Two common founder-related gene mutations that affect the low-density lipoprotein receptor (LDLR) are responsible for -80% of familial hypercholesterolemia (FH) in South African Afrikaners. The FH Afrikaner-1 (FHl) mutation (Asp^-»Glu) in exon 4 results in defective receptors with =20% of normal activity, whereas the FH Afrikaner-2 (FH2) mutation (VaUog-^Met) in exon 9 completely abolishes LDLR activity (<2% normal activity). We analyzed the contribution of these mutations and other factors on the variation of hypercholesterolemia and clinical features in Afrikaner FH heterozygotes. The type of FH mutation, plasma triglyceride levels, and age of patients each contributed significantly to the variation in hypercholesterolemia, whereas smoking status, high-density lipoprotein cholesterol levels, and gender had no influence. Although all FH heterozygotes had frank hypercholesterolemia, patients with the FHl mutation had significantly lower cholesterol levels than those with the FH2 mutation. FHl heterozygotes also tended to have milder clinical features. The differences between the two FH groups could not be explained by a difference in the common apolipoprotein E variants. This study demonstrates that mutational heterogeneity in the LDLR gene influences the phenotypic expression of heterozygous FH. autosomal dominant disease caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Characteristic phenotypic features of FH are raised low-density lipoprotein (LDL) cholesterol (C) levels, the presence of tendon xanthomata, and the premature development of coronary heart disease (CHD). These features are more striking and their age of onset earlier in FH homozygotes who inherit two mutant LDLR gene alleles. The mutational heterogeneity of FH explains most of the phenotypic variation found among FH homozygotes, in whom a strong correlation is found between residual receptor activity and the severity of the disease. The founder-related basis for the high prevalence of FH in the Afrikaans-speaking white population of South Africa (Afrikaners) enabled us to study the degree of phenotypic variability caused by different LDLR gene mutations. Two of the three founder mutations identified in the Afrikaner population, which together account for =80% of FH in Afrikaners, 37 have different effects on the cellular expression of the LDLR. The FH Afrikaner-1 (FHl) mutation (Asp^ to Glu; GAC to GAG) in exon 4 of the LDLR gene results in the formation of functionally distinct forms of the mutant receptor; one form exhibits normal receptor activity, whereas another is unable to bind lipoprotein ligands.8 ' 9 As a result, fully upregulated cells homozygous for this mutation express about 20% of normal receptor activity. The FH Afrikaner-2 (FH2) mutation (VaLwe to Met; GTG to ATG) in exon 9 causes the receptor to be rapidly degraded and results in very low receptor activity (<2% of normal receptor activity).

Three different point mutations were recently identified in South African familial hypercholesterolaemics. These mutations result in the modification of recognition sites of specific restriction endonucleases. This study describes rapid methods for presymptomatic detection of these defects based on restriction enzyme analysis or allele-specific hybridization of enzymatically amplified genomic DNA. These methods were used to determine the frequencies of the three known low-density lipoprotein (LDL) receptor gene mutations in 138 chromosomes of Afrikaner FH patients. It has been shown that a common mutation at the 3' end of exon 4 (base 681) of the LDL receptor gene is present in about 70% of alleles, while the mutations in exons 9 (base 1285) and 4 (base 523) of the gene are present in about 20 and 10% respectively of the genes studied. These mutations were found in approximately 95% of Afrikaner familial hypercholesterolaemic patients studied, indicating at least three founder members for the disease in this population of South Africa.

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We have developed a rapid, nonradioactive screening test enabling the simultaneous analysis of three lowdensity Iipoprotein receptor (LDLR) gene mutations (DIS4N, D206E, and V408M), which together account for familial hypercholesterolemla (FH) in -90% of the South African Afrikaner population. The assay is designed so that FH patients, negative for these founder-related mutations (found in descendants of European settlers), subsequently can be screened for unknown mutations in the mutation-rich exon 4 of the LDLR gene. Our screening assay consists of two steps: (1) multiplex allele-specific PCR amplification of exons 4 and 9, and (2) simultaneous analysis of single-and double-strand conformational polymorphisms in exon 4 by vertical electrophoresis on low crosslinked polyacrylamide gels. The simplicity, specificity, and versatility of the multiplex assay makes it an ideal system for routine screening of FH mutations in large population samples.

SOURCE AND DESCRIPTION OF CLONE: pLDLR-2HH1, a 1.9kb fragment of the 3'end of the LDL receptor c-DNA clone (Yamamoto et al, 1984). POLYMORPHISM: NcoI (CCATGG) identifies invariant bands of 9kb and 7kb, in addition to a simple two allele polymorphisun with a band at either 3.4kb (Ni) or 13kb (N2) (Figure).