Clinical Chemistry
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Clinical Chemistry 48: 1913-1918, 2002;
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(Clinical Chemistry. 2002;48:1913-1918.)
© 2002 American Association for Clinical Chemistry, Inc.

Use of Denaturing HPLC to Provide Efficient Detection of Mutations Causing Familial Hypercholesterolemia

Olaf A. Bodamer1a, Dan Bercovich1,1, Michael Schlabach1, Christie Ballantyne2, Danièle Zoch2 and Arthur L. Beaudet1

Departments of
1 Molecular and Human Genetics and
2 Medicine, Baylor College of Medicine, Houston, TX 77030.

aAddress correspondence to this author at: Biochemical Genetics and National Neonatal Screening Laboratory, Department of Paediatrics, University Children’s Hospital, Währinger Gürtel 18-20, A-1090 Vienna, Austria. Fax 43-1-406-3484; e-mail olaf.bodamer{at}univie.ac.at.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Autosomal dominant familial hypercholesterolemia (FH) attributable to mutations in the LDL receptor (LDLR) gene is one of the most common genetic disorders associated with significant morbidity and mortality. Definitive diagnosis would help to initiate appropriate treatment to prevent premature cardiovascular disease. Currently, clinical diagnosis of FH is imprecise, and molecular diagnosis is labor-intensive and expensive because of the size of the LDLR gene and number of coding exons.

Methods: We used PCR to amplify all exons, including exon/intron boundaries, and the promoter of the LDLR gene. Nine individuals from five families with typical findings for a clinical diagnosis of heterozygous FH, 2 heterozygous FH cell lines, and 50 control individuals were screened for mutations by denaturing HPLC (DHPLC) followed by direct sequencing of aberrantly migrating fragments.

Results: Mutations that were previously reported to be disease causing were identified in eight of nine individuals with FH and both cell lines (V502M, C146X, E207X, C660X, C646Y, and delG197), but none were found in controls. The one individual with FH in whom no mutation was found had a previously unreported change in the 5'-untranslated region of unknown significance. In addition, we identified several previously reported polymorphism both in controls and individuals with FH.

Conclusions: DHPLC can be used to detect mutations causing FH. On the basis of our current experience with DHPLC, this method combined with confirmatory DNA sequencing is likely to be sensitive and efficient.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Familial hypercholesterolemia (FH;2 OMIM 143890) is an autosomal dominant disorder characterized by increased serum cholesterol bound to LDL (1). Mutations in the LDL receptor (LDLR) gene cause FH with a frequency as high as 1 in 500 individuals (1). Approximately 50% of affected heterozygous males show clinical signs of premature cardiovascular disease (CVD) by the age of 50, whereas affected heterozygous females show first symptoms ~10–15 years later (1). In contrast, individuals with homozygous FH present during the first decade, and their life expectancy is severely reduced (1). Early diagnosis of at-risk individuals, such as relatives of known FH patients, will allow early pharmacologic and dietary treatment with the potential of reducing the risk of CVD (1)(2). Diagnosis of FH is currently based on clinical, biochemical, and genetic criteria rather than on the identification of disease-causing mutations (1).

Molecular diagnosis of FH is labor-intensive, time-consuming, and expensive because of the size of the LDLR gene, which spans >45 kb of genomic DNA and contains 18 exons (3). In addition, there are many rare or "private" mutations, including deletions in approximately one-third of patients, that add to the complexity of the molecular genotype (1)(4). Consequently, very few clinical laboratories offer general molecular testing for FH, and those that do limit the analysis to a subset of mutations that are prevalent in certain ethnic groups.

The development and introduction of novel analytical methods such as denaturing HPLC (DHPLC) have substantially aided in the molecular diagnosis of genetic disorders involving large genes (5). DHPLC has been used for the diagnosis of Rett syndrome (6), Duchenne muscular dystrophy (7), hereditary hemochromatosis (8), familial melanomas attributable to mutations in INK4A (9), familial breast cancer attributable to mutations in BRCA1/BRCA2 (10), and neurofibromatosis (11), but not yet for the diagnosis of FH.

The following preliminary study was designed primarily to develop and test the analytical conditions for DHPLC followed by direct sequencing for diagnosis of FH. We did not aim to compare this method with others, such as single-strand conformation polymorphism analysis or denaturing gradient gel electrophoresis, as part of this study. Consequently, only individuals with an unequivocal clinical and biochemical diagnosis of FH were included in the study. Fifty healthy controls were included for comparison.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
We studied a total of nine individuals from five families as well as two cell lines. Five unrelated individuals (patients 1, 2, 3, 5, and 8) with the diagnosis of heterozygous FH (two females and three males; mean age, 50 years; age range, 33–63 years) were recruited through the Atherosclerosis Clinic at The Methodist Hospital, Houston, TX (Dr. C. Ballantyne). Diagnosis of FH was based on (a) the presence of increased plasma total and LDL-cholesterol before lipid-lowering therapy, (b) increased plasma total and LDL-cholesterol in a first-degree relative, (c) tendon xanthomata, and (d) a family history of premature CVD and/or stroke and/or sudden death. Four additional first-degree relatives with increased LDL and total cholesterol were identified (patients 4, 6, 7, and 9; one female and three males; mean age, 19 years; age range, 2–40 years). Patients 3 and 4 (father and daughter); patients 5, 6, and 7 (mother, son, and uncle); and patients 8 and 9 (father and son) were related. The study was approved by the Institutional Review Board of Baylor College of Medicine, Houston, TX (#H-8078). Informed, written consent was obtained from all participants.

DNA from 20 mL of EDTA-anticoagulated blood was extracted by the phenol–chloroform method as described previously (12). In addition, DNA was extracted from two lymphoblast cell lines from FH heterozygotes (GM01448 and GM01460) that were obtained through the NIGMS Human Genetic Cell Repository at the Coriell Institute (Camden, NJ). DNA from 50 healthy individuals that was previously extracted was used as control.

pcr
The primer sequences, sizes of amplicons, and respective annealing temperatures are listed in Table 1 . All primers have been reported previously (13) with the exception of the primers for exon 16, which were 5'-CCT CAC TCT TGC TTC TCT CCT GCA-3' and 5'-CGC TGG GGG ACC GGC CCG CGC TTA C-3'. Exon-intron boundaries were included in all PCR products to detect potential splice site mutations. DNA (100–200 ng in 5 µL of distilled H2O) was mixed with 1.25 mM deoxynucleoside triphosphates, 10x PCR buffer, 25 mM MgCl2, 10 pmol of forward and reverse primer, respectively, and 1 U of Taq polymerase. Distilled H2O was added to a total volume of 50 µL. The samples were run on a GeneAmp PCR System 9700 (PE Applied Biosystems) at 95 °C for 5 min, followed by 35 cycles at 95 °C for 1 min, 57 °C for 1 min (with the exception of exon 16, which was run at 65 °C), and 72 °C for 1 min, respectively, with a final step at 72 °C for 7 min. The samples were then stored at 4 °C until further analysis. Before DHPLC analysis, 5 µL of the PCR reaction was run on a 1% agarose gel with a 1-kb marker run for comparison.


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  		Table 1. FH primers (13) and DHPLC temperatures.

dhplc
Mutation analysis was run on a partially inert analysis system from Transgenomic Inc. The PCR products were denatured at 95 °C for 5 min and cooled to 65 °C at a temperature ramp rate of 1 °C/min. The samples were kept cool until 5 µL was applied to a preheated C18 reversed-phase column based on nonporous poly(styrenedivinylbenzene) particles (DNASep column; Transgenomic Inc.). DNA was eluted within a linear acetonitrile gradient consisting of 0.1 mol/L triethylammonium acetate (PE Applied Biosystems; buffer A) and 0.1 mol/L triethylammonium acetate containing 250 mL/L acetonitrile (buffer B). The temperature at which heteroduplex detection occurred was deducted from the Transgenomic software (Wavemaker 4.0; Transgenomic) and the Stanford DHPLC Melting Program (http://insertion.stanford.edu/melt.html), which analyzes the melting profile of the specific DNA fragment. The DHPLC temperatures for each LDLR exon are listed in Table 1Up .

direct sequencing
PCR fragments from individuals with FH who showed abnormal DHPLC heteroduplex formation compared with controls were sequenced. PCR fragments were sequenced with the ABI PRISM BigDye terminator cycle sequencing ready reaction reagent set (PE Applied Biosystems) according to the manufacturer’s recommendations in conjunction with an ABI PRISM 377 DNA sequencer (PE Applied Biosystems).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previously reported disease-causing FH mutations were identified in all participants but patient 1, as well as in the two lymphoblast cell lines (Table 2 ). Patient 1 carried a previously unreported insertion of a single nucleotide, 220 bases upstream of the start codon. The pathologic significance of this change is not known. After identifying mutations in index patients 3, 5, and 8, we also found the respective mutations in all clinically affected, first-degree relatives; the exon with the mutation was amplified and analyzed by DHPLC and confirmed through direct sequencing. The DHPLC analysis and sequence of one mutation are shown as an example in Fig. 1 .


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  		Table 2. LDLR mutations detected by DHPLC and confirmed by direct sequencing.



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  		Figure 1. Heteroduplex formation of LDLR mutation delG652 compared with controls (A), sequence of delG652 (cell line GM01448; B), and control sequence (C).

(A), mutation delG652 (cell line GM01448) is in lane 5 from the top; lanes 1–4 and 6–8 contain controls sequences. Arrows in B and C indicate the site of the mutation.

Additional polymorphisms were identified in all index patients as well as the two cell lines (Table 3 ).


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  		Table 3. Benign polymorphisms in the LDLR gene detected by DHPLC in patients with FH.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
FH is one of the most frequent genetic disorders with significant morbidity and mortality attributable to vascular events (1). Diagnosis is currently based on the presence of typical clinical symptoms, increased plasma LDL-cholesterol, and positive family history (1). Unfortunately, other genetic disorders may have a similar phenotype with increased total cholesterol, xanthomas, and premature CVD, such as mutations in the genes that encode apolipoprotein B-100 and ß-sitosterolemia (14)(15). Although patients with mutations in apolipoprotein B-100 respond to therapies similar to those for patients with mutations in LDLR, ß-sitosterolemia requires a different medical therapy (14)(15). Routine assessment of mutations in the LDLR gene by DHPLC would in most cases distinguish true diagnoses of FH from false-positive clinical diagnoses and lead to better definition of patients with heterogeneous etiologies for increased LDL-cholesterol. In addition, patients with partial or early clinical features of FH could be diagnosed definitively.

In addition, molecular diagnosis of FH would identify affected relatives and provide the opportunity to assess variation in expression among individuals with the same primary mutation. This would facilitate the definition of modifying genetic and environmental factors. The molecular diagnosis of FH within a family would allow both genetic counseling and early treatment, which could in turn lead to decreases in FH-associated morbidity and mortality (2). However, molecular diagnosis of FH is not widely available because of the size of the LDLR gene, the number of coding exons, and the diversity of mutations (1). The recent development of novel molecular methods such as DHPLC and its combination with direct sequencing has helped in the diagnosis of many other genetic disorders involving large genes (7)(8)(9)(10)(11), but to our knowledge, this approach has not been applied to the molecular diagnosis of FH.

We have PCR-amplified all exons, including the promoter region, of the LDLR gene in five individuals clinically suspected to have heterozygous FH but with unknown mutation status, as well as two heterozygous FH cell lines from a repository. Previously reported disease-causing mutations were found in all but one known FH patient as well as in the two cell lines (16)(17)(18)(19). Among these mutations were different point mutations and a small deletion (16)(17)(18)(19). The single patient in whom we did not identify a known mutation carried an insertion of a single nucleotide 220 bases upstream of the start codon. The significance of this change for LDLR function is not known.

First-degree relatives with increased plasma total cholesterol and LDL-cholesterol were screened after the identification of mutations in the respective index patients. Mutations were identified in all clinically affected relatives after amplification of the exon harboring the mutation, DHPLC analysis, and direct sequencing. Although diagnosis in family members could have been made with DHPLC analysis only because heteroduplex formation is characteristic for the mutation, thus substantially reducing the overall costs involved in screening of families (2)(5), confirmation by direct sequencing is highly desirable. Alternatively, diagnosis in family members with known mutations can be done by PCR followed by restriction analysis. Identification of the precise mutation in a family will greatly facilitate genetic diagnosis, therapeutic intervention, and genetic counseling for large numbers of relatives and future offspring because most cases of FH are caused by inherited rather than de novo mutations.

The sensitivity and specificity of DHPLC appear to be >96% irrespective of sequence variations with the exception of high-melting domains surrounded by lower-melting sequences (5). The optimum amplicon size varies, depending on GC content, between 150 and 700 bp (5). Deletions beyond the amplicon will not be detected by DHPLC, and alternative methods such as Southern blotting would have to be applied.

If DHPLC fails to detect a LDLR mutation, molecular analysis should be expanded to include the apolipoprotein B-100 gene. Southern blotting should be done to exclude large deletions or rearrangements in the LDLR gene, which may be as common as 30% in individuals with FH (17). To reach a diagnosis, the entire LDLR gene may ultimately need to be sequenced, including all introns.

In conclusion, we have demonstrated the feasibility of DHPLC and direct sequencing as a new sensitive and rapid method for the diagnosis of FH. DHPLC may be cost-effective in the diagnosis of FH compared with other analytical methods, such as sequencing all exons without prior screening (5). With DHPLC, improved diagnosis of index cases and family-based screening could lead to a reduction in associated morbidity and mortality. Population-based screening is not likely to be feasible any time soon, if ever, but family-based screening combined with genetic counseling and options such as preimplantation diagnosis could dramatically reduce the incidence of FH in future generations (20).


   Acknowledgments
 
We are indebted to the FH patients and their families for continued support and encouragement in completing this study.


   Footnotes
 
1 Current address: Department of Molecular Genetics, Migal-Galilee Technology Center, Rosh Pinna 12100, Israel.

2 Nonstandard abbreviations: FH, familial hypercholesterolemia; LDLR, LDL receptor; CVD, cardiovascular disease; and DHPLC, denaturing HPLC.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ose L. An update on familial hypercholesterolaemia. Ann Med 1999;31(Suppl 1):13-18.
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