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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:408-418

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:408-418.)
© 1999 American Heart Association, Inc.

Original Contributions

Analysis of LDL Receptor Gene Mutations in Italian Patients With Homozygous Familial Hypercholesterolemia

S. Bertolini; S. Cassanelli; R. Garuti; M. Ghisellini; M. L. Simone; M. Rolleri; P. Masturzo; S. Calandra

From the Centro Prevenzione Arteriosclerosi (S.B., M.R.), Università di Genova, Genova; and the Dipartimento di Scienze Biomediche (S. Cassanelli, R.G., M.G., M.L.S., S. Calandra), Università di Modena, Modena, Italy.

Correspondence to Sebastiano Calandra, MD, Dipartimento di Scienze Biomediche, Università di Modena, Via Campi 287, I-41100 Modena, Italy. E-mail sebcal{at}unimo.it


*    Abstract
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 *Abstract
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Abstract—The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996. The age of the patients ranged from 1 to 64 years; one third of them were older than 30. Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L. The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from <2% to 30% of normal and was inversely correlated with the plasma LDL cholesterol level (r=-0.665; P<0.003). The most severe coronary atherosclerosis was observed in those patients with the lowest residual LDL receptor activity (<=5% of normal) and the highest plasma LDL cholesterol levels. Twenty-nine patients (23 of whom were unrelated) were found to be homozygotes at the LDL receptor locus. In this group we discovered 2 major rearrangements and 12 different point mutations (9 in the coding region and 3 in splice sites). Some mutations (D200G, C358R, V502M, G528D, and P664L) were found in 3 or more unrelated patients. Patients with the same mutation shared the same haplotype at the LDL receptor gene locus and came from the same geographic area. Ten patients (9 of whom were unrelated) were found to be compound heterozygotes. The mutations found in this group consisted of one large deletion and 12 point mutations (11 in the coding sequence and one in a splice site). In 3 compound heterozygotes we failed to identify the second mutant allele at the LDL receptor locus. These observations confirm the allelic heterogeneity underlying familial hypercholesterolemia in the Italian population and indicate that the variability of phenotypic expression of homozygous familial hypercholesterolemia is, to a large extent, related to the type of mutation of the LDL receptor gene.


Key Words: homozygous familial hypercholesterolemia • LDL receptor gene • mutational analysis


*    Introduction
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 *Introduction
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Familial hypercholesterolemia (FH) is an autosomal codominant disorder characterized by an elevated level of plasma LDL cholesterol (LDL-CH), tendon xanthomas, and premature atherosclerosis. The genetic basis of FH is a lack of functional receptors for LDL on the cell surface.1 The heterozygous form of FH is estimated to be 0.2% in most populations.1 However in selected populations, such as Afrikaners of South Africa2 and French Canadians,3 the frequency of FH is much higher because of a founder effect. In European and North American populations homozygous FH is extremely rare, being estimated to occur in about 1 in 1 million births.1 Homozygous FH is characterized by a marked elevation of LDL-CH, cutaneous as well as tendon xanthomas, arcus cornealis, and generalized atherosclerosis developing during childhood. If untreated, death from myocardial infarction or sudden cardiac death is expected to occur by the end of the third decade of life.1

FH is heterogeneous at the molecular level; more than 200 mutations of the LDL receptor gene have been reported so far in FH patients.1 4 5 6 In some cases the effect of these mutations on LDL receptor function has been fully characterized.4 5 These receptor defects have been grouped into 5 major classes: (1) failure to express receptor protein synthesis; (2) defective transport of receptor precursors from the endoplasmic reticulum to the cell surface; (3) impairment of receptor binding to the ligand; (4) impaired internalization of the receptor–ligand complex because of defective clustering of LDL receptors into coated pits; and (5) defective receptor recycling.1 In many cases each functional class is associated with mutations in regions of the gene that encode one specific domain of the receptor protein.

During the past few years we have characterized more than 60 mutations of the LDL receptor gene in Italian FH patients.7 During this study we have had the opportunity to collect DNA samples, fibroblasts, and clinical data from FH homozygotes who have been referred to various Italian lipid clinics during the last decade and who were alive in 1994 or were born afterward. The aim of this study was the characterization of the mutations of the LDL receptor gene in these patients as the basis for future studies on the genotype–phenotype relationship, the response to pharmacological or nonpharmacological treatment, and the identification of suitable candidates for future gene therapy.

We performed DNA analysis in 39 patients, all of them Italians, with the clinical phenotype of homozygous FH. Molecular analysis has revealed that 29 of these patients were homozygotes and 10 were compound heterozygotes.


*    Methods
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FH Patients
In all cases the diagnosis of homozygous FH was based on the following criteria: (1) plasma or serum LDL-CH >10 mmol/L; (2) presence of tendon and cutaneous xanthomas at an early age; (3) an autosomal mode of inheritance of hypercholesterolemia in the kindred; and (4) the presence of primary hypercholesterolemia in the proband's parents. Detailed family studies were conducted specifically in 2 probands (compound heterozygotes 35 and 37 in Tables 3Down and 4Down) who were found to have a relatively high residual LDL receptor activity in fibroblasts and in whom we have been able to identify only one mutant allele at the LDL receptor locus (Table 4Down). In these cases plasma LDL-CH values found in the probands' parents (8.5 mmo/L in the mother and 5.1 mmol/L in the father of proband 35; 6.5 mmol/L in the mother and 6.6 mmol/L in the father of proband 37) suggested that the parents were FH heterozygotes. This hypothesis was supported by the observation that other subjects with primary hypercholesterolemia were present in both maternal and paternal lines of probands' families.


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  		Table 3.


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  		Table 4.

Plasma lipids and lipoproteins were measured as previously reported.8 Informed consent was obtained from the patients or, in the case of children, from their parents. The study protocol was approved by the institutional human investigation committee of each participating institution.

Cardiological Evaluations
At the time of DNA analysis some patients had already experienced a myocardial infarction or were affected by angina pectoris. Most of the patients with overt coronary artery disease (CAD) had undergone coronary angiography as well as cardiac and carotid artery ultrasound examination. All asymptomatic subjects had undergone electrocardiography and, with the exception of some children, ECG stress testing. Most patients have been under treatment with LDL apheresis for several years, and some have also received hypolipidemic drugs (HMG-CoA reductase inhibitors, cholestyramine, and probucol). Several patients had undergone coronary artery bypass graft surgery. Some patients died of ischemic heart disease or sudden cardiac death during the study (1994–1996).

Fibroblast Culture and LDL Receptor Activity
Skin biopsies were taken from 32 patients. The assay of 125I-labeled LDL binding, internalization, and degradation by cultured skin fibroblasts was performed as described by Goldstein et al.9 Residual LDL receptor activity (calculated as the maximum rate of saturable 125I-LDL degradation) was expressed as a percentage of the value obtained in control fibroblasts.

Southern Blot Analysis and Single-Strand Conformation Polymorphism (SSCP)
Genomic DNA was extracted from peripheral blood leukocytes or cultured fibroblasts by a standard procedure.10 All DNA samples were digested using 5 to 10 U/µg of several restriction enzymes, separated by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with various LDL receptor cDNA probes.8 SSCP was performed as specified previously,11 using primers suggested in previous reports.5 12 13 Polymerase chain reaction (PCR)-amplified exons that showed an abnormal SSCP pattern were sequenced directly using the ThermoSequenase sequencing kit (Amersham).

PCR Amplification of Genomic DNA and Direct Sequencing
In one patient (No. 1), who was homozygous for a >15-kb deletion eliminating the 3' end of the gene (downstream from exon 12), exon 12 was amplified by PCR from genomic DNA using flanking primers14 to ascertain whether this exon was involved in the deletion. The incubation conditions were 94°C for 3 minutes, and subsequently 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes for 35 cycles.

In the siblings 30 and 31 (carrying a deletion involving exon 13 to 15 region) we amplified the region spanning from exon 12 to intron 16 using primer 5' ACC GGA AGA CCA TCT TGG AGG ATG A 3' (as forward primer in exon 12) and primer SP85 (complementary to the 5' end of intron 16).12 In this PCR amplification we used high-fidelity Taq polymerase (Boehringer Mannheim) in the presence of 1.5 mmol/L MgCl2 under the following conditions: 94°C for 3 minutes, 94°C for 30 s, and 68°C for 7 minutes (10 cycles) and subsequently 94°C for 30 s and 68°C for 7 minutes (for the initial cycle), with a gradual increase in the elongation time of 20 s in each cycle, for 20 cycles. The PCR fragment corresponding to the deleted allele was purified by Jet-sorb (Genomed) and then sequenced using primer SP85.12

All patients were screened for the presence of the R3500Q mutation in apolipoprotein B by using a selective PCR amplification of the apo B gene.15

Northern Blot Analysis and Reverse Transcription-PCR Amplification (RT-PCR)
Total cellular RNA was isolated by extraction in guanidine-thiocyanate16 from cultured skin fibroblasts that had been maintained in lipoprotein-deficient serum for 48 hours. Northern blot analysis was performed as specified previously.8

RNA (1 µg) from cultured fibroblasts of patients 27 and 28 and from a control subject was reverse-transcribed and amplified by PCR as specified previously.11 In the case of patient 27, RT-PCR was performed using the following primers: 5'-AAT GCA TCA CCC TGG AGA AAG TCT G-3' (6s; forward primer in exon 6) and 5'-GTT GTG GAA GAG GAC CAT ATC-3' (13as; reverse primer in exon 13). In the case of patient 28, RT-PCR was performed using the following primers: 5'-ATG ACA CCG TCA TCA GCA GGG A-3' (10s; forward primer in exon 10) and 5'-CTT GGT GAG ACA TTG TCA CTA TC-3' (15as; reverse primer in exon 15). The conditions were 95°C for 4 minutes and subsequently 95°C for 1 minute, 58°C for 1.30 minutes, and 72°C for 2 minutes, for 30 cycles. RT-PCR products were sequenced directly using the ThermoSequenase sequencing kit.

In 2 compound heterozygotes (No. 31 and 35), the search for the second mutant allele was performed on overlapping cDNA fragments (exons 1 to 9, 6 to 13, and 13 to 18) obtained by RT-PCR. The primers used for RT-PCR were the following: 5'-GCT GGA AAT TGC GCT GGA CCG TCG C-3' (1s; forward primer in exon 1) and 5'-CTG CAG ATC ATT CTC TGG GA-3' (9as; reverse primer in exon 9); primers 6s and 13as (see above); 5'-TTG TTG GCT GAA AAC CTA CTG TCC C-3' (13s; forward primer in exon 13); and 5'-CAA GGC CGG CGA GGT CTC AAG A-3' (18as; reverse primer in exon 18). For the amplification of exons 1 to 9 the conditions were 95°C for 3 minutes and subsequently 95°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes, for 30 cycles; for the amplification of exons 6 to 13 and exons 13 to 18, the conditions were 95°C for 3 minutes, and subsequently 95°C for 1 minute and 70°C for 1.30 minutes for 35 cycles. These fragments were sequenced directly using appropriate primers.

Haplotype Analysis
Haplotype analysis was performed in FH homozygotes carrying the 5 most common mutations (D200G, C358R, V502M, G528D, and P664L). Specific exons were amplified by PCR from genomic DNA using previously reported primers12 13 and incubation conditions.11 In the case of P664L mutation, because of an abnormal NcoI restriction pattern detected by Southern blotting, we also amplified intron 10 (using primers complementary to intron 9 and intron 11)12 to check for the presence of the NcoI restriction site in intron 10. This study included DNA analysis of patients 23, 25, 26, and 38 as well as that of 2 FH heterozygotes of Asian ancestry (carrying the P664L mutation designated FH-Gujerat)17 18 and 2 British FH heterozygotes with the same mutation.19 The DNA samples of these patients were a kind gift from Dr A. Soutar (Royal Post-graduate Medical School, London, UK).

Statistical Analysis
Values were given as the mean±standard deviation of the mean (SD). Statistical comparison between groups was made using the Student's t test.


*    Results
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 *Results
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Tables 1Down and 3Up show some features of the patients with the clinical phenotype of homozygous FH. The type and localization of the mutations in the LDL receptor gene as well as the LDL receptor activity in skin fibroblasts are listed in Tables 2Down and 4Up. Our series included 29 homozygotes (23 of whom were unrelated) (Tables 1Down and 2Down) and 10 compound heterozygotes (9 of whom were unrelated) (Tables 3Up and 4Up). Plasma LDL-CH levels were markedly increased (2.3- to 5.2-fold) above the 95th percentile value for the population. There was no significant difference in the mean plasma LDL-CH level between homozygotes (16.6±3.2 mmol/L) and compound heterozygotes (17.4±4.3 mmol/L). In both groups the mean plasma HDL cholesterol levels (0.80±0.22 in men and 0.81±0.27 in women) were below the fifth percentile for our adult control population (0.85 mmol/L for men and 1.02 mmol/L for women) (Tables 1Down and 3Up).


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  		Table 1.


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  		Table 2.

Severe cardiovascular involvement (as indicated by the presence of 2- or 3-vessel CAD, myocardial infarction, or sudden cardiac death) was observed in 16 of 25 patients older than 10 years. These data are to be regarded as minimal figures, as we did not perform advanced tests of ventricular function or structure, especially in young patients.20 21 The residual LDL receptor activity in cultured skin fibroblasts, measured in 32 patients, varied from <2% to 30% of the values found in fibroblasts from control subjects (Tables 2Up and 4Up). There was a negative correlation between plasma LDL-CH level and the residual LDL receptor activity (r=-0.655; P<0.003) (Figure 1Down). The highest levels of LDL-CH were observed in 6 children (5 to 13 years of age) whose residual LDL receptor activity was <=5%.



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  		Figure 1. Relationship between plasma LDL-CH level and the residual LDL receptor activity in cultured skin fibroblasts of 32 patients with the clinical phenotype of homozygous FH (Tables 1Up and 3Up). LDL receptor activity, measured as the maximum rate of saturable degradation of 125I-labeled LDL by cultured fibroblasts, is given as percentage of the value found in control fibroblasts.

Mutations of LDL Receptor Gene in FH Homozygotes
The mutations found in FH homozygotes are listed in Table 2Up. In 2 patients (1 and 2) major rearrangements of the LDL receptor gene were observed. The mutation found in patient 2 (a 5.5-kb duplication involving exons 16 and 17) was previously reported in detail by our group.8 In patient 1 the hybridization of genomic DNA with a cDNA probe complementary to exons 1 to 11 (Ex 1–11 probe) resulted in normal fragments after DNA digestion with several restriction enzymes, whereas no fragments were detected with a probe complementary to exons 12 to 18 (Ex 12–18) (Figure 2Down). These findings suggested the presence of a large deletion (>15 kb) eliminating the 3' half of the gene. An additional Southern blotting of DNA digested with EcoRV (whose restriction sites are located in exon 8 and at the 5' end of intron 12) and hybridized with the Ex 1–11 probe, showed, besides the expected 26-kb band, an abnormal band of approximately 20 kb, replacing the expected fragment of 7 kb (Figure 2Down). This result suggested that the EcoRV site in intron 12 was deleted. To ascertain whether exon 12 was maintained we performed a PCR amplification of exon 12 using flanking primers. Because this amplification generated a normal fragment (data not shown) we concluded that exon 12 was maintained and the 5' boundary of the deletion was located at the 5' end of intron 12. We were unable to define the 3' boundary of this deletion. However, because a PCR amplification of genomic DNA, using primers complementary to exon 12 and exon 18, was unsuccessful, we assumed that all of exon 18 was deleted.



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  		Figure 2. Southern blot analysis in patient 1 (G.R.). DNA was digested with restriction enzymes and hybridized with 3 LDL receptor cDNA probes: Ex 1–11 (complementary to exons 1 to 11); Ex 8–10 (complementary to exons 8 to 10) and Ex 12–18 (complementary to exons 12 to 18). Abnormal fragments were seen with several enzymes only after DNA hybridization with the Ex 1–11 probe. The complete absence of fragments after DNA hybridization with the Ex 12–18 probe suggested that the patient was homozygous for a large deletion eliminating the 3' half of the LDL receptor gene.

In the other homozygotes we found 12 different point mutations, 9 in the coding region and 3 in splicing sites (Table 2Up).

Mutations of LDL Receptor Gene in FH Compound Heterozygotes
The mutations found in FH compound heterozygotes are listed in Table 4Up. In patients 35, 37, and 39 we failed to identify one of the 2 mutant alleles, despite an extensive analysis of the proximal promoter, the coding sequence, and the exon–intron junctions of the LDL receptor gene. In siblings 30 and 31 Southern blotting revealed abnormal bands after genomic DNA digestion with several restriction enzymes and hybridization with a cDNA probe complementary to exons 12 to 18 (Figure 3Down). This finding was consistent with the presence of an allele carrying a {approx}5-kb deletion eliminating exons 13 to 15. The deletion of exon 15 was also confirmed after PCR amplification of genomic DNA using primers complementary to the 5' end of exon 12 and to intron 16. Digestion of this PCR-amplified fragment (approximately 8 kb) with EcoRV and HindIII demonstrated that the EcoRV site in intron 12 and the HindIII site in intron 15 were maintained. The presence of this mutation was also confirmed by the analysis of LDL receptor mRNA in cultured fibroblasts. RT-PCR amplification of the exon 12 to exon 18 region from LDL receptor mRNA resulted in a fragment of 890 bp in the control and 3 fragments of 890, 424, and 346 bp in patient 31 (Figure 3Down). The nucleotide sequence showed that (1) in the 890-bp fragment exon 12 joined to exon 13 (indicating the presence of a nondeleted allele); (2) in the 424-bp fragment exon 12 joined to exon 16 (indicating the presence of the deleted allele, as expected from Southern blot results); and (3) in the 346-bp fragment exon 12 joined to exon 17 (indicating the occurrence of an exon skipping in mRNA, presumably transcribed from the deleted allele).22



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  		Figure 3. Southern blot analysis in patient 31 (Tables 3Up and 4Up.). DNA was digested with several restriction enzymes and hybridized with cDNA probes complementary to exons 12 to 18 (Ex 12–18) and exons 13 to 14 (Ex 13–14) (A). The presence of abnormal bands suggested that the proband was heterozygous for a {approx}5-kb deletion eliminating exons 13, 14, and 15. B, RT-PCR obtained from mRNA isolated from cultured skin fibroblasts by using primers complementary to exon 12 and exon 18 (see Methods). The nucleotide sequence (data not shown) showed that (1) the 890-bp fragment, found in patient 31 as well as in the control, corresponded to the transcript of the nondeleted allele (Table 4Up); (2) the 424-bp fragment corresponded to the transcript of the mutant allele carrying the deletion of exons 13, 14, and 15; and (3) the 346-bp fragment corresponded to an abnormally spliced mRNA in which exon 12 joined directly to exon 16 (exon skipping).

Among the point mutations found in compound heterozygotes (Table 4Up), 11 were located in the coding sequence and one in a splice site.

Haplotype Analysis
As shown in Table 2Up, some point mutations (D200G, C358R, V502M, G528D, P664L) were observed in more than one apparently unrelated FH homozygote. The haplotype analysis of the LDL receptor gene showed that all patients with the same mutation shared the same haplotype. The following polymorphic restriction sites were examined: SmaI (intron 7); StuI (exon 8); HincII (intron 12); BstEII (intron 12); AvaII (exon13); MspI (exon 15); ApaLI (intron 15); PvuII (intron 15); NcoI (exon 18); and ApaLI (3'FLK). The haplotypes found were the following: D200G (SmaI/-; StuI/+; 1HincII/-; AvaII/+; MspI/+;ApaLI/+; PvuII/-; NcoI/+);C358R (SmaI/+; StuI/+;HincII/+; AvaII/-; MspI/+;ApaLI/-; PvuII/-; NcoI/-);V502M (SmaI/-; StuI/+; HincII/-; AvaII/+; MspI/+;ApaLI/+; PvuII/-; NcoI/+);G528D (StuI/+; HincII/+;BstEIi/-; AvaII/-; ApaLI/-;PvuII/-; NcoI/-; ApaLI/-); and P664L (SmaI/-; StuI/+;HincII/-; AvaII/+; MspI/+;ApaLI/+; PvuII/-; NcoI/+). Furthermore, we discovered that in patients 23, 25, and 26 the P664L mutation cosegregated with the absence of an NcoI restriction site in intron 10 (data not shown) that is normally found in our control subjects. The absence of this site was also found in patient 38 (a compound heterozygote carrying the P664L mutation). In sharp contrast, in 2 FH heterozygotes of Asian descent (FH-Gujerat) and 2 British FH heterozygotes, the P664L mutation was associated with the presence of the NcoI site in intron 10. This suggests that the P664L substitution is a recurrent mutation.

Effect of Splice-Site Mutations on LDL Receptor mRNA
The novel splice-site mutations found in patients 27 and 28 were studied in detail with regard to the species of LDL receptor mRNA generated by the mutant allele. In patient 27 (g->a+1 in intron 10) Northern blot analysis showed that the LDL receptor mRNA in the proband's cells was apparently similar, in terms of size and content, to that found in control cells (Figure 4Down). RT-PCR of the exon 6 to exon 13 region revealed the presence of a single 1109-bp fragment in the control and 2 abnormal fragments of 1175 and 881 bp in patient 27. The sequence of the 1175-bp fragment (corresponding to mRNA1 in Table 5Down) showed that exon 10 was followed by a partially retained intron 10 (the first 66 nucleotides of this intron were retained) (Figure 5Down). This resulted from the activation of a cryptic donor splice site (tg/gt) in intron 10. The sequence of the 881-bp fragment (corresponding to mRNA2 in Table 5Down) showed that exon 9 joined to exon 11 with the complete skipping of exon 10 (Figure 5Down).



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  		Figure 4. Northern blot analysis and RT-PCR of RNA isolated from cultured skin fibroblasts of patient 27 (S.T.) (g->a+1 in intron 10) (Tables 1Up and 2Up) and of a control subject (C). RNA was hybridized with a full-size LDL receptor cDNA and rehybridized with human ß-actin cDNA (left). RT-PCR (right) was performed by using primers complementary to exon 6 and exon 13. LDL-R indicates LDL receptor; mw, molecular size markers.


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  		Table 5.



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  		Figure 5. Nucleotide sequence of the RT-PCR fragments found in patient 27 (S.T.) and in a control subject (C) shown in Figure 4Up. A, Sequence of the 1175-bp fragment in which exon 10 joins to the 5' end of intron 10 (the first 66 nucleotides of this intron are retained in mature mRNA). The novel amino acids encoded by the 5' end of intron 10 are indicated in italics. This mRNA has an in-frame insertion of 22 novel amino acids between Gly508 and Phe509 of the normal receptor protein (Table 5Up). B, Sequence of the 881-bp fragment in which exon 9 is followed by exon 11 with the complete skipping of exon 10. This abnormal mRNA has an in-frame deletion of 76 amino acids from Thr433 to Gly508 of the normal receptor protein (Table 5Up).

In patient 28 (g->a-1 in intron 12) the LDL receptor mRNA was present only in trace amounts in cultured fibroblasts (Figure 6Down). RT-PCR of the exon 10 to exon 15 region generated a single fragment of 918 bp in the control and 3 fragments of 899, 759, and 623 bp in patient 28 (Figure 6Down). The nucleotide sequence of these fragments (Figure 7Down) showed that (1) in the 899-bp fragment (corresponding to mRNA1 in Table 5Up) exon 12 joined to a partially deleted exon 13 (the first 19 nucleotides of this exon being deleted because of the activation of a cryptic acceptor splice site, ag/at); (2) in the 759-bp fragment (corresponding to mRNA2 in Table 5Up) exon 11 joined to the partially deleted exon 13 (see above); and (3) in the 623-bp fragment (corresponding to mRNA3 in Table 5Up) exon 12 joined to exon 15 with the complete skipping of exons 13 and 14 (Figure 6Down). While this study was in progress the same mutation (g->a-1 in intron 12) was reported in a Danish FH heterozygote.23 Analysis of the LDL receptor mRNA in the Danish patient demonstrated the presence of only 2 abnormal mRNAs,24 which were identical to mRNA1 and mRNA3 (Table 5Up) found in the present study.



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  		Figure 6. Northern blot analysis and RT-PCR of RNA isolated from cultured skin fibroblasts of patient 28 (A.F.) (g->a-1 in intron 12) and a control subject (C). RNA was hybridized with a full-size LDL receptor cDNA and rehybridized with human ß-actin cDNA (left). RT-PCR (right) was performed with primers complementary to exon 10 and exon 15. Abbreviations as in Figure 4Up.



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  		Figure 7. Nucleotide sequence of the RT-PCR fragments found in patient 28 (A.F.) and shown in Figure 6Up. A, Sequence of the 899-bp fragment in which exon 12 is followed by a partially deleted exon 13 (the first 19 nucleotides of this exon are deleted). This causes a shift in the reading frame leading to a premature stop codon after a stretch of 42 novel amino acids (in italics). B, Sequence of the 759-bp fragment in which exon 11 joins to the partially deleted exon 13. This abnormal mRNA has an in-frame deletion of 53 amino acids from Asp548 to Thr600 of the normal receptor protein. C, Sequence of the 623-bp fragment in which exon 12 is followed by exon 15. This causes a shift in the reading frame leading to a premature stop codon after a stretch of 15 novel amino acids (in italics).

We have been unable to investigate the effects of the splicing mutation found in patient 39, (g->a+1 in intron 16) as fibroblasts of this patient were not available for this type of study. The disruption of LDL receptor mRNA splicing caused by the mutation found in patient 29 (g->a+1 in intron 15) had been previously reported by our group.11


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The aim of this study was to characterize the mutations of the LDL receptor gene in a group of patients with the clinical phenotype of homozygous FH; these patients had been identified in Italy during the previous decade and were alive in 1994 or they were born in the period 1994 to 1996. It is likely that our series includes almost all the phenotypically "severe" FH homozygotes who were living in Italy during the time of our study. We are aware, however, of the presence of 3 FH homozygotes living in Sardinia who have not been included in this study, as consent to DNA analysis had not been given. With the exception of patients 2 and 29, reported previously by our group,8 11 and patients 11, 21, and, at least in part, 32, who (after we had performed DNA analysis and LDL receptor assay) were discovered to have been investigated by the Dallas group,1 4 5 the data of the patients included in this study were reported for the first time. Our series included 39 patients, 29 of whom were found to be homozygotes and 10, compound heterozygotes. The mean pretreatment plasma LDL-CH levels were similar in the 2 groups and were of the same order of magnitude as those reported in other large series of FH homozygotes.25 26 27 28 29 In 32 patients we were able to perform LDL receptor assay in cultured fibroblasts and define the residual LDL receptor activity. The finding of an inverse correlation between these 2 parameters is in keeping with a previous study conducted in a group of American FH homozygotes with uncharacterized LDL receptor defects,26 and with recent observations made in French Canadian FH homozygotes with molecularly defined mutations.27 Although the relatively "small" number of patients of our series and their wide age distribution do not allow a statistical analysis, the data about the severity of cardiovascular involvement (as judged by clinical criteria) seem to confirm the idea that the more severe the LDL receptor defect and the higher the plasma LDL-CH levels (as found in patients 19, 20, 21, 28, 30, and 31) (Tables 1Up and 3Up), the more rapid appears to be the rate of progression of atherosclerosis and the severity of coronary heart disease.1 30 Although the majority of our patients were younger than 30 years, we were surprised to find a substantial number of patients older than 30 years; it is well-known, in fact, that in the majority of FH homozygotes, if untreated, fatal coronary heart disease occurs before 30 years of age.1 A similar observation was reported by Webb et al29 in FH homozygotes of various ethnic groups. This relatively large proportion of subjects older than 30 years might reflect a combination of factors such as (1) a selection bias owing to the sampling, among living FH homozygotes, of those subjects who had a "relatively mild" LDL receptor defect; (2) the presence, in some individuals, of other genetic and environmental factors capable of slowing the rate of progression of atherosclerosis; (3) the long-term treatment with LDL apheresis (alone or in combination with hypolipidemic drugs); and (4) the availability of advanced surgical techniques (such as coronary artery bypass graft surgery) for the treatment of coronary heart disease. One important factor in reducing the rate of progression of coronary atherosclerosis might have been the use of LDL apheresis, a procedure adopted in several Italian lipid clinics for the treatment of FH homozygotes. Several reports indicate that plasma exchange and LDL apheresis improve the survival of homozygous FH by slowing the progression of atherosclerosis and promoting the regression of intimal lesions.31 32 33 34 35 36 LDL apheresis in FH homozygotes would also reduce the oxidation of LDL, which is thought to play an important role in the development of intimal lesions.37 At the time of DNA analysis a dozen of these patients had been being treated systematically with LDL apheresis for more than 5 years.

Tables 1Up and 3Up also underscore the difficulty in predicting the levels of plasma LDL-CH in homozygotes carrying the same mutation. Although in some patients (19, 20, and 21) plasma LDL-CH levels were within a fairly narrow range, in others there was a large interindividual variation. This is in agreement with previous reports showing a large interindividual variability of plasma LDL-CH levels in French Canadian FH homozygotes carrying the same mutation.27 The reasons for this finding still need to be clarified.

In all homozygotes (Table 2Up) we were able to identify the mutations of the LDL receptor gene, by using a combination of Southern blotting, SSCP analysis, or direct sequencing of the SSCP-positive region or the complete sequencing of the whole coding region. In 3 unrelated compound heterozygotes (Table 4Up) we failed to identify the mutation in the second allele of the LDL receptor gene. The failure to identify the second mutant allele in some FH patients has been reported in other studies.28 29 38 We cannot exclude the possibility that mutations located in introns, far from the intron–exon junctions, or in some regulatory sequences located upstream from the proximal promoter, have been missed in our analysis of the LDL receptor gene. One can also argue that some patients, such as patients 35 and 37 (whose residual LDL receptor activity was 20% to 30% of normal values), were not compound heterozygotes but rather "simple" heterozygotes with a particularly severe expression of FH. Although these "relatively high" values of residual LDL receptor activity in FH homozygotes have been reported by others,29 clinical and family data make this hypothesis unlikely (see Methods). In the case of patient 35, we found that all family members of the maternal line carrying the identified mutant allele (E267X) had lower plasma LDL-CH levels than the proband and did not manifest the same clinical phenotype (ie, tendon xanthomas at early age). Because the proband's father had a moderate elevation of plasma LDL-CH levels, we favor the idea that patient 35 has inherited a severe mutation (producing a truncated LDL receptor) from her mother and a relatively mild mutation from her father. The same considerations can be applied to patient 37 (20% residual LDL receptor activity), as both parents had elevated plasma LDL-CH levels. Although only the father carried the identified mutation found (2-bp deletion in exon 10), the mother, the putative carrier of the unknown mutant allele, had a brother with primary hypercholesterolemia (7.02 mmol/L) and belonged to a family with premature CAD.

Although our data confirm the allelic heterogeneity of FH in Italians as in most European populations, some mutations (D200G, C358R, V502M, G528D, and P664L), found in homozygotes, were found in more than one patient. This observation, as well as the finding that the patients with the same mutation carried the same intragenic haplotype, suggested the presence of a common descent and raised the possibility that these mutations were more frequent in those geographic areas from which these patients came. A systematic screening of 2 mutations (D200G and G528D, found in northern and southern Italy, respectively) among FH heterozygotes living in those geographic areas has led to the identification of 2 large clusters of these mutations in those areas (Bertolini and Calandra, unpublished results).

In this study we report 2 additional examples of FH homozygotes with splice-site mutations, which allowed us to perform a detailed analysis of the disruption of LDL receptor mRNA processing caused by this type of mutation. At present only a small number of splice-site mutations have been reported in the LDL receptor gene, mostly in heterozygote subjects.5 11 39 40 41 42 43 44 Here we show 2 splice-site mutations (one in a donor and the other in an acceptor splice site) that lead to the production of several mRNA species through the activation of cryptic splice sites in introns (whereby intron sequences are retained in mature mRNA) as well as in exons (with partial deletion of the coding sequence) or through the use of an alternative splicing involving canonical splice sites (exon skipping). In patient 28 the g->a-1 in intron 12 is associated with a marked reduction of LDL receptor mRNA in fibroblasts and the presence of 3 abnormal mRNAs. Two of them encode truncated proteins, which are presumably secreted from the cells, being devoid of the transmembrane domain, whereas the third abnormal mRNA encodes a receptor with an in-frame deletion in the epidermal growth factor spacer, a condition that may reduce the intracellular transport or the recycling of the receptor.1 The same mutation was recently reported in some Danish FH heterozygotes.23 24 Nissen et al24 demonstrated the presence of 2 abnormally spliced mRNAs (corresponding to mRNA1 and mRNA3 in Table 5Up) in peripheral blood lymphocytes of these patients. The third abnormal mRNA we found in patient 28 (corresponding to mRNA2 in Table 5Up) was not detected in the Danish patients,24 presumably because this mRNA was present only in trace amounts in FH heterozygotes. In patient 27 (g->a+1 in intron 10) the 2 abnormal mRNAs are predicted to encode 2 receptors: one with an in-frame insertion of 22 amino acids and the other with an in-frame deletion of 76 amino acids in the epidermal growth factor precursor homology domain (Table 5Up). Also in this case these sequence variations may impair either the intracellular transport or recycling of the receptor protein. These results, together with our previous study performed in patient 29,11 confirm the general idea that mutations involving the invariant dinucleotides at the 5' or 3' end of introns result almost invariably in incorrect splicing of the primary transcript, even though the pattern of aberrant splicing varies in different genes.45

In conclusion, this study sheds more light on the molecular defects of the vast majority of FH homozygotes in Italy and sets the stage for further studies on the genotype–phenotype relationship and the contribution of other genetic and environmental factors to the progression of premature atherosclerosis in these patients. The data collected in this study have also provided the clue for extending DNA analysis in probands' relatives and for the screening of specific mutations of the LDL receptor gene in defined geographic areas of the country. Finally, the complete clinical and molecular characterization of these patients will provide the basis for a better understanding of the effect of new pharmacological and nonpharmacological treatments that are presently used46 or will be adopted in the future for the therapy of homozygous FH.


*    Acknowledgments
 
The authors thank the patients and their families for their collaboration in this study. This study was supported by a grant from the Consiglio Nazionale delle Ricerche (Progetto Finalizzato Invecchiamento) to S.B. and a grant from Istituto Superiore di Sanità to S. Calandra.

The following investigators and institutions also participated in the study: A. Avogaro, Dipartimento di Medicina Clinica e Sperimentale, Università di Padova; B. Capurso, Dipartimento di Geriatria, Università di Bari; C. Martini, Istituto di Medicina Interna, Università di Padova; D. Notarbartolo, Dipartimento di Medicina Interna, Università di Palermo; G.F. Pagano, Dipartimento di Medicina Interna, Università di Torino; A. Postiglione and F. Mancini, Istituto di Medicina Interna, Università di Napoli; C. Sirtori, Istituto di Scienze Farmacologiche, Università di Milano; and C. Stefanutti, Istituto di Terapia Medica, Università di Roma, "La Sapienza."

Received February 17, 1998; accepted August 24, 1998.


*    References
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 *References
 

  1. Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disease. 7th ed. New York, NY: McGraw-Hill;1995:1981–2030.
  2. Leitersdorf E, Van der Westhuyzen DR, Coetzee GA, Hobbs HH. Two common low density lipoprotein receptor gene mutations cause familial hypercholesterolemia in Afrikaners. J Clin Invest. 1989;84:954–961.
  3. Moorjani S, Roy M, Gagnè C, Davignon J, Brun D, Toussaint M, Lambert M, Campeau L, Blaichman S, Lupien P. Homozygous familial hypercholesterolemia among French Canadians in Quèbec Province. Arteriosclerosis. 1989;9:211–216.[Abstract/Free Full Text]
  4. Hobbs HH, Russell DW, Brown MS, Goldstein JL. The LDL receptor locus in familial hypercholesterolemia: mutational analysis of a membrane protein. Ann Rev Genet. 1990;24:133–170.[Medline] [Order article via Infotrieve]
  5. Hobbs HH, Brown MS, Goldstein JL. Molecular genetics of the LDL receptor gene in familial hypercholesterolemia. Hum Mut. 1992;1:445–466.[Medline] [Order article via Infotrieve]
  6. Varret M, Rabès JP, Collod-Bèroud G, Junien C, Boileau C, Bèreoud C. Software and database for the analysis of mutations in the human LDL receptor gene. Nucl Acid Res. 1997;25:172–180.[Abstract/Free Full Text]
  7. Calandra S, Bertolini S. The low density lipoprotein (LDL) receptor and familial hypercholesterolemia. In: Swallow DM, Edwards YH, eds. Protein dysfunction in human genetic disease. Oxford: ßios Scientific Publishers; 1997:123–143.
  8. Lelli N, Ghisellini M, Gualdi R, Tiozzo R, Calandra S, Gaddi A, Ciarrocchi A, Arca M, Fazio S, Bertolini S. Characterization of three mutations of the low density lipoprotein receptor gene in Italian patients with familial hypercholesterolemia. Arterioscler Thromb. 1991;11:234–243.[Abstract/Free Full Text]
  9. Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260.[Medline] [Order article via Infotrieve]
  10. Sambrook JE, Fritsch F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  11. Lelli N, Garuti R, Ghisellini M, Tiozzo R, Rolleri M, Aimale V, Ginocchio E, Naselli A, Bertolini S, Calandra S. Occurrence of multiple aberrantly spliced mRNAs of the LDL-receptor gene upon a donor splice site mutation that causes familial hypercholesterolemia (FHBenevento). J Lipid Res. 1995;36:1315–1324.[Abstract]
  12. Leitersdorf E, Tobin EJ, Davignon J, Hobbs HH. Common low-density lipoprotein receptor mutations in the French Canadian population. J Clin Invest. 1990;85:1014–1023.
  13. Gudnason V, King-Underwood L, Seed M, Sun X-M, Soutar AK, Humphries SE. Identification of recurrent and novel mutations in exon 4 of the LDL receptor gene in patients with familial hypercholesterolemia in the United Kingdom. Arterioscler Thromb. 1993;13:56–63.[Abstract/Free Full Text]
  14. Jensen HK, Jensen LG, Hansen PS, Faegerman O, Gregersen N. High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing. Clin Chem. 1996;42:1140–1146.[Abstract/Free Full Text]
  15. Hansen PS, Rudiger N, Tybjaerg-Hansen A, Faegerman O, Gregersen N. Detection of the apo B-3500 mutation (glutamine for arginine) by gene amplification and cleavage with MspI. J Lipid Res. 1991;32:1229–1233.[Abstract]
  16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
  17. Soutar AK, Knight BL, Patel DD. Identification of a point mutation in growth factor repeat C of the low density lipoprotein-receptor gene in a patient with homozygous familial hypercholesterolemia that affects ligand binding and intracellular movement of receptors. Proc Natl Acad Sci U S A. 1989;86:4166–4170.[Abstract/Free Full Text]
  18. Rubinsztein DC, Coetzee GA, Marais AD, Leitersdorf E, Seftel HC, van der Westhuyzen DR. Identification and properties of the proline664-leucine mutant LDL receptor in South Africans of Indian origin. J Lipid Res. 1992;33:1647–1655.[Abstract]
  19. King-Underwood L, Gudnason V, Humphries S, Seed M, Patel D, Knight B, Soutar A. Identification of the 664 proline to leucine mutation in the low density lipoprotein receptor in four unrelated patients with familial hypercholesterolemia in the UK. Clin Genet. 1991;40:17–28.[Medline] [Order article via Infotrieve]
  20. Mouratidis B, Vaughan-Neil EF, Gilday DL, Ash JM, Cullen-Dean G, McIntyre S, MacMillan JH, Rose V. Detection of silent coronary artery disease in adolescents and young adults with familial hypercholesterolemia by single-photon emission computed tomography thallium-201 scanning. Am J Cardiol. 1992;70:1109–1112.[Medline] [Order article via Infotrieve]
  21. Hoeg JM, Feuerstein IM, Tucker EE. Detection and quantitation of calcific atherosclerosis by ultrafast computed tomography in children and young adults with homozygous familial hypercholesterolemia. Arterioscler Thromb. 1994;14:1066–1074.[Abstract/Free Full Text]
  22. Lelli N, Garuti R, Zambelli F, Cassanelli S, Tiozzo R, Corsini A, Bertolini, Riva E, Ortisi MT, Bellù R, Calandra S. Alternative splicing of mutant LDL-receptor mRNA in an Italian patient with familial hypercholesterolemia due to a partial deletion of LDL-receptor gene (FHPotenza). J Lipid Res. 1993;34:1347–1354.[Abstract]
  23. Jensen HK, Jensen LG, Hansen PS, Bolund L, Faegerman L, Gregersen NA. A G-1-to-A acceptor splice site LDLR mutant allele leads to reduced relative transcript levels in patients with heterozygous familial hypercholesterolemia. Clin Genet. 1996;49:175–179.[Medline] [Order article via Infotrieve]
  24. Nissen H, Hansen AB, Guldberg P, Petersen NE, Hansen TS, Horder M. Detection and characterization of a novel splice mutation in the LDL receptor intron 12 resulting in two different mutant mRNA variants. Atherosclerosis. 1997;128:75–83.[Medline] [Order article via Infotrieve]
  25. Seftel HC, Baker SG, Sandler MP, Forman MB, Joffe BI, Mendelsohn D, Jenkins T, Mieny C. A host of hypercholesterolemic homozygotes in South Africa. BMJ. 1980;281:633–636.
  26. Sprecher DL, Hoeg JM, Schaefer EJ, Zech LA, Gregg RE, Lakatos E, Brewer BH. The association of LDL receptor activity, LDL cholesterol level, and clinical course in homozygous familial hypercholesterolemia. Metabolism. 1985;34:294–299.[Medline] [Order article via Infotrieve]
  27. Moorjani S, Roy M, Torres A, Bétard C, Gagnè C, Lambert M, Brun D, Davignon J, Lupien P. Mutations of low-density-lipoprotein-receptor gene, variation in plasma cholesterol, and expression of coronary heart disease in homozygous familial hypercholesterolemia. Lancet.. 1993;341:1303–1306.[Medline] [Order article via Infotrieve]
  28. Sun X-M, Patel DD, Webb JC, Knight BL, Fa L-M, Cai H-J, Soutar AK. Familial hypercholesterolemia in China: identification of mutations in the LDL-receptor gene that result in a receptor-negative phenotype. Arterioscler Thromb. 1994;14:85–94.[Abstract/Free Full Text]
  29. Webb JC, Sun X-M, McCarthy SN, Neuwirth C, Thompson GR, Knight BL, Soutar AK Characterization of mutations in the low density lipoprotein (LDL)-receptor gene in patients with homozygous familial hypercholesterolemia, and frequency of these mutations in FH patients in the United Kingdom. J Lipid Res. 1996;37:368–381.[Abstract]
  30. Sprecher DL, Schaefer EJ, Kent KM, Gregg RE, Zech LA, Hoeg JM, McManus B, Roberts WC, Brewer BH. Cardiovascular features of homozygous familial hypercholesterolemia: analysis of 16 patients. Am J Cardiol. 1984;54:20–30.[Medline] [Order article via Infotrieve]
  31. Thompson GR, Miller JP, Breslow JL. Improved survival of patients with homozygous familial hypercholesterolemia treated by plasma exchange. BMJ. 1985;291:1671–1673.
  32. Koga N, Iwata Y. Pathological and angiographic regression of coronary atherosclerosis by LDL-apheresis in a patient with familial hypercholesterolemia. Atherosclerosis. 1991;90:9–21.[Medline] [Order article via Infotrieve]
  33. Tatami R, Inoue N, Itoh H, Kishino B, Koga N, Nakashima T, Nishide T, Okamura K, Saito Y, Teramoto T, Yasugi T, Yamamoto A, Goto Y. Regression of coronary atherosclerosis by combined LDL-apheresis and lipid-lowering drug therapy in patients with familial hypercholesterolemia: a multicenter study. The LARS Investigators. Atherosclerosis. 1992;95:1–13.[Medline] [Order article via Infotrieve]
  34. Gordon BR, Saal SD. Advances in LDL-apheresis for the treatment of severe hypercholesterolemia. Curr Opin Lipidol. 1994;69–73.
  35. Gordon BR, Saal SD. Clinical experience and future directions for low density lipoprotein apheresis in the United States. Ther Apheresis. 1997;3:249–252.
  36. Koga N. The retardation of progression, stabilization, and regression of coronary and carotid atherosclerosis by low density lipoprotein apheresis in patients with familial hypercholesterolemia. Ther Apheresis. 1997;3:260–270.
  37. Napoli C, Ambrosio G, Scarpato N, Corso F, Palumbo, G, D'Armiento FP, Mancini FP, Malorni A, Formisano S, Ruocco A, Calì A, Chiariello M. Decreased low-density lipoprotein oxidation after repeated selective apheresis in homozygous familial hypercholesterolemia. Am Heart J. 1997;133:585–595.[Medline] [Order article via Infotrieve]
  38. Sun Xi-M, Patel DD, Knight BL, Soutar AK. Comparison of the genetic defect with LDL-receptor activity in cultured cells from patients with a clinical diagnosis of heterozygous familial hypercholesterolemia. Arteriocler Thromb Vasc Biol. 1997;17:3092–3101.[Abstract/Free Full Text]
  39. Top B, van der Zee A, Havekes LM, van 't Hooft FM, Frants R. Identification of a splice-site mutation in the low density lipoprotein receptor gene by denaturing gradient gel electrophoresis. Hum Genet. 1993;91:480–484.[Medline] [Order article via Infotrieve]
  40. Lombardi P, Hoffer MJV, Top B, de Wit E, Gevers Leuven JAG, Frants RR, Havekes LM. An acceptor splice site mutation in intron 16 of the low density lipoprotein receptor gene leads to an elongated, internalization defective receptor. Atherosclerosis. 1993;104:117–128.[Medline] [Order article via Infotrieve]
  41. Lombardi P, Sijbrands EJG, van de Giessen K, Smelt AHM, Kastelein JJP, Frants RR, Havekes LM. Mutations in the low density lipoprotein receptor gene of familial hypercholesterolemic patients by denaturing gel electrophoresis and direct sequencing. J Lipid Res. 1995;36:860–867.[Abstract]
  42. Leren TP, Solberg K, Rodningen OK, Tonstad S, Ose L. Two founder mutations in the LDL-receptor gene in Norwegian familial hypercholesterolemia subjects. Atherosclerosis. 1994;111:175–182.[Medline] [Order article via Infotrieve]
  43. Sun XM, Patel DD, Bhatnagar D, Knight BL, Soutar AK. Characterization of a splice-site mutation in the gene for the LDL receptor associated with an unpredictably severe clinical phenotype in English patients with heterozygous FH. Arterioscler Thromb Vasc Biol. 1995;15:219–227.[Abstract/Free Full Text]
  44. Webb JC, Patel DD, Soulders CC, Knight BL, and Soutar AK. Genetic variation at a splicing branch point in intron 9 of the low density (LDL)-receptor gene: a rare mutation that disrupts mRNA splicing in a patient with familial hypercholesterolemia and a common polymorphism. Hum Mol Genet. 1996;5:1325–1331.[Abstract/Free Full Text]
  45. Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet.. 1992;90:41–54.[Medline] [Order article via Infotrieve]
  46. Marais AD, Naoumova RP, Firth JC, Penny C, Neuwirth CKY, Thompson GR. Decreased production of low density lipoprotein by atorvastatin after apheresis in homozygous familial hypercholesterolemia. J Lipid Res. 1997;38:2071–2078.[Abstract]
  47. Schuster H, Keller C, Wolfram G, Zollner N. Ten LDL receptor mutants explain one third of familial hypercholesterolemia in a German sample. Arterioscler Thromb Vasc Biol. 1995;15:2176–2180.[Abstract/Free Full Text]



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