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Published on: September 15, 2018
A de novo LDLR mutation in severe familial hypercholesterolemia: case report, functional characterization, and a
Shuran Zhang1,2, Wenming Huang1, Haoqiang Chen1
1Medical Genetics Department of The First People's Hospital of Yunnan Province/Affiliated Hospital of Kunming University of Science and Technology, Key Laboratory Reproductive Health and Birth Defects Prevention and Control in Western China, National Health Commission, Key Laboratory of Birth Defects and Genetic Diseases of Yan Province, Kunming, Yunnan, China.
Insights
A novel familial hypercholesterolemia (FH) mutation, LDLR c.331C>T, impairs LDLR protein expression. Prime editing technology precisely corrected this mutation in vitro, offering a potential cure for homozygous FH (HoFH).
Area of Science:
- Genetics
- Molecular Biology
- Cardiovascular Disease Research
Background:
- Familial hypercholesterolemia (FH) is a genetic lipid metabolism disorder causing cardiovascular disease (CVD).
- Mutations in the LDLR gene are the primary cause of FH, with no effective treatments for homozygous FH (HoFH).
- Many FH mutations remain unvalidated, lacking gene correction studies.
Purpose of the Study:
- To assess the pathogenicity of the novel LDLR c.331C>T (p.Gln111Ter) mutation.
- To explore gene correction strategies for this FH-associated mutation.
Main Methods:
- Systematic evaluation of an FH patient and family.
- Construction of a Huh7 cell line with the LDLR c.331C>T mutation using CRISPR/Cas9.
- Validation of mutation impact on LDLR protein expression via qPCR, Western blot, and immunofluorescence.
- Development of a prime editing (PE) system for precise gene correction.
Main Results:
- The HoFH patient presented with biallelic LDLR mutations, including a de novo LDLR c.331C>T.
- In vitro studies confirmed the mutation impairs LDLR protein expression.
- The prime editing system achieved approximately 98% correction efficiency for the LDLR c.331C>T mutation.
Conclusions:
- LDLR c.331C>T is identified as a likely pathogenic mutation.
- Prime editing technology offers precise correction for this mutation.
- This research expands the spectrum of pathogenic FH mutations and shows promise for personalized gene therapy for HoFH.
Background:
Familial hypercholesterolemia (FH) is a genetic disorder of lipid metabolism characterized by elevated plasma low-density lipoprotein resulting in cardiovascular disease (CVD). The harmful mutations of LDLR are the main cause of FH. Especially, there is no effective treatment options for homozygous FH (HoFH) patients. Numerous FH cases have been reported, but most mutations remain unvalidated and lack gene correction studies. The study aims to assess the pathogenicity of a novel mutation, LDLR c.331C>T (p.Gln111Ter), and seek its gene correction strategy.
Methods:
The study systematically evaluated a female HoFH patient and her family. Using CRISPR/Cas9 technology, a Huh7 cell line carrying the point mutation was constructed. The impact of this mutation on LDLR protein expression was confirmed by qPCR, Western blot (WB), and immunofluorescence. A high-fidelity gene correction system targeting the LDLR c.331C>T point mutation was established based on the prime editing (PE) technology.
Results:
The HoFH patient exhibited a biallelic LDLR mutation comprising an LDLR c.1693_1696 del GGCA inherited from her mather and a de novo LDLR c.331C>T (p.Gln111Ter) mutation. In vitro validation indicated that the mutation impaired normal LDLR protein expression, and the candidate gene editing system achieved approximately 98% correction efficiency.
Conclusion:
LDLR c.331C>T is a likely pathogenic mutation, which canbe precisely corrected by PE technology. The study expands the spectrum of likely pathogenic mutations in FH and holds promise for personalized, precise gene therapy through customized therapeutic systems, potentially alleviating or curing HoFH-a current challenge in conventional clinical management.
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