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.