- Email: [email protected]
Familial hypercholesterolaemia: Mutations in the gene for the low-density-lipoprotein receptor
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Familial hypercholesterolaemia: mutations in the gene for the low-density, lipoprotein receptor
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/ Anne K. Soutar
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Lipoprotein Team. / / MRC Clinical Sciences Centre. / / Royal Postgraduate Medical School. / Hammersmith Hospital. / / Du Cane Road, London, UK W 12 0NN. / / Tel: +44 181 740 3262 Fax: +44 181 746 0586 / /
e-mail: [email protected]
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Familial hypercholesterolaemia is a co-dominant inherited disorder of lipoprotein metabolism, in which defects in the gene for the low-density-lipoprotein (LDL) receptor result in a twofold increase in the plasma concentration of cholesterol and moderate-to-severe premature coronary heart disease. Many mutations in the gene for the LDL receptor that have different effects on the structure and function of this multifunctional protein have been found, but it is not yet clear whether the nature of the mutation determines the severity of the disorder. This question is being / / answered by comparing patients with well-characterized mutations, and / recent work suggests that other genetic / or environmental factors may be / important in modulating the effect / of the defect in LDL-receptor function in / / patients who are heterozygous for / the disorder. / /
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© 1995, Elsevier Science Ltd
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MOLECULAR MEDICINE TODAY
THE recent results of the 4S study (the Scandinavian Simvastatin Survival Study) on the effects of cholesterol-lowering treatment in patients with coronary heart disease (CHD) should now convince even the most sceptical that reducing plasma concentrations of low-density-lipoprotein (LDL) cholesterol markedly improves survival in such individuals j. Thus, not only is a raised plasma cholesterol level a strong risk factor for developing early atherosclerosis, but lowering plasma cholesterol levels, even in a mildly hypercholesterolaemic individual, reduces their risk of another cardiovascular event. The 4S study is also reassuring in that there was no mysterious increase in 'non-cardiovascular deaths', so a low (or lowered) plasma cholesterol does not, after all, make an individual more likely 'to be run over by a bus', despite suggestions to this effect from some analyses of earlier trials. What determines plasma cholesterol concentration and the likelihood that someone with a particular cholesterol concentration will, or will not, suffer from early CHD is complex, but inherited factors clearly play a dominant role. In most individuals, these will include contributions from several gene variants, which may each have an imperceptible effect on a measurable biochemical process, but which interact with each other and with the environment to produce a definite increase in risk. Unravelling these interactions and identifying the individual gene variants themselves is proving to be rather difficult, although a glance at the biochemistry of cholesterol metabolism and the cell biology of atherosclerosis shows that there is no shortage of candidate genes.
Familial hypercholesterolaemia Although relatively few in number, individuals with a known genetic defect in one such candidate gene are important because they can shed light not only on the precise role in the overall process of the gene product concerned, but also on how the effects of the known gene defect are either mitigated or exacerbated by other genetic and environmental factors2. One such human genetic disorder of cholesterol metabolism that has received much attention is familial hypercholesterolaemia (FI-I), which affects about 1 in 400 to 500 individuals. FH is caused by mutations in the gene for the LDL receptor, which plays a central role in determining the concentration of LDL in the human circulation 3. Heterozygous FH patients, with one defective and one normal LDL-receptor gene, have a twofold increase in the concentration of LDL in plasma, which puts them at considerably greater risk of premature CHD than the general population. Homozygous patients, in which both copies of the gene are defective, are very rare, but are much more severely affected. Although it was shown in 1964 that FH is a single-gene defect, there is wide variation in the severity of the disorder in different heterozygous patients. Before any evidence to the contrary was available, this variation was ascribed to differences in the precise nature of the mutation in the gene. Measurement of LDL uptake and catabolism by cells cultured from homozygous FI-I patients and analysis of the LDL-receptor protein show considerable differences between patients; some have virtually undetectable LDL-receptor activity, while others have considerable residual activity. Thus, it was likely that there were many different mutations that affected the receptor differently. Not surprisingly, those homozygous patients whose cells retain some LDL-receptor activity in vitro tend to have less severe hypercholesterolaemia and a better clinical prognosis than those without3. However, the picture is less clear in the more common heterozygous FH patients, and it will take longer to be certain whether or not the type of mutation alone determines the severity of the hypercholesterolaemia and its associated risk of early CHD. If the nature of the genetic defect is not the major determinant, then it is worthwhile trying to find out what is, because the information
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Glossary Arcus - Visible opacity around the outer edge of the cornea caused by lipid deposition. Atherosclerosis - A degenerative disease of blood vessels, defined as a variable combination of changes in the intima (inner lining) of the arteries, consisting of accumulated lipids, other blood constituents and fibrous tissue, accompanied by proliferative changes in the media (middle layer). Chylomicrons - Large, triglyceride-rich lipoprotein particles secreted from the intestine after ingestion of a fatty meal. Familial hypercholesterolaemia (FH) - A genetic disorder of lipoprotein metabolism, characterized clinically by a raised concentration of Iow-density-lipoprotein (LDL) cholesterol, the presence of tendon xanthoma and a family history of raised LDL cholesterol, tendon xanthoma or premature coronary heart disease in a first-degree relative. Genotype/phenotype - The genotype of an individual denotes the genes that they have inherited; the phenotype denotes the effect that those genes have on the physiology of the individual. Homozygous/heterozygous familial hypercholesterolaemia (FH) - A homozygous FH patient has two identical defective copies of the gene; a heterozygous FH patient has one normal and one defective copy. A compound heterozygous FH patient has the clinical characteristics of a homozygous patient, but has two different defective copies of the gene. Xanthoma - Visible yellowish deposits of lipid, either flat (planar) or prominent (tuberous), in skin and tendons.
should be applicable to the general population, where most of the premature ~ occurs. Identification of those individuals with a moderately raised plasma cholesterol level who ~ at high risk so that they can be treated, preferably before, rather than after they have overt CHD, is still a major goal.
The LDL-reeeptor pathway Several reasons why mutations in different parts of the LDL-'receptor gene might be expected to have rather different effects on its function are apparent when the role played by the LDL receptor in lipoprotein metabolism is considered4. Lipoproteins function primarily to transport lipids, mainly triglycerides and cholesteryl esters, from their sites of synthesis and absorption to sites of utilization and storage. Following a fatty meal, chylomicrons are secreted from the intestine into the circulation where the triglycerides are hydrolysed to release fatty acids that are taken up by muscles or adipose tissue, leaving a lipiddepleted chylomicron remnant that is cleared rapidly by the liver. The liver synthesizes and secretes very-low-densitylipoproteins (VLDL), which enter the plasma and, initially, have the same fate as chylomicrons. However, the process differs in that not all the remnants are elearod immediately, but are subjected to fur~er modification in plasma to form intermediate-density lipoproteins (IDL) and eventually LDL, which is cleared only slowly and accumulates in plasma. V'trtually al! the triglyceride originally in the VLDL particle is replaced with cholesteryl esters in LDL by exchange and transfer from other lipoproteins, and the m
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sole protein component in LDL is apoBt0o. The LDL receptor in the liver is responsible for the uptake and removal of LDL from plasma ,and 'also VLDL remnants and IDL. The ligands recognized by the LDL receptor are apoBt0o in LDL, and apoE in IDL and VLDL remnants, although they also contain apoB~oo. It is not clear what determines the proportion of VLDL remnants and IDL that are taken up, rather than being converted
to LDL, but it is clear that defective LDL-receptor activity in patients with FH both decreases the rate of clearance of LDL and increases its rate of formation in plasma, as IDL is the immediate precursor of LDL (Fig. 1). Once the LDL-receptor gene had been cloned, it was possible to predict from its sequence and gene structure that the protein comprises several different structural domains 5 (Fig. 2). The newly synthesized
Cholesteryl ester
(( v,o, ))
.o, Triglyceride
v \ S y n t h e s i s of a p o B l o o and triglycerides
Liver
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O x i d a t i o n and uptake by scavenger receptor
Concentration in p l a s m a J
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Non-receptor-mediated u p t a k e in peripheral t i s s u e ~ / /
I
LPL
ApoB 48
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Figure 1. Effectof defectiveIow-density-lipoprotein(LDL)-receptorfunction on the metabolismof triglyceride-richlipoproteins.The LDL receptor normally mediates the specific uptake, mainly by the liver, of the remnants of the triglyceride-rich lipoproteins,very-low-densitylipoproteins (VLDL) and chylomicrons, which have been depleted of triglyceride by the action of the enzymelipoprotein lipase (LPL).VLDL remnants, or intermediate-densitylipoproteins (IDL), are further depleted of triglyceride by LPL and hepatic lipase (HL) to form LDL Exchangeand transfer of lipids between lipoproteinsalso occurs by the action of the cholesterylestertransfer protein (CETP).When LDL-receptoractivity is defective,lessVLDL remnantsare cleared,and more are therefore availableto be convertedto LDL, and the removal of LDL is impaired.This results in an increasedconcentration of LDL in plasma and an increasein its half-life in plasmawhich, in turn, resultsin the deposition of cholesterolin the peripheraltissuesas xanthomata and an acceleratedrate of atherosclerosis, possibly pertly becauseof oxidative damageto the LDL and its subsequent uptake by macrophages.
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Exon
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2
3
4 5 6
7 8 9 10 11 12 13 14
15
16 17
18
Gene -45 kb
5.3 kb mRNA
Protein
NH2 Ii
Signal peptide (cleaved from mature protein)
Ligand-binding domain with disulphide-rich repeats I-VII
EGF-precursor domain with growth-factor-like disulphide-rich repeats A, B and C
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O-linked Cytoplasmic sugars tail domain domain with internalization signal X Transmembrane domain
Figure 2. The Iow-density-lipoprotein (LDL) receptor. Diagram of the LDL-receptor gene (shown in blue) with the 18 exons shown as vertical bars. The primary transcript of the mRNA (not shown) is spliced to remove the introns to form the spliced cytoplasmic mRNA (shown in green). The regions of the protein (shown in red) coded for by each exon of the gene are indicated by the vertical dotted lines between the mRNA and the protein. The protein comprises five structural domains, as indicated, some of which have homology to other functionally unrelated proteins; important functional or structural features of each domain of the protein are indicated below.
precursor form of the LDL receptor is glycosylated co-translationally with O- and N-linked sugars, which are trimmed and modified during the passage of the protein through the Golgi to form the mature glycoprotein. The mature receptor is transported to the cell surface, where it spans the cell membrane, exposing its ligand-binding domain to the exterior. The LDL receptor on the cell surface clusters into clathrin-coated pits, which constantly invaginate and internalize molecules bound to the cell surface, so that LDL receptors and their bound ligands are delivered to endosomes. Here, sorting of different receptors occurs, and the LDL receptor and its ligand dissociate as a result of the rapid fall in pH. The receptor molecule recycles to the cell surface, while the lipoprotein ligand is degraded in lysosomes, forming amino acids from the protein component, and cholesterol and fatty acids from the cholesteryl esters in the lipidcore of the lipoprotein particle. The free cholesterol released in this way regulates transcription of the LDL-receptor gene and the genes for key enzymes in the pathway of cholesterol synthesis. Excess free cholesterol is esterified by the enzyme acyl:cholesterol-acyltransferase (ACAT) to form cholesteryl esters that can be stored safely in the cytoplasm as lipid droplets. Thus, LDL-receptor-mediated uptake of cholesterol regulates both further uptake and the rate of de nero synthesis of cholesterol, and functions to maintain intracellular cholesterol homeostasis. This entire process is referred to as the LDL-receptor pathway 6 (Fig. 3).
Effects of mutations on LDL-receptor function It is not surprising that a multistep pathway of this type demands a mullifunctional protein. Analysis of the effect of different mutations, both
naturally occurring and those specifically introduced by mutagenesis, on LDL-receptor expression and activity in cultured cells has revealed that each structural domain of the protein is important for different aspects of its function. Mutations in the gene in patients with F[-I have been broadly classified into different classes based on LDL-receptor function in cultured cells, and mutations in one particular domain are often associated with each class 7 (Table 1). However, there are exceptions, and as many mutations do not fall clearly into one single category, a~signing a newly identified mutant allele to a particular class without supporting biochemical data is prone to error. Mutations in genes can obviously cause a wide range of defects in the function of a protein, ranging from a point mutation that results in the substitution of a single amino acid residue with one whose properties are so similar that the mutation has a negligible effect on the structure and function of the protein, to a major deletion that wipes out a large section of the coding region of the gene. In between are a variety of minor nucleotide changes, the precise effect of which on structure and function may be difficult to predict, depending on the particular protein. One or two aspects of the LDL receptor are interesting in this respect. First of all, the protein structure appears to be exquisitely sensitive to even the most conservative amino acid substitutions. This is not necessarily because such changes affect the ability o'f the receptor to bind and internalize lipoprotein ligands, but because the post-translational glycosylation of the protein and its transport to the cell surface are disrupted. Point mutations that introduce a premature stop codon anywhere in the gene frequently destabilize the mRNA so that little or no truncated mutant m
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Endosomes Lysosomes ~ 13 Utilization 9 10 12 | or esterification Internalization Dissociation Degradation • ~, • ~Cholesterol ~ ~ R e g u l a t i o n /
8 Clustering in coated pits 7 Ligand binding
~11 Recycling 6 Insertion in 5 Intracellular 4 Protein cell membrane transport maturation Cytoplasm
Golgi
3 Protein synthesis
2~mRNA I Gene splicing transcriptionJ
Endoplasmic reticulum
Nucleus
Cell membrane Rgure 3. The Iow-density-lipoprotein (LDL)-receptor pathway. Diagram showing the different stages (1-11) at which mutations in the LDL-receptor gene can disrupt its function, Mutations can impair the sterol-regulated transcription of the gene (1) and splicing of the primary mRNA transcript (2), which occur in the nucleus and can result in reduced amounts of mRNA and newly synthesized protein (3). The protein is synthesized as a precursor form (3), with co-translational addition of sugars that are trimmed and modified as the protein passes through the Golgi (4) to the cell surface (5). Many single amino acid substitutions impair this part of the pathway. The mature receptor protein is inserted in the cell membrane (6), where it binds ligands containing apoB or apoE (7). The receptor clusters in coated pits (8), which invaginate to form intracellular endosomes (9) that fuse with lysosomes, where dissociation of the receptor-ligand complex occurs (10). The receptor recycles to the cell surface (11), while the lipoprotein ligand is degraded (12). Several mutations have been described that impair these steps in the pathway. The free cholesterol released from the cholesteryl esters in the core of the particle is used for synthesis of the cell membrane, as a substrate for biosynthetic pathways, or is esterified and stored in the cell (13). It also exerts a negative regulatory effect on the transcription of the LDL-receptor gene and other genes in the biosynthetic pathway of cholesterol biosynthesis (14).
receptor protein is synthesized. Furthermore, truncated proteins are usually recognized as foreign by the cell and degraded rapidly; even if some does persist, it is unlikely to reach the cell surface and cannot function in receptor-mediated endocytosis. The ligand-binding domain is also of interest because it comprises seven 40-residue cysteine-rich repeats, each with a cluster of negatively charged residues, most of which are encoded by a single exon. Mutations that cause single amino acid substitutions in the two central repeats severely impair the ability of the receptor to bind lipoproteins containing both apoB and apoE, while mutations in the other repeats are much less deleterious 8. If they have any effect, such mutations generally impair recognition of apoB, but not of apoE. If the mutant receptor cannot recotmi7e apoB or apoE, then clearance of both LDL and VLDL remnants will be impaired and LDL production in plasma will be increased. By contrast, if the receptor retains some ability to bind apoE, then VLDL remnants will be cleared and, although LDL catabolism will still be impaired, the supply of precursors for LDL production will not be increased and LDL should not accumulate to such high levels in plasma. One other aspect that is of relevance is the structure of the LDLreceptor gene. Many of its intron-exon junctions, particularly those in the binding domain, are in phase. This means that a deletion encompassing one or two exons will result in an mRNA species in which those residues encoding the exons concerned will be neatly spliced out, leaving the rest of the protein intact, rather than introducing a frameshift that generally results in the addition of a number of foreign residues to the protein at the deletion joint before a premature stop codon is encountered. Sometimes, but not always,deletion of a whole domain, or even part of a domain, leaves the remainder of the protein structure intact and the mutant protein is not recognized as foreign; it may also retain some function. Thus, a major deletion will not necessarily result in a null phenotype. m
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Relationship between genotype and phenotype in FH Despite the wealth of information already available about the LDL receptor, answering the question of whether or not the specific mutation in the LDL-receptor gene determines the clinical severity of the disorder in patients with FH is not as straightforward as it may appear. First, the effects of a variety of specific mutations in the gene on the function of the LDL receptor have to be determined. Second, sufficiently large groups of patients with these mutations need to be identified and their clinical characteristics compared. The availability of new methods that speed up the process of finding mutations in genes has allowed the identification of an ever-increasing number of LDL-receptor-gene variants that cause FH. Determining the precise effect of the mutation on LDL-receptor function is more demanding, but has been achieved for a sufficient variety of mutations to allow some comparisons to be made. As described above, it is possible, in some cases, to predict with reasonable accuracy what the effect of a particular mutation will be on receptor function but, in others, it may be necessary to express the mutant gene in heterologous cells in vitro. The major problem in many populations is finding sufficient unrelated patients with each mutation. For example, in a group of 200 apparently unrelated patients with FH in the London area, we have already found more than 35 different mutations in the LDL-receptor gene. Of these, the four or five commonest each occur in -3-5% of the patients 9-~2, while the majority, by far, have each been found in a single individual. This diversity'is partly the result of the heterogeneity of the population in the London area and, in arararararar~ of Britain where the population has been more stable, some mutations may be more common. For example, two mutations that are present in 1% and 2.5% of the London group occur in 5% and 14% of a group of patients with FI-I from the Manchester area. In some countries where the population has remained stable for many generations, either because of geographical isolation, as in Finland, or
MOLECULAR MEDICINETODAY
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cultural isolation, as in the Afrikaners in South Africa, one or two mutations may account for 80-90% of the incidence of FH. Indeed, it is in these populations that some of the first comparisons between patients with different mutations have been made. However, it has to be borne in mind that such isolated groups will also have many other genetic variants in common and will share very similar environmental influences, which may permit the nature of the genetic mutation to have an apparently overriding effect that would not be observed in a more diverse racial and cultural group. Even when sufficient patients with different defined defects have been assembled, the question remains of how the severity of the disease should be measured. Although the clinician may have the impression that one patient with FH is less severely affected than another, providing a quantitative measure that can be subjected to statistical analysis is quite another matter. The obvious initial choice is to compare the total cholesterol or LDL-cholesterol value in the patient before treatment commences, but a reliable value for this is not always available. There is the added complication that an individual's plasma cholesterol concentration normally increases with age, so that values have to be adjusted for age when comparisons are made. There is no consensus as to whether this correction should be made for patients with FH, as at least one study has shown that this relationship between LDL cholesterol concentration and increasing age does not hold in all FH patients. Similarly, comparisons should be strictly limited to groups of individuals of the same gender, as premenopausal women have lower levels of plasma cholesterol than men, and this difference appears to be maintained in patients with FH. Other parameters that can be assessed are the presence or absence of corneal arcus and xanthoma and, as has been done in some studies, the thickness of tendon xanthoma, which are all indicators of cholesterol deposition in peripheral tissues. The age of onset of manifestations of overt CHD, if any, in the patient is another important indicator of the severity of the disorder, but again this information is not always available. Another variable of interest is the extent to which the hypercholesterolaemia and CHD respond to cholesterol-lowering therapy in a particular individual. A number of recent studies have addressed the question of whether there is a direct correlation between the type of mutation and the clinical
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phenotype (summarized in Table 2). There is strong evidence from the one study t3 of homozygous patients that the mutation in the LDL-receptot gene has an overriding influence on the disease, whether measured in terms of the associated hypercholesterolaemia, or from clinical observations. Two founder genes in French Canadians have resulted in an unusually high number of patients with FH in this region who are homozygous for one of two mutations, one of which is a deletion of the promoter region so that no mRNA or protein can be produced. The second is a single base change in exon 2, which is predicted to result in substitution of glycine for the normal tryptophan residue at position 66 in the protein. This residue lies in repeat 2 of the ligand-binding domain and has a relatively minor effect on lipoprotein uptake. Patients with the major deletion had significantly more severe hypercholesterolaemia, which resulted in earlier onset of CHD and earlier death from CHD. The differences were much less marked in their heterozygous relatives, however, showing that other factors also come into play when there is only one defective copy of the gene. This is also the case in several other studies of heterozygous FH patients. One exception is the study in Finland ~5'~6,where a mutation that results in the deletion of the O-linked sugars domain has been found. When this same mutation was introduced into the LDL receptor cDNA and expressed h7 vitro, receptor function in cultured cells was indistin~ishable from normal 19. Clearly, the mutation has some effect in vivo, but the patients have very mild hypercholesterolaemia and less severe clinical manifestations of the disorder; for example, the thickness of tendon xanthoma was significantly less than in another group of Finnish patients with a large deletion causing an essentially null mutation. It should be noted, however, that all the patients with the exon 15 mutation were members of a single large kindred and would have other factors in common. In two other studies, one in South Africa 17 and one in Israel ~4, there were no significant differences in plasma cholesterol concentration between groups of patients with the same mutation that could be correlated with the type of mutation. In a recent study from the UK ~8,patients with different mutations were grouped together according to the type of mutation, and it was found that the group with point mutations in the fifth repeat of the binding domain, which is essential for function, had a higher mean cholesterol
Table 1. Phenotype classification system for mutant LDL-receptor genes in cultured cells from patients with familial hypercholesterolaemia Class of mutant allele"
Phenotype
Location of mutations b
Class 1 - Null Class 2c - Transport-defective
No LDL receptor protein or mRNA detectable Precursor fails to be converted to mature protein, or to be transported to cell surface Mature protein reaches cell surface, but fails to bind ligand Mature protein binds ligand on cell surface, but fails to localize in clathrin-coated pits Receptor-ligand complex fails to dissociate in lysosomes; receptor fails to return to cell surface and is degraded
Premature termination codons in all domains Primarily in epidermal growth factor (EGF)precursor-like and binding domains Clustered in binding domain
Class 3 - Binding-defective Class 4 - Internalization-defective Class 5 - Recycling-defective
One, in cytoplasmic tail Clustered in EGF-precursor-like domain
8Datafrom Ref. 6. bpoint mutations and small deletions/insertions. Data from Ref. 7. cSubdivided into Class 2A (total - no mature receptor protein reachesthe cell surface) and 2B (partial - maturation of precursor to mature protein delayed).
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concentration than those with point mutations elsewhere. However, the difference was rather small. In the Israeli study, one group of patients did have a significantly lower mean plasma cholesterol concentration, but as the mutation concerned results in an essentially null phenotype and cannot be considered as mild, it is possible that strong cultural or environmental influences are at work in this group.
Other factors that influence the FH phenotype Evidence that other factors influence the FH phenotype comes also from studies of FH patients in China 9 and Tunlsm-, where homozygous FH is found at the same frequency as in most countries, but heterozygous FH is not recognized as a disorder. Even the obligate heterozygous parents of homozygous children in China frequently have plasma cholesterol values that would be considered in the normal range in the USA and most of Europe. This is not because of the presence of particularly mild mutations in Cl{inese patients, as the same spectrum of mutations has been found in the Chinese patients as in those elsewhere, and the hyper•
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cholesterolaemia in the homozygotes is just as severe. Presumably, some environmental factor, probably related to their healthy diet, is protecting Cbinese and Tunisian heterozygous FH patients against the worst effects of one copy of a defective LDL-receptor gene, but is insufficient to override the effect of two defective genes. In Western countries, the hypercholesterolaemia of FH has always been considered refractory to treatment with lipid-lowering diets, so an alternative possibility is that there are other genes in these racial groups that influence the phenotype. It would be of interest to know whether Chinese or Tunisian heterozygous FH individuals exposed to a western lifestyle retain, or lose, their protection against CHD. The conclusion from these studies to date appears to be that in heterozygous FH, the nature of the mutation in the LDL-receptor gene, while causing the disorder, interacts with other factors to produce the particular phenotype in an individual patient. As yet, we have little idea about what these factors might be, and it is quite likely that they may not be the same in all patients. Several studies have attempted to investigate the influence
Table 2. Effect of different mutations in the Iow-density-lipoprotein (LDL) receptor gene on plasma cholesterol in patients with heterozygous familial hypercholesterolaemia (FH) LDL-receptor activity in cells b (% of normal)
Mean plasma cholesterol (mmol I-~)°
Mean age of Number patients of patients ° (years) Refs
Mutation
Trivial name
Phenotype in cultured cellsa
I
Deletion of promoter and exon 1 Trp~Gly
French Canadian-1 French Canadian-4
Null 3 or 5
<2 25-100%
8.1 (26.7) 7.2(16.1) d
20 (11) 20 (10)
7.8 8.6
II
Asp147His Deletion of Gly197 Cys66oStop
FH Sephardic FH Lithuania FH Lebanese
2B 2B 2A
<2 <2 <2
10.21 9.61 7.82d
15 5 21
No data 14 No data No data
Ill
Deletion of exons 16-18 (two groups) Deletion of exon 15 Deletion of 7 bp in exon 6
FH Helsinki
4B
<2
No data 1
Mild e No data (? none)
66 23 10 69
47 38 33 47
15,16
FH Espoo FH North Karelia
11.7 9.7 7.3d 12.1
IV
Asp2o6Glu Val40aMet
FH Afrikaner-1 FH Afrikaner-2
2B 5
5-15 <2
9.4~ 10.8
112 36
40 40
17
V
Single amino acid substitutions in repeat 5 Other single amino acid substitutions Stop codons in exon 4
Variousf
Various
<2-5
11.2
17
41
18
Various °
Various
<2-30
9.6d
11
42
Various h
Null
<2
11.3
12
40
Study number
13
"From the classificationsystem describedin Table1. bBasedon binding, uptakeand degradationof 12Sl-labelledLDL by cultured skin fibroblastsfrom patientswho are homozygousfor the mutation. Datafrom Ref. 7. cValuesin parenthesesrepresentpatientswho are homozygousfor two mutations. dValuesignificantly lower than others in samestudy. eDatafromstudies in stimulated lymphocytes,not fibroblasts'6. rlncludesAsp2~Gly,Asp2~Gluand deletion of Gly19v ~lncludesGlumLys, Pro~4Leu,Seq~Leuand 'two gross deletionscausing defectiveprotein'~8. hlncludesGlu2oTStop, Cys2,0Stop and a frameshift in codon 206 that introduces a prematurestop codon.
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of oflaer candidate genes on the FI-I phenotype (for review, see Ref. 2), but these have all been carried out on groups of patients in whom the mutation has not been defined. However, the answers to these questions should continue to be sought. The ability to identify the mutation in' the LDL-receptor gene in an FH patient that attends a lipid clinic allows relatives who carry the mutation to be identified, many of whom may not previously have been diagnosed ,as having FH. The increasing availability of such technology could result in the identification of many more such FH patients and information about what determines their likelihood of suffering from premature CHD is obviously of importance for counselling and treatment. There may also be variants of the LDL-receptor gene that can function quite normally when in a favourable environment, whether genetic or otherwise, but which may result in hypercholesterolaemia in an adverse environment. Such gene variants would not give rise to classical FH, but could be a common underlying cause of the mild hypercholesterolaemia that is so prevalent in western countries.
The outstanding questions • Are the factors that influence the clinical phenotype of a patient with familial hypercholesterolaemia (FI-I) with a particul~ gene defect different from the known risk factors for coronary, heart disease (CHD) in tile general population? • Does the mutant Iow-density-lipoprotein (LDL)-receptor gene product in a heterozygous FH patient influence the activity of the protein from the normal copy of the gene? • Does the behaviour of tile normal LDL-receptor gene allele in heterozygous FH patients vary and, if so, does this affect their clinical phenotype, particularly their response to treatment? • Are there nlild mutations in the LDL-receptor gene in individuals with a modestly raised, or even normal, concentration of LDL cholesterol that put them at increased risk of coronary disease'? • Are there mutations in genes other than the structural gene for the LDL-receptor protein that can influence its expression or function?
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9 Sun, X.M. et al. (1992) Characterization of deletions in the LDL-reeeptorgene in patients with familial hypercholesterolemiain the United Kingdom,Arterioscler. Thromb. 12, 762-770 10 King. U.L. et al. ( 199 l) Identificationof the 664 proline to leucinemutation in the Iow-density-lipoproteinreceptor in four unrelated patients with familialhypercholesterolaemia in the UK, Clin. Genet. 40, 17-28 11 Webb, J.C. et al. (1992) Characterization of two new point mutations in the low. density-lipoprotein-receptorgenes of an English patient with homozygousfamilial hypercholesterolemia,Z Lipid Res. 33,689-698 12 Gudnason, V. et al. (1993) Identification of recurrent and novel mutations in exon 4 of the LDL-receptorgene in patients with familial hypercholesterolaemia in the United Kingdom,Arterioscler. Thromb. 13, 56--63 13 Moorjani,S. et al. 0993) Mutations ofthe Iow-density-lipoprotein-receptorgene, variation in plasma cholesterol, and expression of coronary heart disease in homozygous familial hypercholesterolemia,Lancet 341, 1303-1306 14 Leitersdorf,E. el al. (1993) Genetic determinants of responsiveness to the HMGCoA reduetase inhibitor fluvastatin in patients with molecularlydefined heterozygous familial hypereholesterolemia,Circulation 87 (Suppl. III), 35--44 15 Koivisto, U.M. et aL (1992) The familial hypereholesterolemia (FH)-North Karelia mutation of the low density lipoprotein receptor gene deletesseven nucleotidesof exon 6 and is a common cause of FH in Finland, J. Clin. Invest. 90, 219-228 16 Koivisto, P.V.l. et aL (1993) Deletion of exon15 nf the LDL.receptor gene is asso. ciated with a mild form of familialhyperchalesterolemia-FH-Espoo,Arterioscler Thromb. 13, 1680--1688 17 Kotze, M.J. et aL (1993) Phenotypic variation among familial hypercholesterolemics heterozygous for either one of two Afrikaner founder LDL-reeeptor mutations, Arteriosc/er. Thromb. 13, 1460-1468 18 Gudnason,V., Day,I.N.M.and Humphries,S.E. (1994) Effect on plasma lipid levels of different classes of mutations in the Iow-density-lipoprotein-reeeptorgene in patients with familial hypereholesterolemia,Arterioscler, Thromb. 14, 1717-1722 19 Davis,C.G. et aL (1986) Deletionof clustered O-linked carbohydrates does not impair function of Iow-density-lipoproteinreceptor in transfected fibroblasts, J. Biol. Chem. 261,2828-2838 20 Slimane,M.N.etaL (1993) Phenotypic expressionof familialhypercholesterolemia in central and southern Tunisia, Atherosclerosis 104, 153-158
Next month's Round up in IVIMT References 1 ScandinavianSimvastatin SurvivalStudy Group (1994) Randomized trial of cholesterol loweringin 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S), Lancet 344, 1383-1389 2 Sout,'u,A.K. (1994) FamilialhyperCholcsterolaemia,in From Genotype to Phenotype (Humphfies, S.E. and Malcolm,S., eds), pp. 83-109, Bios Scientific 3 Goldstein,J.L. and Brown, M.S. (1989) Familial hypercholesterolemia, !n The Metabolic Basis of hlherited Disease (Scriver, C.R. et aL, eds), pp. 1215-1250, McGraw-i-lill 4 Havel,R.J. and Kane,J.P. (1989) Structure and metabolism of plasma lipoproteins, in The Metabolic Basis of Inherited Disease (Scriver,C.R. et al., eds), pp. 1129-1138, McGraw-Hill 5 Sudhof, 1~.C.,Goldstein, J.L., Brown, M.S. and Russell, D.W. (1985) The LDLreceptor gene: a mosaic of exons shared with different proteins, Science 228, 815-822 6 Brown,M.S.and Goldstein,J.L. (1986) A receptor-mediatedpathway for cholesterol homeostasis, Science 232, 34-47 7 Hobbs,H.H.,Brown,M.S.and Goldstein,J.L. (1992) Moleculargeneticsof the LDLreceptor gene in familial hypercholesterolemia,Hum. Mutation I, 445-466 8 lesser,V.et al. (1988) Mutational analysis of the ligand-bindingdomain of the Iowdensity-lipoprotein receptor, J. Biol. Chem. 263, 13282-13290
The meetings: • DNA Vaccines: A N e w Era in Vaccinology • Gene and Nucleic Acid Vaccine Strategies The books: • Protocols for Gene Analysis • Protein Expression in Animal Cells • RNA: Isolation and Analysis • Molecular Biology: Current Innovations and Future Trends
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Familial hypercholesterolaemia: mutations in the gene for the low-density, lipoprotein receptor
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/ Anne K. Soutar
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Lipoprotein Team. / / MRC Clinical Sciences Centre. / / Royal Postgraduate Medical School. / Hammersmith Hospital. / / Du Cane Road, London, UK W 12 0NN. / / Tel: +44 181 740 3262 Fax: +44 181 746 0586 / /
e-mail: [email protected]
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Familial hypercholesterolaemia is a co-dominant inherited disorder of lipoprotein metabolism, in which defects in the gene for the low-density-lipoprotein (LDL) receptor result in a twofold increase in the plasma concentration of cholesterol and moderate-to-severe premature coronary heart disease. Many mutations in the gene for the LDL receptor that have different effects on the structure and function of this multifunctional protein have been found, but it is not yet clear whether the nature of the mutation determines the severity of the disorder. This question is being / / answered by comparing patients with well-characterized mutations, and / recent work suggests that other genetic / or environmental factors may be / important in modulating the effect / of the defect in LDL-receptor function in / / patients who are heterozygous for / the disorder. / /
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© 1995, Elsevier Science Ltd
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MOLECULAR MEDICINE TODAY
THE recent results of the 4S study (the Scandinavian Simvastatin Survival Study) on the effects of cholesterol-lowering treatment in patients with coronary heart disease (CHD) should now convince even the most sceptical that reducing plasma concentrations of low-density-lipoprotein (LDL) cholesterol markedly improves survival in such individuals j. Thus, not only is a raised plasma cholesterol level a strong risk factor for developing early atherosclerosis, but lowering plasma cholesterol levels, even in a mildly hypercholesterolaemic individual, reduces their risk of another cardiovascular event. The 4S study is also reassuring in that there was no mysterious increase in 'non-cardiovascular deaths', so a low (or lowered) plasma cholesterol does not, after all, make an individual more likely 'to be run over by a bus', despite suggestions to this effect from some analyses of earlier trials. What determines plasma cholesterol concentration and the likelihood that someone with a particular cholesterol concentration will, or will not, suffer from early CHD is complex, but inherited factors clearly play a dominant role. In most individuals, these will include contributions from several gene variants, which may each have an imperceptible effect on a measurable biochemical process, but which interact with each other and with the environment to produce a definite increase in risk. Unravelling these interactions and identifying the individual gene variants themselves is proving to be rather difficult, although a glance at the biochemistry of cholesterol metabolism and the cell biology of atherosclerosis shows that there is no shortage of candidate genes.
Familial hypercholesterolaemia Although relatively few in number, individuals with a known genetic defect in one such candidate gene are important because they can shed light not only on the precise role in the overall process of the gene product concerned, but also on how the effects of the known gene defect are either mitigated or exacerbated by other genetic and environmental factors2. One such human genetic disorder of cholesterol metabolism that has received much attention is familial hypercholesterolaemia (FI-I), which affects about 1 in 400 to 500 individuals. FH is caused by mutations in the gene for the LDL receptor, which plays a central role in determining the concentration of LDL in the human circulation 3. Heterozygous FH patients, with one defective and one normal LDL-receptor gene, have a twofold increase in the concentration of LDL in plasma, which puts them at considerably greater risk of premature CHD than the general population. Homozygous patients, in which both copies of the gene are defective, are very rare, but are much more severely affected. Although it was shown in 1964 that FH is a single-gene defect, there is wide variation in the severity of the disorder in different heterozygous patients. Before any evidence to the contrary was available, this variation was ascribed to differences in the precise nature of the mutation in the gene. Measurement of LDL uptake and catabolism by cells cultured from homozygous FI-I patients and analysis of the LDL-receptor protein show considerable differences between patients; some have virtually undetectable LDL-receptor activity, while others have considerable residual activity. Thus, it was likely that there were many different mutations that affected the receptor differently. Not surprisingly, those homozygous patients whose cells retain some LDL-receptor activity in vitro tend to have less severe hypercholesterolaemia and a better clinical prognosis than those without3. However, the picture is less clear in the more common heterozygous FH patients, and it will take longer to be certain whether or not the type of mutation alone determines the severity of the hypercholesterolaemia and its associated risk of early CHD. If the nature of the genetic defect is not the major determinant, then it is worthwhile trying to find out what is, because the information
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Glossary Arcus - Visible opacity around the outer edge of the cornea caused by lipid deposition. Atherosclerosis - A degenerative disease of blood vessels, defined as a variable combination of changes in the intima (inner lining) of the arteries, consisting of accumulated lipids, other blood constituents and fibrous tissue, accompanied by proliferative changes in the media (middle layer). Chylomicrons - Large, triglyceride-rich lipoprotein particles secreted from the intestine after ingestion of a fatty meal. Familial hypercholesterolaemia (FH) - A genetic disorder of lipoprotein metabolism, characterized clinically by a raised concentration of Iow-density-lipoprotein (LDL) cholesterol, the presence of tendon xanthoma and a family history of raised LDL cholesterol, tendon xanthoma or premature coronary heart disease in a first-degree relative. Genotype/phenotype - The genotype of an individual denotes the genes that they have inherited; the phenotype denotes the effect that those genes have on the physiology of the individual. Homozygous/heterozygous familial hypercholesterolaemia (FH) - A homozygous FH patient has two identical defective copies of the gene; a heterozygous FH patient has one normal and one defective copy. A compound heterozygous FH patient has the clinical characteristics of a homozygous patient, but has two different defective copies of the gene. Xanthoma - Visible yellowish deposits of lipid, either flat (planar) or prominent (tuberous), in skin and tendons.
should be applicable to the general population, where most of the premature ~ occurs. Identification of those individuals with a moderately raised plasma cholesterol level who ~ at high risk so that they can be treated, preferably before, rather than after they have overt CHD, is still a major goal.
The LDL-reeeptor pathway Several reasons why mutations in different parts of the LDL-'receptor gene might be expected to have rather different effects on its function are apparent when the role played by the LDL receptor in lipoprotein metabolism is considered4. Lipoproteins function primarily to transport lipids, mainly triglycerides and cholesteryl esters, from their sites of synthesis and absorption to sites of utilization and storage. Following a fatty meal, chylomicrons are secreted from the intestine into the circulation where the triglycerides are hydrolysed to release fatty acids that are taken up by muscles or adipose tissue, leaving a lipiddepleted chylomicron remnant that is cleared rapidly by the liver. The liver synthesizes and secretes very-low-densitylipoproteins (VLDL), which enter the plasma and, initially, have the same fate as chylomicrons. However, the process differs in that not all the remnants are elearod immediately, but are subjected to fur~er modification in plasma to form intermediate-density lipoproteins (IDL) and eventually LDL, which is cleared only slowly and accumulates in plasma. V'trtually al! the triglyceride originally in the VLDL particle is replaced with cholesteryl esters in LDL by exchange and transfer from other lipoproteins, and the m
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sole protein component in LDL is apoBt0o. The LDL receptor in the liver is responsible for the uptake and removal of LDL from plasma ,and 'also VLDL remnants and IDL. The ligands recognized by the LDL receptor are apoBt0o in LDL, and apoE in IDL and VLDL remnants, although they also contain apoB~oo. It is not clear what determines the proportion of VLDL remnants and IDL that are taken up, rather than being converted
to LDL, but it is clear that defective LDL-receptor activity in patients with FH both decreases the rate of clearance of LDL and increases its rate of formation in plasma, as IDL is the immediate precursor of LDL (Fig. 1). Once the LDL-receptor gene had been cloned, it was possible to predict from its sequence and gene structure that the protein comprises several different structural domains 5 (Fig. 2). The newly synthesized
Cholesteryl ester
(( v,o, ))
.o, Triglyceride
v \ S y n t h e s i s of a p o B l o o and triglycerides
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Concentration in p l a s m a J
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Non-receptor-mediated u p t a k e in peripheral t i s s u e ~ / /
I
LPL
ApoB 48
/
synthesis
Figure 1. Effectof defectiveIow-density-lipoprotein(LDL)-receptorfunction on the metabolismof triglyceride-richlipoproteins.The LDL receptor normally mediates the specific uptake, mainly by the liver, of the remnants of the triglyceride-rich lipoproteins,very-low-densitylipoproteins (VLDL) and chylomicrons, which have been depleted of triglyceride by the action of the enzymelipoprotein lipase (LPL).VLDL remnants, or intermediate-densitylipoproteins (IDL), are further depleted of triglyceride by LPL and hepatic lipase (HL) to form LDL Exchangeand transfer of lipids between lipoproteinsalso occurs by the action of the cholesterylestertransfer protein (CETP).When LDL-receptoractivity is defective,lessVLDL remnantsare cleared,and more are therefore availableto be convertedto LDL, and the removal of LDL is impaired.This results in an increasedconcentration of LDL in plasma and an increasein its half-life in plasmawhich, in turn, resultsin the deposition of cholesterolin the peripheraltissuesas xanthomata and an acceleratedrate of atherosclerosis, possibly pertly becauseof oxidative damageto the LDL and its subsequent uptake by macrophages.
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Exon
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4 5 6
7 8 9 10 11 12 13 14
15
16 17
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Gene -45 kb
5.3 kb mRNA
Protein
NH2 Ii
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EGF-precursor domain with growth-factor-like disulphide-rich repeats A, B and C
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O-linked Cytoplasmic sugars tail domain domain with internalization signal X Transmembrane domain
Figure 2. The Iow-density-lipoprotein (LDL) receptor. Diagram of the LDL-receptor gene (shown in blue) with the 18 exons shown as vertical bars. The primary transcript of the mRNA (not shown) is spliced to remove the introns to form the spliced cytoplasmic mRNA (shown in green). The regions of the protein (shown in red) coded for by each exon of the gene are indicated by the vertical dotted lines between the mRNA and the protein. The protein comprises five structural domains, as indicated, some of which have homology to other functionally unrelated proteins; important functional or structural features of each domain of the protein are indicated below.
precursor form of the LDL receptor is glycosylated co-translationally with O- and N-linked sugars, which are trimmed and modified during the passage of the protein through the Golgi to form the mature glycoprotein. The mature receptor is transported to the cell surface, where it spans the cell membrane, exposing its ligand-binding domain to the exterior. The LDL receptor on the cell surface clusters into clathrin-coated pits, which constantly invaginate and internalize molecules bound to the cell surface, so that LDL receptors and their bound ligands are delivered to endosomes. Here, sorting of different receptors occurs, and the LDL receptor and its ligand dissociate as a result of the rapid fall in pH. The receptor molecule recycles to the cell surface, while the lipoprotein ligand is degraded in lysosomes, forming amino acids from the protein component, and cholesterol and fatty acids from the cholesteryl esters in the lipidcore of the lipoprotein particle. The free cholesterol released in this way regulates transcription of the LDL-receptor gene and the genes for key enzymes in the pathway of cholesterol synthesis. Excess free cholesterol is esterified by the enzyme acyl:cholesterol-acyltransferase (ACAT) to form cholesteryl esters that can be stored safely in the cytoplasm as lipid droplets. Thus, LDL-receptor-mediated uptake of cholesterol regulates both further uptake and the rate of de nero synthesis of cholesterol, and functions to maintain intracellular cholesterol homeostasis. This entire process is referred to as the LDL-receptor pathway 6 (Fig. 3).
Effects of mutations on LDL-receptor function It is not surprising that a multistep pathway of this type demands a mullifunctional protein. Analysis of the effect of different mutations, both
naturally occurring and those specifically introduced by mutagenesis, on LDL-receptor expression and activity in cultured cells has revealed that each structural domain of the protein is important for different aspects of its function. Mutations in the gene in patients with F[-I have been broadly classified into different classes based on LDL-receptor function in cultured cells, and mutations in one particular domain are often associated with each class 7 (Table 1). However, there are exceptions, and as many mutations do not fall clearly into one single category, a~signing a newly identified mutant allele to a particular class without supporting biochemical data is prone to error. Mutations in genes can obviously cause a wide range of defects in the function of a protein, ranging from a point mutation that results in the substitution of a single amino acid residue with one whose properties are so similar that the mutation has a negligible effect on the structure and function of the protein, to a major deletion that wipes out a large section of the coding region of the gene. In between are a variety of minor nucleotide changes, the precise effect of which on structure and function may be difficult to predict, depending on the particular protein. One or two aspects of the LDL receptor are interesting in this respect. First of all, the protein structure appears to be exquisitely sensitive to even the most conservative amino acid substitutions. This is not necessarily because such changes affect the ability o'f the receptor to bind and internalize lipoprotein ligands, but because the post-translational glycosylation of the protein and its transport to the cell surface are disrupted. Point mutations that introduce a premature stop codon anywhere in the gene frequently destabilize the mRNA so that little or no truncated mutant m
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Endosomes Lysosomes ~ 13 Utilization 9 10 12 | or esterification Internalization Dissociation Degradation • ~, • ~Cholesterol ~ ~ R e g u l a t i o n /
8 Clustering in coated pits 7 Ligand binding
~11 Recycling 6 Insertion in 5 Intracellular 4 Protein cell membrane transport maturation Cytoplasm
Golgi
3 Protein synthesis
2~mRNA I Gene splicing transcriptionJ
Endoplasmic reticulum
Nucleus
Cell membrane Rgure 3. The Iow-density-lipoprotein (LDL)-receptor pathway. Diagram showing the different stages (1-11) at which mutations in the LDL-receptor gene can disrupt its function, Mutations can impair the sterol-regulated transcription of the gene (1) and splicing of the primary mRNA transcript (2), which occur in the nucleus and can result in reduced amounts of mRNA and newly synthesized protein (3). The protein is synthesized as a precursor form (3), with co-translational addition of sugars that are trimmed and modified as the protein passes through the Golgi (4) to the cell surface (5). Many single amino acid substitutions impair this part of the pathway. The mature receptor protein is inserted in the cell membrane (6), where it binds ligands containing apoB or apoE (7). The receptor clusters in coated pits (8), which invaginate to form intracellular endosomes (9) that fuse with lysosomes, where dissociation of the receptor-ligand complex occurs (10). The receptor recycles to the cell surface (11), while the lipoprotein ligand is degraded (12). Several mutations have been described that impair these steps in the pathway. The free cholesterol released from the cholesteryl esters in the core of the particle is used for synthesis of the cell membrane, as a substrate for biosynthetic pathways, or is esterified and stored in the cell (13). It also exerts a negative regulatory effect on the transcription of the LDL-receptor gene and other genes in the biosynthetic pathway of cholesterol biosynthesis (14).
receptor protein is synthesized. Furthermore, truncated proteins are usually recognized as foreign by the cell and degraded rapidly; even if some does persist, it is unlikely to reach the cell surface and cannot function in receptor-mediated endocytosis. The ligand-binding domain is also of interest because it comprises seven 40-residue cysteine-rich repeats, each with a cluster of negatively charged residues, most of which are encoded by a single exon. Mutations that cause single amino acid substitutions in the two central repeats severely impair the ability of the receptor to bind lipoproteins containing both apoB and apoE, while mutations in the other repeats are much less deleterious 8. If they have any effect, such mutations generally impair recognition of apoB, but not of apoE. If the mutant receptor cannot recotmi7e apoB or apoE, then clearance of both LDL and VLDL remnants will be impaired and LDL production in plasma will be increased. By contrast, if the receptor retains some ability to bind apoE, then VLDL remnants will be cleared and, although LDL catabolism will still be impaired, the supply of precursors for LDL production will not be increased and LDL should not accumulate to such high levels in plasma. One other aspect that is of relevance is the structure of the LDLreceptor gene. Many of its intron-exon junctions, particularly those in the binding domain, are in phase. This means that a deletion encompassing one or two exons will result in an mRNA species in which those residues encoding the exons concerned will be neatly spliced out, leaving the rest of the protein intact, rather than introducing a frameshift that generally results in the addition of a number of foreign residues to the protein at the deletion joint before a premature stop codon is encountered. Sometimes, but not always,deletion of a whole domain, or even part of a domain, leaves the remainder of the protein structure intact and the mutant protein is not recognized as foreign; it may also retain some function. Thus, a major deletion will not necessarily result in a null phenotype. m
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Relationship between genotype and phenotype in FH Despite the wealth of information already available about the LDL receptor, answering the question of whether or not the specific mutation in the LDL-receptor gene determines the clinical severity of the disorder in patients with FH is not as straightforward as it may appear. First, the effects of a variety of specific mutations in the gene on the function of the LDL receptor have to be determined. Second, sufficiently large groups of patients with these mutations need to be identified and their clinical characteristics compared. The availability of new methods that speed up the process of finding mutations in genes has allowed the identification of an ever-increasing number of LDL-receptor-gene variants that cause FH. Determining the precise effect of the mutation on LDL-receptor function is more demanding, but has been achieved for a sufficient variety of mutations to allow some comparisons to be made. As described above, it is possible, in some cases, to predict with reasonable accuracy what the effect of a particular mutation will be on receptor function but, in others, it may be necessary to express the mutant gene in heterologous cells in vitro. The major problem in many populations is finding sufficient unrelated patients with each mutation. For example, in a group of 200 apparently unrelated patients with FH in the London area, we have already found more than 35 different mutations in the LDL-receptor gene. Of these, the four or five commonest each occur in -3-5% of the patients 9-~2, while the majority, by far, have each been found in a single individual. This diversity'is partly the result of the heterogeneity of the population in the London area and, in arararararar~ of Britain where the population has been more stable, some mutations may be more common. For example, two mutations that are present in 1% and 2.5% of the London group occur in 5% and 14% of a group of patients with FI-I from the Manchester area. In some countries where the population has remained stable for many generations, either because of geographical isolation, as in Finland, or
MOLECULAR MEDICINETODAY
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cultural isolation, as in the Afrikaners in South Africa, one or two mutations may account for 80-90% of the incidence of FH. Indeed, it is in these populations that some of the first comparisons between patients with different mutations have been made. However, it has to be borne in mind that such isolated groups will also have many other genetic variants in common and will share very similar environmental influences, which may permit the nature of the genetic mutation to have an apparently overriding effect that would not be observed in a more diverse racial and cultural group. Even when sufficient patients with different defined defects have been assembled, the question remains of how the severity of the disease should be measured. Although the clinician may have the impression that one patient with FH is less severely affected than another, providing a quantitative measure that can be subjected to statistical analysis is quite another matter. The obvious initial choice is to compare the total cholesterol or LDL-cholesterol value in the patient before treatment commences, but a reliable value for this is not always available. There is the added complication that an individual's plasma cholesterol concentration normally increases with age, so that values have to be adjusted for age when comparisons are made. There is no consensus as to whether this correction should be made for patients with FH, as at least one study has shown that this relationship between LDL cholesterol concentration and increasing age does not hold in all FH patients. Similarly, comparisons should be strictly limited to groups of individuals of the same gender, as premenopausal women have lower levels of plasma cholesterol than men, and this difference appears to be maintained in patients with FH. Other parameters that can be assessed are the presence or absence of corneal arcus and xanthoma and, as has been done in some studies, the thickness of tendon xanthoma, which are all indicators of cholesterol deposition in peripheral tissues. The age of onset of manifestations of overt CHD, if any, in the patient is another important indicator of the severity of the disorder, but again this information is not always available. Another variable of interest is the extent to which the hypercholesterolaemia and CHD respond to cholesterol-lowering therapy in a particular individual. A number of recent studies have addressed the question of whether there is a direct correlation between the type of mutation and the clinical
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phenotype (summarized in Table 2). There is strong evidence from the one study t3 of homozygous patients that the mutation in the LDL-receptot gene has an overriding influence on the disease, whether measured in terms of the associated hypercholesterolaemia, or from clinical observations. Two founder genes in French Canadians have resulted in an unusually high number of patients with FH in this region who are homozygous for one of two mutations, one of which is a deletion of the promoter region so that no mRNA or protein can be produced. The second is a single base change in exon 2, which is predicted to result in substitution of glycine for the normal tryptophan residue at position 66 in the protein. This residue lies in repeat 2 of the ligand-binding domain and has a relatively minor effect on lipoprotein uptake. Patients with the major deletion had significantly more severe hypercholesterolaemia, which resulted in earlier onset of CHD and earlier death from CHD. The differences were much less marked in their heterozygous relatives, however, showing that other factors also come into play when there is only one defective copy of the gene. This is also the case in several other studies of heterozygous FH patients. One exception is the study in Finland ~5'~6,where a mutation that results in the deletion of the O-linked sugars domain has been found. When this same mutation was introduced into the LDL receptor cDNA and expressed h7 vitro, receptor function in cultured cells was indistin~ishable from normal 19. Clearly, the mutation has some effect in vivo, but the patients have very mild hypercholesterolaemia and less severe clinical manifestations of the disorder; for example, the thickness of tendon xanthoma was significantly less than in another group of Finnish patients with a large deletion causing an essentially null mutation. It should be noted, however, that all the patients with the exon 15 mutation were members of a single large kindred and would have other factors in common. In two other studies, one in South Africa 17 and one in Israel ~4, there were no significant differences in plasma cholesterol concentration between groups of patients with the same mutation that could be correlated with the type of mutation. In a recent study from the UK ~8,patients with different mutations were grouped together according to the type of mutation, and it was found that the group with point mutations in the fifth repeat of the binding domain, which is essential for function, had a higher mean cholesterol
Table 1. Phenotype classification system for mutant LDL-receptor genes in cultured cells from patients with familial hypercholesterolaemia Class of mutant allele"
Phenotype
Location of mutations b
Class 1 - Null Class 2c - Transport-defective
No LDL receptor protein or mRNA detectable Precursor fails to be converted to mature protein, or to be transported to cell surface Mature protein reaches cell surface, but fails to bind ligand Mature protein binds ligand on cell surface, but fails to localize in clathrin-coated pits Receptor-ligand complex fails to dissociate in lysosomes; receptor fails to return to cell surface and is degraded
Premature termination codons in all domains Primarily in epidermal growth factor (EGF)precursor-like and binding domains Clustered in binding domain
Class 3 - Binding-defective Class 4 - Internalization-defective Class 5 - Recycling-defective
One, in cytoplasmic tail Clustered in EGF-precursor-like domain
8Datafrom Ref. 6. bpoint mutations and small deletions/insertions. Data from Ref. 7. cSubdivided into Class 2A (total - no mature receptor protein reachesthe cell surface) and 2B (partial - maturation of precursor to mature protein delayed).
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concentration than those with point mutations elsewhere. However, the difference was rather small. In the Israeli study, one group of patients did have a significantly lower mean plasma cholesterol concentration, but as the mutation concerned results in an essentially null phenotype and cannot be considered as mild, it is possible that strong cultural or environmental influences are at work in this group.
Other factors that influence the FH phenotype Evidence that other factors influence the FH phenotype comes also from studies of FH patients in China 9 and Tunlsm-, where homozygous FH is found at the same frequency as in most countries, but heterozygous FH is not recognized as a disorder. Even the obligate heterozygous parents of homozygous children in China frequently have plasma cholesterol values that would be considered in the normal range in the USA and most of Europe. This is not because of the presence of particularly mild mutations in Cl{inese patients, as the same spectrum of mutations has been found in the Chinese patients as in those elsewhere, and the hyper•
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cholesterolaemia in the homozygotes is just as severe. Presumably, some environmental factor, probably related to their healthy diet, is protecting Cbinese and Tunisian heterozygous FH patients against the worst effects of one copy of a defective LDL-receptor gene, but is insufficient to override the effect of two defective genes. In Western countries, the hypercholesterolaemia of FH has always been considered refractory to treatment with lipid-lowering diets, so an alternative possibility is that there are other genes in these racial groups that influence the phenotype. It would be of interest to know whether Chinese or Tunisian heterozygous FH individuals exposed to a western lifestyle retain, or lose, their protection against CHD. The conclusion from these studies to date appears to be that in heterozygous FH, the nature of the mutation in the LDL-receptor gene, while causing the disorder, interacts with other factors to produce the particular phenotype in an individual patient. As yet, we have little idea about what these factors might be, and it is quite likely that they may not be the same in all patients. Several studies have attempted to investigate the influence
Table 2. Effect of different mutations in the Iow-density-lipoprotein (LDL) receptor gene on plasma cholesterol in patients with heterozygous familial hypercholesterolaemia (FH) LDL-receptor activity in cells b (% of normal)
Mean plasma cholesterol (mmol I-~)°
Mean age of Number patients of patients ° (years) Refs
Mutation
Trivial name
Phenotype in cultured cellsa
I
Deletion of promoter and exon 1 Trp~Gly
French Canadian-1 French Canadian-4
Null 3 or 5
<2 25-100%
8.1 (26.7) 7.2(16.1) d
20 (11) 20 (10)
7.8 8.6
II
Asp147His Deletion of Gly197 Cys66oStop
FH Sephardic FH Lithuania FH Lebanese
2B 2B 2A
<2 <2 <2
10.21 9.61 7.82d
15 5 21
No data 14 No data No data
Ill
Deletion of exons 16-18 (two groups) Deletion of exon 15 Deletion of 7 bp in exon 6
FH Helsinki
4B
<2
No data 1
Mild e No data (? none)
66 23 10 69
47 38 33 47
15,16
FH Espoo FH North Karelia
11.7 9.7 7.3d 12.1
IV
Asp2o6Glu Val40aMet
FH Afrikaner-1 FH Afrikaner-2
2B 5
5-15 <2
9.4~ 10.8
112 36
40 40
17
V
Single amino acid substitutions in repeat 5 Other single amino acid substitutions Stop codons in exon 4
Variousf
Various
<2-5
11.2
17
41
18
Various °
Various
<2-30
9.6d
11
42
Various h
Null
<2
11.3
12
40
Study number
13
"From the classificationsystem describedin Table1. bBasedon binding, uptakeand degradationof 12Sl-labelledLDL by cultured skin fibroblastsfrom patientswho are homozygousfor the mutation. Datafrom Ref. 7. cValuesin parenthesesrepresentpatientswho are homozygousfor two mutations. dValuesignificantly lower than others in samestudy. eDatafromstudies in stimulated lymphocytes,not fibroblasts'6. rlncludesAsp2~Gly,Asp2~Gluand deletion of Gly19v ~lncludesGlumLys, Pro~4Leu,Seq~Leuand 'two gross deletionscausing defectiveprotein'~8. hlncludesGlu2oTStop, Cys2,0Stop and a frameshift in codon 206 that introduces a prematurestop codon.
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MOLECULAR MEDICINE TODAY
of oflaer candidate genes on the FI-I phenotype (for review, see Ref. 2), but these have all been carried out on groups of patients in whom the mutation has not been defined. However, the answers to these questions should continue to be sought. The ability to identify the mutation in' the LDL-receptor gene in an FH patient that attends a lipid clinic allows relatives who carry the mutation to be identified, many of whom may not previously have been diagnosed ,as having FH. The increasing availability of such technology could result in the identification of many more such FH patients and information about what determines their likelihood of suffering from premature CHD is obviously of importance for counselling and treatment. There may also be variants of the LDL-receptor gene that can function quite normally when in a favourable environment, whether genetic or otherwise, but which may result in hypercholesterolaemia in an adverse environment. Such gene variants would not give rise to classical FH, but could be a common underlying cause of the mild hypercholesterolaemia that is so prevalent in western countries.
The outstanding questions • Are the factors that influence the clinical phenotype of a patient with familial hypercholesterolaemia (FI-I) with a particul~ gene defect different from the known risk factors for coronary, heart disease (CHD) in tile general population? • Does the mutant Iow-density-lipoprotein (LDL)-receptor gene product in a heterozygous FH patient influence the activity of the protein from the normal copy of the gene? • Does the behaviour of tile normal LDL-receptor gene allele in heterozygous FH patients vary and, if so, does this affect their clinical phenotype, particularly their response to treatment? • Are there nlild mutations in the LDL-receptor gene in individuals with a modestly raised, or even normal, concentration of LDL cholesterol that put them at increased risk of coronary disease'? • Are there mutations in genes other than the structural gene for the LDL-receptor protein that can influence its expression or function?
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s
9 Sun, X.M. et al. (1992) Characterization of deletions in the LDL-reeeptorgene in patients with familial hypercholesterolemiain the United Kingdom,Arterioscler. Thromb. 12, 762-770 10 King. U.L. et al. ( 199 l) Identificationof the 664 proline to leucinemutation in the Iow-density-lipoproteinreceptor in four unrelated patients with familialhypercholesterolaemia in the UK, Clin. Genet. 40, 17-28 11 Webb, J.C. et al. (1992) Characterization of two new point mutations in the low. density-lipoprotein-receptorgenes of an English patient with homozygousfamilial hypercholesterolemia,Z Lipid Res. 33,689-698 12 Gudnason, V. et al. (1993) Identification of recurrent and novel mutations in exon 4 of the LDL-receptorgene in patients with familial hypercholesterolaemia in the United Kingdom,Arterioscler. Thromb. 13, 56--63 13 Moorjani,S. et al. 0993) Mutations ofthe Iow-density-lipoprotein-receptorgene, variation in plasma cholesterol, and expression of coronary heart disease in homozygous familial hypercholesterolemia,Lancet 341, 1303-1306 14 Leitersdorf,E. el al. (1993) Genetic determinants of responsiveness to the HMGCoA reduetase inhibitor fluvastatin in patients with molecularlydefined heterozygous familial hypereholesterolemia,Circulation 87 (Suppl. III), 35--44 15 Koivisto, U.M. et aL (1992) The familial hypereholesterolemia (FH)-North Karelia mutation of the low density lipoprotein receptor gene deletesseven nucleotidesof exon 6 and is a common cause of FH in Finland, J. Clin. Invest. 90, 219-228 16 Koivisto, P.V.l. et aL (1993) Deletion of exon15 nf the LDL.receptor gene is asso. ciated with a mild form of familialhyperchalesterolemia-FH-Espoo,Arterioscler Thromb. 13, 1680--1688 17 Kotze, M.J. et aL (1993) Phenotypic variation among familial hypercholesterolemics heterozygous for either one of two Afrikaner founder LDL-reeeptor mutations, Arteriosc/er. Thromb. 13, 1460-1468 18 Gudnason,V., Day,I.N.M.and Humphries,S.E. (1994) Effect on plasma lipid levels of different classes of mutations in the Iow-density-lipoprotein-reeeptorgene in patients with familial hypereholesterolemia,Arterioscler, Thromb. 14, 1717-1722 19 Davis,C.G. et aL (1986) Deletionof clustered O-linked carbohydrates does not impair function of Iow-density-lipoproteinreceptor in transfected fibroblasts, J. Biol. Chem. 261,2828-2838 20 Slimane,M.N.etaL (1993) Phenotypic expressionof familialhypercholesterolemia in central and southern Tunisia, Atherosclerosis 104, 153-158
Next month's Round up in IVIMT References 1 ScandinavianSimvastatin SurvivalStudy Group (1994) Randomized trial of cholesterol loweringin 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S), Lancet 344, 1383-1389 2 Sout,'u,A.K. (1994) FamilialhyperCholcsterolaemia,in From Genotype to Phenotype (Humphfies, S.E. and Malcolm,S., eds), pp. 83-109, Bios Scientific 3 Goldstein,J.L. and Brown, M.S. (1989) Familial hypercholesterolemia, !n The Metabolic Basis of hlherited Disease (Scriver, C.R. et aL, eds), pp. 1215-1250, McGraw-i-lill 4 Havel,R.J. and Kane,J.P. (1989) Structure and metabolism of plasma lipoproteins, in The Metabolic Basis of Inherited Disease (Scriver,C.R. et al., eds), pp. 1129-1138, McGraw-Hill 5 Sudhof, 1~.C.,Goldstein, J.L., Brown, M.S. and Russell, D.W. (1985) The LDLreceptor gene: a mosaic of exons shared with different proteins, Science 228, 815-822 6 Brown,M.S.and Goldstein,J.L. (1986) A receptor-mediatedpathway for cholesterol homeostasis, Science 232, 34-47 7 Hobbs,H.H.,Brown,M.S.and Goldstein,J.L. (1992) Moleculargeneticsof the LDLreceptor gene in familial hypercholesterolemia,Hum. Mutation I, 445-466 8 lesser,V.et al. (1988) Mutational analysis of the ligand-bindingdomain of the Iowdensity-lipoprotein receptor, J. Biol. Chem. 263, 13282-13290
The meetings: • DNA Vaccines: A N e w Era in Vaccinology • Gene and Nucleic Acid Vaccine Strategies The books: • Protocols for Gene Analysis • Protein Expression in Animal Cells • RNA: Isolation and Analysis • Molecular Biology: Current Innovations and Future Trends
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