Familial hypercholesterolaemia in Finland: common, rare and mild mutations of the LDL receptor and their clinical consequences. Finnish FH-group.

Familial hypercholesterolaemia (FH) is an autosomal co-dominantly inherited condition resulting from mutations of the low-density lipoprotein (LDL) receptor which occur in heterozygous form in approximately one in 500 individuals. Clinically, FH is characterized by 2-3-fold elevation of serum LDL cholesterol levels, accelerated development of atherosclerotic vascular disease, and, if untreated, shortened lifespan. The Finnish population, which represents a genetic isolate, offers exceptional possibilities for genetic-epidemiological studies on FH, as a handful of founder gene mutations account for the majority of FH cases in Finland. This review summarizes data from our FH studies carried out since 1985. We wish to emphasize the continuum of genotype-phenotype relationships, the importance of molecular diagnosis, the detection of novel risk factors of vascular disease, and innovations inhibiting cholesterol absorption for the modern treatment of FH.

A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor.

Basolateral targeting of membrane proteins in polarized epithelial cells typically requires cytoplasmic domain sorting signals. In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. Consequently, virally encoded mutant receptor fails to mediate cholesterol clearance in LDL receptor-deficient mice, suggesting that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele. This evidence directly links the pathogenesis of a human disease to defects in basolateral targeting signals, providing a genetic confirmation of these signals in maintaining epithelial cell polarity.

Familial moderate hypercholesterolemia caused by Asp235-->Glu mutation of the LDL receptor gene and co-occurrence of a de novo deletion of the LDL receptor gene in the same family.

We identified a large family in which a hitherto unreported point mutation of the LDL receptor gene (Asp235-->Glu) cosegregated with moderately elevated serum LDL cholesterol concentration. Within one generation, the mean serum total and LDL cholesterol levels in four heterozygous carriers of this mutation (7.76 +/- 1.46 and 5.89 +/- 1.56 mmol/L, respectively) were significantly (P < .05) higher than the corresponding concentrations in their five nonaffected siblings (5.81 +/- 0.57 and 3.77 +/- 0.54 mmol/L, respectively). Lipid levels in carriers of the Asp235-->Glu mutation were, however, markedly lower than the corresponding total and LDL cholesterol levels (about 12 and 10 mmol/L, respectively) in heterozygous patients with the two common LDL receptor mutations (FH-Helsinki and FH-North Karelia). None of the four siblings in the age range of 54 to 69 years had experienced a myocardial infarction, although symptoms suggestive of coronary artery disease were present in two and tendon xanthomas were found in one. Expression of the mutant receptor in COS cells indicated an approximately 50% to 70% reduction of LDL-binding activity compared with the normal receptor. One patient (female, aged 39 years) had severe hypercholesterolemia in the range of 13 to 20 mmol/L when untreated, extensive coronary artery disease as demonstrated by angiography, and extensor tendon xanthomatosis. In addition to the Asp235-->Glu mutation, she was found to have a de novo deletion of exons 14 and 15 in her other LDL receptor allele. In this subject, the total LDL receptor activity of mitogen-stimulated blood lymphocytes was very low. In conclusion, along with another LDL receptor gene mutation (FH-Espoo or deletion of exon 15) described by us previously, the Asp235-->Glu mutation (designated as FH-Keuruu) indicates that moderate varieties of inherited hypercholesterolemia may result from LDL receptor gene mutations of mild expression.

Polymorphisms of the genes encoding apoproteins A-I, B, C-III, and E and LDL receptor, and cholesterol and LDL metabolism during increased cholesterol intake. Common alleles of the apoprotein E gene show the greatest regulatory impact.

Genetic and dietary factors regulate serum cholesterol level, but detailed investigations into their interactions have not been established. We assessed the effects of apoprotein (apo) E phenotype and polymorphic alleles of the apo A-I, apo B, apo C-III, and LDL receptor genes, separately and together, on regulation of serum LDL cholesterol level. The study group consisted of 29 middle-aged men, and cholesterol absorption, bile acid, and cholesterol synthesis and LDL apo B kinetics were studied in these men during low- and high-cholesterol diets. The six apo B alleles were identified on the basis of Xba I, EcoRI, and Msp I restriction fragment length polymorphism (RFLP), the apo A-I alleles with the Msp I RFLP, and the apo C-III and LDL receptor alleles corresponded to the Sst I and PvuII RPLPs of these genes, respectively. During low cholesterol intake, LDL cholesterol levels were similar in all of the genetic groups except for men with apo E2 phenotype. They had significantly (P < .05) lower levels of LDL apo B and cholesterol than men without the epsilon 2 allele. The low values were caused by a significantly higher removal of LDL apo B (apo E2, 0.453 +/- 0.03 versus apo E3, 0.312 +/- 0.01 pools per day, P < .05). High cholesterol intake increased LDL cholesterol levels in all genetic categories except in the apo E2 phenotype irrespective of the combinations with other polymorphisms. Carriers of the apo B R+ allele (EcoRI site present) presented with the most prominent LDL cholesterol rise (from 2.71 +/- 0.14 to 3.37 +/- 0.29 mmol/L). In multiple stepwise regression analysis, apo B EcoRI RFLP and apo E phenotypes were the only variables that explained the variability of high cholesterol intake-induced change in LDL cholesterol levels. In summary, in any genetic combination, individuals with the epsilon 2 allele had the lowest LDL cholesterol values and were nonresponders to dietary cholesterol, whereas subjects with the apo B R+ allele had marked LDL elevations, especially in combination with the epsilon 4.

A novel deletion/inversion mutation in the low-density lipoprotein receptor gene as a cause of heterozygous familial hypercholesterolemia.

A combined deletion/inversion rearrangement of the LDL receptor gene was discovered in a Finnish patient with heterozygous familial hypercholesterolemia (FH). Sequence analysis of the mutated allele revealed an insertion of 4 nucleotides in exon 11, caused by a combined deletion and insertion event replacing a 13-bp segment of the normal exon 11 sequence of the LDL receptor gene by a 17-bp stretch of new sequence at the deletion breakpoint. The inserted sequence was identical to the normal exon 9 sequence of the LDL receptor gene from nt1225 to nt1241 inserted in an inverted orientation. This defect causes a translational frameshift after amino acid 525 (glycine) and leads to a premature termination codon at amino acid position 538. Analysis of reverse transcriptase-PCR products from total RNA extracted from cultured fibroblasts revealed only transcripts encoded by the normal allele. This finding was consistent with the reduced functional activity of the LDL receptor found in the fibroblasts of the patient to levels less than 50% of those in normal cells. In conclusion, we have identified a complex and hitherto unreported type of rearrangement of the human LDL receptor gene. The precise mechanism of this mutation (designated as FH-Jalasjärvi) remains obscure, although it may involve complex loop formation by interaction of complementary sequences present in the mutation breakpoints and their immediate flanking regions.

Molecular characterization of minor gene rearrangements in Finnish patients with heterozygous familial hypercholesterolemia: identification of two common missense mutations (Gly823-->Asp and Leu380-->His) and eight rare mutations of the LDL receptor gene.

Two deletions of the low-density lipoprotein (LDL) receptor gene were previously shown to account for about two thirds of all mutations causing familial hypercholesterolemia (FH) in Finland. We screened the DNA samples from a cohort representing the remaining 30% of Finnish heterozygous FH patients by amplifying all the 18 exons of the receptor gene by PCR and searching for DNA variations with the SSCP technique. Ten novel mutations were identified, comprising two nonsense and seven missense mutations as well as one frameshift mutation caused by a 13-bp deletion. A single nucleotide change, substituting adenine for guanidine at position 2533 and resulting in an amino acid change of glycine to aspartic acid at codon 823, was found in DNA samples from 14 unrelated FH probands. This mutation (FH-Turku) affects the sequence encoding the putative basolateral sorting signal of the LDL receptor protein; however, the exact functional consequences of this mutation are yet to be examined. The FH-Turku gene and another point mutation (Leu380-->His or FH-Pori) together account for approximately 8% of the FH-causing genes in Finland and are particularly common among FH patients from the southwestern part of the country (combined, 30%). Primer-introduced restriction analysis was applied for convenient assay of the FH-Turku and FH-Pori point mutations. In conclusion, this paper demonstrates the unique genetic background of FH in Finland and describes a commonly occurring FH gene with a missense mutation closest to the C terminus thus far reported.

A single-base substitution in the proximal Sp1 site of the human low density lipoprotein receptor promoter as a cause of heterozygous familial hypercholesterolemia.

We have identified a Finnish family with a typical phenotype of heterozygous familial hypercholesterolemia (FH) due to a single-base substitution in the proximal Sp1 binding site of the low density lipoprotein (LDL) receptor gene promoter. The mutation, a C-->T substitution at nucleotide -43, cosegregated with the FH phenotype in six available family members and abolished binding of Sp1 transcription factor to this site. As a consequence, transcriptional activity of the mutated LDL receptor promoter was only about 1/20th of that of the wild-type promoter, as judged by transfection studies in HeLa cells. Studies of primary fibroblast cultures established from a family member revealed a markedly reduced LDL receptor mRNA concentration as well as reduction of binding, internalization, and degradation of 125I-labeled LDL to values < 50% of those in normal fibroblasts. This DNA alteration is thus a naturally occurring promoter mutation causing a severe disorder of human lipoprotein metabolism.

Deletion of exon 15 of the LDL receptor gene is associated with a mild form of familial hypercholesterolemia. FH-Espoo.

We describe a mutation of the low-density lipoprotein (LDL) receptor gene, designated familial hypercholesterolemia (FH)-Espoo, which deletes exon 15 of the LDL receptor gene. The mutant receptor is predicted to lack 57 amino acids, including 18 serine and threonine residues, which are the sites of the clustered O-linked sugars of the receptor. Studies on 10 carriers of this gene revealed that FH-Espoo is associated with an exceptionally mild form of FH. Thus, in conditions in which cell proliferation was rendered dependent on the function of LDL receptors, lymphocytes from the patients with the FH-Espoo allele had a growth rate intermediate between those from healthy subjects and patients with the FH-Helsinki gene, a mutation known to abolish LDL receptor function. The in vivo fractional catabolic rate of LDL apolipoprotein B was lower than normal in the two FH-Espoo heterozygotes studied. Although higher than those in healthy controls, the serum LDL cholesterol concentrations in patients with the FH-Espoo gene were significantly lower than those in patients with the FH-Helsinki mutation. The thickness of the Achilles tendons was within the normal limits in subjects with the FH-Espoo gene. Our study suggests that moderate varieties of hypercholesterolemia, ie, those not considered to represent FH, may occasionally be due to subtle LDL receptor gene mutations.

Prevalence of familial hypercholesterolemia among young north Karelian patients with coronary heart disease: a study based on diagnosis by polymerase chain reaction.

Two deletions of the low density lipoprotein (LDL) receptor gene account for about 90% of the mutations that cause familial hypercholesterolemia (FH) in eastern Finland. The FH-Helsinki mutation deletes exons 16, 17 and a portion of exon 18, while the FH-North Karelia allele is characterized by a deletion of seven nucleotides from exon 6 of the LDL receptor gene. We developed a DNA assay based on the use of polymerase chain reaction (PCR) which simultaneously detects both of these mutations. We have screened 90 young (< 45 years) eastern Finns with symptomatic coronary heart disease (CHD) for the presence of these FH genes. One or the other of the mutations was present in 4 out of 55 survivors of acute myocardial infarction (AMI) and 4 out of 35 patients with angina pectoris (AP), but in none of 50 healthy controls of similar age. These data show a relatively high prevalence of confirmed FH in young CHD patients (AMI and MI combined: 8/90, or 9%), and also demonstrate the feasibility of PCR techniques in diagnosis of FH among populations with enrichment of specific types of LDL receptor gene mutations.

Molecular genetics of familial hypercholesterolaemia: common and rare mutations of the low density lipoprotein receptor gene.

Mutations of the low density lipoprotein (LDL) receptor gene give rise to familial hypercholesterolaemia (FH), one of the most common single-gene diseases in the world. Approximately 150 different LDL receptor gene mutations have been reported until now and the list seems to be continuously growing. Although hampering molecular genetic diagnosis of FH, this wide variability at the DNA level provides a useful tool to population genetics and may ultimately lead to better understanding of the variation in disease manifestations from family to family. The Finns are among the few populations in which one or two mutant LDL receptor genes explain the majority of FH cases. Either of the two 'Finnish-type' LDL receptor gene deletions, FH-Helsinki or FH-North Karelia, is present in more than 60% of the Finnish FH patients; there are no reports on their existence in other ethnic groups. Assays for these mutations were shown to markedly complement clinical diagnosis of FH in Finland.

The familial hypercholesterolemia (FH)-North Karelia mutation of the low density lipoprotein receptor gene deletes seven nucleotides of exon 6 and is a common cause of FH in Finland.

A mutation of the LDL receptor gene very common among Finnish patients with heterozygous familial hypercholesterolemia (FH) was identified. This mutation, designated as FH-North Karelia, deletes seven nucleotides from exon 6 of the LDL receptor gene, causes a translational frameshift, and is predicted to result in a truncated receptor protein. Only minute quantities of mRNA corresponding to the deleted gene were detected. Functional studies using cultured fibroblasts from the patients revealed that the FH-North Karelia gene is associated with a receptor-negative (or binding-defective) phenotype of FH. Carriers of the FH-North Karelia gene showed a typical xanthomatous form of FH, with mean serum total and LDL cholesterol levels of 12 and 10 mmol/liter, respectively. This mutation was found in 69 (34%) out of 201 nonrelated Finnish FH patients and was especially abundant (prevalence 79%) in patients from the eastern Finland. These results, combined with our earlier data on another LDL receptor gene deletion (FH-Helsinki), demonstrate that two "Finnish-type" mutant LDL receptor genes make up about two thirds of FH mutations in this country, reflecting a founder gene effect. This background provides good possibilities to examine whether genetic heterogeneity affects the clinical presentation or responsiveness to therapeutic interventions in FH.

Laminin and its neurite outgrowth-promoting domain in the brain in Alzheimer's disease and Down's syndrome patients.

Immunocytochemistry, radioimmunoassay, immunoblotting, Northern analysis, and polymerase chain reaction (PCR) technique were applied to investigate the distribution of laminin and its neurite outgrowth domain in brains of neuropathologically verified cases of Alzheimer's disease and Down's syndrome. New antibodies against a neurite outgrowth domain of laminin were characterized and were used in localization of this peptide antigen in the human brain. Laminin was found as large punctate deposits in all plaques in the affected brains. Laminin synthesis was increased as assessed by RNA blotting and immunoblotting, and glial cells were heavily immunoreactive with antibodies for a neurite outgrowth-promoting peptide antigen of the B2 chain of laminin. This peptide antigen not only was produced by glial cells but also was deposited in the brain tissue. As this peptide antigen promotes neurite outgrowth at low concentrations, and is specifically neurotoxic at high concentrations, it may play a synergistic role with other molecules in inducing the sprouting and neurodegeneration occurring in brains of patients with either Alzheimer's disease or Down's syndrome.

Diagnosis of heterozygous familial hypercholesterolemia. DNA analysis complements clinical examination and analysis of serum lipid levels.

The concordance of clinical and molecular genetic diagnoses of heterozygous familial hypercholesterolemia (FH) was studied in 65 subjects (10 propositi and 55 first-degree relatives) from 10 families with FH. Nine propositi were carriers of the FH-Helsinki deletion of the low density lipoprotein (LDL) receptor gene, prevalent in the Finnish population, while a new deletion, extending from intron 14 to intron 15 of the LDL receptor gene, was identified in one family. Serum LDL cholesterol levels used in the clinical diagnosis (less than 5.0 mmol/l, not FH; 5.0-5.9 mmol/l, possible FH; greater than or equal to 6.0 mmol/l, FH; limits are 1 mmol/l lower for those less than 18 years) were derived from an authoritative recommendation. Tendon xanthomas constituted an additional criterion. With the DNA analysis as the reference, 55 (85%) subjects could be correctly classified clinically as FH patients or subjects without FH. The remaining 10 subjects were misclassified or were in the "possible FH" category. When the age- and sex-specific 95th percentile LDL cholesterol levels were used instead of the rigid values for both adults and children, the percentage of correct diagnoses rose to 95%. Common genetic polymorphisms of apolipoproteins E and B did not markedly affect LDL cholesterol levels in FH patients, whereas increasing age and obesity were associated with elevated LDL levels. In conclusion, DNA analysis is a valuable adjunct to the diagnosis of FH that is applicable to families with a known mutation of the LDL receptor gene. If DNA methods are not available, age- and sex-specific LDL levels should be used as an aid in the clinical diagnosis of FH.

Prevalence and geographical distribution of major LDL receptor gene rearrangements in Finland.

In order to determine the prevalence of major rearrangements of the low density lipoprotein (LDL) receptor gene in Finland, DNA samples of 199 unrelated Finnish patients with the heterozygous form of familial hypercholesterolaemia (FH) were examined by Southern blot analysis. The FH-Helsinki mutation, characterized by a 9.5-kb deletion in the 3'-end of the LDL receptor gene, was found in 75 (38%) of the patients. The prevalence of this mutation ranged from 26-58% in different areas of Finland. A striking exception was the North Karelia region, where only one out of 26 (4%) FH patients was found to carry the FH-Helsinki allele. Two patients were found to carry other types of large nucleotide rearrangements of the LDL receptor gene. One mutation was a 7.5-kb deletion eliminating exons 7 to 10, and the other was a 13-kb deletion covering exons 11 to 16 of the LDL receptor gene. Serum lipoprotein levels were very similar in each category of mutation, i.e. in patients with the FH-Helsinki gene, those with the two other types of deletion, and the remaining patients with as yet unknown types of LDL receptor gene defects. These results show that, even in genetically uniform populations, FH may be heterogeneous at the DNA level. DNA techniques enable an unequivocal diagnosis for almost 40% of the Finnish patients with the heterozygous form of FH.

Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture.

The regulation by glucose and insulin of the muscle-specific facilitative glucose transport system GLUT-4 was investigated in L6 muscle cells in culture. Hexose transport activity, mRNA expression, and the subcellular localization of the GLUT-4 protein were analyzed. As observed previously (Walker, P. S., Ramlal, T., Sarabia, V., Koivisto, U.-M., Bilan, P. J., Pessin, J. E., and Klip, A. (1990) J. Biol. Chem. 265, 1516-1523), 24 h of glucose starvation and 24 h of insulin treatment each increase glucose transport activity severalfold. Here we report a differential regulation of the GLUT-4 and GLUT-1 transport systems under these conditions. (a) The level of GLUT-4 mRNA was not affected by glucose starvation and was diminished by prolonged (24 h) administration of insulin; in contrast, the level of GLUT-1 mRNA was elevated under both conditions. (b) Glucose starvation and prolonged insulin administration increased the amount of both GLUT-4 and GLUT-1 proteins in the plasma membrane. (c) In intracellular membranes, glucose starvation elevated, and prolonged insulin administration reduced, the GLUT-4 protein content. In contrast, the GLUT-1 protein content in these membranes decreased with glucose starvation and increased with insulin treatment. Glucose transport was rapidly curbed upon refeeding glucose to glucose-starved cells, with half-maximal reversal after 30 min and maximal reversal after 4 h. This was followed by a marked decrease in the levels of GLUT-1 mRNA without major changes in GLUT-4 mRNA. Neither 2-deoxy-D-glucose nor 3-O-methyl-D-glucose could substitute for D-glucose in these effects. It is proposed that glucose and insulin differentially regulate the two glucose transport systems in L6 muscle cells and that the rapid down-regulation of hexose transport activity by glucose is regulated by post-translational mechanisms.

Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription.

Chronic (24 h) insulin treatment and/or glucose deprivation of differentiated rat L6 skeletal muscle cells resulted in an increase in glucose transport activity and a 2-3-fold increase in the number of plasma membrane-associated cytochalasin B binding sites and immunoreactive glucose transporters. In contrast to the acute effect of insulin, chronic treatment did not decrease the number of cytochalasin B binding sites or immunoreactive glucose transporter proteins present in intracellular low density microsomes. Although acute insulin stimulation of glucose transport activity was not affected by cycloheximide, chronic insulin stimulation of glucose transport activity and glucose transporter protein were decreased. In contrast, the stimulation of glucose transport activity by both acute and chronic glucose deprivation were cycloheximide-insensitive. Previously we have reported that chronic insulin treatment transiently induces the rat brain/HepG2 glucose transporter subtype (GLUT-1) mRNA, whereas glucose deprivation induces a substained increase (Walker, P. S., Ramlal, T., Donovan, J. A., Doering, T. P., Sandra, A., Klip, A., and Pessin, J. E. (1989) J. Biol. Chem. 264, 6587-6595). Consistent with these data, nuclear run-on analysis demonstrated a transient 3-fold increase in the rate of GLUT-1 glucose transporter mRNA transcription induced by either chronic insulin treatment or glucose deprivation. The combination of chronic insulin treatment with glucose deprivation resulted in a more persistent 3-4-fold increase in transcription rate than either treatment alone. These data demonstrate that prolonged insulin- and glucose-dependent regulation of glucose transporter function occurs by a complex mechanism which includes enhanced GLUT-1 mRNA transcription and glucose transporter synthesis, as well as changes in the subcellular distribution of glucose transporter proteins.

Stimulation of Na+/H+ exchange by insulin and phorbol ester during differentiation of 3T3-L1 cells. Relation to hexose uptake.

Acute exposure of 3T3-L1 undifferentiated fibroblasts to insulin or 4 beta-phorbol-12,13-dibutyrate (PDB) produced a moderate but significant stimulation of hexose transport (100% stimulation). In differentiated 3T3-L1 adipocytes, stimulation by insulin increased significantly (to 340%), while that by PDB remained at 130%. Total protein kinase C activity was 3-fold higher in 3T3-L1 fibroblast than adipocyte homogenates. PDB, but not insulin, induced migration of protein kinase C from the cytosol to the membrane, in both fibroblasts and adipocytes. Moreover, the hormone increased by 15% the protein kinase C activity of the cytosol. In 3T3-L1 fibroblasts, both insulin and PDB elicited a rapid (2 min lag) cytoplasmic alkalinization, measured with the fluorescent pH indicator bis-carboxyethyl carboxyfluorescein trapped in the cytoplasm. In 3T3-L1 adipocytes, PDB but not insulin elicited the cytoplasmic alkalinization. The alkalinization was prevented by amiloride or by replacing Na+ with either N-methylglucamine+ or K+. Stimulation of hexose transport by insulin or PDB was not affected by amiloride or Na+ substitution. It is concluded that: 1) Insulin and PDB have different effects on protein kinase C activity and subcellular distribution; 2) the responses of Na+/H+ exchange and hexose transport to insulin and PDB develop independently during differentiation of 3T3-L1 cells; 3) stimulation of Na+/H+ exchange and of hexose transport occur in parallel rather than in series in 3T3-L1 cells.