'n Ondersoek na die hiperlipidemie-veroorsakende mutasies en hul geassosieerde haplotipes in Suid-Afrikaanse hiperlipidemiese kleurlinge

[摘要] ENGLISH ABSTRACT: Coronary heart disease (CHD), common among South African whites, isincreasingly manifesting itself in the coloured population of South Africa.More than 70% of coloureds participating in the CRISIC I study, a studyundertaken to investigate CHD risk factors in the coloureds of the CapePeninsula, had serum cholesterol levels imparting CHD risk.Increased levels of plasma low-density lipoprotein cholesterol (LDL-C) are thehallmark of the disease familial hypercholesterolemia (FH). The underlyingmolecular defect of FH consists of mutations in the low-density lipoprotein(LDL) receptor gene.The coloured study population was screened for the presence of the threefounder related point mutations in the LDL receptor gene that account formore than 90% of FH cases in the Afrikaner population of South Africa. Theprevalence of the FH Afrikaner mutations was determined in 31 FHheterozygotes and 39 hyperlipidemic coloureds. The 39 hyperlipidemiccoloureds did not meet the criteria for heterozygous FH. The FH Afrikaner-1 (Asp₂₀₆→Glu , FH-1) en FH-Afrikaner-2 (Val₄₀₈→MetFH-2) mutations hadthe same prevalence, 12.9% (4 individuals), in the FH group. The FHAfrikaner-3 (Asp₁₅₄→Asn, FH-3) mutation was absent in the FH group. The three FH Afrikaner mutations were not detected in the hyperlipidemic group of39 individuals. Haplotype analysis in the families with the FH-1 mutationshowed that the chromosomal background of the mutation was compatible with that described in the Afrikaner patients. In the FH-2 heterozygouspatients the mutation was found on a haplotype that differed from theAfrikaner haplotype at polymorphic sites at both sides of the defect in exon 9.These results indicate that identical LDL-receptor gene mutations originatedin two different South African population groups due to independent events ata potential CpG dinucleotide hotspot. The clinical effects of the FH-1 andFH-2 mutations were the same as described in the Afrikaners.Heteroduplex analysis detected a 3 bp deletion that causes deletion of aminoacid Gly₁₉₇ (FH Lithuania) in one of 23 FH heterozygous coloured patients(4.3 %) and in one indiviaual of 90 hyperlipidemic coloureds (1.1%) alreadyscreened for the FH Afrikaner mutations , by means of heteroduplex analysis.A hyperlipidemic group of 66 coloureds, screened for the first time formutations in exon 4 of the LDL-receptor gene by means of heteroduplexanalysis, showed one individual (1.5%) with the FH Lithuania mutation. In allcases, the haplotype of the mutant allele was compatible with that describedin Ashkenazi Jews. The heteroduplex screening method was sensitiveenough to detect the FH-1 mutation, as 5 individuals of the group of 66hyperlipidemic coloureds were found to have the FH-1 mutation.A total of 22 FH heterozygous coloured patients, negative for the abovementioned mutations, were screened for new mutations in exon 4 of the LDL-receptorgene by means of the single-strand-conformation-polymorphism(SSCP) technique and the heteroduplex method. A new mutation, hithertonot described, was detected in one patient by means of the SSCP technique. A point mutation (A to C) at nucleotide 671 in exon 4 of the LDL receptorcDNA results in an amino acid change from aspartic acid to alanine at residue203 in the cysteine rich ligand binding domain of the LDL receptor. Themutation gives rise to an additional Hae Ill restriction site in the DNA ofaffected subjects. The accurate diagnosis of this so-called FH Tygerbergmutation in subjects with FH is possible now by means of the amplification ofgenomic DNA (using the polymerase chain reaction) and restriction enzymeanalysis.The allele frequencies of four restriction-fragment-length-polymorphisms(RFLP's) in the LDL-receptor gene were determined for 17 FH heterozygouspatients and 21 control individuals. Statistical analysis indicated a significantrise in the frequency of the least frequent allele of the Ava 11 polymorphism (x2= 7.14, P<0.01). This indicates population association of the allele with agene that causes FH. The heterozygosities and PIC values of each RFLPsite were determined, and these values disclosed that the Sma I site would bethe most informative in genetic linkage studies, followed by the Ava II andNco I sites. These three sites were all moderately polymorphic. The Stu I site was the least polymorphic and therefore would be the least informative ofthe four RFLP sites in genetic studies.A 478 bp deletion in exon 18 of the LDL-receptor gene was detected in 2 FHheterozygous coloured patients. One of the two patients was heterozygousfor the FH-1 mutation. This deletion is most likely a polymorphism as it was detected in the 3'-nontranslated region of the LDL-receptor gene andtherefore it may be used in genetic marker analysis.This study suggests that the moderately informative RFLP sites (Sma I, Ava IIand Nco I) must be combined with the TA dinucleotide repeat locus, alsomoderately informative, to indirectly diagnose FH in families where the directmolecular diagnosis of FH, by means of the detection of the FH associatedmutations, is not possible.A better molecular diagnostic service is now possible for the coloureds with ahigh risk for CHD by combining the screening for FH associated mutationswith RFLP and dinucleotide repeat polymorphism studies.

[发布机构] Stellenbosch University