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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:219-227.)
© 1995 American Heart Association, Inc.

Articles

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

X.-M. Sun; D.D. Patel; D. Bhatnagar; B.L. Knight; A.K. Soutar

From the Lipoprotein Team, Medical Research Council Clinical Sciences Centre, Hammersmith Hospital, London, and the Department of Medicine, The Royal Infirmary, Manchester (D.B.), England.

Correspondence to Dr A.K. Soutar, Lipoprotein Team, MRC Clinical Sciences Centre, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN.

	Abstract

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Abstract We have identified a substitution of G to A in the first base pair of intron 3 in the LDL receptor gene of an English heterozygous familial hypercholesterolemia (FH) patient. Reverse transcription, amplification, and nucleotide sequencing of the LDL receptor mRNA from mononuclear blood cells showed both the normal mRNA and one that lacked the nucleotides encoded by exon 3, which codes for repeat 2 of the ligand-binding domain. The same mutant allele was identified in 2/200 unrelated FH patients from the London area and 4/77 from Manchester. Immunoblotting of cultured lymphoblasts from the index patient revealed the normal receptor protein and smaller amounts of a receptor protein with electrophoretic mobility consistent with a deletion of the 41 amino acid residues encoded by exon 3. Normal amounts of a similar protein were observed when the mutant cDNA was expressed in heterologous cells; this protein showed reduced binding affinity for LDL but bound apoprotein E–containing lipoproteins normally. Despite these and other observations that repeat 2 of the binding domain is relatively unimportant for receptor function in vitro, carriers of this allele exhibit a severe clinical phenotype, typical of FH. Thus, the relationship between genotype and phenotype in heterozygous FH is not always predictable.

Key Words: mutant protein • immunoblotting • LDL binding • single-strand conformational polymorphism • in vitro expression

	Introduction

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Familial hypercholesterolemia (FH) is a relatively common hereditary metabolic disorder resulting from defective LDL receptor activity caused by mutations in the gene for the LDL receptor. So far, more than 150 different mutant alleles of the LDL receptor gene in patients with FH have been described,^{1 2} and for a number of these, the mutant LDL receptor protein has been characterized, either in cultured cells derived from the patient or by mutagenesis and expression of the mutant allele in heterologous cells in vitro. The LDL receptor is a multifunctional protein with distinct structural domains,³ so that different mutations result in mutant proteins whose structure and function are impaired in different ways and to different extents.⁴ Patients with heterozygous FH show a wide variation in the severity of the disorder,⁵ in both the degree of their hypercholesterolemia and the age of onset of clinical symptoms of coronary artery disease, but the underlying causes of this variation, whether genetic or environmental, are not well understood.

Attempts to determine the extent to which the precise nature of the defect in the LDL receptor gene is important in determining the phenotype of a patient depend on the ability to identify and characterize the underlying mutation in a sufficient number of individuals to permit comparisons to be made. Many studies have shown that in the mutation spectrum of the LDL receptor gene, major rearrangements account for fewer than 5% of the mutant alleles in most populations,¹ including that in the United Kingdom,^{6 7} and that most mutations in the LDL receptor gene are either point mutations or minor rearrangements involving a few nucleotides, which are more difficult to identify. The ability to detect and characterize such mutations has been revolutionized by the development of gene amplification by the polymerase chain reaction (PCR)⁸ and of procedures for detection of single base changes, eg, chemical modification and cleavage of nucleic acid heteroduplexes,⁹ altered electrophoretic mobility of heteroduplexes,¹⁰ denaturing gradient gel electrophoresis,¹¹ and more recently, analysis of single-strand conformational polymorphisms (SSCPs) developed by Orita et al.¹²

In this article we describe the use of SSCP and direct nucleotide sequencing of amplified fragments of the LDL receptor gene and its mRNA to identify a single base substitution (G to A) in the first base of the 5' donor site of intron 3 in a heterozyous FH patient. We show that the mutation results in aberrant splicing of the mRNA, producing a mutant receptor protein that lacks the second disulfide-rich repeat of the binding domain.¹³

	Methods

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Subjects
The index patient (designated I,1 in the Table and Fig 2) described in this study had a plasma cholesterol concentration of 11.2 mmol/L after treatment with diet alone for 3 months, has tendon xanthomas, and suffered a nonfatal myocardial infarction at the age of 43 years. He also has a family history of hypercholesterolemia and premature coronary heart disease. Treatment with a statin (40 mg/d) and cholestyramine (16 g/d) lowered his plasma cholesterol level to 8.3 mmol/L, and he currently (at age 54 years) has no chest pain. Other FH patients in this study, including those attending lipid clinics in London or Manchester, have been described elsewhere.^{7 14}

View this table: [in this window] [in a new window]   Table 1. Clinical Details of Individuals With Splice-Site Mutations

View larger version (22K): [in this window] [in a new window]   Figure 2. Analysis of the haplotype of the mutant allele in two families with the G+1-to-A mutation in intron 3. The haplotype of the mutant allele in family I (A) and family II (B) was determined from the pattern of inheritance at 10 restriction fragment length polymorphism sites in the LDL receptor gene (C); clinical details of affected individuals are shown in the Table. In family I, two affected individuals, I,1 and his son (I,2), were studied. Sites at which one individual (I,2) was heterozygous are shown on the family tree; both individuals were homozygous at all other sites. In family II, five affected and two unaffected individuals were available for study; at least one member of the family carrying the mutant allele (indicated by half-filled symbols) was homozygous for each polymorphic site; polymorphisms shown on the family tree are those that were informative and for which the haplotype in this family differs from that in family I. The two different haplotypes could have been generated by a single recombination (crossover) between the original mutant allele and another allele, as indicated in C. denotes females; , males; /, deceased.

Isolation of DNA
Genomic DNA was extracted from frozen EDTA-blood and subjected to Southern blotting with specific cDNA probes as described previously.⁷

PCR-dependent amplification of fragments of the LDL receptor gene from genomic DNA was performed as described before.¹⁵ SSCP was performed essentially as described by Orita et al¹² with the modifications described previously.¹⁵ To further characterize the mutations, samples that showed a variant pattern after SSCP analysis were analyzed by direct sequencing of the PCR product by the dideoxy chain-termination method as described previously.¹⁴

Analysis of polymorphic sites in the LDL receptor gene was determined by PCR, followed by restriction enzyme digestion (Taq I in intron 4,^{16 17} Stu I in exon 8,¹⁸ Ava II in exon 13,¹⁹ HincII in exon 12,²⁰ Msp I in exon 15,²¹ and Nco I in exon 18²² ) or hybridization with allele-specific oligonucleotides (SfaNI in exon 2²³ ), and Southern blotting (Bsm I in the 5' flanking region,²⁴ Pvu II in intron 15,²⁵ and ApaLI in exon 18).²⁶ In some cases, polymorphisms were detected by SSCP of the PCR fragments (Ava II, Msp I, HincII, and Stu I).

Cell Culture
Human mononuclear cells were isolated from 30 mL of peripheral blood as described previously²⁷ and seeded at a density of approximately 10⁷ cells per dish in 9-cm dishes. Adherent monocytes were cultured for 3 days in RPMI-1640 medium containing 20% (vol/vol) autologous serum. Epstein-Barr virus–transformed lymphoblastoid cell lines were maintained in Falcon flasks in RPMI-1640 medium containing 10% (vol/vol) heat-inactivated fetal calf serum at approximately 10⁶ cells/mL.

Preparation of RNA and Reverse Transcription Coupled With PCR Amplification (RT-PCR)
Mononuclear cells or lymphoblasts were incubated for 15 hours with medium containing 10% (vol/vol) human lipoprotein-deficient serum (LPDS) and 0.3 µg/mL compactin to induce synthesis of LDL receptor mRNA. Total cellular RNA was extracted using a commercial kit (RNAgents Total RNA Isolation System, Promega) according to the manufacturer's instructions and stored at -70°C.

Total RNA from 10⁵ cells (1 µg) was preincubated for 10 minutes at 65°C and then incubated for 45 minutes at 42°C in a reaction mixture (20 µL) containing 50 mmol/L Tris (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl₂, 10 mmol/L dithiothreitol, 0.625 mmol/L of each deoxynucleoside triphosphate, 1 U of ribonuclease inhibitor (Amersham), 100 pmol of random hexanucleotide primers, and 25 U of avian myoblastosis virus reverse transcriptase (Promega). The reaction was terminated by incubation at 95°C for 3 minutes. Fragments of the LDL receptor cDNA were amplified by PCR by addition of 40 µL of the PCR reaction solution to 10 µL of the RT reaction mix.¹⁵ Details of the oligonucleotide primers are given in the legends to the figures.

Expression of the Mutant LDL Receptor Protein
An LDL receptor cDNA expressing the splice-site mutation was constructed by subcloning an EcoRI–Sac II restriction fragment of the mutant cDNA containing the deletion into an expression vector containing the human LDL receptor cDNA (pLDLR4, kindly provided by Dr David Russell, Department of Molecular Genetics, Dallas, Tex). The mutant EcoRI–Sac II fragment was excised from a fragment amplified RT-PCR from the patient's RNA as described above. The presence of the desired deletion was confirmed by nucleotide sequencing of the large-scale preparation of the plasmid. The normal and mutant LDL receptor cDNA plasmids were transfected into COS cells as DEAE-dextran complexes, and the transiently expressed proteins were analyzed by immunoblotting with specific monoclonal antibodies to the LDL receptor. The method used was essentially as that described previously,¹⁵ with the exception that the monoclonal antibodies were unlabeled and that bound antibody was detected by chemiluminescence of peroxidase-labeled anti-mouse IgG with use of a commercially available kit and the manufacturer's instructions (Amersham International). The efficiency of transfection was highly reproducible, as determined from experiments in which luciferase activity in cell extracts was determined after cotransfection of a plasmid carrying a simian virus 40 promoter–driven luciferase gene construct (pGL2-promoter vector and luciferase assay system, Promega).

Binding, uptake, and degradation of ¹²⁵I-labeled lipoproteins by COS cells expressing normal and mutant cDNA were carried out essentially as described for cultured human skin fibrobasts.^{28 29} COS cells (8.5x10⁴ cells per well in six-place, multiwell, 3.5-cm-diameter dishes) were transfected with 5 µg of plasmid DNA per well on day 2. On day 3, the cells were refed and incubated for 24 hours with medium containing 10% (vol/vol) human LPDS and sterols (30 µg/mL cholesterol and 6 µg/mL 25-hydroxycholesterol). On day 4 the cells were incubated in medium containing 5% LPDS, to which ¹²⁵I-labeled lipoproteins were added to give the final concentrations indicated in the figures, in the presence or absence of an excess of the same unlabeled lipoprotein. After incubation for 4 hours at 37°C, heparin-releaseable binding, cellular uptake, and degradation of the ¹²⁵I-labeled lipoproteins were determined by standard techniques.^{28 29} Degradation of ¹²⁵I-LDL by lymphoblasts was determined essentially as described above with the following modifications. Cells (1.5x10⁶ cells in 1 mL of medium in 2.4-cm, 12-place, multiwell dishes) were preincubated for 14 hours with medium containing 10% (vol/vol) LPDS and then incubated for 4 hours with labeled LDL. The cells plus medium were transferred to a microfuge tube and pelleted by microcentrifugation for 20 seconds; the medium was then removed to a fresh tube for measurement of degradation products. The cell pellet was washed twice by resuspension in washing buffer without albumin and recentrifugation and then solubilized in 1 mL 0.1N NaOH for measurement of cell protein.

Remnants of triglyceride-rich lipoproteins were isolated from the plasma of a normolipemic individual 2 hours after ingestion of a fat-rich meal. Chylomicrons were removed from the plasma by centrifugation for 30 minutes at 32 000 rpm in a Beckman 70Ti rotor and discarded, after which time the d=1.006 to 1.019 g/mL fraction was isolated.³⁰ LDL was isolated and lipoproteins were labeled with ¹²⁵I as described previously.³¹

	Results

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Analysis of Genomic DNA
Southern blotting of genomic DNA from patient I,1 did not reveal any major deletions or rearrangements in the LDL receptor gene. Fragments of genomic DNA comprising the entire coding sequence of the LDL receptor gene and the probable 5' regulatory region were amplified and examined for the presence of any minor nucleotide sequence variations by SSCP analysis. The SSCP pattern observed with the amplified fragment containing exon 3 in patient I,1 differed from that in other patients, in that two extra bands were observed in addition to the two bands observed in all other individuals (Fig 1A). Direct nucleotide sequencing of the PCR product comprising exon 3 and its flanking regions showed that this patient was heterozygous for a single base substitution of G to A in the first base of intron 3 (Fig 1B). The pattern obtained with fragments comprising all other exons and the 5' untranslated region did not differ from that obtained with the majority of individuals, either those with FH or those unaffected.

View larger version (39K): [in this window] [in a new window]   Figure 1. Analysis of exon 3 of the LDL receptor gene. A, Single-strand conformational polymorphism (SSCP) analysis of exon 3 of the LDL receptor gene, which was amplified from genomic DNA with primers located in adjacent introns, as follows: 5'-aaagtcgacTGACAGTTCAATCCTGTCTC in intron 2 and 5'-aaagtcgacAATAGCAAAGGCAGGGCCACA in intron 3, where lower-case letters represent nucleotides containing a restriction enzyme site not present in the gene. 32P-labeled dCTP was included in the final three cycles of amplification, and the labeled polymerase chain reaction (PCR) product was subjected to SSCP analysis on a 5% nondenaturing polyacrylamide MDE gel at ambient temperature, as described in "Methods." I,1 indicates a patient heterozygous for a mutation in the PCR fragment of exon 3; lanes 1 through 4, different heterozygous familial hypercholesterolemia patients with no detectable variant in exon 3. Extra bands visible for patient I,1 are indicated by arrows. B, Nucleotide sequence of part of exon 3. The nucleotide sequences (noncoding strand) of amplified fragments of exon 3 of the LDL receptor gene from patient I,1 and a normal control subject (N) were determined directly with the 3' amplification primer shown in the legend to Fig 1A. Arrow indicates the variant sequence in patient I,1, who is heterozygous for a substitution of C to T (G for A in the coding sequence) in the first nucleotide of intron 3. In the sequence shown below, the coding region is indicated by upper-case letters and the intron sequence by lower-case letters, with the site of mutation shown in bold-face type.

Screening for the Mutation in Other FH Patients
Genomic DNA samples from 200 unrelated FH patients attending lipid clinics in the London area and from 77 FH patients in the Manchester area were analyzed for the presence of the mutation by hybridization with allele-specific oligonucleotides of the amplified genomic fragment comprising exon 3. The allele was identified in 2/200 (1%) of the patients from London and in 4/77 (5%) of the patients from Manchester. Analysis of the inheritance of 10 restriction fragment length polymorphism sites in the LDL receptor gene and of the mutation in the families revealed that the haplotype of the mutant allele differed in the two patients from London (Fig 2). The difference could have resulted from a single crossover, as shown in the figure, or from recurrence of the same mutation.

Clinical details of the patients carrying the splice-site mutation and their affected relatives are shown in the Table. All had severe hypercholesterolemia before undergoing lipid-lowering treatment, and several showed signs of coronary heart disease at an early age.

Characterization of the LDL Receptor mRNA
To investigate the effect of the G-to-A substitution in the splice-site junction of exon 3, the structure of the LDL receptor mRNA in patient I,1 was examined by amplification by RT-PCR of the region of mRNA comprising exon 2 to exon 6. Total RNA was isolated from monocyte-derived macrophages cultured from peripheral blood cells of patient I,1, and cDNA was prepared by RT with random primers. The LDL receptor cDNA was then amplified by nested PCR as shown in Fig 3. Gel electrophoresis of the second-round PCR products revealed a single major band of the expected size (394 bp) for a normal subject, indicating that the target region was amplified accurately. In contrast, amplification of the same region of cDNA from patient I,1 with the G-to-A substitution revealed an additional fragment of about 274 bp, as well as the normal band of 394 bp (Fig 3B). The PCR fragments were subjected to direct nucleotide sequencing in both orientations. The shorter RT-PCR fragment from the patient was shown to contain a precise deletion of exon 3, with exon 2 spliced to exon 4, whereas the fragment of 394 kb from both the control subject and the patient contained the expected sequence of exons 2, 3, and 4 (Fig 3A and 3C).

View larger version (43K): [in this window] [in a new window]   Figure 3. Analysis of the LDL receptor mRNA from familial hypercholesterolemia patient I,1. Total RNA was isolated from monocyte-derived macrophages that had been cultured for 4 days and then incubated for 15 hours in medium containing lipoprotein-deficient serum. A fragment of the LDL receptor mRNA was amplified by nested polymerase chain reaction with reverse transcription as indicated in A, which shows a diagram of part of the LDL receptor mRNA. First-round polymerase chain reaction (PCR) was with primer A, located in exon 1 (5'-ACACTGCCTGGCAGAGGCTGC) and primer D, located in exon 6 (5'-TCTCTAGCCATGTTGCAGACT); the second-round reaction contained 5 µL of the first-round PCR reaction mix in a total volume of 50 µL and was with primer AKS-31, located in exon 2 (5'-ACAGATGCGAAAGA) and primer B, located in exon 4 (5'-CTGTTGCACTGGAAGCTGGCG). Second-round PCR products were analyzed by agarose gel electrophoresis and detected with ethidium bromide (B). M indicates molecular weight markers; N, mRNA from normal human skin fibroblasts; I,1, mRNA from monocyte/macrophages from patient I,1. The 394-bp and 274-bp PCR products from the patient were eluted from the gel and sequenced directly with primer AKS-31 (C).

The relative concentration of mRNA derived from mutant and normal alleles of a second patient heterozygous for the mutation was determined. Total RNA was isolated from lymphoblastoid cells established from patient II,6 and from her unaffected mother (II,7 in Fig 2), both of whom were heterozygous for the Ava II restriction fragment length polymorphism in exon 13 of the LDL receptor gene. After RT-PCR of the appropriate fragment of mRNA with use of a ³²P end-labeled primer, the amplified products were digested with Ava II and analyzed by nondenaturing polyacrylamide gel electrophoresis and autoradiography (Fig 4). From knowledge of the haplotype of the mutant allele in II,6 (Fig 2), it is clear that the RNA derived from the mutant allele (Ava II+) was present in the heterozygous FH cells at a lower concentration than that derived from the normal (Ava II-) allele, whereas in cells from the unaffected mother and other normolipemic individuals, mRNA from each allele was present in approximately equal amounts. The same ratio of Ava II+ to Ava II- product in a number of individuals was observed when the number of PCR cycles was varied between 20 and 30, in which range the reaction was apparently linear (data not shown). Similar results were obtained with two independent preparations of RNA from cells heterozygous for the G-to-A mutation, and the same apparent difference in the relative amounts of mutant and normal mRNA species was observed in RNA isolated from monocyte-derived macrophages from patient I,1.

View larger version (25K): [in this window] [in a new window]   Figure 4. Quantitation of relative amounts of mRNA produced from each allele in lymphoblasts from a patient heterozygous for the G+1-to-A mutation in intron 3. A fragment of the LDL receptor mRNA was amplified by polymerase chain reaction with reverse transcription from total RNA from lymphoblasts of heterozygous (genotype +-) or homozygous (genotype ++ or --) individuals, where + indicates the presence and - the absence of the Ava II restriction fragment length polymorphism (marked # in upper diagram) in exon 13 of the LDL receptor gene. Primers 5'-TTGCTGCCTGTTTAGGACAAA (M) and 5'-CCACACCTGTGAGGCAGCTCC (N) were located as indicated in the diagram; primer I was end-labeled with 32P. Polymerase chain reaction products were digested with Ava II and analyzed by electrophoresis on 10% acrylamide followed by autoradiography. Arrows indicate positions of fragments obtained when the mRNA contains (lower band) or does not contain (upper band) the Ava II site. Lane 1, patient II,6 (heterozygous for G+1 to A in intron 3); lane 2, her unaffected mother II,7; lanes 3 through 7, non–familial hypercholesterolemia individuals; the Ava II genotype of each individual is indicated below; the genotype of the mutant allele in individual II,6 is Ava II+.

Characterization of the Mutant Protein
Extracts of lymphoblastoid cells from individual II,6, who is heterozygous for the mutation, and from her unaffected mother were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with monoclonal antibodies specific to the LDL receptor. The normal LDL receptor protein of apparent molecular weight 130 kD was visible in extracts of both cell types, but less was present in the cells from the patient. In addition, a much fainter band of apparent molecular weight of approximately 126 kD was visible on the immunoblot of cells from the patient but not in those from her mother (Fig 5, lanes 1 and 2).

View larger version (46K): [in this window] [in a new window]   Figure 5. Detection of mutant LDL receptor protein by immunoblotting. Cells that had been preincubated for 15 hours in medium containing 10% lipoprotein-deficient serum and 0.3 µg/mL compactin (for lymphoblasts) or transfected 48 hours previously and incubated in medium containing lipoprotein-deficient serum and sterols for 24 hours (COS cells) were solubilized and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting with antibody 10A2 or 125I-labeled antibody 4B3; bound antibody was detected by chemiluminescence after incubation with peroxidase-labeled anti-mouse IgG (10A2). Each lane contained 100 µg of cell protein. Lane 1, lymphoblasts (Nor) from the unaffected mother (II,7) of patient II,6; lane 2, lymphoblasts from patient II,6 (Mut) (heterozygous for G+1 to A in intron 3); lane 3, COS cells mock-transfected with salmon sperm DNA (None); lane 4, COS cells transfected with plasmid containing the mutant LDL receptor cDNA (Mut) derived from mRNA transcribed from the allele with the intron 3 G+1-to-A mutation; lane 5, COS cells transfected with a plasmid containing the normal human LDL-receptor cDNA (Nor). The plain arrows indicate the position of the normal mature receptor protein in each cell type; the dashed arrow indicates "band X" (see text for details). The apparent molecular weights indicated were estimated from the position of standard proteins run on the same gel.

The observed deletion in the patient's mRNA caused by the splice-site mutation in the gene was introduced into a full-length cDNA for the human LDL receptor, and the mutant cDNA was expressed transiently in COS cells in culture. With antibody 10A2, a major band of LDL receptor protein with an apparent molecular weight of 126 kD (slightly smaller than that of the receptor protein in cells expressing the normal receptor cDNA) was observed on immunoblots of extracts of cells expressing the mutant receptor, as well as a second band described as "band X" (Fig 5, lanes 4 and 5). The amount of mutant receptor protein observed was consistently similar to or slightly greater than the amount of normal protein detected in cells expressing the same amount of cDNA plasmid, as determined from cotransfection of a plasmid containing a luciferase reporter gene construct. In contrast, antibody 4B3, which also does not detect band X, barely detected any protein in cells expressing mutant cDNA (data not shown). The mobility of the band X protein, which we believe to be a breakdown product of the mature receptor (D.D.P. et al, unpublished observations), was the same in extracts of cells expressing either normal or mutant cDNA, and the proportion of the total receptor protein present as band X was the same.

The ability of the mutant protein to catalyze receptor-mediated endocytosis of lipoproteins was assessed in COS cells transiently expressing mutant cDNA (Fig 6). Cells expressing the mutant protein showed reduced affinity for binding, uptake, and degradation of ¹²⁵I-labeled LDL compared with those cells expressing the normal receptor but showed identical affinity for apoprotein E–containing remnant lipoproteins. The efficiency of transfection with each plasmid was essentially identical, as assessed by cotransfection with the luciferase plasmid, and the total amounts of LDL receptor activity with saturating concentrations of ligand were similar.

View larger version (26K): [in this window] [in a new window]   Figure 6. Plots showing binding, uptake, and degradation of lipoproteins by COS cells expressing the normal and mutant LDL receptor cDNA. Cells were preincubated for 24 hours in medium containing 10% lipoprotein-deficient serum and sterols, after which time the medium containing 5% lipoprotein-deficient serum and the indicated concentration of 125I-labeled LDL (specific activity, 90 cpm/mg protein) or 125I-labeled remnant lipoproteins (specific activity, 650 cpm/mg protein) was added. Heparin-releasable binding, uptake, and degradation of labeled lipoproteins were determined after 4-hour incubation at 37°C. Values shown are corrected for nonspecific binding, uptake, and degradation, as determined in cells incubated with labeled lipoprotein together with an excess of the appropriate unlabeled lipoprotein, and are the mean of duplicate wells. , COS cells mock-transfected with salmon sperm DNA; , COS cells transfected with the normal LDL receptor cDNA; , COS cells transfected with the mutant LDL receptor cDNA, derived from mRNA transcribed from the allele with the intron 3 G+1-to-A mutation.

Although there was no difference between the stabilities of mutant and normal mRNA and the protein expressed in COS cells, the immunoblot of cell extracts from the patient's lymphoblasts showed clearly that there was less mature, mutant, receptor protein in heterozygous cells, and this finding was confirmed by scanning the immunoblots (Fig 7A). The amount of receptor protein detected on the immunoblots was proportional to the amount applied to the gel in each case, and the amount of total receptor protein in mutant cells was approximately 70% of that in cells from an unaffected individual. The resolution was not sufficient to allow separate assessment of mutant and normal proteins in the same cell. Degradation of labeled LDL by lymphoblastoid cells was also determined. In cells from the patient heterozygous for the mutation (II,6), the apparent affinity for degradation of ¹²⁵I-LDL was lower than that in normal cells (Fig 7B), but the difference was less marked than in transfected COS cells. The maximum rate of degradation of LDL by heterozygous cells was approximately 70% of that of normal cells. Measurement of the incorporation of ³⁵S-labeled methionine into normal and mutant receptor proteins in cells from a heterozygous patient also suggested that less mutant than normal protein was synthesized, but that there was little difference in the rate of breakdown of the two proteins (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 7. Plots of measurement of LDL receptor protein and activity in cultured lymphoblasts. A, Extracts of lymphoblasts that had been preincubated for 14 hours with 10% lipoprotein-deficient serum and 0.3 µg/mL compactin were subjected to nonreduced sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotting with antibody 10A2, and detection by chemiluminescence. The films were scanned, and the relative amount of total LDL receptor protein in each lane was determined as the area under the peak, expressed in arbitrary units. B, Lymphoblasts that had been preincubated for 14 hours with 10% lipoprotein-deficient serum and 0.3 µg/mL compactin were incubated with 125I-labeled LDL in the presence (nonsaturable) or absence (total) of excess unlabeled LDL. Results are shown for saturable degradation of LDL (total minus nonsaturable) and are the means of duplicate incubations. Nonsaturable degradation was always less than 5% of total degradation. Dotted lines indicate the concentration of 125I-labeled LDL (x axis) at which half-maximum degradation of LDL (y axis) occurred with each cell line. , Cells from II,7 (unaffected); , cells from II,6 (heterozygous for G-to-A mutation in intron 3).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Nucleotide sequencing of amplified genomic DNA from FH patient I,1 revealed the presence of a G-to-A transition at position 1 of intron 3 of the LDL receptor gene. This single base substitution changed the highly conserved dinucleotide GT, which forms part of the GT(A/G)AGT recognition signal at the donor splice site,³³ to AT. Mutations involving the GT dinucleotide at the 5' ends of introns have almost invariably been shown to result in incorrect splicing of the primary transcript, but the pattern of aberrent splicing varies in different genes and includes exon skipping, activation of cryptic splice sites, and retention of the unspliced intron in the mature transcripts (for a review, see Reference 34³⁴ ).

The sequencing data from the RT-PCR–amplified fragment of mRNA in cells from one of our patients show that the single base change in the conserved sequence at the 5' end of intron 3 causes exon skipping, with exon 3 deleted and exon 2 spliced to exon 4. The amount of mutant mRNA detected in cells from patients heterozygous for the mutation was less than that of normal mRNA, although the mutant mRNA was apparently as stable as normal mRNA when expressed in heterologous cells. This suggested that splicing of mutant mRNA was less efficient than normal. However, PCR amplification with other potential primers did not reveal any different products (data not shown), so that if other splicing reactions had occurred, they presumably resulted in insignificant quantities of alternatively spliced mRNA. Several other examples of 100% exon skipping due to a mutation in the invariant G at the donor splice junction have been described, eg, in mutant genes causing phenylketonuria,³⁵ retinoblastoma,³⁶ and cutaneous porphyria,³⁷ but not previously in the LDL receptor gene.

Even with the limited number of FH patients with the intron 3 mutation who are available to us, it is clear from the information in the Table that this mutant allele is associated with a relatively severe clinical phenotype, in terms of both the associated hypercholesterolemia and the age of onset of symptoms of coronary heart disease. Deletion from the mRNA of sequences encoded by exon 3 was predicted to result in loss of the 41 amino acids encoded by this exon and substitution of a single amino acid residue at the new splice junction (Pro₈₄Ser). Previous studies in vitro³⁸ have suggested that deletion from the protein of amino acid residues encoded in exon 3 has only a minimal effect on the function of the LDL receptor in heterologous cells, ie, reducing the binding of LDL to some extent but having little or no effect on binding of lipoproteins containing apoprotein E. This is supported by descriptions elsewhere of FH patients in whom deletions encompassing exons 2 and 3 apparently result in a mild clinical phenotype.³⁹ Thus, we were originally puzzled by the relatively severe clinical phenotype in our index patient and in the majority of other individuals that we have identified who carry this mutation. One possibility was that the single proline residue inserted at the new splice site in the naturally occurring receptor (which was not present in the receptor resulting from site-directed mutagenesis to remove exon 3³⁸ ) destabilized the protein in some way. This encouraged us to determine the effect of this particular mutation on LDL receptor protein structure and function in both homologous and heterologous cells.

The predicted effect of the mutation on the primary structure of the protein was supported by our observation of the slightly faster mobility on SDS-PAGE of the mutant receptor protein compared with that of the normal receptor in the same cells, both in lymphoblasts from the patient and in heterologous cells expressing the mutant or normal cDNA. One monoclonal antibody to the LDL receptor, antibody 10A2, detects an additional form of the LDL receptor protein. This protein, which we believe to be a degradation product of the mature receptor, is more prominent in heterologous cells expressing the receptor protein than it is in human skin fibroblasts in culture.¹⁵ Band X in cells expressing the mutant protein lacking repeat 2 has the same mobility as band X in cells expressing the normal receptor, suggesting that band X is identical in both cell types, whereas the mature forms of the normal and mutant proteins have distinctly different mobilities. The most likely explanation for this observation is that band X is a form of the protein from which a portion of the binding domain of the LDL receptor that includes repeat 2 has been cleaved. Since antibody 4B3 detects the mutant receptor poorly and does not detect band X, part of the antigenic site is probably located in the region of the binding domain that has been cleaved.

Interpretation of the effects of mutations on receptor protein function in cells from heterozygous patients expressing the normal and the mutant allele is difficult, and thus the ability of the mutant receptor to catalyze receptor-mediated endocytosis of lipoproteins was assessed by expressing the mutant gene in heterologous cells. As predicted from other studies on the effect of introducing mutations into repeat 2 of the binding domain of the LDL receptor,³⁸ the mutant protein bound LDL with a reduced apparent affinity, ie, approximately half that of the normal receptor, but bound triglyceride-rich, apoprotein E–containing lipoproteins as efficiently as did the normal receptor expressed in vitro. Thus, the mutant receptor in a heterozygous patient would be predicted to show only slightly impaired function compared with normal. However, although the same amount of LDL receptor mRNA was apparently produced in COS cells transiently expressing either the mutant or the normal LDL receptor cDNA, cells from the patient heterozygous for the mutation contained much less aberrantly spliced mRNA than normally spliced mRNA from the other allele, most probably due to a decrease in splicing efficiency. Cells from a patient heterozygous for the mutation also contained about 30% less total mature receptor protein and had about 70% of receptor activity, with a reduced affinity for LDL compared with that of cells from an unaffected relative.

Thus, although the mutation might at first sight have been predicted to be associated with a mildly receptor-defective FH phenotype, in practice it results in a typically severe phenotype in the majority of patients available to us. Presumably the combination of a reduced amount of protein and the reduced affinity for lipoprotein ligands is sufficient to markedly impair LDL clearance in vivo. Nonetheless, at least one male patient from Manchester was not so severely affected, which stresses the importance of other factors in addition to the structural defect in the LDL receptor in determining the phenotype in heterozygous FH patients.

	Acknowledgments

 
This work was supported in part by the British Heart Foundation (project grant 93/0005). We thank Dr David Russell, Department of Molecular Genetics, Dallas, Tex, for providing plasmid pLDLR4; Dr B. Harding, MRC Molecular Medicine, Hammersmith Hospital, for establishing lymphoblastoid cell lines; and Dr G.R. Thompson (MRC Lipoprotein Team), Dr P. Durrington (Manchester Royal Infirmary), and Dr P. Miller (Withington Hospital, Manchester) for access to information about their patients.

Received July 15, 1994; accepted December 7, 1994.

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