(Circulation. 1995;91:1641-1646.)
© 1995 American Heart Association, Inc.
Articles |
Genetic Diagnosis With the Denaturing Gradient Gel Electrophoresis Technique Improves Diagnostic Precision in Familial Hypercholesterolemia
From the Department of Clinical Chemistry (H.N., A.B.H., N.E.P., M.H.) and the Department of Cardiology (M.L.L., T.H.), Odense University Hospital; the Department of Molecular Biology, Odense University (K.K.), Odense; and the John F. Kennedy Institute (P.G.), Glostrup, Denmark.
Correspondence to Henrik Nissen, MD, Department of Clinical Chemistry, Odense University Hospital, 5000 Odense C, Denmark.
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Abstract |
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Background Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder of lipid metabolism caused by mutations in the LDL receptor gene. FH is characterized clinically by elevated LDL cholesterol level and premature coronary disease. Diagnosing FH on clinical grounds may be difficult, and previous genetic methods are too cumbersome for routine use except in the few populations with FH-founder mutations. A simple mutation screening technique based on denaturing gradient gel electrophoresis (DGGE) has been highly useful in detecting mutations in other genes, and in the present study we evaluated the diagnostic potential of this method for the diagnosis of FH.
Methods and Results Conditions for screening exon 3 of the LDL receptor gene using the DGGE technique were established and 14 Danish FH families were examined. An index patient from 1 family had an abnormal DGGE pattern; consequently, an examination of exon 3 of the LDL receptor gene in 21 members of this patient's family was done. The DGGE pattern was seen only in patients with a definite clinical diagnosis of FH. Subsequent sequencing of exon 3 of the LDL receptor gene in these individuals revealed the presence of the French-Canadian type 4 Trp66-Gly mutation. However, in 4 of 11 cases in which a definite clinical diagnosis of FH had been made, the inheritance of the French-Canadian type 4 mutation could be rejected on the basis of genetic analysis.
Conclusions Introduction of a simple genetic analysis based on DGGE may improve the precision of diagnosis in FH families.
Key Words: hypercholesterolemia • diagnosis • genes • molecular biology • arteriosclerosis
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder of lipid metabolism caused by mutations in the gene encoding the low-density lipoprotein (LDL) receptor (LDLR) that lead to elevated levels of LDL cholesterol in plasma and an increased risk of premature atherosclerosis.1 A prevalence of FH of approximately 1 in 500 is estimated for most populations in the Western world.1 In some populations, a few mutations have been enriched through founder effects: examples include the Finns,2 Lebanese Christian Arabs,3 French-Canadians,4 and South African Afrikaners,5 but in general different families are expected to have different mutations. As recently reviewed by Hobbs et al,6 the diversity of mutations is so great that screening for specific mutations in FH families, apart from populations with founder mutations, is not feasible. Because larger LDLR gene rearrangements detectable by Southern blotting techniques account for only 2% to 17% of FH families in the populations studied,7 8 9 the majority of mutations in the LDLR gene are probably point mutations or minor rearrangements undetectable by Southern blotting. The clinical expression of the different LDLR mutations varies,10 11 and in FH families there is a significant overlap between cholesterol levels in children with or without FH.12 Methods permitting early detection of FH gene carrier status will allow primary prevention to be instituted at a young age in children who have FH without the risk of misclassification of FH status. The difficulties in securing accurate diagnosis in FH families using traditional clinical and biochemical diagnostic criteria13 indicate the need for simple diagnostic tools based on genetic analysis that can also be used in populations without FH founder mutations. Because of the lack of mutational hot spots in the LDLR gene, simple methods of examining the entire coding region of the LDLR gene are needed. In phenylketonuria, another autosomal inherited disease with multiple underlying mutations and a varying phenotypic expression, more than 99% of the underlying mutations in the phenylalanine hydroxylase gene have been detected14 by use of guanine-cytosine–clamped (GC-clamped) denaturing gradient gel electrophoresis (DGGE).15 We therefore decided to use the DGGE technique16 to screen for the presence of mutations in the LDLR gene in 14 Danish FH families that have been followed up with clinical observations for several decades.17 In screening these families for mutations in exon 3 of the LDL receptor, we detected the French-Canadian type 4 Trp66-Gly mutation4 in one family. A young French-Canadian woman homozygous for this particular mutation has recently been the subject of the first attempted gene therapy for FH.18 The mutation in the Danish population, however, may have arisen on a different genetic background, because the haplotype differs from the original description.4 The clinical picture of the heterozygous state of this mutation in a Danish family and the contributions of genetic analysis for improved diagnostic accuracy in FH are presented here.
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Methods |
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Patients
We examined 14 families with FH who have been followed up since 1969 at the Lipid Clinic of the Departments of Endocrinology and Cardiology at Odense University Hospital, Odense, Denmark. A total of 305 family members are registered; 222 are alive today. The diagnosis of FH in these families was based on (1) serum levels of total cholesterol or, if known, LDL cholesterol above the 95th percentile for their sex and age in the Danish population,19 (2) a family history positive for hypercholesterolemia, and (3) the presence of tendon xanthomas in members of the family.
Blood Sampling and Lipid Analysis
After approval of the study
protocol by the regional ethical
committee on human research and after subjects' informed consent was
obtained, blood sampling for DNA isolation and lipid analysis was
performed in adults from the 14 families; in a few cases it was also
performed in children during routine blood sampling for hospital
admission. Subsamples of the children's blood samples were used for
this study after informed consent had been given by the parents. DNA
for genetic analysis was isolated from peripheral blood leukocytes
according to standard procedures.20 Lipid analysis was
performed after subjects had fasted overnight. Serum cholesterol, serum
HDL cholesterol, and serum triglyceride concentrations were measured
according to routine methods of the clinical laboratory. The
concentrations of LDL cholesterol were calculated using the Friedewald
formula21 when serum triglyceride level was 
5 mmol/L. To
exclude causes of secondary hypercholesterolemia, levels of serum
creatinine, serum 
-glutamyltransferase, serum thyroid-stimulating
hormone, and blood glucose were determined using standard techniques of
the clinical laboratory. For deceased individuals and individuals not
wishing to participate in blood sampling (pedigree identification
numbers I1, II1, II3, and III4), the biochemical values were obtained
from the medical records, if available.
Southern Blotting
The haplotype of the LDLR gene was
determined at three variable
restriction enzyme sites for BsmI, PstI, and
SpeI.22 DNA (6 µg) was digested with the
appropriate restriction enzymes under conditions recommended by the
manufacturer (Boehringer Mannheim). Digests were size fractionated by
gel electrophoresis through a 0.7% agarose gel. Southern blotting was
performed essentially as described by Rüdiger et
al.23 Two cDNA probes were used for hybridization: a
1.7-kilobase HindIII/BglII fragment containing
sequences from exon 1-11 and a 1.9-kilobase BamHI fragment
encompassing sequences from exon 11 to 18 of the cDNA clone pLDLR3
(American Type Culture Collection, product #57.004). Haplotypes were
constructed with the assumption that there had been no recombination
within the LDLR locus.
Polymerase Chain Reaction
A one-step polymerase chain
reaction (PCR) technique of
introducing a GC-clamp to the end of a PCR product was
used.15 The PCR primers had the following sequences:
5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGTCGGCCTCAGTGGGTCTTTC-3'
(primer A) and 5'-ACTCCCCAGGACTCAGATAGGC-3' (primer B). PCR
conditions
were as follows: 0.8 µmol/L of primer A; 0.8 µmol/L of primer B;
100 ng of genomic DNA; 10 mmol/L Tris HCl, pH 8.3; 1.5 mmol/L
MgCl2; 50 mmol/L KCl; 0.01% gelatin; 200 µmol/L of each
deoxyribonucleotide; and 1 U Thermus Aquaticus DNA polymerase
(Amplitaq, Perkin-Elmer-Cetus) in a volume of 50 µL. The thermal
cycling protocol was 95°C for 5 minutes, 94°C for 1 minute, and
68°C for 5 minutes for 40 cycles followed by a last step of 72°C
for 10 minutes before a denaturation/renaturation program of 99°C for
7 minutes, 65°C for 60 minutes, and 37°C for 60 minutes. The
protocol ended with cooling to 4°C. The expected length and purity of
the GC-clamped PCR products was verified by electrophoresis in a 3%
Nusieve gel (FMC).
DGGE and Sequencing
Linearly increasing denaturing gradient
polyacrylamide gels
(16x20x0.1 cm) of 20%-60% denaturant were made with a gradient gel
mixer (Hoeffer). Stock solutions in 0.04 mol/L Tris-acetate and 1
mmol/L EDTA, pH 8.0, of (1) 6% polyacrylamide without denaturant and
(2) 6% polyacrylamide with 80% denaturant (100% denaturant, 7 mol/L
urea and 40% formamide) were used. Aliquots of 12 µL of the
GC-clamped PCR products were loaded on the gels. Electrophoresis was
performed for 5 hours at 150 V in a buffer bath of 0.04 mol/L
Tris-acetate and 1 mmol/L EDTA, pH 8.0, heated to 60°C using a
modified PROTEAN II Slab electrophoresis cell (Bio-Rad) to ensure a
uniform temperature in the gels during the run. After electrophoresis,
gels were stained with ethidium bromide and photographed under
ultraviolet translumination.
Double-stranded DNA for sequencing was made using PCR under the same conditions as in the first round of PCR, except that primers A and B were used in concentrations of 0.2 µmol/L, nucleotides were used in concentrations of 20 µmol/L, and the number of cycles was reduced to 30. Direct sequencing of the PCR products was performed as described24 using a 32P–end-labeled primer B as the sequencing primer.
Melting Profile Calculations
The melting profile for the
GC-clamped DNA fragments covering
exon 3 of the LDLR gene was calculated using the computer algorithm
MELT87.25 With this program the theoretical
melting temperature of a DNA segment can be calculated. The specific
melting profile of a given DNA fragment is dependent on its composition
and nucleotide sequence. The higher the melting temperature, the longer
the DNA fragment will travel in a DGGE gel before it is halted by the
steric hindrance to further movement that is the result of the melting
process, during which the transition from a compact double-stranded
structure to a partly single-stranded structure takes place. The
melting profile of the GC-clamped PCR fragment containing exon 3 and
bordering intron sequences of the LDLR gene is shown in Fig 1
.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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In an ongoing study aimed at characterizing the mutational spectrum of the LDLR gene in the Danish population with FH, we applied an exon-screening technique based on DGGE to the study of the LDLR gene mutations in 14 Danish FH families. Detection of mutations using DGGE is based on the fact that a change in the base sequence of a given DNA fragment induces a change in its melting profile and consequently alters the electrophoretic characteristics of the fragment in a denaturing gradient gel compared with the wild-type sequence.
We used a set of PCR primers that in a single PCR reaction amplified a
228–base pair fragment of the LDLR gene covering the 123–base pair
exon 3 sequences and adjoining 55– and 50–base pair intron 2 and 3
sequences, and simultaneously introduced a 40–base pair GC-clamp to
the 5' end of the PCR product. An artificial high–melting
temperature
domain was thereby created so that all of the exon 3 sequences were in
a single low–melting temperature domain (Fig 1
), which
ensured maximum
sensitivity in detection of mutations in this sequence region.
When the DGGE technique was applied to the index patients of the
families, the denaturing gradient gel generally showed the single-band
pattern expected in individuals with two identical alleles. In one
index subject, however, the presence of a four-band pattern in the DGGE
gel indicated the existence of two different alleles in the exon 3
region of the LDLR gene, the lower set of bands being caused by the PCR
products from the two different alleles and the two upper bands being
caused by formation of heteroduplexes between the PCR products from the
two alleles. Consequently, DGGE screening of exon 3 of the LDLR gene in
another 20 members from the family of the index subject was performed.
Fig 2 shows the pedigree indicating which family members
have FH as established by clinical criteria, and Fig 3
shows the results of the DGGE screening of exon 3 of the LDLR gene in
this family. In another 6 family members the four-band pattern
identical to the one in the index subject, subject IV1, was seen.
Because all individuals with this pattern had a definite or strongly
suspected clinical diagnosis of FH, this finding indicated that the
DGGE pattern was caused by an underlying mutation in exon 3 of the LDLR
gene.
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This region was therefore sequenced and a thymine-to-guanine transversion was found that corresponded to the first position of codon 66 in the LDLR gene.26 This transversion results in an amino acid change from tryptophan to glycine in the ligand-binding region of the LDL receptor and is identical to the French-Canadian Trp66-Gly mutation.4
By comparing the results of the genetic screening from Fig 3
with the
clinical data from Tables 1 and 2 and the
resultant pedigree in Fig 2, we observed a discrepancy between
the
genetic and clinical diagnoses of FH in this family. Subjects II2,
III6, IV8, and IV11 all had LDL cholesterol levels above the 95th
percentile for their sex and age, and in subjects II2, III6, and IV8
this was also the case for total cholesterol levels. Secondary
hyperlipidemia could not be demonstrated in any of these patients.
After DGGE-based mutation screening, however, inheritance of the
French-Canadian type 4 mutation could be rejected, as illustrated in
Fig 3.
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In this FH family the Trp66-Gly LDLR gene mutation cosegregated with the haplotype BsmI-, PstI-, and SpeI+ (+ indicates the presence and - the absence of a cutting site). Of the 4 individuals with clinical FH but absence of the French-Canadian Trp66-Gly mutation, SpeI- was seen in subjects II2, III6, and IV8; however, SpeI+ was seen in subject IV11. The absence of the mutation in this individual makes it highly likely that the SpeI+ site was inherited from her father, from whom blood samples could not be obtained.
Only one male subject with FH was more than 30 years old at the time of data collection, and he had already suffered his first myocardial infarction at the age of 38 years. Genetic testing was not performed in this patient, but because his daughter has the mutation it must have been inherited from him. All women in this study more than 55 years old who had definite FH had already had their first myocardial infarction. Thus, the high risk of cardiovascular disease caused by the French-Canadian Trp66-Gly LDLR gene mutation in this family is clearly demonstrated.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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To date, more than 150 different mutations in the LDLR gene have been described,6 and in most populations different families can be expected to have different mutations. After the cloning of the human LDLR by Yamamoto et al26 in 1984, most of the LDLR gene mutations described during the first years were larger gene rearrangements detectable by the Southern blotting technique.27 However, in recent years the development of the PCR technique28 has allowed the design of a range of different mutation screening techniques29 30 that detect minor gene rearrangements and point mutations with high sensitivities. In our laboratory we use the GC-clamped DGGE technique15 for screening splice sites and exons of the LDLR gene for mutations in a Danish FH population.
Using this DGGE technique, we analyzed exon 3 in a Danish FH family and found the French-Canadian Trp66-Gly mutation in codon 66 of the LDLR gene,4 which results in a tryptophan-to-glycine amino acid change in the ligand-binding domain of the LDLR protein. This is the first description of this mutation in a non-Canadian population. The mutation does not occur at a CpG dimer hot spot for point mutations.31 Haplotype analysis in the Danish family revealed the haplotype BsmI-, PstI-, and SpeI+, which is different from that in the French-Canadian FH population,4 in whom the mutation cosegregates with BsmI+, PstI-, and SpeI+. The difference indicates that perhaps the mutation detected in the two populations cannot be traced to a common ancestor but may have arisen in each population independently.
Detailed information of the clinical picture of heterozygotes for the French-Canadian Trp66-Gly LDLR gene mutation has not, to our knowledge, been presented, and analysis of the environmental effect on a common mutation in Danish and French-Canadian FH patients is therefore not possible. Moorjani et al11 have, however, reported a mean cholesterol level in 20 children heterozygous for the French-Canadian Trp66-Gly FH mutation of 7.2 mmol/L, compared with a mean of 8.3 mmol/L in the 2 patients less than 18 years old in the Danish family. Thus, the cholesterol levels seems to be at least within the same range.
Hypercholesterolemia is a multifactorial condition; it has been estimated that FH is the causative factor in only 5% of persons with the type II lipoprotein pattern.1 In accordance with findings from examination of Finnish FH families,32 the multifactorial etiology of hypercholesterolemia is likely to blur the clinical diagnosis in some FH families, as demonstrated in this paper. We found that as many as 4 of 21 persons in the family we examined were misclassified as having FH when traditional clinical criteria were used to make the diagnosis, thus documenting the improved diagnostic precision obtained by introducing genetic diagnosis in FH families. According to the probability assessments of Williams et al33 for diagnosing FH in FH families, subjects II2, III6, IV8, and IV11 have probabilities of 79.7%, 99.8%, 97.1%, and 97.1%, respectively, for having FH when total cholesterol levels were used and 90.0%, 99.3%, 97.1%, and 48.1%, respectively, when LDL cholesterol levels were used (lipid values for the population less than 18 years old were reference values for subject IV8). These high probabilities despite the absence of the mutation argues for establishing simple and efficient methods for genetic diagnosis that can also be used in populations without FH-founder mutations.2 3 4 5 The DGGE-based methodology described could be a candidate, because preliminary results indicate that DGGE allows screening of the LDLR gene exons in an index patient within 24 hours without much effort; sequencing of a possible mutation-bearing exon in family members can then be performed. Final evaluation of the DGGE approach described must, however, await the results of ongoing studies evaluating the sensitivity of the method to detect mutations after all 18 exons of the LDLR gene are examined.
Presently, the therapeutic consequences of differentiating between the different causes of hypercholesterolemia are limited. However, the first reports comparing the results of medical treatment of different LDLR gene mutations indicate that knowledge of the underlying mutations may be a tool allowing better predictions of clinical response to treatment.34 35 Likewise, different mutations are known to be associated with different prognosis with respect to cardiovascular disease.1 11 Studies like the present one that give detailed descriptions of the phenotypic presentation of the different LDLR mutations are therefore needed so we can create a framework from which knowledge of the underlying mutation in FH families can be used to improve prognostic and therapeutic evaluation. Until these data become available, the ability to make an unambiguous diagnosis of FH carrier status in FH families is highly relevant because it allows both tailoring of early interventions in young FH carriers and alleviates the psychological stress of uncertainty about carrier status in members of FH families.
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Acknowledgments |
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This study was supported by grants from The Danish Heart Foundation.
Received July 18, 1994; revision received October 5, 1994; accepted October 24, 1994.
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