A double mutant [N543H+2393del9] allele in the LDL receptor gene in familial hypercholesterolemia: effect on plasma cholesterol levels and cardiovascular disease

Familial hypercholesterolemia is a genetic disorder caused by mutations in the LDL receptor gene. During a survey of mutations of LDL receptor gene in Spanish FH patients we found two mutations in the same allele: a missense N543H mutation in exon 11 and a 9bp inframe deletion (2393del9) located in exon 17. This double mutant allele was founded in 10 out of 458 unrelated patients: one homozygous FH [N543H+2393del9] + [N543H+2393del9], one compound heterozygote [N543H+2393del9] + [W-18X+E256K] and 8 heterozygotes. Flow cytometric analysis showed a defective LDL binding (20% of normal value) and internalization (23%) in lymphocytes from the homozygous patient; furthermore, studies of mitogen-stimulated lymphocytes demonstrated that the ability of LDL to support cell proliferation was impaired. Unexpectedly, not all carriers of the double mutant allele develop hypercholesterolemia and, furthermore, cholesterol-lowering treatment of the homozygous patient resulted in a 58% LDL cholesterol reduction. In conclusion, the phenotypic expression in the homozygous and heterozygous patients presented here, as well as the LDL-receptor residual activity, allowed the classification of this mutation as mild extending the group of mild mutations found at homozygosity.     

Mutation analysis in 46 German families with familial hypercholesterolemia: Identification of 8 new mutations

In order to obtain a survey of the mutations being prevalent in Northern Germany and to enable molecular genetic testing for families with clinically diagnosed familial hypercholesterolemia (FH), we screened 46 unrelated German individuals with elevated LDL levels for mutations in the 18 exons and their flanking intron sequences including the promotor region of the LDL receptor (LDLR) gene. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B‐100 (apoB‐100). We detected 15 mutations affecting the LDLR gene, 8 of which, designated A29S, 195insAT, 313+1insG, 553insG, 680insGGACAAATCTG, D200N, E267K and L411V have not yet been reported. One patient is heterozygous for the double mutant N543H and 2393del9Bp. Two patients carried the mutation R3500Q (Arg→Glu) within the apoB‐100 gene. © 1999 Wiley‐Liss, Inc.     

Molecular genetic testing for familial hypercholesterolemia: spectrum of LDL receptor gene mutations in The Netherlands

Mutations in the LDL receptor are responsible for familial hypercholesterolemia (FH). At present, more than 600 mutations of the LDL receptor gene are known to underlie FH. However, the array of mutations varies considerably in different populations. Therefore, the delineation of essentially all LDL receptor gene mutations in a population represents a prerequisite for the implementation of nation-wide genetic testing for FH. In this study, the frequency and geographical distribution of 13 known mutations were evaluated in a cohort of 1223 FH patients. We identified 358 mutation carriers, representing 29% of the FH cohort. Four mutations (N543H-2393de19, 1359--1G-->A, 313 + 1 G-->A and W23X) occurred with a relatively high frequency, accounting for 22.4% of the entire study cohort. Two of these common FH mutations (N543H-2393de19 and 1359 - 1G-->A) showed a preferential geographic distribution. Second, to further expand the array of LDL receptor gene mutations, we conducted mutation analysis by denaturing gradient gel electrophoresis (DGGE) in 141 children with definite FH. A mutation was identified in 111 patients, involving 16 new single base substitutions and four small deletions and insertions, which brings the number of different FH-causing mutations in our country up to 61. Our data indicate that an estimate of the prevalence of specific mutations, as well as the compilation of a database of all FH-causing mutations in a given country, can facilitate selection of the most appropriate molecular diagnostic approach.     

Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online

We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.     

Mutation analysis in 46 German families with familial hypercholesterolemia: identification of 8 new mutations. Mutations in brief no. 226. Online

In order to obtain a survey of the mutations being prevalent in Northern Germany and to enable molecular genetic testing for families with clinically diagnosed familial hypercholesterolemia (FH), we screened 46 unrelated German individuals with elevated LDL levels for mutations in the 18 exons and their flanking intron sequences including the promotor region of the LDL receptor (LDLR) gene. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B-100 (apoB-100). We detected 15 mutations affecting the LDLR gene, 8 of which, designated A29S, 195insAT, 313+1insG, 553insG, 680insGGACAAATCTG, D200N, E267K and L411V have not yet been reported. One patient is heterozygous for the double mutant N543H and 2393del9Bp. Two patients carried the mutation R3500Q (Arg-->Glu) within the apoB-100 gene.     

A novel point mutation in a splice acceptor site of intron 1 of the human low density lipoprotein receptor gene which causes severe hypercholesterolemia: an unexpected absence of exon skipping. Mutations in brief no. 139. Online

Familial hypercholesterolemia (FH) is a genetic disorder caused by mutations in the low density lipoprotein (LDL)-receptor gene. We found a new mutation in the splice acceptor site of intron 1 of the LDL receptor gene, which is designated as 68-1 G->C according to the nomenclature suggested by Beaudet and Tsui (1993), in a Japanese FH homozygote. She was born from consanguineous marriage and has this mutation as a true homozygous form. Her cultured fibroblasts showed no LDL receptor protein synthesis. This mutation caused activation of a cryptic splice acceptor side in the downstream exon 2, leading to frameshift and appearance of premature in-frame stop codon. The mutation was detected by Dde I restriction enzyme. The identical mutation was not found among 24 patients with homozygous and 120 patients with heterozygous FH. The mutation was very rare among the Japanese population.     

The molecular genetic basis and diagnosis of familial hypercholesterolemia in Denmark

Normal function of the hepatic low-density lipoprotein (LDL) receptor is obligate for normal levels of plasma LDL cholesterol. The LDL receptor regulates the concentration of plasma LDL cholesterol by internalizing apolipoprotein B-100- and apolipoprotein E-containing lipoproteins by receptor-mediated endocytosis. Mutations in the gene encoding the LDL receptor protein give rise to one of the most common classical autosomal dominant inherited disorders in man, familial hypercholesterolemia (FH). The estimated prevalence of heterozygous FH is 0.2% (1:500) in most populations of the world including the Danish. Worldwide, an estimated ten million people are afflicted with FH and in Denmark there are approximately 10,000 subjects with heterozygous FH. Persons with heterozygous FH are characterized by a severely elevated concentration of LDL cholesterol in plasma starting in early childhood, tendon xanthomas and a markedly increased risk of premature coronary heart disease (CHD). Adequate control of plasma LDL cholesterol levels can be achieved in most patients with heterozygous FH, and to a lesser extent in the very rare cases with homozygous FH, using combinations of diet, drug therapy and selective LDL-apheresis. So, it is very important that physicians be aware of this relatively common disorder since there is good evidence that early diagnosis and cholesterol-lowering therapy will delay or even prevent CHD in persons with FH. A large majority of these persons, however, are still not diagnosed or adequately treated. It is believed that the diagnostic abilities molecular biology has to offer will provide the impetus for correcting this situation. The aims of the studies behind the present thesis, therefore, were to obtain important knowledge about current mutation detection technology, prevalence and spectrum of LDL receptor gene mutations in Denmark, methods to evaluate pathogenicity of LDL receptor gene mutations, relationship between FH genotype-phenotype, and clinical versus DNA diagnosis in the Danish FH population. Among different relative laborious and expensive scanning methods for unknown gene mutations we have shown that the polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) analysis is a highly efficient and sensitive technique for detection of mutations in the 18 exons including intronic splice-site sequences and the promoter region of the LDL receptor gene, reserving DNA sequencing to the exons revealing variant SSCP patterns. Southern blot analysis or long distance PCR analysis are necessary to identify large gene re-arrangements in the LDL receptor gene in FH patients in whom SSCP analysis did not reveal any smaller sequence alterations. Worldwide, about 700 different mutations in the LDL receptor gene have been reported and in the Danish FH population we have so far identified 60 different mutations localized throughout the LDL receptor gene. In certain populations a small number of mutations predominate due to founder effects. The spectrum of LDL receptor mutations in Danish FH patients is intermediate between such specific founder populations with 5 predominant mutations (W23X, W66G, W556S, 313 + 1G-->A, 1846-1G-->A) accounting for about 40-50% of FH. These frequent mutations can easily and inexpensively be tested for by specific PCR based assays using restriction enzyme cleavage. Future analysis of LDL receptor mutations in heterozygous FH subjects, therefore, should be based on the mutational spectrum present in each relevant specific subset. Most mutations in the LDL receptor gene cause the classical heterozygous form of FH, but a small proportion seem to result in mild or moderate forms of autosomal, dominantly inherited hypercholesterolemia. Differentiation between harmless sequence variations and disease-causing mutations is not always easy without additional work. We have experienced that large re-arrangements, frame-shift and nonsense mutations obviously are pathogenic, but full pathogenicity should not be ascribed to missense mutations and small in-frame deletions, e.g. the N543H and 2393del9 mutations, unless in vitro gene expression in eukaryotic cells have been studied, or to splice-site mutations, e.g. the 1592 + 5G-->A mutation, before mRNA studies in patient cells have been performed. The cumulated LDL cholesterol exposure, mainly determined by the defect LDL receptor, plays a crucial role for the clinical manifestation of FH. The phenotypic expression of homozygous FH appears to be dominated by the consequences of the LDL receptor gene mutations. In heterozygous FH, however, the underlying mutational LDL receptor type determines only to a much lesser extent, if any, the variable phenotypic expression as seen in Danish patients. Extreme low fat dietary habits or major gene interactions may influence the lipid profile and the excess cardiovascular mortality observed in heterozygous FH, whereas minor gene determinants do not seem to play any significant role. The clinical diagnosis of heterozygous FH should be based on an elevated plasma LDL cholesterol concentration above the 95th percentiles for the general population together with either the presence of tendon xanthomas or an autosomal dominant transmission of hypercholesterolemia in the family or a child with hypercholesterolemia. Our studies illustrate clearly that molecular genetics can strengthen an equivocal clinical diagnosis and assist decision-making in diagnosis and tracing family members. If demonstration of a pathogenic mutation in the LDL receptor gene fails, other causes of autosomal dominant inherited hypercholesterolemia should be sought. Familial defective apolipoprotein B (FDB) caused by the R3500Q apolipoprotein B gene mutation may mimic FH but the clinical course, however, is often milder than that seen in patients with LDL receptor gene mutations. A newly discovered third major locus at chromosome 1 may also be of future diagnostic importance although the exact gene remains to be identified. The overall molecular genetic knowledge obtained about FH in Denmark forms the basis for the implementation and use of molecular genetic diagnostics of FH in daily clinical practice.     

The molecular basis of familial hypercholesterolemia in Lebanon: Spectrum of LDLR mutations and role of PCSK9 as a modifier gene

Autosomal dominant hypercholesterolemia (ADH), a major risk for coronary heart disease, is associated with mutations in the genes encoding the low‐density lipoproteins receptor (LDLR), its ligand apolipoprotein B (APOB) or PCSK9 (Proprotein Convertase Subtilin Kexin 9). Familial hypercholesterolemia (FH) caused by mutation in the LDLR gene is the most frequent form of ADH. The incidence of FH is particularly high in the Lebanese population presumably as a result of a founder effect. In this study we characterize the spectrum of the mutations causing FH in Lebanon: we confirm the very high frequency of the LDLR p.Cys681X mutation that accounts for 81.5 % of the FH Lebanese probands recruited and identify other less frequent mutations in the LDLR. Finally, we show that the p.Leu21dup, an in frame insertion of one leucine to the stretch of 9 leucines in exon 1 of PCSK9, known to be associated with lower LDL‐cholesterol levels in general populations, is also associated with a reduction of LDL‐cholesterol levels in FH patients sharing the p.C681X mutation in the LDLR. Thus, by studying for the first time the impact of PCSK9 polymorphism on LDL‐cholesterol levels of FH patients carrying a same LDLR mutation, we show that PCSK9 might constitute a modifier gene in familial hypercholesterolemia. © 2009 Wiley‐Liss, Inc.     

Mutation analysis in 36 unrelated Spanish subjects with familial hypercholesterolemia: identification of 3 novel mutations in the LDL receptor gene

We used the single strand conformation polymorphism (SSCP) method to investigate 36 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein receptor (LDLR) gene. Nineteen aberrant SSCP patterns were found, and the underlying mutations were characterized by DNA sequencing. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B (apo B). Five missense mutations (Q71E, S156L, E256K, N543H and T705I), four nonsense mutations (W(-18)X, E10X, Q133X and C255X), six frameshift mutations (211delG, 518delG, 1045delC, 2085del19, 2207insT and 2393del9) and five splicing mutations (313+1G->C, 1061-8T->C, 1845+1G->C, 2140+5G->A and 2390-1G->C) were identified in the LDLR gene. In total, we detected 20 mutations, 3 of which, designated 1045delC, 1845+1G->C and 2207insT, have not been previously described. Seven patients were found to carry two different mutations in the same allele: W(-18)X and E256K (one patient), Q71E and 313+1G->C (two patients), 1061-8T->C and T705I (two patients), 518delG and 2140+5G->A (one patient) and N543H and 2393del9 (one patient). As we expected, there is a broad spectrum of mutations in the LDLR gene, given the genetic heterogeneity of the Spanish population.     

Molecular characterization of familial hypercholesterolemia in German and Greek patients

We used the denaturing gradient gel electrophoresis (DGGE) method to define mutations in the promoter region, the 18 exons, and their flanking intronic sequences of the low-density lipoprotein (LDL) receptor gene LDLR, causing familial hypercholesterolemia (FH) phenotype in 100 German and in 100 Greek hypercholesterolemic individuals. In addition, we tested all patients for the presence of mutations in codons 3456-3553 of the gene encoding apolipoprotein B-100 (APOB). Twenty-six aberrant DGGE patterns were identified and subsequently directly sequenced. In LDLR, two novel missense mutations (c.1957G>T/p.V653F, c.647 G>A/p.C216Y) and one novel homozygous base substitution c.1-156 C>T in the repeat 2 of the promoter region were identified among German FH patients; one novel splice site c.1060+10C>G was identified among Greek FH patients. One of the German FH patients was a carrier for the mutations c.1171G>A/p.A391T and p.V653F, and two of the Greek FH patients were compound heterozygotes for the mutations c.1150C>T/p.Q384X and c.1158C>G/p.D386E. Two German FH patients carried the mutation p.R3500Q within APOB. Comparing the mutations within the LDLR gene of the two European FH populations, the German population seems to be more heterogeneous than the Greek cohort. Further studies in progress are trying to elucidate the responsiveness to drug therapy in association with LDLR genotype and the nutritional habits of the two FH populations.     

Identification of four novel mutations of the low-density lipoprotein receptor gene in Korean patients with familial hypercholesterolemia

To obtain insight into the genetic variation of the low-density lipoprotein (LDL) receptor gene in Korean patients with familial hypercholesterolemia (FH), we used single-strand conformation polymorphism to screen all 18 exons and a promotor of the LDL receptor gene in 20 unrelated Korean FH patients. Four novel point mutations were detected in 5 FH patients and were characterized by sequence analysis. Of them, one is a nonsense mutation, a Glu-->Stop (CAG-->TAG) at codon 161, and results in a large deletion. The other three, which were a Ala-->Glu (GCG-->GAG) mutation at signal peptide, Cys-->Tyr (TGC-->TAC) at codon 210, and Pro-->Leu (CTG-->CCG) at codon 584, were novel missense mutations, which modified the highly conserved region of the LDL receptor gene. All these mutations were absent in normolipidemic controls and were associated in heterozygote carriers with clinical signs of FH. Identification of these novel mutations provides another example of the molecular heterogeneity of the LDL receptor gene mutations causing FH.     

Evaluation of a clinically applicable mutation screening technique for genetic diagnosis of familial hypercholesterolemia and familial defective apolipoprotein B

We have recently developed a simple mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) technique for detection of mutations in the coding and regulatory regions of the low density lipoprotein receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene leading to familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. To evaluate the assay, 14 Danish families suspected of FH were studied. In ten families, the DGGE assay detected seven different point mutations, including mutations undescribed prior to establishing the assay. In addition, in one of these ten families and in one of the remaining four families, Southern blotting detected the FH-DK3 exon 5 deletion. Based on segregation analysis and clinical data, the FH diagnosis was dubious in the remaining three families without DGGE or Southern blotting detectable mutations.
In conclusion, a simple DGGE based mutation screening assay may detect underlying mutations in most FH/FDB families, thus allowing its routine use in genetic counselling of FH-families.     

Use of the denaturing gradient gel electrophoresis (DGGE) method for mutational screening of patients with familial hypercholesterolaemia (FH) and Familial defective apolipoprotein B100 (FDB)

Familial hypercholesterolaemia (FH) and Familial defective apolipoprotein B100 (FDB) are autosomal dominant inherited diseases of lipid metabolism caused by mutations in the low density lipoprotein (LDL) receptor and apolipoprotein B 100 genes. FH is clinically characterised by elevated concentrations of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), presence of xanthomata and premature atherosclerosis. Both conditions are associated with coronary artery disease but may be clinically indistinguishable. Seventy-two (72) FH patients were diagnosed based on the Simon Broome's criteria. Mutational screening was performed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Positive mutations were subjected to DNA sequencing for confirmation of the mutation. We successfully amplified all exons in the LDL receptor and apo B100 genes. DGGE was performed in all exons of the LDL receptor (except for exons 4-3', 18 and promoter region) and apo B100 genes. We have identified four different mutations in the LDL receptor gene but no mutation was detected in the apo B 100 gene. The apo B100 gene mutation was not detected on DGGE screening as sequencing was not performed for negative cases on DGGE technique. To our knowledge, the C234S mutation (exon 5) is a novel mutation worldwide. The D69N mutation (exon 3) has been reported locally while the R385W (exon 9) and R716G (exon 15) mutations have not been reported locally. However, only 4 mutations have been identified among 14/72 patients (19.4%) in 39 FH families. Specificity (1-false positive) of this technique was 44.7% based on the fact that 42/76 (55.3%) samples with band shifts showed normal DNA sequencing results. A more sensitive method needs to be addressed in future studies in order to fully characterise the LDLR and apo B100 genes such as denaturing high performance liquid chromatography. In conclusion, we have developed the DNA analysis for FH patients using PCR-DGGE technique. DNA analysis plays an important role to characterise the type of mutations and forms an adjunct to clinical diagnosis.     

Molecular genetics of the LDL receptor gene in familial hypercholesterolemia.

The low density lipoprotein (LDL) receptor is a cell surface transmembrane protein that mediates the uptake and lysosomal degradation of plasma LDL, thereby providing cholesterol to cells. Mutations disrupting the function of this receptor produce autosomal dominant familial hypercholesterolemia (FH). Affected individuals have elevated plasma levels of LDL, which causes premature coronary atherosclerosis. To date, 71 mutations in the LDL receptor gene have been characterized at a molecular level. In this report, we describe 79 additional mutations and review the insights that all 150 mutations have provided into the structure/function relationship of the receptor protein and the clinical manifestations of FH.     

HDL cholesterol levels in patients with molecularly defined familial hypercholesterolemia

Familial hypercholesterolemia (FH) is the most common genetic disorder leading to premature atherosclerosis. Typically, it is due to mutations in the LDL receptor gene resulting in elevated total and LDL cholesterol levels. The type of the LDL receptor gene mutations may affect the severity of hypercholesterolemia and consequently the incidence of coronary atherosclerosis. Furthermore, high-density lipoprotein (HDL) cholesterol levels have been recently shown to be an independent risk factor for coronary heart disease in this population. We examined the effect of the type of the LDL receptor gene mutations and of common gene polymorphisms possibly affecting HDL metabolism [cholesterol ester transfer protein (CETP), apolipoprotein A-IV (ApoA-IV), angiotensin converting enzyme (ACE), and apolipoprotein E (ApoE)] on HDL cholesterol levels in patients with molecularly defined heterozygous FH who were attending our lipid clinic (n=84). The nature of the LDL receptor gene mutation (81T>G, n=12; 858C>A, n=13; 1285G>A, n=12; 1646G>A, n=22; and 1775G>A, n=25) did not significantly influence HDL cholesterol levels. Unlike other gene polymorphisms, the apolipoprotein (apo) E gene polymorphism did significantly affect these levels. In fact, the presence of the E4 allele was associated with lower HDL cholesterol levels compared to patients not carrying this allele. We conclude that HDL cholesterol levels in heterozygous FH patients may be affected by the apoE gene polymorphism.     

LDL receptor cDNA sequence analysis in familial hypercholesterolemia patients: 5 novel mutations with high prevalence in families originating from southern Italy

We screened a group of patients from southern Italy with clinically diagnosed familial hypercholesterolemia (FH) for mutations of the LDL receptor (LDLR) gene. RNA from each proband was analysed by RT-PCR followed by complete cDNA sequencing. Among 51 unrelated FH families we detected 17 mutations affecting the coding region of the LDLR gene. Five of these mutations, designated R395P, L783fsinsG, IVS15-3C>A, IVS3+5G>A, and 1698-1704delCACCCTAinsGCCCAAT (ITL545MPN), have not yet been reported in the literature. Interestingly, the novel IVS15-3C>A splicing mutation was detected in 20% of our unrelated FH families, suggesting an unusually high prevalence in our local population. Hum Mutat 17:433, 2001.     

Two mutations in the same low-density lipoprotein receptor allele act in synergy to reduce receptor function in heterozygous familial hypercholesterolemia

Mutations in genes are not necessarily pathogenic. Expression of mutant genes in cells can therefore be required to demonstrate that mutations in fact disturb protein function. This applies especially to missense mutations, which cause an amino acid to be replaced by another amino acid. In the present study of two families with familial hypercholesterolemia in the heterozygous form, we found two mutations in the same allele of the low-density lipoprotein (LDL) receptor gene: a missense Asn⁵⁴³-His mutation (N543H) in exon 11, and an in-frame 9-bp deletion (2393del9) in exon 17. The two mutations were identified in heterozygous FH index patients in whom no other pathogenic mutations were detected by SSCP analysis of the remaining 16 exons and the promoter region. Both mutations cosegregated with hypercholesterolemia within the families. Each of these mutations had little or no effect on receptor function in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric measurement of fluorescent LDL uptake in cells, was reduced by 75%. Immunostainable receptors on the cell surface were decreased by 80% as measured by flow cytometry. The two mutations therefore acted in synergy to affect receptor function, possibly during intracellular receptor transport, since Northern blot analysis suggested that mRNA levels were unaffected. Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected. Hum Mutat 9:437–444, 1997. © 1997 Wiley-Liss, Inc.     

An individual with a healthy phenotype in spite of a pathogenic LDL receptor mutation (C240F).

Familial hypercholesterolemia (FH) is caused by a defect in the function of the low density lipoprotein (LDL) receptor and inherited in an autosomal, codominant way. In this study we present a 13-year-old girl, compound heterozygote for the LDL receptor mutations C240F and Y167X. Fibroblasts from the patient showed very low cholesterol esterification rate, LDL uptake, and degradation compared to normal fibroblasts (< 2%, 8%, and < 2%, respectively). The C240F mutant was expressed in LDL receptor deficient CHOMldlA7 cells. Analysis of cell extracts by immunoblotting demonstrated delayed processing of the mutated LDL receptor, which was accumulated as a precursor protein of normal size. A high molecular weight form of the receptor was also detectable in these cells, which probably reflects cross-linking through the unpaired cysteine residue in the binding domain. Cells expressing the C240F mutant protein were unable to mediate uptake and degradation of LDL. The two siblings of the index case also carried the C240F mutation, but surprisingly one of them (a 17-year-old brother) showed no signs of hypercholesterolemia. This observation is consistent with the view that there may be cholesterol lowering mechanisms that can be activated, perhaps by mutations in known or hitherto unknown genes.     

Clinically applicable mutation screening in familial hypercholesterolemia

Mutations in the LDL receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene result in the clinically indistinguishable phenotypes designated familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. Introduction of genetic diagnosis in phenotypic FH families may remove the diagnostic inaccuracies known from traditional clinical/biochemical FH diagnosis and allow more differentiated prognostic evaluations and genetic counseling of FH/FDB families. Previous genetic screening methods for FH have, however, been too cumbersome for routine use, however. To overcome these problems, we designed a mutation screening assay based on the highly sensitive denaturing gradient gel electrophoresis (DGGE) technique. The setup allows within 24 hr to pinpoint if and where a potential mutation is located in the LDLR promoter, the 18 LDLR gene exons and corresponding intronic splice site sequences, or in the codon 3500 region of apo B-100. The pinpointed region is subsequently sequenced. As an evaluation of the sensitivity, we demonstrated the ability of the assay to detect 27 different mutations or polymorphisms covering all the examined regions, except LDLR exon 16. In conclusion, a simple, but sensitive, clinically applicable mutation screening assay based on the DGGE principle may reveal the underlying mutation in most FH/FDB families and offer a tool for a more differentiated prognostic and therapeutic evaluation in FH/FDB. © 1996 Wiley-Liss, Inc.