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Clinical Chemistry 52: 1021-1025, 2006. First published April 20, 2006; 10.1373/clinchem.2006.067645
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(Clinical Chemistry. 2006;52:1021-1025.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Analysis of Sequence Variations in the LDL Receptor Gene in Spain: General Gene Screening or Search for Specific Alterations?

Sebastian Blesa1, Ana Barbara Garcia-Garcia1, Sergio Martinez-Hervas2, Maria Luisa Mansego1, Veronica Gonzalez-Albert1, Juan Francisco Ascaso2, Rafael Carmena2, Jose Tomas Real2 and Felipe Javier Chaves1,a

1 Laboratorio de Estudios Genéticos, Fundación de Investigación HCUV, Hospital Clínico Universitario de Valencia, Valencia, Spain.
2 Service of Endocrinology and Nutrition, Hospital Clínico Universitario de Valencia, University of Valencia, Valencia, Spain.

aAddress correspondence to this author at: Fundación de Investigación Hospital Clínico Universitario, Avda. Blasco Ibáñez 17, E-46010 Valencia, Spain. Fax 34-96-3862665; e-mail felipe.chaves{at}uv.es.


   Abstract
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 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Familial hypercholesterolemia (FH) is a frequent form of autosomal-dominant hypercholesterolemia that predisposes to premature coronary atherosclerosis. FH is caused by sequence variations in the gene coding for the LDL receptor (LDLR). This gene has a wide spectrum of sequence variations, and genetic diagnosis can be performed by 2 strategies.

Methods: Point variations and large rearrangements were screened along all the LDLR gene (promoter, exons, and flanking intron sequences).

Results: We screened a sample of 129 FH probands from the Valencian Community, Spain, and identified 54 different LDLR sequence variations. The most frequent (10% of cases) was 111insA, and 60% of the variants had a frequency as low as 1%. A previously described method for detection of known sequence variations in the Spanish population by DNA array analysis allowed the identification of only ~50% of patients with a variant LDLR gene and ~40% of the screened samples.

Conclusion: Our results indicate that the adequate procedure to identify LDLR sequence variations in outbreed populations should include screening of the entire gene.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Autosomal-dominant hypercholesterolemias (ADHs; 1 MIM603776) are frequently occurring lipid disorders characterized by increased plasma total cholesterol (TC) and LDL-cholesterol (LDL-C) concentrations and premature coronary atherosclerosis. The frequency is ~1 heterozygote in 500 persons worldwide, and 1 homozygote in 1 million persons. Cholesterol concentrations of heterozygous patients are twice as high as those in healthy people, and patients present with tendon xanthomas and atherosclerosis in their second or third decade of life. Homozygotes can present in early childhood with a 6-fold increase in LDL-C concentrations, extensive tendon and cutaneous xanthomas, and coronary atherosclerosis, and they frequently die from myocardial infarction before age 20 (1)(2). ADHs have been classified according to the responsible genetic defect. Familial hypercholesterolemia (FH; MIM 143890) is caused by sequence variations in the gene coding for the receptor that clears LDL-C from plasma (LDLR 2 gene). More than 900 sequence variations in this gene have been described (3)(4)(5) (databases accessible at http//www.umd.necker.fr and http//www.ucl.ak.uk/fh) and are distributed along the entire length of the gene. When the defect is found in the gene that codes for the LDL receptor ligand, APOB, the ADH is known as familial defective apolipoprotein B100 (FDB). Only 3 sequence variants have been described, and they restrict the binding of apolipoprotein B to the LDL receptor (6)(7)(8). A third gene causing ADH is the one coding for the proprotein convertase subtilisin kexin 9 (PCSK9), the role of which is not yet well understood. To date, 4 different PCSK9 sequence variations have been described and have led to the identification of a new form of ADH (9).

The clinical features of the different ADHs are very similar, although patients with FDB tend to show a milder phenotype than do FH patients (10)(11). Furthermore, it is known that in FH the type of genetic variation correlates with the severity of the phenotype and response to treatment (12)(13)(14)(15); therefore, identification of genetic variations causing ADHs has acquired great clinical importance.

With the development of new and rapid technologies with high throughput, 2 different strategies to perform a genetic diagnosis are now available: (a) general screening of target genes; or (b) searching for known pathogenic sequence variations. Several studies have recently been conducted in Spain aiming at the molecular characterization of ADHs (16)(17)(18). As an example of the second strategy, Tejedor et al.(19) developed a microarray for population screening that included 117 LDLR sequence variations and APOB R3500Q. The selection of these variants was based on those found in previous studies in patients from different areas of Spain. However, the use of this strategy in open populations with high genetic variability is controversial. Thus, to acquire molecular information on ADH and to test the usefulness of screening for known sequence variations, we screened for the LDLR and APOB genes in a sample of 129 probands from the Valencian Community in Spain.


   Patients and Methods
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 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
patients
The study was conducted in 129 ADH probands attending our Lipid Clinic at the Valencia Hospital Clínico Universitario. ADH was diagnosed according to the Med Ped criteria, summarized as follows: plasma concentrations of TC and LDL-C above the 95th percentiles corrected for both age and sex, together with presence of tendon xanthomas, coronary artery disease in the proband or in a first-degree relative, and bimodal distribution of TC and LDL-C plasma concentrations in the family (autosomal-dominant pattern of lipid IIa phenotype). All patients were Caucasian and lived in the Valencian Community (Spain). The protocol was approved by the institutional ethics committee, and all patients gave their written consent.

dna analysis
DNA was extracted as described by Tilzer et al. (20) from whole blood. Large rearrangements in the LDLR gene were studied by Southern blot as described previously (13) and a semiquantitative fragment analysis based on the one described by Heath et al. (21) covering the entire LDLR gene(22). Small sequence variations in genes were detected by amplification of fragments containing individual exons and their splice intron-exon junctions and sequencing in an ABI3730 system (Applied Biosystems).


   Results
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 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Two patients of the 129 studied probands carried APOB R3500Q; 89 carried an LDLR sequence variations, and in 38 participants, no sequence variants were identified for either of these genes. The identified sequence variations and their type, and the number of probands carrying them are shown in Table 1 . Some patients were double mutant: D280G and G528W, E10X and C358Y, Q71E and 313 + 1G>C, N407S and 1868del3 (1 carrier each), and N543H and 2393del9 (3 carriers). Patients carrying D280G + G528W and E10X + C358Y had been previously clinical and biochemically diagnosed as homozygotes, whereas the remaining double mutants were diagnosed as heterozygotes. In addition, Table 1 shows the sequence variations found in our population that could also be detected by the microchip designed by Tejedor et al. (19). As can be noted, only 26 of the 117 sequence variations included in the microchip are present in our population.


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  		Table 1. LDLR sequence variations present in patients from the Valencian Community.

According to our results, only ~50% of patients carrying a sequence variation in the LDLR gene can be identified by the microchip designed for the Spanish population. In total, the DNA array will allow a diagnosis in ~40% of the clinically diagnosed ADH patients.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Autosomal, monogenic forms of hypercholesterolemia (ADHs) are common diseases, mainly caused by sequence variations in the LDLR and APOB genes. Approximately 70%–75% of sequence variations causing ADH are found in the LDLR gene implicated in FH. Worldwide screening of LDLR gene sequence variations has led to the identification of >900 such variations responsible for FH. As is known, open populations show a wide spectrum of LDLR gene variations. Spain is a good example of an open population, as is the Valencian Community, located on the eastern shore of the country, which is the home of the patients who took part in the present study.

We investigated 129 patients with the FH phenotype and found 2 carrying a sequence variation in the APOB gene (responsible for FDB), whereas 89 (69%) carried a variant LDLR gene. In the remaining 38 patients (29%), our screening found no sequence variations in these 2 genes. The 89 carriers of a variant LDLR gene carried 54 different sequence variations: 5 large rearrangements, 6 frameshift, 7 splicing, and 31 missense variants, and 5 sequence variations that produced base changes that encoded a stop codon or a truncated protein (Table 1Up ). The most frequent sequence variation found in our sample was 111insA, which was carried by 9 patients, representing a frequency of 10%, followed by C95R (5 patients; 6%) and S156L and C358Y (4 patients; 4.5%). Six sequence variations (884delT, I467N, Q133X, W-18X, N543H, and 2393del9) were carried by 3 patients (3.4%), 12 by 2 patients (2.2%), and the remaining 32 were present only in 1 proband (1%). Altogether, 59% of the LDLR sequence variations identified had a frequency as low as 1%.

Our data show the substantial genetic heterogeneity in the ADH population from the Valencian Community and support the value of the strategy followed to identify LDLR and APOB variant genes in open populations. Shown in Table 2 are the sequence variations that can be detected with the array described by Tejedor et al. (19), which is promoted as a general screening tool for ADH in Spain. If we compare Tables 1Up and 2 , our population shares only 26 of the 117 LDLR sequence variations included in the array. If we had applied this array to study our sample of FH patients from the Valencian Community, we would have detected only 54% of the patients carrying variations in the APOB or LDLR genes. On the other hand, when we included our 129 probands, the percentage of carriers detected with the array decreased to 41%. Of even greater importance, the sequence variation found most frequently in our study, 111insA, would have been missed. A laboratory using a mid-sized DNA sequencer (i.e., an ABI Prism 3730 system with 48 capillaries) can analyze 1000 sequences, or more than 20 000 fragments per day, which means that the LDLR gene can be sequenced in 25 patients, and large rearrangements with semiquantitative procedures can be detected in 300 patients in a day (by use of 6 different multiplexes). Therefore, the cost of the procedure is much less than that for analysis by the array, in which patients with a negative result should also have their LDLR gene screened, delaying the final result and increasing the cost of the genetic diagnosis. The cost of the semiquantitative procedure involving detection of large rearrangements and screening of the entire LDLR gene for point variations is approximately {euro}250 (approximately US $297.00), whereas the price for the genetic diagnosis involving the array [commercialized by Lacer S.A., Barcelona, Spain (www.lacer.es) is {euro}425 (approximately US $505.00).


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  		Table 2. LDLR gene variations present in the array described by Tejedor et al. (19) not found in our Valencian population.

In addition, if 1 sequence variation is found by this array method, it will not exclude the presence of other variations not included in the array. Overall, previous observation is important if the array includes sequence variations that may not cause the disease (23)(24) and the rest of the gene is not analyzed. By contrast, our procedure can effectively detect all variations present in sequenced regions as well as large rearrangements. On the basis of these data, we recommend a diagnosis strategy involving screening of the complete gene; we consider this option superior to the use of an array designed to detect predetermined sequence variations.

Thirty-eight of the 129 ADH patients studied did not show any sequence variations in the LDLR or APOB genes. Consequently, further analysis of other genes involved in cholesterol metabolism will be required to identify the genetic cause of ADH in these patients.

In conclusion, the present work shows the extensive variability of the LDLR gene sequence in an open population. The prevalence of each LDLR sequence variation and the percentage each represents in the total gene pool supports the possibility that almost every FH family could exhibit a different variant of the LDLR gene. An important consequence of this genetic variability is the requirement of a diagnostic strategy that analyzes the entire gene, looking for point variations and large rearrangements, as we have done.


   Acknowledgments
 
This work was supported by grants from Fondo de Investigaciones Sanitarias, Spanish Ministry of Health (FIS 01/3047 and 02/1875), Instituto Carlos III Red de Metabolismo y Nutrición (C03/08) and Red de Hiperlipemias Primarias (G03/181), and Generalidad Valenciana (CTIDIA 2002/65 and GV04/255).


   Footnotes
 
1 Nonstandard abbreviations: ADH, autosomal-dominant hypercholesterolemia; TC, total cholesterol; LDL-C, LDL-cholesterol; FH, familial hypercholesterolemia; and FDB, familial defective apolipoprotein B100.

2 Human genes: LDLR, low-density lipoprotein receptor (familial hypercholesterolemia); APOB, apolipoprotein B [including Ag(x) antigen]; and PCSK9, proprotein convertase subtilisin/kexin type 9.


   References
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 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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