective allele Ch46Y missense mutation (8.18 + 1.53 and 6.65 + 1.50 mmol/l) compared to the group with the defective allele W66G missensemutation (7.19 f 1.23 and 5.62 f 1.16 mmol/l). Comparisons of other lipoprotein-lipid parameters between FH heterozygotes and normolipemic (n = 120) children indicated that all mutation groups had significantly (P= 0.0001) lower plasma HDL-cholesterol (HDL-C) levels and a higher total cholesterol (TC) to HDL-C ratio (P <: 0.05). Among FH heterozygote groups, the W66G group had the lowest TC to HDL-C ratio. Multivariate analyses revealed that in FH heterozygotes as well as in controls, HDL-C levels contributed to a greater proportion of the variation in TC to HDL-C ratio than TC. In order to examine the age effect, control and FH heterozygote A > 15 kb groups were then subdivided into four groups (l-4; 5-8; 9-13, and 14-19 years). The variation in HDL-C and triglycerides with age in heterozygous FH children showed a pattern which was similar to the one noted in the control group. In conclusion, the present study demonstrated that the overall contribution of age to variation in the lipoprotein profile of heterozygous FH children is similar to the effect observed among healthy children. The effect of LDLR gene in FH is dominant and there was no difference in plasma TC and LDL-C

* Correspondingauthor. Tel.: + 1 418 6542133;fax: + 1 418 6542714. ’ Deceased I October 1995

0021-9150/96/!Z15.00 0 1996ElsevierScience Ireland Ltd. Ail rights reserved PII SOO21-9150(96)05907-2

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due to gender. Finally, this study indicates that the LDLR gene type mutations are a modulator of the magnitude of the increase in plasma TC and LDL-C levels noted among FH heterozygote children. Keywords: Familial hypercholesterolemia; LDL-receptor gene; Children; Genetic epidemiology

1. Introduction Familial hypercholesterolemia (FH) is an autosomal codominant gene disorder with a gene dosage effect. The primary defect is caused by mutations in gene coding for the low density lipoprotein receptor (LDLR); the defective protein fails to catabolize LDL particles causing high plasma levels of total and LDL-cholesterol [l]. Epidemiological studies showed that elevated LDL-cholesterol (LDL-C) levels are one of the principal factors responsible for premature atherosclerosis, this process beginning early in life [2-41. In FH heterozygotes, plasma concentrations of total cholesterol (TC) and LDL-C are 2 to 3 fold higher than normal and these elevated levels are already noted in childhood [5]. However, the phenotypic expression of FH is quite variable and levels of TC and LDL-C show great interindividual variation in FH heterozygotes [6], which may be attributable to several environmental and genetic factors. FH is heterogeneous at the molecular level. Several mutations in the LDLR gene cause the disease and more than 150 mutations have been documented [6,7]. However, in some populations, such as Afrikaners [8], Lebanese [9], Ashkenazi Jews [lo], Finns [ll], and Tunisians [12], FH is more common and is caused by a small number of mutations due to special cultural and geographic characteristics causing isolation which resulted in a rapid expansion of an original limited genetic pool. This is also the case for Qutbec, a Canadian province, where only six mutations are responsible for the majority of FH cases [13- 161.These special population characteristics allow the diagnostic at molecular level in FH heterozygote children. Since environmental factors play an important role in plasma lipoprotein-lipid levels variation among adults, the study of children provided an opportunity to examine phenotypic

variation at a time where environmental factors may presumably have had lesser influences than in adults. It is therefore important to determine factors modulating plasma lipoprotein-lipid concentrations in children, which may later have an impact on the severity of clinical manifestations in adulthood. The availability of a large number of subjects in each mutation group made possible the comparative study of phenotypic expression of these mutations affecting the LDLR function. The purpose of the present study was to investigate whether different mutations at the LDLR gene locus play a major role in the modulation of plasma TC and LDL-C levels in a sample of FH heterozygote children and adolescents. 2. Material and methods 2.1. Subjects The present sample came from a cohort of three hundred forty-three children (1 - 19 years old) suspected for heterozygous FH by clinical criteria. Most children studied were first degree relatives of patients with FH followed at our Lipid Research Clinic in QuCbecCity. Diagnosis of FH was made according to previously described criteria [ 171 which included: (a) plasma cholesterol levels above 95th percentile (age and sex adjusted); (b) presence of tendinous xanthomas; and (c) in absence of tendinous xanthomas due to young age of subjects, LDL-C > 95th percentile and presence of tendinous xanthomas in at least one first degree relative. Subjects with secondary causesof hypercholesterolemia were excluded from the study. After diagnosis was confirmed at molecular level by analysis of genomic DNA for the six French Canadian most common mutations, a sample of 266 FH heterozygote children with whole lipoprotein-lipid profile and bearing the A > 15 kb (n = 188), W66G missense mutation in exon 3

A.L. Torres ef al. /)Atherosclerosis

(n = 57) and C646Y missense mutation in exon 14 (n = 21) were used for this study. The control group included 120 healthy siblings and unrelated children. In order to study the age effect on lipoprotein-lipid levels, FH heterozygotes and controls were divided into four subgroups: 1, children < S years; 2, children between 5 and 8 years; 3, children between 9 and 13 years; and 4, adolescents between 14 and 19 years. 2.2. Plasma lipids and lipoproteins

Blood samples were obtained in the morning after a 12 h fast from an antecubital vein into Vacutainer tubes (Becton Dickinson) containing EDTA. Cholesterol and triglyceride (TG) levels in plasma were measured enzymatically on an RA-1000 analyzer (Technicon Instruments, Tarrytown, N.Y.). The HDL fraction was obtained after precipitation from plasma of apo B-containing lipoproteins with heparin and MnCl, [18]. The LDL-C was calculated by the Friedewald formula [19]. The overall coefficient of variation for the measurement of control sera was 2.1% for high cholesterol value, 2.7% for low cholesterol value, and 3.0% for TG. Duplicate analysis of 55 se:rafor HDL,-C determinations on different days yielded analytical variation of 3.3%. 2.3. DNA analy:ris DNA was extracted from peripheral blood leucocytes by standard methods [20]. For the detection of the :+ 15 kb and 5 kb deletions, 10 pug of genomic DNA were digested with XbaI and EcoRV restriction enzymes using 10 units of enzyme per pg DNA, in the buffer provided by manufacturer Promega. Resulting fragments were size separated by electrophoresis on 0.8% agarose gels and transferred to Hybond-N + nylon membranes (Amersham) by Southern blotting [15]. The probe used to detect the specific fragments was prepared from the clone pLDLR3 (American type culture collection, Rockville, Maryland, USA), which contains the entire cDNA sequence of the LDL-receptor gene. A 650 bp Pst I-EcoRI fragment was radiolabelled

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with a-32P-dCTP by random priming [21] with the oligolabelling kit (Pharmacia) and used as a probe to detect the presence of > 15 kb and 5 kb deletions. Point mutations in exons 3, 4 and 14 were detected by allele-specific oligonucleotide hybridization. The exons 3, 4 and 14 were amplified by the polymerase chain reaction (PCR) in a DNA Thermal Cycler (Perkin-Elmer Cetus) with 25-mers primers SP-59 and SP-60 for exon 3, SP-51A and SP-15A for exon 4 and SP-80 and SP-81 for exon 14 [14]. After amplification, PCR products were denatured with 0.4 M NaOH/25 mmol EDTA and dot-blotted on nylon membranes (Hybond-N + , Amersham) folhybridization with 20-mers lowed by oligonucleotides probes specific to the mutant and the normal allele [14]. Oligonucleotides were labelled using Y-‘~P-ATP and T4 polynucleotide kinase (Pharmacia). The stop 468 nonsense mutation in exon 10 was detected by PCR using primers previously described by Vohl et al. [22]. After amplification PCR products were digested with BfuI restriction enzyme [22]. The resulting fragments were size separated by electrophoresis on a 8% nondenaturing polyacrylamide gel. 2.4. Statistical analyses

Comparisons of lipoprotein and lipids among mutation and age groups were performed by using multiple comparison ANOVA analysis and the Duncan post hoc test when a significant group effect was observed. Sex differences in plasma lipoprotein and lipid levels were assessed by t-test. Associations between lipoproteins and lipids were analyzed by using Pearson’s correlation coefficient. Prior to statistical analyses, plasma TG levels were log(n) transformed in order to reduce the skewness of the distribution but untransformed values are shown in figures and tables. TG were normally distributed after log(n) transformation. Multivariate analyses were performed to estimate the independent contribution of the lipoprotein-lipid levels to the increase in the TC to HDL-C ratio. Statistical analyses were performed using the SAS software package.

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Table 1 Characteristics of controls and FH heterozygote children according to LDL receptor gene mutations Controls

n

Age (years) BMI” (kg/m*) TC (mmol/l) LDL-C (mmol/l) HDL-C (mmol/l) TG (mmoljl) TC/HDL-C ratio

120 9.05 k 4.63 17.94+ 2.61 4.32 5 0.60 2.60 _+0.56 1.26& 0.29 I .04 f 0.40 3.56 -fr0.82

FH heterozygotes A>15 kb

C646Y

W66G

188 8.21 _+4.14 16.98+ 2.52 8.17 rt 1.45* 6.58 + 1.42* 1.11*0.23*** 1.09* 0.49 7.70 f 2.15*

21 7.06 k 4.09 16.55) 2.13 8.18 * 1.53* 6.65 + 1.50* 1.08k 0.28*** 1.24_+0.76 8.25 + 3.35*

57 8.00 + 4.12 17.79f 3.67 7.19 k 1.23*.** 5.62 + 1.16*.** 1.14;0.20*** 1.01* 0.43 6.48 + 1.57*,**

The values are expressed as mean k S.D. a Number of subjects for BMI: controls = 56; A> 15 kb = 174; W66G = 52. * Significantly different (PiO.05) from controls; ** P~0.05 from group A> 15 kb and C646Y; *** P = 0.0001 from controls.

3. Results

In this cohort of 343 children the frequency of the A > 15 kb reached 56.0%, 17.8% of subjects had the W66G mutation in exon 3, whereas 6.4% had the C646Y mutation in exon 14. The last 20% include subjects with the E207K mutation in exon 4, the stop 468 mutation in exon 10 and subjects with unclassified mutations that were excluded for the present analyses. Phenotypic characteristics in the three different mutation groups of FH heterozygous children and in controls are shown in Table 1. All groups were similar for age, BMI, and TG levels. Compared to controls, FH heterozygote children showed a two to three-fold increase in plasma TC and LDL-C levels irrespective of the LDLR mutations. Furthermore, the W66G group showed the lowest plasma TC and LDL-C levels compared to the two other mutation groups (P < 0.05) whereas A > 15 kb and C646Y showed similar levels. HDL-C levels were significantly decreased in the three mutation groups compared to controls. TC to HDL-C ratio has consistently been reported to be a strong predictor of coronary heart disease risk [23] and this ratio was significantly increased in the three FH heterozygote groups compared to controls (P < 0.05). However, among FH heterozygote groups, the W66G group had the lowest TC to HDL-C ratio compared to the two other mutation groups (P < 0.05). Multivariate analyses were

performed to examine whether the decreased HDL-C levels also found among FH heterozygotes children contribute to increase the TC to HDL-C ratio. Fig. 1 shows the model including HDL-C, TC and TG levels. HDL-C contributed to a larger proportion of the variance of the TC to HDL-C ratio in FH heterozygotes (A > 15 kb = 52%; W66G = 54%) as well as in controls (57%) compared to the TC (A > 15 kb = 42%; W66G = 43%; controls = 32%). There was no substantial contribution of TG to the variance of the TC to HDL-C ratio in FH heterozygotes as well as in controls. The effect of gender on lipoprotein-lipid profile in A > 15 kb and W66G groups is shown in Table 2. There was no gender difference despite the fact that boys were younger than girls in the A > 15 kb group (P = 0.007). Plasma lipoprotein and lipid concentrations were similar between boys and girls. It is also relevant to note that the differences in the plasma TC and LDL-C levels between the two mutation groups remained significant when the analyses were performed by gender (not shown). The age-related variation of lipoprotein-lipid levels in the A > 15 kb group is shown in Table 3. The plasma TC and LDL-C levels were slightly decreasedin the 14 to 19 age group but this trend did not reach statistical significance. A significant age-effect was observed for plasma HDL-C and TG concentrations. In the fourth age group (14-

A.L. Torres et al. i Atherosclerosis

19 years), HDL-C levels were significantly lower (P < 0.01) and the TG concentrations were significantly higher (P < 0.05) than in the other three groups. When the effect of age was examined in boys and girls separately, no gender difference in TC and LDL-C levels was noted. In the age group 14-19 years, boys had mean HDL-C levels of 0.88 + 0.22 mmol/l whereas mean HDL-C levels reached 0.98 & 0.13 mmol/l in girls. However, plasma HDL-C levels were significantly lower in girls aged 14-19 years compared to the 5-8 years group (0.98 + 0.13 versus 1.17 f 0.26, P = 0.045). Significant lower HDL-C levels in girls in the group 14-19 ye:ars compared with those in the 5-8 years can re:sult from influences of confounding environmental factors. Indeed, two girls in this small group of 15 subjects were current smok-

TC. 42% (p=O 0001) ,/--\

A >15

kb

TC. 43% (p=0 0001) /--\

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ers, one girl was obese (BMI = 27), whereas three girls were oral contraceptive users. Similar age related variation was noted when the control group was subdivided into similar age groups. As shown in Fig. 2, the effect of age on HDL-C (panel A) and TG (panel B) levels in the A > 15 kb and control groups was similar, but the A > 15 kb showed significantly lower HDL-C levels at any age (Fig. 2A). When comparable analyses were performed in the W66G group, essentially similar results were obtained (not shown). As expected the strongest association between lipoprotein lipid variables in A > 15 kb and in normolipemic controls was found between TC and LDL-C. Age showed a significant negative correlation with HDL-C levels (A > 15 kb, r = 0.16, PcO.05; control, r= -0.21, PiO.05) and a positive association with TG levels (A > 15 kb, r = 0.14; control, Y= 0.15, P = N.S.). No correlation was observed between age and TC and LDLC. Plasma TG levels were negatively correlated with HDL-C concentrations in both groups (A > 15 kb, r = - 0.37, P= 0.0001; control, r = 0.38, P = O.OOOl)(datanot shown). In FH heterozygote children, the LDL-receptor gene type mutation (A > 15 kb or W66G) explained 8.1 and 8.2 percent of the variance in plasma TC and LDL-C levels, respectively. The percent of sample variance in TC and LDL-C attributable to LDLR type mutation was similar between the two sexes(TC, boys 9.3% girls 8.3%; LDL-C, boys 8.3%, girls 9.4%). The distribution of plasma LDL-C levels in the A > 15 kb and W66G groups of FH heterozygote children is shown in Fig. 3. In the two groups, plasma LDLC levels were normally distributed, a three-fold variation within each mutation group being observed (A > 15kb, 3.73-11.42 mmol/l; W66G, 3.08-8.35 mmol/l). The distribution of LDL-C levels in the W66G group was, however, slightly shifted to the left compared to A > 15 kb heterozygotes. 4. Discussion

Fig. 1. Proportion of the variance in the TC to HDL-C ratio explained by TC, HDL-C and TG levels in FH heterozygote (A > 15 kb and W66G) and normolipemic controls.

The present study examined the influence of three LDLR gene mutations on the variation of

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Table 2 Plasma lipoprotein-lipid profile in relation to LDL-receptor gene mutations: effect of gender A>15 kb

n Age (years) BMI” (kg/m’) TC LDL-C HDL-C TG TC/HDL-C

W66G

Boys

Girls

Boys

Girls

105 7.49 * 3.11 16.97+ 2.34 8.06 5 1.41 6.47 + 1.37 1.12kO.23 1.03k 0.46 7.47 + 2.03

83 9.13*4.41* 16.99+ 2.74 8.31 f 1.49 6.72 k 1.48 1.09f 0.23 1.16kO.53 7.98 & 2.28

23 8.54 + 3.49 18.28i 2.98 6.93 + 1.33 5.44 k 1.28 1.09*0.17 0.94 f 0.38 6.53 _+1.74

34 1.63 k 4.52 17.49+ 4.05 7.37 + 1.15 5.74 + 1.08 1.18+0.22 1.06+ 0.46 6.45 k 1.46

“Number of subiects for BMI. A> 15 kb: bow = 97. girls = 77; W66G: boys = 20, girls = 32. By t-test: * P =‘6.007 from boys A> 15 kb. . -

plasma lipoprotein and lipid levels among children with heterozygous FH. The frequency of the A > 15 kb in this cohort (56%) was similar to frequencies of 63 and 59% reported in two previous studies of patients studied in the Montreal area [13,14]. The frequency of 6.4% for the C646Y mutation is also similar to the 5% frequency reported previously [14]. However, the frequency of the W66G mutation has been reported to reach 7%, this mutation being much more prevalent in our cohort where it reached 17.8% [16]. These three groups of mutations alter the LDLR function differently. In two of these mutations, the LDLR protein is completely absent from the cell surface; in the A > 15 kb, there is no production of mRNA due to the deletion of the promoter region and of exon 1, (class 1, null allele) [14]. The C646Y missense mutation in exon 14 included in the EGF-homology region causes an impairment in protein transport, the protein not reaching the Golgi complex and thus being rapidly degraded (class 2A, transport) [14]. In the W66G missense mutation contained in the second cysteine repeat of the ligand binding domain of the LDLR, the protein can be expressed at the cell surface but this protein fails to bind LDL particles (class 3, binding defective allele) [14]. Plasma TC and LDL-C levels were significantly lower in mutation W66G, which is a defective mutation compared to A > 15 kb and C646Y. In the two latter groups, TC and LDL-C levels were essentially similar. This significant difference in TC and LDL-C lev-

els can be explained by the greater capacity of the W66G resulting protein to accept LDL particles from plasma. Site-directed mutagenesis studies have demonstrated that point mutations or deletion of the entire second cysteine repeat, such as the W66G mutation, reduced LDL binding capacity by nearly 32% [24]. Studies on the activity of LDLR in class 1 (null alleles) such as the A > 15 kb mutation showed less than 2% of the normal LDL-receptor activity. On the other hand, the class 2A defective-transport mutation, such as the C646Y also showed a LDL-receptor activity of less than 2% [25]. Thus, it is not surprising that these two mutation types resulted in a similar phenotypic expression of the disease. The dominant effect of LDLR gene defect in FH is well demonstrated by the absenceof gender differences in plasma TC and LDL-C levels within each mutation group and by the fact that these significant differences between mutation groups remained when results were analyzed by gender. Moreover, in heterozygous FH children, variance of plasma TC and LDL-C attributable to mutations A > 15 kb and W66G was similar between the two sexes(TC, boys 9.3%, girls 8.3%; LDL-C, boys 8.3%, girls 9.4%). Heterozygous FH is a disease associated with an increased risk for coronary heart disease (CHD). Despite the dominant character of FH, other factors can cause variation in the expression and severity of CHD. In this sample of FH heterozygote children, age-related changes in TG and

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Table 3 Effect of age on plasma lipoprotein-lipid profile in children with A> 15 kb of the LDL-receptor gene

n

BMP (kg/m’) TC LDL-C HDL-C TG TC/HDL-C

Age groups (years) l-4

5-8

9-13

14-19

48 16.35+ 1.27* 8.41 +_1.40 6.82 & 1.35 1.11+0.24 I .07 + 0.35 7.94 + 2.06

62 15.89ri: 1.39* 8.16 + 1.28 6.57 k 1.24 1.16~0.22 0.98 k 0.48 7.35 + 2.10

58 17.33+ 2.44** 8.15 + 1.51 6.54 + 1.53 1.11kO.22 1.11kO.59 7.70 f 2.36

20 21.38 + 3.41 7.69 + 1.84 6.17 + 1.73 0.96 k 0.16*** I .37 4 0.44*** 8.15 It. 1.83

ABMI: number of subjects in groups: l-4 = 45; 5-8 = 60; 9- I3 = 52; 14- 19 = 17. * Different (PcO.05) from the groups 9-13 and 14-19: ** PcO.05 from the group 14-19; *** PcO.05 from the three other groups.

HDL-C levels were similar to controls. Similar results were reported in a larger epidemiological study [26]. According to the Lipid Research Clinics program reference values [27], 41% of our group of FH heterozygote children had TG levels > 90th percentile, which is similar to the 39% proportion found in controls. However, 36% of FH heterozygote children had HDL-C levels < 10th percentile whereas this phenotype was found in 16% of controls. Thus, among these FH heterozygotes there is a subgroup with another alteration (high TG and low HDL-C levels) contributing to increase further the risk of CHD. In population studies, this trait of high TG and low HDL-C was associated with a significantly higher rate of coronary artery disease [28,29] and with metabolic disorders indicative of insulin-resistant state [30,31]. The association between high TG and low HDL-C levels and CHD has been documented in FH heterozygote adults by Moorjani et al. [32]. In this previous study performed on adult men (mean age of 38 + 11 years; range 21-71), subjects with the type IIb phenotype (elevated LDL-C levels and TG levels 2 200 mg/dl or 2 2.26 mmol/l) and subjects with the type IIa phenotype (elevated LDL-C levels and TG I: 2.26 mmol/l) presented similar levels of LDL-C, but there was a difference between these two groups in mean HDL-C concentrations as men with the IIb phenotype had a 15% decreasein their HDLC concentrations compared to men with IIa phenotype. These alterations in lipoprotein-lipid profile were associated with a three-fold increase

in the incidence of myocardial infarction in men with IIb phenotype compared to men with the IIa phenotype (25 versus 8.5%). Considering the increased TC to HDL-C ratio, our group of FH heterozygote children is particularly at risk for CHD. Moreover, there was significant differences in the increase of this ratio between the mutation groups. Multivariate analyses revealed that in FH heterozygotes, low HDL-C in addition of high TC levels contributed to the increased TC to HDL-C ratio found in these children. We observed a significant inverse correlation between TG and HDL-C levels in A > 15 kb and W66G mutations groups. These associations have also been found in large school-aged children population studies [26,33]. There was variation in the strength of relationships between lipoproteins and lipids in each mutation group. These differences could be possibly due to alterations in the interaction of LDL particles with the LDL receptor. In other words, the LDLR gene type mutations may affect the dynamic of the lipoprotein precursors of LDL and other lipoproteins in the chain of events leading to the catabolism of lipoproteins. In conclusion, the present study demonstrated that the effect of age in heterozygous FH children was similar to healthy children, HDL-C contribute in FH children as in controls, at the same extent to the TC to HDL-C ratio, suggesting that genetic and environmental factors influencing the risk for CHD in the general population may also be present in children with FH. However, the

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effect of LDLR gene is dominant and suppresses differences in plasma TC and LDL-C levels due to gender. Finally, this study demonstrated that the LDLR gene type mutations found in heterozygous FH children modulate the magnitude of elevation in plasma TC and LDL-C and hence may have important implications for the progression of the diseaseand for the onset of manifestation of atherosclerotic disease in adult life.

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7

Lx

Acknowledgements

6

LDL-C

This study was supported by grant from “Fonds de la Recherche en Sante du Quebec”/ Hydro-Quebec (FRSQ-Hydro). M.C. Vohl and B. Lamarche are fellows of the Medical Research A 1..5-

7

8

A > 15Kb

9

(mmol/L)

Fig. 3. Frequency distribution of plasma LDL-C (mmolj) levels in FH heterozygote children according to LDL-receptor gene mutations (A > 15 kb and W66G).

Council bf Canada (MRC). We would also like to express our gratitude to the staff of the Lipid Research Center for their dedicated work.

0 Control 0 A>lSkb

References

Omg

l-4

’ 5-8

’ 9-13

‘14-19’

AGE GROUP B 1.5

b 0 Control

la4 2

0 A>15kb

,d

1.3

!

g 0.s

1.1 1.2 i

AGE GROUP

Fig. 2. Effect of age on plasma HDL-C (panel A) and TG (panel B) levels (mean + SE.). Comparison between FH heterozygote children bearing the A z 15 kb and control group of children; a, P < 0.05 (control vs. FH bearing the A > 15 kb); and b, P < 0.05 (compared to other age groups within control and FH bearing the A > 15 kb).

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