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Clinical Chemistry 43: 1630-1634, 1997;
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(Clinical Chemistry. 1997;43:1630-1634.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Detecting familial defective apolipoprotein B-100: three molecular scanning methods compared

Bryony G. Henderson, Philip R. Wenhama, J. Peter Ashby and Gillian Blundell

Department of Clinical Biochemistry, Western General Hospital, Edinburgh EH4 2XU, UK.
a Author for correspondence. Fax 44-131-537-1023.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Familial defective apolipoprotein (apo) B-100 (FDB), a condition that may give rise to hypercholesterolemia, is caused by mutations around codon 3500 of the apo B gene. We have compared the ability of three molecular-scanning techniques, heteroduplex analysis, single-strand conformation polymorphism (SSCP) analysis, and denaturing gradient gel electrophoresis (DGGE), to detect these mutations in a cohort of 432 hypercholesterolemic individuals. Heteroduplex analysis and DGGE detected 11 individuals with apo B mutations, 9 of whom were heterozygous for apo B R3500Q and 2 who were heterozygous for apo B R3531C. Whereas DGGE was able to distinguish between these two mutations, heteroduplex analysis was technically simpler and gave a higher sample throughput. In contrast, SSCP analysis detected only 7 of the R3500Q and none of the R3531C heterozygotes and was the most complex of the three techniques. We believe heteroduplex analysis to be the method of choice for screening large numbers of samples for FDB.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Familial defective apolipoprotein B-100 (FDB)1 is an autosomal codominant disorder associated with hypercholesterolemia (1)(2)(3), caused by mutations in and around codon 3500 of the apolipoprotein (apo) B gene, which encodes apo B-100. This is the main protein of low-density lipoprotein (LDL) and is the ligand through which LDL binds to its receptor in the process of receptor-mediated endocytosis (4).

The mutations all occur in arginine codons and result in an apo B-100 molecule that exhibits defective binding to the LDL receptor, leading to impaired uptake of LDL into the cell and consequently, hypercholesterolemia. The first to be described, and the most characterized, is caused by a G->A transition at nucleotide 10 708 and results in the substitution of arginine by glutamine at codon 3500 (apo B R3500Q) (5). The other two, both recent discoveries, are each caused by a C->T transition, one at nucleotide 10 800 and the other at nucleotide 10 707. These result, respectively, in the substitution of arginine by cysteine at codon 3531 (apo B R3531C) (6) and arginine by tryptophan at codon 3500 (apo B R3500W) (7).

The estimated heterozygote frequency of FDB in the UK, based on the R3500Q mutation, is believed to be ~1 in 500 (8). However, the prevalence could possibly be greater with the discovery of these two other mutations.

Traditionally, studies of FDB have centered on the R3500Q mutation and use of detection methods specific for this mutation (9)(10)(11). Because at least two other mutations are known to cause FDB, it is more appropriate to use a method that would detect all such mutations in a single analysis. Three procedures that may be suitable for this are the molecular-scanning techniques of heteroduplex analysis (12)(13), single-strand conformation polymorphism (SSCP) analysis (14)(15), and denaturing gradient gel electrophoresis (DGGE) (16). In this study, we have compared the ability of these three techniques to detect the mutations in and around codon 3500 of the apo B gene that cause FDB in a cohort of 432 hypercholesterolemic individuals. The aim was to ascertain which, if any, was the most appropriate method to detect FDB in the diagnostic setting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects.
Hypercholesterolemic individuals attending lipid clinics in Scotland (Edinburgh) and Wales (Newport), 425 subjects, were screened with the three techniques. In addition, to test thoroughly the ability of each method to detect apo B mutations, an additional seven individuals who were known to be heterozygous for apo B R3500Q (17) were also studied. All 432 patient samples were identified by a number, and only one of us (P. R. W.) knew the patient identity of the numbered samples. To remove any possible bias during the performance of each run, the operators (B. G. H. and G. B.) identified samples by number only; i.e., all analyses were performed blind.

Samples.
Whole-blood samples for DNA analysis were anticoagulated with potassium-EDTA (1.2 mg/mL).

DNA extraction.
This was performed by the method of Miller et al. (18)

Heteroduplex analysis.
Heteroduplex analysis was performed according to the method of Kotze et al. (19). Briefly, a 345-bp fragment that spanned codon 3500 of the apo B gene was first amplified by the polymerase chain reaction (PCR), with the oligonucleotide primers and reaction conditions of Tybjærg-Hansen et al. (8). PCR product (5 µL) was then mixed with an equal volume of gel-loading buffer (950 mL/L formamide, 0.5 g/L bromphenol blue, 0.5 g/L xylene cyanol), and the complete mixture was loaded on the gel. To maximize throughput, up to 5 sample loadings were applied to each track of the gel, and each application was separated by a 15-min, 800-V electrophoresis step. Electrophoresis was then performed at 300 V for 17 h overnight, on a low-cross-linking 40 x 21 x 0.1 cm polyacrylamide gel (10% polyacrylamide, 0.1% N,N'-methylenebisacrylamide, containing 150 g/L urea in 0.6x TBE (1x TBE = 89 mmol/L Tris, 89 mmol/L H3BO4, 2 mmol/L EDTA). The homo- and heteroduplex DNA bands were then stained with ethidium bromide and viewed over ultraviolet light.

SSCP analysis.
A method for SSCP analysis was developed that gave the clearest bandshift when used to analyze a sample known to be heterozygous for apo B R3500Q. This required the systematic optimization of all of the variables known to influence the efficiency of SSCP detection, namely, PCR-product size, percentage acrylamide, percentage cross-linker, temperature of electrophoresis, and presence/absence of glycerol. These studies lead to the choice of the following procedure and conditions.

DNA was first amplified by the PCR with the same primers and conditions as described above (8) and the addition of 1 µCi of [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham International) to the reaction mixture before PCR. The radiolabeled PCR product was then digested with 10 U of PstI in a final volume of 10.5 µL at 37 °C for 1 h. One microliter of 10 g/L SDS was then added followed by 10 µL of gel-loading buffer (950 mL/L formamide, 0.5 g/L bromphenol blue, 0.5 g/L xylene cyanol). Samples were denatured at 99 °C for 2 min and quenched in an ice-water bath, and then 2–3 µL was applied to the gel. The use of a sharks-tooth comb allowed up to 48 samples to be run per gel. Electrophoresis was performed at room temperature on a 40 x 21 x 0.04 cm polyacrylamide gel (7.5% polyacrylamide, 0.15% N,N-methylenebisacrylamide in 50 mL/L glycerol, and 1x TBE) for 17 h overnight at 450 V. To maintain as constant a temperature as possible during the electrophoresis step, a cooling fan was directed at the gel for the duration of electrophoresis. After fixing for 20 min in methanol/acetic acid (100 mL/L methanol, 100 mL/L acetic acid) solution, the gel was dried, and the bands were detected after autoradiography for 24–48 h at -70 °C.

DGGE.
This was performed with the D Gene DGGE system (Bio-Rad Laboratories). A region spanning codons 3457–3553 of the apo B gene was amplified by the PCR with the primers and conditions described by Nissen et al. (20). The resulting products were then separated on a 20 x 16 x 0.1 cm gel of 6% polyacrylamide containing a linearly increasing gradient of 20–60% denaturant (100% denaturant = 7 mol/L urea, 400 mL/L formamide) in 1x TAE buffer (40 mmol/L Tris acetate, 1 mmol/L EDTA, pH 8.0) (20). Electrophoresis was performed at 150 V for 5 h in 1x TAE buffer heated to 60 °C. The gels were stained in ethidium bromide, and the homo- and heteroduplex bands were viewed over ultraviolet light.

Mutation-specific method for detection of apo B R3500Q.
This was performed by PCR with mutagenic primers followed by digestion with MspI according to the method of Motti et al. (11).

Sequence analysis of the apo B gene.
Direct DNA sequencing was performed with the Sequenase PCR product sequencing kit (Amersham International). PCR product was generated with the primer pairs of either Tybjærg-Hansen et al. (8) or Motti et al. (11). PCR amplification mixture (7 µL) containing ~0.5 pmol of PCR product was incubated with 1 µL each of exonuclease I (10.0 U) and shrimp alkaline phosphatase (2.0 U) for 15 min at 37 °C. These enzymes were then inactivated by heating for 15 min at 80 °C, and sequencing was performed in both directions with the primers that generated the PCR product, according to the manufacturer's instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Heteroduplex analysis.
During PCR amplification of DNA, which is heterozygous for a nucleotide substitution, both homo- and heteroduplexes are formed (12)(19)(21). The latter are distorted at the point of substitution and have a slower electrophoretic mobility than homoduplexes. After electrophoresis of a sample heterozygous for a mutation, these heteroduplexes may be observed as slower-migrating bands.

Heteroduplex bands were observed in all 7 samples known to be heterozygous for the R3500Q mutation and also in 4 of the 425 samples of unknown genotype. A typical example of a heteroduplex gel is shown in Fig. 1 .



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  		Figure 1. Photograph of part of a heteroduplex gel, stained with ethidium bromide and viewed over ultraviolet light.

Row A: tracks 2–4, samples heterozygous for apo B R3500Q; track 5, sample heterozygous for apo B R3531C; tracks 6–15, samples without either mutation. Row B: track 1, no DNA (negative control); tracks 3–15, samples without either mutation. Positive controls, consisting of samples known to be heterozygous for apo B R3500Q, are present in track 1, row A, and track 2, row B. In those instances where poor amplification was observed, e.g., in row B, track 15, the analysis must be repeated.

SSCP analysis.
Under nondenaturing conditions, single-stranded DNA has a folded structure, or conformation, that arises from intramolecular interactions that are a consequence of its sequence. Substitution of one nucleotide with another, within a piece of DNA, results in a change in this conformation and consequently its electrophoretic mobility through a polyacrylamide gel, which may be observed as a bandshift.

A bandshift was observed after SSCP analysis of six of the seven known R3500Q heterozygotes and one of the remaining four samples of unknown genotype that also tested positive by heteroduplex analysis. An example of an SSCP run showing the mobility shift of a positive sample heterozygous for the R3500Q mutation is shown in Fig. 2 .



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  		Figure 2. Autoradiograph of part of an SSCP gel obtained after analysis of the region spanning codon 3500 of the apo B gene.

The arrows indicate the mobility shifts observed in track 1 [DNA from an R3500Q heterozygote (positive control)] and track 22 (DNA from an unknown R3500Q heterozygote). The direction of electrophoresis was towards the anode. ss DNA, single-stranded DNA.

DGGE.
During electrophoresis, double-stranded DNA amplified by the PCR migrates through a gel containing a gradient of denaturant until it melts; i.e., the strands separate. At this point, no further migration occurs. Because the melting behavior of double-stranded DNA is a function of its base sequence, the presence of a mutation within a fragment of DNA will alter the melting behavior of the molecule. This in turn will alter the position where the fragment is halted when electrophoresis is performed in a denaturing gradient gel. The usual pattern obtained on analysis of DNA heterozygous for a point mutation is four bands, consisting of two faster-moving homoduplex and two slower-moving heteroduplex molecules.

With DGGE, all seven of the individuals heterozygous for apo B R3500Q were identified by the presence of two homo- and two heteroduplex bands. In addition, the same four samples, of unknown genotype, that tested positive by the heteroduplex method also tested positive by DGGE. Two of these four gave the identical four-band DGGE pattern as the seven R3500Q heterozygotes; however, in two of the samples, the homo- and heteroduplex bands were spaced much farther apart (Fig. 3 ).



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  		Figure 3. Photograph of a DGGE gel containing amplified DNA from six individuals.

Lanes 1 and 2, DNA containing no mutation in the region spanning codon 3500 of the apo B gene; lanes 3 and 4, samples from two individuals heterozygous for apo B R3500Q; lanes 5 and 6, samples from two R3531C heterozygotes. For clarity, only part of the complete gel is shown.

Mutation-specific method for the detection of apo B R3500Q.
With the MspI-based mutagenic primer assay (11), two of the four samples of unknown genotype, both of which screened positive by heteroduplex analysis and DGGE, were shown to be R3500Q heterozygotes (only one of these two samples had been detected by SSCP analysis).

Sequence analysis of the apo B gene.
The remaining two samples of unknown genotype that were identified by both the heteroduplex and DGGE methods were shown by direct DNA sequencing to contain a C->T transition at nucleotide 10 800 in codon 3531; i.e., they were both heterozygous for apo B R3531C. A detailed description of the biochemical features associated with this mutation in these individuals has been described elsewhere (22). Neither of these samples was detected by SSCP analysis. An example of an SSCP gel containing undetected R3500Q and R3531C heterozygotes is shown in Fig. 4 . The findings of the method comparison are summarized in Table 1 for clarity.



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  		Figure 4. Autoradiograph of part of an SSCP gel in which two samples, one heterozygous for apo B R3500Q and one heterozygous for apo B R3531C were undetected.

Lane 48, R3500Q heterozygote (positive control); lane 41, R3531C heterozygote; lane 43, R3500Q heterozygote. The arrow indicates the mobility shift observed in the positive control. ss DNA, single-stranded DNA.


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  		Table 1. Mutations detected with the various molecular-scanning methods.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have compared the ability of three molecular scanning methods, heteroduplex analysis, SSCP analysis, and DGGE to detect known and unknown mutations in the apo B gene. All three techniques rely on a change in the electrophoretic mobility of a piece of DNA in a polyacrylamide gel after an alteration to its sequence.

Nine samples heterozygous for apo B R3500Q (seven known and two unknown) were detected by both heteroduplex analysis and DGGE. In contrast, SSCP analysis identified only seven of these individuals. The finding that SSCP analysis detected some, but not all, of the R3500Q heterozygotes is difficult to explain, especially as all of the method variables, such as temperature of analysis, gel characteristics, and size of PCR product, were optimized to detect this mutation. Whereas we did observe some day-to-day variability in the mobility of the single strands between assays (e.g., compare Fig. 2Up with Fig. 4Up ), these were small, and the polymorphism in the positive controls was always detected. Humphries et al. have suggested (23) that the small regions of base-pairing responsible for the conformations of the single strands and therefore the potential polymorphisms are likely to have a melting temperature close to the average room temperature. They further reported that during hot weather the migration of bands changes considerably so that some SSCPs are no longer detected (23). Therefore during hot weather, some of our SSCP gel assays, which were supposedly performed at room temperature, may have become too warm, resulting in nondetection of the polymorphism. However, we think this explanation unlikely because the bandshift in the R3500Q positive controls was always observed. The reason for this apparent inconsistency in R3500Q detection by SSCP remains to be determined.

Unlike heteroduplex analysis and DGGE, SSCP analysis failed to detect the two R3531C heterozygotes present in this cohort, probably because the method was performed under conditions optimized for the detection of apo B R3500Q. In practice, to maximize mutation detection, SSCP analysis is usually performed under at least two different sets of conditions, e.g., with or without glycerol (24), at 4 °C or at room temperature. Therefore had we followed this procedure, the R3531C heterozygotes probably would have been detected. This approach, however, is much more labor-intensive and therefore unsuitable for screening many samples for diagnostic purposes, where in practice a one-pass technique is preferable.

SSCP analysis was the most laborious of the three techniques to perform in practice. Its main disadvantages were the small size (<200 bp) of the PCR product required for maximum efficiency of mutation detection (25), which necessitated digestion with the restriction endonuclease PstI before analysis, the use of radioactivity, the requirement of gels to be dried before autoradiography, the time taken for autoradiography, usually 24–48 h, and the detection of only 64% of the mutations identified by the other two techniques.

In contrast, DGGE was not only highly efficient and simple to perform, but it also appeared able to differentiate between the mutation types. The four-band pattern observed in the nine R3500Q heterozygotes was quite distinct from that observed in the two R3531C carriers. In the former, the bands were closely spaced whereas in the latter, they were further apart. Moreover, the patterns were identical in all individuals with the same genotype, a finding in agreement with Nissen et al. (20). Thus, by including a control R3500Q sample in each run of new samples of unknown genotype, any R3500Q heterozygotes should readily be identified by inspection of their four-band pattern. Samples that give rise to variant patterns may be subjected to further analysis to identify the nature of the nucleotide substitution. The two main disadvantages of DGGE were the requirement for specialized equipment and the finding that this method had the lowest throughput, 32 samples per electrophoretic run.

The heteroduplex method used in this study was the simplest of the three techniques to perform and was as efficient as DGGE in detecting mutations in this region of the apo B gene. Furthermore, by multiple sample loadings, up to 100 samples could be analyzed in each run, giving it the biggest throughput of all three techniques. It does have two minor drawbacks, however. Firstly, unlike DGGE, it cannot distinguish DNA homozygous for a mutation from wild-type DNA. Because only a handful of individuals homozygous for FDB have been described worldwide, this is unlikely to be a problem in practice (26)(27). Secondly, it is unable to distinguish between different mutations, so any samples that give rise to heteroduplexes require further analysis by another method. This should not prove too much of a problem given the relatively low incidence of FDB, even in hypercholesterolemic populations (17).

Because we did not perform sequence analysis on every sample studied, we cannot exclude the presence of other undetected mutations in this population. However, because DGGE is believed to be almost 100% effective in mutation detection (28), we believe it unlikely that there are any. In conclusion, we found both DGGE and heteroduplex analysis to be equally effective in detecting mutations in and around codon 3500 of the apo B gene. Furthermore, both were more effective and less laborious to perform than SSCP analysis. However, in view of its greater ease, throughput, and absence of a requirement for specialized equipment, we believe that heteroduplex analysis is the method of choice for detection of mutations in this region of the apo B gene that give rise to FDB.


   Acknowledgments
 
B.G.H was employed as a Grade A Trainee Clinical Biochemist, as part of the training scheme of the Association of Clinical Biochemists, and acknowledges financial support from the National Services Division of the National Health Service in Scotland. This work was supported by the Endowment Fund of the Department of Clinical Biochemistry, Western General Hospital, Edinburgh. The assistance of the Department of Medical Illustration at the Western General Hospital is also gratefully acknowledged.


   Footnotes
 
1 Nonstandard abbreviations: FDB, familial defective apolipoprotein B-100; apo, apolipoprotein; SSCP, single-strand conformation polymorphism; DGGE, denaturing gradient gel electrophoresis.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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