MOLECULAR GENETICS OF FAMILIAL HYPERCHOLESTEROLEMIA

MOLECULAR GENETICS OF FAMILIAL HYPERCHOLESTEROLEMIA

INTRODUCTION

More than fifty years years ago first reports of an inherited disease with cholesterol clustering in tendons and the presence of coronary heart disease at an early age were reported1. This

disease, called familial hypercholesterolemia (FH), has been found to be a single-gene, autosomally dominantly inherited disease2. The cause of FH has been demonstrated to be a defect in the amount or functioning of low density lipoprotein (LDL) receptors2. The function of these receptors is to carry cholesterol-rich particles from the bloodstream into hepatocytes and peripheral cells for synthesis of cell membranes and steroid hormones3.

FH is characterized by a gene dosage effect2: those who are carrying one mutant and one normal LDL receptor allele have the milder (heterozygous) form of the disease, whereas those with two mutant LDL receptor alleles (the homozygotes) have a more severe form of the disease with the onset of cardiovascular symptoms already in the childhood. Serum LDL cholesterol concentration is elevated from birth on. Compared to the normal level, it is 2-3 times higher in heterozygotes and 5-8 times higher in homozygotes. Clinical manifestations of the disease include cholesterol accumulation in extensor tendons and arterial wall and, most importantly, a greatly increased risk of myocardial infarction. Typically, heterozygotes get their first myocardial infarctions at the age of 40-50 years and homozygotes during their two first decades of life.

At the cellular level FH was found to be a heterogeneous disease and the existence of four different types of defective LDL receptor alleles were demonstrated: null alleles, transport deficient allelles, binding-deficient aleles and internalization-defective alleles2, 3. This favored the idea that multiple types of DNA alterations would ultimately be detected to explain the heterogeneity of FH.

STRUCTURE OF THE NORMAL LDL RECEPTOR AND ITS GENE

The LDL receptor gene is located on chromosome 19 and comprises about 45 000 base pairs4. The gene contains 18 exons, 13 of which share homology to other known genes.

Molecular cloning of eDNA for the LDL receptor revealed that the LDL receptor mANA is about 5 300 nucleotides in length and the LDL receptor contains five functional domains5. The first domain is negatively charged with a number of loop-forming cysteine residues. The function of this domain is to bind the positively charged ligand (apolipoprotein B of the LDL particle). The second domain, homologous to epidermal growth factor precursor, has been found to be essential for the normal recycling of the receptors and for the normal dissociation of the receptor from its ligand at the acidic pH in an endosome6. The third domain contains multiple carbohydrate chains and is proposed to increase the stability of LDL receptors?. The fourth domain contains 22 hydrophobic amino acids and spans the cell membrane anchoring the receptor to the cell. The fifth domain, the cytoplasmic part of the receptor, is needed for the clustering of the LDL receptors into coated pits along the cell membrane and for the normal internalization of the receptors into cells.

MOLECULAR BASIS OF FH

A vast number of mutations of the LDL receptor gene, ranging from single base changes to large deletions or insertions, have been reported2, 3. Only 2-6% of these mutations have been found to involve major gene rearrangements that are readily detectable by routine Southern blot analysis8, 9. Some of the mutations have been found to result in the production of mRNA molecules with an altered molecular size10. The LDL receptor has been shown to contain a large number of DNA polymorphisms. These apparently harmless alterations in functional terms provide a valuable tool to examine the inheritance of FH in affected families 11.

ENRICHMENT OF SPECIFIC LDL RECEPTOR GENE MUTATIONS IN GENETICALLY HOMOGENEOUS POPULATIONS

In most populations, the frequency of the heterozygous form of FH is approximately 1 in 500 whereas only 1 in 1 million suffer from the homozygous form of the disease2. However, there are

several examples of populations in which FH has been enriched by an apparent founder gene effect, including the Lebanese, French Canadians and South Africans. In each case, one or two mutations alone seem to explain the increased prevalence of the disease. A fourth example of populations with a characteristic LDL receptor mutation is Finland.

The Finnish population forms a genetic isolate with a curious panel of inherited diseases and a virtual lack of genetic diseases common in other parts of the world, such as cystic fibrosis and phenylketonuria 12. When DNA samples from Finnish patients with FH were examined, about 40% of them were found to carry the same type of LDL receptor mutation13, 14. Subsequent to the cloning of a portion of the mutant receptor gene and the corresponding eDNA 14, 15, the mutation was defined as a 9.5 kb deletion in the 3' coding region of the LDL receptor gene. The deletion involves exons 16 and 17 and a part of exon 18 (Fig. 1). These exons normally encode the carboxy terminus, i.e. the transmembrane and the cytoplasmic domains of the receptor. The carboxyterminal part of the mutant LDL receptor is encoded by 163 bases at the 5' end of intron 15 and thus shares no homology to the corresponding domain of the normal LDL receptor 14. LDL binding studies with fibroblasts from FH patients with this gene deletion revealed anint ernalization-defective phenotype of FH14. When lipid and lipoprotein levels were compared in FH patients carrying the deleted allele and those Finnish FH patients with yet an unknown LDL receptor mutation, no significant differencies were found14. This mutation of the LDL receptor gene, very common in the Finnish population and designated as FH-Helsinki according to the residence of the first proband examined in detail, has not been reported in other populations. In the lack of exact epidemiological studies we do not know yet whether the enrichment of the FH­ Helsinki allele in the Finnish population also signifies an increased prevalence of FH in Finland.

In Lebanon the frequency of FH is high, i.e. about one patient in 100 subjects. Molecular genetic studies of the LDL receptor alleles from Lebanese FH patients revealed that more than 90% of these patients carry an identical LDL receptor mutation16. This mutation is characterized by a single nucleotide change at the codon for amino acid 660, causing the appearance of a premature inframe stop codon (Fig. 1). The resulted truncated LDL receptor is thus normal up to the last portion of the domain homologous to the epidermal growth factor precursor. This mutant receptor is not matured normally in the Golgi complex but it is degraded rapidly intracellularly.

A specific mutation of the LDL receptor gene explaines about two-thirds of FH cases in the . French speaking population around Montreal area17, 18. This mutation is characterized by a large deletion of more than 10 kb in size eliminating the promotor region and the first exon of the LDL receptor gene (Fig. 1). Apparently this mutant gene is not transcribed at all and accordingly patients homozygotes for this type of LDL receptor mutation do not synthesize any LDL receptors. The other French Canadian mutations (one deletion and three point mutations) characterized so far account together less than 20% of the defective LDL receptor alleles in this population. The frequency of FH has been estimated to be two or three times higher among French Canadians than in an average Caucasian population18.

Similar to Lebanon, the prevalence of FH is very high among South African Africaners 19. Two LDL receptor mutations alone explain the presence of FH in about 95% of the South African FH patients19. Both mutations involve single nucleotide alterations causing an inframe amino acid change. One mutation affects the codon for amino acid 206 causing a substitution of glutamine for asparagine (Fig. 1). The change of the amino acid at position 206 impairs the maturation of the resultant LDL receptors which are rapidly degraded. Previous studies suggest that abnormal number or spacing of the cysteine residues may affect the normal maturation of LDL receptor20-22. It appears, however, that the South African LDL receptor mutation does not disturb the normal number or spacing of the amino acids in the first domain. The other type of South African mutation affects the codon for amino acid 408, resulting in a substitution of methionine

for valine (Fig. 1). Amino acid 408 is located in the domain homologous to the epidermal growth factor precursor which is known to be essential for the normal dissociation of the receptor from

its ligand at the acidic pH in an endosome6. With disturbance of this dissociation the half-life of this mutant receptor is shorter than that of the normal one.

MOLECULAR BIOLOGY OF THE LDL RECEPTOR: THEORETICAL AND PRACTICAL IMPLICATIONS

Detailed characterization of the structure of the LDL receptor gene has greatly increased our understanding, not only on fundamentals of lipoprotein metabolism in particular, but also on the functioning of cell membrane receptors in general. On the basis of the pioneering studies of Brown, Goldstein and collaborators it came as no surprise that FH proved to be heterogeneous at the DNA level. Patients with FH seem to comprise an almost endless repertoir of living laboratories: abnormal structures combined with defects in function often permit strong reasoning of the essentials of the normal structure. Today, DNA techniques offer definite diagnostic tools for FH only in a few selected populations discussed above. It may be anticipated, however, that the rapidly progressing technology based on DNA amplification by polymerase chain reaction (PCR) as well as automated DNA sequencing will eventually provide means for molecular diagnosis of most, if not all, patients with FH.

REFERENCES

1. C. Muller, Xanthomata, hypercholesterolemia, angina pectoris. Acta Med. Scand. (Suppl.) 89:75, (1938).

2. J. L. Goldstein, and M.S. Brown, Familial hypercholesterolemia. j!}: 'The Metabolic Basis of Inherited Diseases,' J. B. Stanbury, J. B. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown, eds. McGraw-Hill Book Co, New York (1983).

3. M. S. Brown, and J. L. Goldstein, A receptor mediated pathway for cholesterol homeostasis. Science 232: 34 (1986).

4. T. C. Sudhof, J. L. Goldstein, M. S. Brown, and D. W. Russell, The LDL receptor gene: a mosaic of exons shared with different proteins. Science 228: 815 (1985).

5. T. Yamamoto, C. G. Davis, M. S. Brown, W. J. Schneider, M. L. Casey, J. L. Goldstein, and D. W. Russell, The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mANA. Cell 39: 27 (1984).

6. C. G. Davis, J. L. Goldstein, T. C. Sudhof, R. G. W. Anderson, D. W. Russell, and M. S.

Brown, Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 326: 760 (1987).

7. K. Kajinami, H. Mabuchi, H. ltoh, I. Michishita, M. Takeda, T. Wakasugi, J. Koizumi, and R.

Takeda, New variant of low density lipoprotein receptor gene: FH-Tonami. Arteriosclerosis 8: 187 (1988).

8. B. Horsthemke, A. Dunning, and S. Humphries, Identification of deletions in the human low density lipoprotein receptor gene. J. Med. Genet 24: 144 (1987).

9. S. Langlois, J. J. P. Kastelein, and M. R. Hayden, Characterization of six partial deletions in the low-density-lipoprotein (LDL) receptor gene causing familial hypercholesterolemia (FH). Am. J. Hum. Genet. 43: 60 (1988).

10. H. H. Hobbs, E. Leitersdorf, J. L. Goldstein, M. S. Brown, and D. W. Russell, Multiple crm­ mutations in familial hypercholesterolemia: evidence for 13 alleles including four deletions. J. Clin. Invest. 81: 909 (1988).

11. E. Leitersdorf, A. Chakravarti, and H. H. Hobbs, Polymorphic DNA haplotypes at the LDL receptor locus. Am. J. Hum. Genet. 44: 409 (1989).

12. R. Norio, H. R. Nevanlinna, and J. Perheentupa, Hereditary diseases in Finland; rare flora in rare soil. Ann. Clin. Res. 5: 109 (1973).

13. K. Aalto-Setala, H. Gylling, T. Miettinen, and K. Kontula, Identification of a deletion in the LDL receptor gene: a Finnish type of mutation. FEBS Lett. 230:31 (1988).

14. K. Aalto-Setala, E. Helve, P. T. Kovanen, and K Kontula, The Finnish type of LDL receptor gene mutation (FH-HelsinkO deletes exons encoding the carboxy-terminal part of the receptor and creates an internalization-defective phenotype. J. Clin. Invest. 84:499 (1989).

15. K Aalto-Setala, The Finnish type of the LDL receptor gene mutation: molecular characterization of the deleted gene and the corresponding mANA. FEBS Lett. 234: 411 (1988).

16. M. A. Lehrman, W. J. Schneider, M. S. Brown, C. G. Davis, A. Elhammer, D. W. Russell, and J. L. Goldstein, The Lebanese allele at the low density lipoprotein receptor locus: nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum. J. Bioi. Chern. 262:401 (1987).

17. H. H. Hobbs, M.S. Brown, D. W. Russell, J. Davignon, and J. L. Goldstein, Deletion in the gene for the low-density-lipoprotein receptor in a majority of French Canadians with familial hypercholesterolemia. N. Engl. J. Med. 317: 734 (1987).

18. E. Leitersdorf, E. J. Tobin, J. Davignon, and H. H. Hobbs, Common low-density lipoprotein receptor mutations in the Freilch Canadian population. J. Clin. Invest. 85: 1014 (1990).

19. E. Leitersdorf, D. R. Van ber Westhuyzen, C. A. Coetzee, and H. H. Hobbs, Two common low density lipoprotein receptor gene mutations cause familial hypercholesterolemia in Africaners. Clin. Invest. 84: 954 (1989).

20. T. Yamamoto, R. W. Bishop, M. S. Brown, J. L. Goldstein, and D. W. Russell, Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. Science 232: 1230 (1986).

21. V. Esser, and D. W. Russell, Transport-deficient mutations in the low density lipoprotein receptor: alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellular transport. J. Bioi. Chern. 263: 13276 (1988).

22. E. Leitersdorf, H. H. Hobbs, A. M. Fourie, M. Jacobs, D. R. Van Der Westhuyzen, and G. A.

Coetzee, Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia. Proc. Natl. Acad. Sci USA 85: 7912 (1988).