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(Clinical Chemistry. 1997;43:916-923.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Denaturing gradient-gel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed Asian cohort: two cases of arginine3500 -> tryptophan mutation associated with a unique haplotype

Meng-Ling Choong1, Evelyn S. C. Koay1,2,a, Kah-Lin Khoo3, Min-Cheh Khaw2 and Sunil K. Sethi1,2

1 Department of Pathology, National University of Singapore, Singapore 119260.

2 Department of Laboratory Medicine, National University Hospital, Singapore 119074.

3 Pantai Medical Centre, Kuala Lumpur, Malaysia.
a Author for correspondence. Fax (065)7780671; e-mail patkoaye{at}nus.sg


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Arg-to-Trp substitution at codon 3500 in the apolipoprotein (apo) B-100 gene is established as a cause of familial defective apo B-100 (FDB), a functional mutation, resulting in reduced LDL receptor binding and manifest hypercholesterolemia. In a search for similar mutations in 163 Malaysians, we screened the putative receptor-binding region (codons 3456–3553) of the apo B-100 gene by PCR amplification and denaturing gradient-gel electrophoresis. Four single-base mutations were detected and confirmed by DNA sequencing. Two females, a Chinese and a Malay, had the same CGG3500 -> TGG mutation, resulting in an Arg3500-to-Trp substitution. This is the second published report of such an independent mutation involving the same codon as the established Arg3500-to-Gln mutation. The two other mutations detected, CTT3517 -> CTG and GCC3527 -> GCT, resulted in degenerate codons with no amino acid substitutions. All four mutations were associated with a unique apo B haplotype, different from those found in Caucasian FDB patients but concurring with that previously reported for two other Asians with FDB.


Key Words: indexing terms: population screening • genetic screening • heritable disorders


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LDL particles are the principal carriers of cholesterol in the bloodstream, and LDL-associated cholesterol is atherogenic. The plasma concentration of cholesterol is regulated by LDL receptor-mediated endocytosis by which circulating LDL is taken primarily into the hepatocytes (1). Apolipoprotein (apo)1 B-100 is the major protein associated with the LDL particle and contains the ligand that binds LDL to its receptor. Gene mutations in the LDL receptor or in the receptor-binding zone of the apo B-100 can disrupt binding and impair removal of circulating LDL. More than 150 mutations exist in the LDL receptor gene, associated with familial hypercholesterolemia (FH), an autosomal dominant inherited disorder characterized by severe hypercholesterolemia, frequent presence of tendon xanthomas, and premature coronary heart disease (2).

That a defect in the ligand apo B-100 gene could produce a phenotype like that of clinical FH was first observed by Vega and Grundy in 1986 (3) in five subjects with moderate hypercholesterolemia. These subjects had substantially reduced fractional catabolic rates of autologous LDL but average catabolic rates of homologous LDL, indicating the presence of functional LDL receptors. The LDL from one of these patients was subsequently shown to possess only 32% of average binding affinity to LDL receptors on cultured fibroblasts (4); this condition, familial defective apolipoprotein B-100 (FDB), is a genetic disorder associated with moderately increased plasma LDL cholesterol (LDL-c) concentrations and accelerated atherosclerosis (5)(6)(7). The point mutation arising from a single base change in the apo B-100 gene sequence, a CGG -> CAG change in codon 3500, with resulting Arg-to-Gln switch in the apo B-100 protein, has been established as the cause of FDB (4)(8). FDB is transmitted by autosomal codominant inheritance; the incidence of heterozygotes in most populations is 1:500–1:700, similar to that of FH (5)(7).

LDL particles containing apo B-100 with Gln at amino acid residue 3500 located within the putative receptor-binding region (9) have reduced binding affinity for the LDL receptor (4)(10). The resulting diminished rate of clearance of plasma LDL leads to hypercholesterolemia. Three other mutations in the LDL-receptor–binding region of the apo B-100 protein, at codons 3480, 3531, and 3500, have been described: Arg3480 -> Pro (11); Arg3531 -> Cys (12); and Arg3500 -> Trp (13). The latter two mutations resulted in decreased LDL-receptor–binding affinity of the altered apo B-100 protein ligand (12)(13). The importance of Arg at position 3500 and its flanking regions to the functional integrity of apo B-100 as the ligand moiety for LDL receptor recognition is confirmed by studies with monoclonal antibodies to specific regions of the apo B-100 protein to investigate the available epitopes when LDL is bound to its receptor (14).

Subjects with FDB are clinically indistinguishable from those with FH, although the former tend to have less severe hypercholesterolemia (7)(15)(16). In heterozygous FDB subjects, residual binding affinity of the unaltered LDL particles (only half of their LDL carries the defective apo B-100 protein) permits substantial LDL catabolism via the LDL receptor pathway. Also, the rate of removal of VLDL remnants, the precursors of LDL, by the hepatic LDL receptor is not affected, given that the interaction of the VLDL remnants is mediated by recognition of the apo E ligand (17). Hence the proportion of VLDL remnants converted to LDL should not change.

We systematically screened a mixed Asian cohort for underlying genetic causes by the denaturing gradient-gel electrophoresis (DGGE) method (11). The putative receptor-binding region from codon 3456 to codon 3553 of the apo B-100 gene was screened, in an attempt to identify previously established and any new functional mutations causally linked to FDB. We also characterized the apo B-100 haplotype on the basis of the inherited traits at five polymorphic sites on the gene, so as to establish the founder origins of any mutations detected. The genotypes at the apo E and the LDL receptor gene exon 8 StuI polymorphic loci on chromosome 19, two interrelated inherited traits, were also studied to assess their effect, if any, on LDL-binding affinity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
study population
We studied a group of 163 unrelated subjects, ages 5–85 years (mean, 46 years), attending a lipid clinic in Kuala Lumpur, Malaysia. This mixed cohort represented the main ethnic groups in the Malaysian population: 131 Chinese (73 males, 58 females); 24 Malays (16 males, 8 females); and 8 Indians (5 males, 3 females). The patients had been referred to the specialist cardiologist for problems such as abnormal plasma lipid concentrations or the presence of tendon xanthomas, arcus, gross obesity, or breathlessness on exertion. All had hypercholesterolemic plasma cholesterol pretreatment concentrations of >6.2 mmol/L. Nine patients with FH were included in the study, because mutations in the apo B-100 and the LDL receptor genes are not mutually exclusive. Whole blood (EDTA-anticoagulated) and serum were collected after an overnight fast. All procedures with the study subjects were in accordance with the Helsinki Declaration of 1975, as revised in 1983.

lipid determinations
Serum (total) cholesterol and triglyceride were measured by commercial enzymatic assays on the Ektachem 750 automated analyzer (Johnson and Johnson, Rochester, NY). HDL cholesterol (HDL-c) was measured on the Reflotron (Boehringer Mannheim, Mannheim, Germany), after dextran sulfate precipitation and removal of other lipid fractions. Apo A-I and apo B were quantified by immunonephelometry on the Beckman Array 2.0 protein analyzer (Beckman, Brea, CA), and lipoprotein(a) was quantified by the Macra enzyme immunoassay from Strategic Diagnostics (Newark, DE). LDL-c was estimated indirectly by application of the Friedewald formula (18).

dna extraction
DNA was extracted from leukocytes, as previously described (19). The DNA pellet was redissolved in 10 mmol/L Tris-HCl, 0.1 mmol/L EDTA buffer, pH 8.0, to a final concentration of ~300 ng/µL and stored at -70 °C.

pcr amplification and dgge screening
The region containing nucleotides 10 551–10 892 (corresponding to codons 3448–3561) of exon 26 of the apo B-100 gene was amplified as described by Nissen et al. (11), with slight modification. The sequences of the two primers are 5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGGGAGCAGTTGACCACAAGCTTAGC-3', with a 40-bp GC clamp at the 5' end, and 5'-GGTGGCTTTGCTTGTATGTTCTCC-3'. Only the region from codon 3456 to codon 3553 lies between the primers and is screened for mutations by the DGGE technique. Briefly, the PCR mixture (final volume, 25 µL) contained 300 ng of DNA, 60 pmol of each primer, 200 µmol/L of each deoxynucleotide triphosphate, 1.5 mmol/L MgCl2, and 1 U of the Taq polymerase (Promega, Madison, WI) in a buffer supplied by the same manufacturer. The mixture was heated in a Perkin-Elmer-Cetus (Norwalk, CT) TL 480 thermocycler to 95 °C for 5 min, then 40 cycles of 94 °C (1 min) and 68 °C (1 min), followed by a final 10-min stage at 72 °C and then incubated at 65 °C for 1 h and at 37 °C for 1 h longer to assist formation of heteroduplexes.

The PCR products were analyzed with a 6% polyacrylamide gel (acrylamide:bis-acrylamide ratio, 37.5:1) on a 20–60% denaturant gradient (100% denaturant consists of 400 mL/L formamide and 7 mol/L urea). Electrophoresis was performed for 6 h at 150 V in a bath of Tris-acetate–EDTA buffer (Tris-acetate 40 mmol/L, EDTA 1 mmol/L, pH 8.0) heated to 60 °C. The gradient-gel former and the electrophoresis apparatus were part of the Bio-Rad D-Gene system (Hercules, CA).

dna sequencing
Mutations detected by the DGGE screen were confirmed by DNA sequencing. Genomic DNA was amplified with the same primers but without the 40-bp GC clamp. The PCR mixture (as described above) was denatured at 95 °C for 5 min, followed by 25 cycles of 94 °C (1 min), 60 °C (1 min), and 72 °C (1 min), with a final 10-min elongation step at 72 °C. The PCR products were cleaned with Prep-A-Gene (Bio-Rad) and cycle-sequenced on a ABI Prism 377 automated sequencer (Perkin-Elmer) according to the protocol described by the manufacturer.

apo b-100 haplotype analysis
Five polymorphic loci on the apo B-100 gene were studied. The apo B signal peptide insertion/deletion (I/D) polymorphism in exon 1 of the gene was studied as previously described (20). The XbaI and MspI polymorphisms in exon 26 and the EcoRI polymorphism in exon 29 were studied with primers by Boerwinkle et al. (21). The minisatellite-length polymorphism caused by variable number of tandem repeats (VNTR) located ~200 bp 3' of the apo B-100 gene was analyzed as previously described (21).

apo e genotyping and ldl receptor stui polymorphism analysis
The genomic DNA samples were analyzed for apo E genotypes and the LDL receptor gene exon 8 StuI polymorphism, two inherited traits that may influence the phenotypic expression of FDB. Apo E was genotyped by the method of Hixson and Vernier (22), and LDL receptor exon 8 StuI polymorphism was genotyped as described by Gudnason et al. (23).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table 1 shows the ethnic composition of the study population (n = 163) and their plasma lipid concentrations. There were no substantial differences in the distribution of cholesterol and of LDL-c concentrations among the ethnic groups. Table 2 shows the lipid profiles and DNA polymorphic traits of the four affected subjects, all of whom had the {epsilon}3{epsilon}3 genotype (wild-type) for the apo E polymorphism and Ala/Ala for the LDL receptor gene exon 8 StuI polymorphism. The small number of subjects precluded statistical computation of the effects of the mutations on plasma lipid concentrations.


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  		Table 1. Plasma lipid profiles, age, and race of the study population (n = 163).


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  		Table 2. Plasma lipid1 profiles and genotypes of the four affected subjects detected by DGGE.

Subject 1 was diagnosed with hypertriglyceridemia. Subject 2 had hypercholesterolemia (6.9 and 7.8 mmol/L on two occasions in 1990), diabetes since 1981, high blood pressure, right coronary artery lesion, and a positive treadmill test. She responded well to treatment and reverted to an average plasma lipid profile within 6 months. Subject 3 had no overt clinical symptoms, despite high plasma cholesterol, triglyceride, and apo B concentrations. His parents, two brothers, and a sister were healthy. Subject 4 had xanthelasma, and several members of her family were hypercholesterolemic (>6.2 mmol/L), with one sibling (plasma total cholesterol, 8.0 mmol/L) having undergone coronary artery bypass surgery before age 50. Except for subject 2, none of the other subjects had coronary heart disease, as documented by negative coronary angiograms.

The DGGE-based screening showed a single-band pattern (indicating no mutations within the codon 3456–3550 region) for all but 4 subjects (n = 159) who had 4-band patterns (indicating the presence of mutations in this region). Different mutations gave rise to migration rates of the four component bands (Fig. 1 ). The mutations in the four subjects were confirmed by DNA sequencing (Fig. 2 ). The location and effect of the mutation in each case are shown in Table 3 . The mutation in subject 1 was found in codon 3517 (CTT -> CTG). This was a degeneracy codon change that did not result in a change of the coded amino acid leucine. Subjects 2 and 4 have the same mutation in codon 3500 (CGG -> TGG), which produced a change from the positively charged Arg to the nonpolar Trp in the encoded amino acid sequence. The mutation in subject 3 again involved a degeneracy codon (GCC -> GCT), both coding for the amino acid alanine. Because base substitutions in subjects 1 and 3 did not involve changes in amino acids in the translated apo B-100 protein, these subjects could not be considered as having FDB.



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  		Figure 1. DGGE screening of the codon 3456–3553 region of the apolipoprotein B-100 gene.

Lane 1, negative control (without DNA); lane 2, subject 1, heterozygous, CTT -> CTG at codon 3517 (Leu3517 -> Leu); lane 3, subject 2, heterozygous, CGG -> TGG at codon 3500 (Arg3500 -> Trp); lane 4, subject 3, heterozygous, GCC -> GCT at codon 3527 (Ala3527 -> Ala); lane 5, subject 4, heterozygous, CGG -> TGG at codon 3500 (Arg3500 -> Trp); lane 6, heterozygous, CGG -> CCG at codon 3480 (Arg3480 -> Pro); lane 7, heterozygous, CGC -> TGC at codon 3531 (Arg3531 -> Cys); lane 8, heterozygous, CGG -> CAG at codon 3500 (Arg3500 -> Gln); lane 9, apparently healthy control, no mutation in the region studied. Subjects 2 and 4 have the same point mutation as determined by sequencing. Electrophoretic conditions were 150 V for 6 h on a 6% polyacrylamide gel containing a 20–60% denaturing gradient.



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  		Figure 2. Electrophoretogram and sequence of the four subjects with mutations detected by DGGE.

Heterozygosity in a nucleotide position of the sequence is represented by two peaks at the same location. DNA sequencing was performed on a Perkin-Elmer ABI Prism 377 automated sequencer.


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  		Table 3. Mutations in the apo B-100 gene in four subjects detected by DGGE.

The first two (slower-moving) bands in the four-band pattern are heteroduplexes formed by the pairing of a mutant strand with a normal/wild-type strand. The single-basepair mismatch lowered the melting temperature (Tm) of the heteroduplexes. Earlier denaturation of the less-than-100%-homologous heteroduplexes in a denaturing gel retarded their migration during electrophoresis. The lowest (fastest-moving) band in subjects 2, 3, and 4 corresponds to the single band found in the apparently healthy control, a homoduplex comprising two wild-type strands. Mutations found in subjects 2, 3, and 4 involved substitution of a C with a T resulting in a lower Tm and earlier denaturation. Thus, the homoduplex mutant band is at a higher position in the gel than the average/wild-type band because of its slower migration rate. Subject 1 differed in having a substitution of a T by a G causing a rise in the Tm of the homoduplex. The mutant strands in this case are therefore harder to denature and thus migrate further in a denaturing gel.

haplotype analysis
All four subjects had the same haplotype, I-DIns/XbaI-/MspI+/EcoRI+/VNTRS, based on five polymorphic sites on the apo B gene (Table 4 ), where + and - indicate the presence or absence of the restriction sites, and S refers to alleles with <43 repeats (L stands for alleles with >=43 repeats). The alleles of three subjects had 35 and 35 repeats at the apo B-100 3'-VNTR; subject 3 had alleles with 35 and 37 repeats. Three were heterozygous with regard to the 9-bp insertion/deletion polymorphism in the apo B-100 signal peptide; subject 3 was homozygous for the insertion alleles.


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  		Table 4. Apo B haplotype in subjects with FDB-causal mutations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The LDL-receptor–binding site of apo B-100 has been crudely localized to codon 3000–4000 in exon 26 of the gene (5). Besides the original CGG -> CAG mutation at codon 3500 (8), which has been established as a cause for FDB in several populations (5), three other mutations have been identified in this region. Only two of these have been linked to functionally reduced LDL-receptor–binding affinity and consequent hypercholesterolemia: a CGG -> TGG coding change at codon 3500 (13); and a CGC -> TGC change at codon 3531 (12). The clinical import of the third single-base change, CGG -> CCG at codon 3480 (11), is yet to be determined. Another mutation, a C-to-T change on codon 4016 in exon 29 found in one kindred and known as apo B-100 Hopkins, was not linked to a hyperlipidemic phenotype (24). Its location outside the putative LDL-receptor–binding domain of the apo B-100 gene may provide some explanation for the lack of atherogenic phenotypic expression.

We used the GC-clamped DGGE method described by Nissen et al. (11) to screen for unknown sequence mutations within codons 3456 and 3553. The specificity and sensitivity of this method are discussed elsewhere (25)(26).

Two of our three mutations, a CCT -> CTG in codon 3517 and a GCC -> GCT in codon 3527, although not clinically important because they are degenerate codon mutations, have nonetheless not been previously reported. The third mutation, a CGG -> TGG at codon 3500 causing a substitution of Arg to Trp, is similar to that reported by Gaffney et al. (13), involving the same codon as the original FDB-coding mutation, CGG -> CAG or CAA. The latter, producing an Arg-to-Gln change, is the predominant mutation reported in Caucasians (27).

The codon for Arg, CGG, contains the hypermutable CG dinucleotide, frequently associated with point mutations (CG -> TG or CG -> CA) in the same codon of various genes, such as the hemoglobin gene cluster and the Factor III gene (28). Likewise, in Alzheimer disease, two clinically important mutations map to the same codon 717 of the ß-amyloid precursor protein gene (29). Similar mutations involving other Arg residues in the LDL-receptor–binding region of the apo B-100 gene may also exist.

Our study population did not include a subject with the classic Arg3500-to-Gln mutation, but we found two subjects with Arg3500-to-Trp mutations, suggesting that the prevalent mutation in the Malaysian population is the Arg3500-to-Trp type. Our subjects are the third and fourth index cases of this new mutation reported to date. The first cases, reported in 1995 (13), were from Scotland; one was of Asian and the other of Scottish descent (Table 4Up ).

Substitution of the Arg (with a positively charged polar side chain) by Gln or Trp or Cys (with an uncharged nonpolar side chain) reduces the binding affinity of the apo B-100 to the LDL receptor (4)(12)(13), as demonstrated by a dual-label fibroblast binding assay (12), a cell culture expression system for the synthesis of recombinant human LDL containing apo B with the mutation at codon 3500 (30), or the U937 monocyte proliferation assay (31)(32). Reduced LDL binding affinity in subjects 2 and 4 in our study is thus expected, as reported by Gaffney et al. (13). Detailed characterization of the phenotypic expression of this mutation in our two affected individuals is in progress.

The apo E polymorphism has been associated with variation in plasma lipid concentrations (33)(34). Compared with the {epsilon}3 allele, reduced LDL-c concentrations frequently accompanied the presence of the {epsilon}2 allele, and higher LDL-c concentrations, the {epsilon}4 allele [33, 34, and our unpublished data]. The LDL receptor gene exon 8 StuI polymorphism is caused by substitution of a G for an A in codon 370 (35). This changes the amino acid Ala to Thr. The Thr allele is associated with high plasma total cholesterol and LDL-c in certain populations (23). Because our four subjects have {epsilon}3{epsilon}3 and Ala/Ala genotypes, their increased cholesterol concentrations are not attributable to the apo E or the LDL receptor gene StuI polymorphisms.

Two recent trials have shown that treatment with a hydroxymethylglutaryl-CoA reductase inhibitor, pravastatin, reduces plasma total cholesterol and LDL-c (20–25%) in FDB patients more than does a fibrate, gemfibrozil (4–6%, P <0.0001) (17). However, the fibrate lowered triglycerides by 25% and elevated HDL-c in these subjects by 11% (36), while the effects of pravastatin on these two interrelated variables were significantly smaller (P <0.0001). These studies also showed that the LDL-c-lowering effect of pravastatin in patients with FDB was similar to that observed in patients with FH. Because of the deleterious effects of untreated familial forms of isolated hypercholesterolemia, it is important to identify FDB and FH subjects early and to treat them with suitable lipid-lowering drugs and a lipid-lowering diet. The DGGE-based screening method we have adopted (11) appears to be a good choice for detection of FDB in high-risk groups.

FDB has been associated with a certain haplotype of the apo B-100 gene, 194 (37). The Arg3500-to-Gln causal mutation was identified on this same haplotype in several Western populations (38)(39)(40)(41), suggesting that this mutation occurred on a single ancestral gene. Haplotype 194 is characterized by XbaI-, MspI+, EcoRI+, the presence of the 5' signal peptide insertion (Ins) allele, and the 49-repeat (according to the nomenclature by Boerwinkle et al. (21) used here) allele of the 3'-VNTR (37). The apo B 3'-VNTR has a 13-allele frequency distribution that follows a bimodal distribution pattern, with peaks at 35 and 49 repeats representing the two main alleles; the other alleles arise from slippage during DNA replication of these two alleles (21). The larger 49 repeat seems to be associated with FDB in Caucasians (37) but not in Asians (13)(42).

The apo B-100 haplotype of our four subjects, I-DIns/XbaI-/MspI+/EcoRI+/VNTRS, differed from haplotype 194 but was similar to (though not identical with) haplotype 195 (42) and reported in a Chinese man residing in California but born in China, one of two previously reported FDB cases of Asian descent (13)(42). Two possible explanations for the unique haplotype associated with the FDB mutations found in the Asians (Table 4Up ) are: (a) the mutations may have arisen from the same ancestral founder gene, but a rare recombination event at the extreme 3' end of the apo B gene could have changed the haplotype (42); similar such recombinations have not been observed in this region of the apo B gene since the genesis of the Ag (x/y) polymorphism (43), making it unlikely that either haplotype could have arisen from the other by recombination between homologous chromosomes; (b) the causal mutations for FDB associated with the Asian haplotype 195 might have arisen independently at any of the hypervariable CG dinucleotides located in codons 3000–4000. Most point mutations in the hemoglobin gene cluster, for example, are associated with a single haplotype, but a few point mutations were observed on a different haplotype from subjects in Southeast Asia (44). In the mutations causing FDB, Asian patients are probably descended from a different founder ancestor in whom the particular mutation had occurred independently on a different haplotype on one or several occasions, quite unrelated to the mutational event(s) that had occurred on the haplotype in most Caucasians.

Specific methods detecting only mutations in codon 3500 (such as allele-specific oligonucleotide hybridization or MspI digestion of PCR products) would not detect mutations at other sites within the receptor-binding region. This may explain the low frequency of FDB in the African (45) and Indian (46) populations and the rarity of FDB in the Japanese population (47).

The DGGE-based assay for detecting mutations in the flanking regions of codon 3500 of the apo B-100 gene allows screening of known and yet unidentified mutations of FDB. Detection of the Arg3500 -> Trp mutation associated with a unique haplotype in two Malaysian women of different ethnic descent, shortly after the first report of its occurrence in two subjects of Scottish and Asian descent, respectively (13), confirms that at least two independent mutations arising from the same codon with the hypermutable CG dinucleotide is causally associated with FDB. Our results suggest that: (a) this may signal a high prevalence of FDB in the Malaysian population; (b) rather than the classic FDB mutation, perhaps the alternative codon 3500 mutation occurs more frequently in Asian patientsthree of four patients reported thus far as having the Arg3500 -> Trp mutation (one from the Scotland study and two from this study) are Asians; (c) all three Asians affected had the same (or a very similar) apo B haplotype, suggesting that they may all have descended from the same founder mutation. The mutations in the two female subjects in our study could also have occurred independently, given that the afflicted individuals are from different racial origins. The ethnicity and country of origin of the Asian patient in the Scotland study was not stated. Larger studies involving larger numbers are needed, and more Asian ethnic groups should be screened for this mutation to confirm the above observations and to provide answers to the questions they raise.


   Acknowledgments
 
This work is supported by a grant to E.S.C.K. (RP930321) by the National Medical Research Council, Singapore, and the National University of Singapore. The DNA sample with the Arg3500 -> Gln mutation was a gift from Ian Day, Division of Cardiovascular Genetics, Department of Medicine, University College London Medical School, The Rayne Institute, London, UK. Clive R. Pullinger, Cardiovascular Research Institute, University of California, San Francisco, CA, generously supplied DNA samples with the Arg3531 -> Cys and the Arg3480 -> Pro mutations. Henrik Nissen, Department of Clinical Chemistry, Odense University, Odense C, Denmark, provided the technical expertise to refine the DGGE electrophoretogram shown in Fig. 1Up , and we thank him for his kind and invaluable advice.


   Footnotes
 
1 Nonstandard abbreviations: -c, cholesterol; apo, apolipoprotein; DGGE, denaturing gradient-gel electrophoresis; FDB, familial defective apolipoprotein B-100; FH, familial hypercholesterolemia; VNTR, variable number of tandem repeats.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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