© 1995 American Heart Association, Inc.
Articles |
Ten LDL Receptor Mutants Explain One Third of Familial Hypercholesterolemia in a German Sample
From Medizinische Poliklinik, Ludwig-Maximillians-Universität (C.K., G.W., N.Z.), Munich, Germany, and Franz-Volhard-Klinik, Rudolf Virchow Klinikum, Max-Delbrück-Centrum für Molekulare Medizin, Humboldt Universität zu Berlin (H.S.), Germany.
Correspondence to Herbert Schuster, MD, Franz-Volhard-Klinik, Wiltberg Strasse 50, 13122 Berlin, Germany.
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Abstract |
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Abstract Mutational defects in the LDL receptor are responsible for familial hypercholesterolemia (FH); thus far more than 150 mutations have been described. Nevertheless, systematic searches among the Germans have not been conducted. We used single-strand conformational polymorphism and polymerase chain reaction to find mutations in 10 index patients and in 40 other individuals with heterozygous FH. Our screen in the 10 index patients revealed 7 hitherto undescribed mutations. A screen of the 40 additional FH patients disclosed 20 defective of 54 total alleles and allowed specific diagnoses in 88 family members. We also found two families in which the children were markedly affected by FH, but the expected parental expression of the trait was not manifest. This observation suggests a role for additional environmental and genetic influences. Our report represents the first comprehensive effort to identify FH mutations in Germany. We found 10 mutations and these mutations explain 37% of FH cases. Our data may have relevance to expected FH patterns in central Europe.
Key Words: familial hypercholesterolemia • gene mutations • coronary heart disease • LDL cholesterol • LDL receptor
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Introduction |
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Familial hypercholesterolemia (FH) is a dominantly inherited defect in LDL receptors on the short arm of chromosome 19. With one normal and one defective gene at the LDL receptor locus, single gene carriers have only one half the normal number of hepatic LDL receptors to remove circulating LDL cholesterol from the bloodstream. As a result, these heterozygotes typically have twice normal LDL cholesterol levels.1 In most countries, heterozygous FH occurs once in every 500 persons; about 10.7 million persons in the world suffer from the disease. Heterozygous FH is present in 5% to 10% of individuals who exhibit coronary artery disease before the age of 60.2 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, which are new drugs that serve to upregulate LDL receptors, are being used as a specific treatment for this disease. They restore the capacity to clear LDL cholesterol to about one half normal levels in heterozygous FH patients.3 HMG-CoA reductase inhibitors are known to reduce morbidity and mortality in patients with prior myocardial infarction.4 Unfortunately, neither those FH patients who are homozygous nor those who have two separate defects involving the LDL receptor benefit from medication.5
To date over 150 different mutations at the LDL receptor locus have been reported.6 Founder effects, which can now be accurately diagnosed by DNA testing, may increase the rate of FH in some locations.7 Only two rare mutations have been identified in the German population. Thus, it is not yet possible to use DNA techniques for direct FH diagnosis in Germany.8 9 We used single-strand conformational polymorphism (SSCP) to detect genetic variation in genomic DNA fragments in 10 representative subjects with FH and their families.10 We extended our observations to 40 additional unrelated FH patients and their family members. Our results give insight into FH mutations in Germany, since we identified hitherto undescribed FH mutations.
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Methods |
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Patients
We selected 10 persons from a previous study of 31 unrelated FH pedigrees. All patients were recruited from a lipid clinic in Munich and all were of German origin. In all cases the LDL receptor defect was diagnosed by cosegregation analysis with four restriction fragment length polymorphism haplotypes of the gene with hypercholesterolemia as previously described.11 Patients were included in this study by standard clinical definition, age, sex-adjusted cholesterol levels above the 90th percentile for the German population,12 tendon xanthomas, and/or premature coronary artery disease in the patient or a first degree relative. We excluded patients with the familial defective apoB mutation, an FH-like syndrome.13 We characterized the individuals as being either heterozygous, homozygous, or compound heterozygous on the basis of cosegregation analysis. The 10 individuals were selected under the assumption that each haplotype likely carries a different mutation. Since the most common alleles may carry more than one mutation, several individuals with the same haplotype were selected from different geographic areas of the country.
SSCP Analysis
A nonradioactive, high-resolution, SSCP protocol was used
that has been previously described.14 Each exon of the LDL
receptor gene was amplified using the polymerase chain reaction (PCR)
with flanking primers described by Hobbs et al.6 Since
these primers are close to the exon intron borders, they may not
identify splice site mutations. For nonradioactive detection of DNA
fragments, the 5' end was biotinylated during DNA synthesis of
oligonucleotides. The PCR reactions were performed in a
total volume of 50 µL and subjected to 1 cycle at 95°C for 5
minutes and 68°C for 2 minutes, subsequently followed by 30 cycles at
95°C for 1 minute and 68°C for 2 minutes. Three-microliter
aliquots of each PCR product were mixed with 5 µL of formamide
dye (95% formamide, 20 mmol/L EDTA, 0.05% bromophenol blue, 0.05%
xylene cyanol), boiled at 95°C for 3 minutes to denature the dsDNA to
ssDNA, and snap-cooled on ice. A 0.5-µL aliquot was loaded on
0.5xMDE gel matrix onto a TE 2000 direct DNA blotting electrophoresis
unit. Electrophoresis was carried out under a single condition at 6 W
and at room temperature for 18 hours without glycerol in gels exceeding
25 cm in length. After electrophoresis and simultaneous
transfer of DNA onto the nylon membrane, the DNA was
UV-cross-linked to the membrane and detected using a
chemiluminescent detection system. Then the membrane was exposed to a
standard roentgen film for 2 hours.
Direct Sequencing
The sequencing reaction was carried out using T7 DNA polymerase
and alpha 35S-labeled dATP as previously
described.15 ssDNA was produced by asymmetric PCR as
described above. The limiting primer was used as sequencing primer.
Annealing was performed after the sample was warmed to 80°C for 3
minutes and the specimen was cooled to room temperature for 20 to 30
minutes. The labeling reaction was carried out using 10 µL of the
template primer solution, 1 µL 1,4-dithiothreitol 0.1 mol/L, 2 µL
of diluted labeling mix, 0.75 µL 35S-labeled dATP, and 2
µL diluted T7 polymerase. The dGTP labeling mix was used in a 1:5
dilution. The termination reaction was performed using 3.7 µL of each
ddNTP and 5.2 µL of the labeling reaction product, which was
incubated at 37°C for 3 to 4 minutes. The labeling reaction was
stopped with 5 µL of formamide dye, and the specimen was stored at
4°C on wet ice until ready to load onto the gel. Electrophoresis was
performed in 6% polyacrylamide gels at approximately 2000 V
for 2 hours. Prior to loading, the samples were heated to 80°C for 2
minutes. Autoradiography was performed after the
gel was dried at 80°C for 2 hours without soaking in methanol and
acetic acid. The dried gel was exposed directly to the film.
Mutation Analysis
For direct mutation analysis, two different methods were
used. In cases in which the nucleotide exchange creates or
destroys a restriction site, restriction digestion was used after PCR
with flanking oligonucleotides as described by Hobbs et
al.6 Restriction fragments were separated by standard
agarose gel electrophoresis techniques as previously
described.9 In all other cases allele-specific
oligonucleotides were designed, which carried the
mutant base change at their 3' end. A second mismatch mutation was
introduced three or four bases from the 3' end to increase specificity
of the amplification reaction. The detailed protocol was published
previously.8 For a 7-bp deletion, neither of these methods
was necessary, since the mutation was detected by simple agarose gel
electrophoresis after PCR probably because of heteroduplex
conformation.
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Results |
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Fourteen LDL receptor–defective alleles (6 heterozygous patients, 3 homozygous patients, and 1 compound heterozygous patient) were screened for mutations in the 18 exons of the LDL receptor gene. In all cases, a different pattern of bands was identified by SSCP in at least one PCR fragment, and subsequently the mutational event was ruled out by direct sequencing (Fig 1
). As expected from
cosegregation analysis with haplotypes, 3 different mutations
were found in homozygous individuals, and 2 different mutations were
identified in the compound heterozygous individual. Within the 6
heterozygous individuals, 5 different mutations were identified; 2
individuals carried the same mutant. Thus, 10 different mutations were
identified. The results are summarized in the Table.
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In a second step, the entire sample of the 50 unrelated FH patients was
screened for these mutations. This approach led to the identification
of 6 additional heterozygous individuals within this sample, including
20 defective of 54 total alleles. This observation led to the
subsequent identification of 88 patients, including the index
cases, in the families of these 10 individuals. In two families
exceptional phenotypic phenomena were observed. One example is shown in
Fig 2 (left). The index case, a 9-year-old boy with
definite signs of homozygous FH and untreated cholesterol
concentrations >500 mg/dL, was identified as compound heterozygous for
two different mutations, both of which were found in his heterozygous
parents. The codon 303 1-bp deletion led to a twofold elevation in
cholesterol levels in the mother. However, the father had
normal cholesterol values despite identification of the
proline678 to leucine mutation, which also cosegregates
with hypercholesterolemia in a second family.
The second exceptional finding is illustrated in the other family shown
in Fig 2 (right). The index case, a 15-year-old boy who had
clinical signs of homozygous FH, was homozygous for the
serine285 to leucine mutation. However, both of his parents
and affected siblings had only borderline
hypercholesterolemia.
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Discussion |
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We used SSCP as described previously10 to identify genetic variation in large DNA fragments of FH patients who were screened for LDL receptor mutations. Of 10 mutations, 7 were found to be new and not described previously. Three mutations that we identified have been described previously: namely, the serine156 to leucine mutation in Puerto Rican patients by Hobbs et al,16 the serine285 to leucine substitution in the Dutch and Danish populations,17 and the codon 469 stop mutation in the Norwegian population.17 Six of the 7 point mutations were caused by a G to A transition. Two of the point mutations occurred in a CpG island. Together with the 3 minor deletions between 1 bp and 7 bp and the premature stop codons in 5 cases, these alleles most likely represent null alleles at the functional level.
A similar approach was used to investigate the molecular basis of FH in France.18 In this study, 7 patients were tested and 9 mutant alleles identified. Two thirds of the possible mutations were detected by SSCP analysis, the others by direct sequencing. Seven new mutations were described, indicating heterogeneity in the French population as well. However, nothing is known about the prevalence of these mutations in the French FH population. Two-hundred patients with FH from lipid clinics in the London area were screened for mutations in exon 4 of the LDL receptor gene. Three different tests were sufficient to detect 8% of the molecular defects.19 The largest collection of FH patients was studied by Hobbs et al,6 who maintained fibroblast cultures from 170 unrelated FH individuals, including 157 homozygotes from 14 different countries. In their report, they describe 150 different mutations, providing evidence that in most populations molecular diagnosis is not feasible by direct DNA testing because in the majority of patients the disorder is caused by different mutations. However, in some countries with populations that have been isolated by cultural or geographic boundaries, the frequency of particular mutations is much higher. For example, in Afrikaners living in South Africa, 2 mutations are responsible for more than 95% of FH patients.20
SSCP provided substantial technical advantages in approaching this problem. The sensitivity of the SSCP protocols has been estimated to approach 97%.21 We achieved 100% sensitivity, perhaps because of the special modified gel matrix (MDE) we employed. Second, the high-resolution electrophoresis protocol, which relied on a blotting device, allowed separation of entire DNA fragments throughout the entire length of the gel. However, the mutational event per se influences the ability of forming SSCPs. In our study, 3 of 10 mutations represent small deletions for which the SSCP technique may be particularly favorable.
Our results confirm earlier observations, that FH is a heterogeneous disorder, caused by at least 150 different mutations worldwide.6 In Germany, only scattered case reports have been published,8 9 and little is known about FH population genetics. Therefore, some of our findings may be influenced by ascertainment bias. However, identification of the major mutations causing a genetic disorder is crucial for the development of diagnostic strategies for DNA testing. This fact has been demonstrated for the cystic fibrosis gene, in which multiplex PCR was used for rapid detection of 3 mutations in the Italian population.22 Presently, screening for known mutations in Germany has not been efficient. Thus, we currently use a two-step diagnostic procedure, namely, family studies to confirm clinical diagnosis of autosomal dominant inheritance followed by DNA screening.
Molecular analysis promises insight above and beyond mere diagnosis. A number of clinically important questions about FH remain unanswered that can be solved with the molecular techniques currently available. Among FH patients there is a great deal of variation in terms of untreated lipid levels and of the age of onset of coronary artery disease. Some individuals develop disease at a very early age, whereas others who survive to age 50 years have a standardized mortality ratio only slightly higher than that of the general population. The age of onset of coronary heart disease in FH patients aggregates within families.23 This phenomenon may be related to the presence of environmental influences or other genetic factors, such as genes for higher levels of lipoprotein(a).24 However, different mutations in the LDL receptor gene may also be responsible in part for these differences.17 If such associations could be established for specific mutations, more active therapeutic strategies could be recommended to patients and their relatives who have inherited a mutation that carries a greater risk.
The classic approach to FH is based on the assumption that FH is inherited in a dominant fashion with high penetrance and therefore is classified as either heterozygous or homozygous. The two unique families we identified support the notion that a nonlinear gene-dose effect exists for some alleles. This phenomenon is well known for disorders in which dominant and recessive forms of the same gene result in different mutations in the same gene.25 Other genetic factors may not only modulate the intermediate phenotype of FH, namely hypercholesterolemia, but also may be essential for susceptibility to develop premature atherosclerosis. In this fashion the LDL receptor defect may continue to serve as a paradigm for coronary heart disease research in the future.
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Acknowledgments |
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This work was supported by a grant-in-aid to Dr Schuster from the Deutsche Forschungsgemeinschaft in the form of a Hess Fellowship.
Received April 4, 1995; accepted October 10, 1995.
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References |
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