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Two mutations in the same low-density lipoprotein receptor allele act in synergy to reduce receptor function in
H K Jensen1, T G Jensen, O Faergeman
1Center for Medical Molecular Biology, Skejby Sygehus University Hospital, Arhus, Denmark.
Abstract:
Mutations in genes are not necessarily pathogenic. Expression of mutant genes in cells can therefore be required to demonstrate that mutations in fact disturb protein function. This applies especially to missense mutations, which cause an amino acid to be replaced by another amino acid. In the present study of two families with familial hypercholesterolemia in the heterozygous form, we found two mutations in the same allele of the low-density lipoprotein (LDL) receptor gene: a missense Asn543. His mutation (N543H) in exon 11, and an in-frame 9-bp deletion (2393del9) in exon 17. The two mutations were identified in heterozygous FH index patients in whom no other pathogenic mutations were detected by SSCP analysis of the remaining 16 exons and the promoter region. Both mutations cosegregated with hypercholesterolemia within the families. Each of these mutations had little or no effect on receptor function in transfected COS cells, but when both mutations were present simultaneously, receptor function, as assessed by flow cytometric measurement of fluorescent LDL uptake in cells, was reduced by 75%. Immunostainable receptors on the cell surface were decreased by 80% as measured by flow cytometry. The two mutations therefore acted in synergy to affect receptor function, possibly during intracellular receptor transport, since Northern blot analysis suggested that mRNA levels were unaffected. Without screening of the entire coding regions of the gene, the synergistic action of these two LDL receptor mutations would not have been detected.
Insights
Two low-density lipoprotein (LDL) receptor gene mutations, individually mild, synergistically caused severe familial hypercholesterolemia by impairing receptor function. This highlights the need for comprehensive genetic screening to detect combined mutation effects.
Area of Science:
- Genetics
- Molecular Biology
- Cardiovascular Disease
Background:
- Genetic mutations do not always cause disease; functional studies are crucial.
- Missense mutations alter protein sequences, and their pathogenicity requires experimental validation.
- Familial hypercholesterolemia (FH) is a genetic disorder characterized by high LDL cholesterol levels.
Purpose of the Study:
- To investigate the functional impact of two novel mutations in the low-density lipoprotein (LDL) receptor gene found in heterozygous FH patients.
- To determine if these mutations, individually or combined, affect LDL receptor function and contribute to hypercholesterolemia.
Main Methods:
- Identified two mutations (N543H missense, 2393del9 in-frame deletion) in the LDL receptor gene in FH families.
- Expressed mutant LDL receptors in COS cells and assessed function via fluorescent LDL uptake using flow cytometry.
- Measured cell surface receptor levels using flow cytometry and analyzed mRNA levels using Northern blot.
Main Results:
- Each mutation alone had minimal impact on LDL receptor function and cell surface expression.
- The simultaneous presence of both mutations significantly reduced LDL receptor function by 75% and cell surface receptors by 80%.
- Mutations acted synergistically, likely affecting intracellular receptor transport, with unaffected mRNA levels.
Conclusions:
- The identified N543H and 2393del9 mutations in the LDL receptor gene exhibit synergistic effects on receptor function.
- Comprehensive genetic screening of entire coding regions is essential for detecting such synergistic mutations in familial hypercholesterolemia.
- These findings underscore the complexity of genotype-phenotype correlations in genetic disorders.