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Analysis of sequence variations in the LDL receptor gene in Spain: general gene screening or search for specific alterations?

Autosomal-dominant hypercholesterolemias (ADHs 3 MlM603776) 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 (LDLR4 gene). More than 900 sequence variations in this gene have been described (3-5) (databases accessible at http// and http// 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 (6-8). 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 (12-15); 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 (16-18). 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


The study was conducted in 129 ADH probands attending our Lipid Clinic at the Valencia Hospital Clinico 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 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).


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 1868de13 (1 carrier each), and N543H and 2393de19 (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.

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.


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 1). 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 (884de1T, I467N, Q133X, W-18X, N543H, and 2393de19) 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 1 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 250 [euro] (approximately US $297.00), whereas the price for the genetic diagnosis involving the array [commercialized by Lacer S.A., Barcelona, Spain (www. is 425 [euro] (approximately US $505.00).

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.

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 Nutricion (C03/08) and Red de Hiperlipemias Primarias (G03/181), and Generalidad Valenciana (CTIDIA 2002/65 and GV04/255).

Received January 27, 2006; accepted March 24, 2006.

Previously published online at DOI: 10.1373/clinchem.2006.067645


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[1] Laboratorio de Estudios Geneticos, Fundacion de Investigacion HCUV, Hospital Clinico Universitario de Valencia, Valencia, Spain.

[2] Service of Endocrinology and Nutrition, Hospital Clinico Universitario de Valencia, University of Valencia, Valencia, Spain.

* Address correspondence to this author at: Fundacion de Investigacion Hospital Clinico Universitario, Avda. Blasco Ibanez 17, E-46010 Valencia, Spain. Fax 34-96-3862665; e-mail

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

[4] Human genes: LDLR, low-density lipoprotein receptor (familial hypercholesterolemia); APOB, apolipoprotein B [including Ag(x) antigen]; and PCSK9, proprotein convertase subtilisin/kexin type 9.
Table 1. LDLR sequence variations present in patients from
the Valencian Community.

LDLR variation Patients, n Exon/Intron

W-18X (a) 3 Exon 1
M-21L 1 Exon 1
E10X (a) 1 Exon 2
111insA 9 Exon 2
C68R 1 Exon 3
C68W (a) 2 Exon 3
Q71E (a) 2 Exon 3
C74G (a) 1 Exon 3
E80K (a) 1 Exon 3
313 + 1G>C (a) 2 Intron 3
C95R (a) 5 Exon 4
E119K 1 Exon 4
Q133X (a) 3 Exon 4
S156L (a) 4 Exon 4
P160R 1 Exon 4
D200G (a) 1 Exon 4
D203N 1 Exon 4
S205P (a) 1 Exon 4
694 + 25C>T 1 Intron 4
E246A(a) 1 Exon 5
790delATG (a) 1 Exon 5
D280G (a) 1 Exon 6
I289T Exon 6
884delT (a) 3 Exon 6
920insTCAG (a) 1 Exon 6
S305F 1 Exon 7
Q328X 1 Exon 7
Y354C 1 Exon 8
C358Y 4 Exon 8
R395W 2 Exon 9
N407S 1 Exon 9
V408M (a) 1 Exon 9
T413R 2 Exon 9
Q427X (a) 1 Exon 9
1358 + 1G>A (a) 2 Intron 9
I467N 3 Exon 10
1359-1G>A (a) 2 Intron 10
G528W 1 Exon 11
N543H (a) 3 Exon 11
1698-1704 delCACCCTA/ 1 Exon 11
1845 + 1G>C (a) 2 Intron 12
1868del3 1 Exon 13
G642E 1 Exon 14
D679G (a) 1 Exon 14
2140 + 5G>A (a) 2 Intron 14
2312-3C>A 2 Intron 15
V779M 1 Exon 17
N804K (a) 1 Exon 17
2393del9 (a) 3 Exon 17
Valencia-1 1 Deletion, promoter to exon 2
Valencia-2 1 Insertion, exons 3 to 14
Valencia-3 1 Insertion exon 3 to 4
Valencia-4 1 Deletion, exon 4 to 6
Valencia-5 2 Deletion, promoter to exon 1

(a) Sequence variation that would also be detected by the array
described by Tejedor et al. (19).

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

 LDLR sequence variation

-42C>G C127R C255G
-49C>T 451del3 C255X
->23A C N418N E256K
7delC 509insC 872delC
Q12X D151N C281Y
108delC 518delG E291X
191-2delAinsCT D157G 941-39C>T
211delC C195R C297F
231delC D200Y C317S
N59K 675del15 C319Y
313 + 1G>A 681ins21 G322S
313 + 1insT E207K 1045delC
338del16 684dupl12 R329X
C113W G248D 1054del11
C122X 818del8 1061-8T>C

 LDLR sequence variation

-42C>G C347Y T433N R612C
-49C>T G352D 1423delGCinsA N619N
->23A C C356Y 1432delG L621S
7delC Q357P 1502insC D630N
Q12X C358R V502M F634L
108delC C371X W515X H635N
191-2delAinsCT A378T G516X C646Y
211delC 1197del9 G518D 2088del19
231delC Y379X A519T C677Y
N59K 1204insT L534P 2140 + 1G>A
313 + 1G>A 1207delT W541X T705I
313 + 1insT R395Q 1706-10G A 2184delG
338del16 D412H G571E 2207insT
C113W T413K R574W 2389 + 4G>A
C122X Y421X 1815del11 2390-1G>C
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Title Annotation:Molecular Diagnostics and Genetics
Author:Blesa, Sebastian; Garcia-Garcia, Ana Barbara; Martinez-Hervas, Sergio; Mansego, Maria Luisa; Gonzale
Publication:Clinical Chemistry
Date:Jun 1, 2006
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