Identification of c.146G > A mutation in a Fabry patient and its correction by customized Cas9 base editors in vitro

Int J Biol Macromol. 2024 Dec;282(Pt 3):136922.

PMID: 27473102

Zhi Yang, Hao Li, Mei Luo, Haonan Yi, Xinyu Han, Enze Liu, Shaohua Yao, Zhangxue Hu

Highlights: This study identifies a significant mutation (c.146G>A) in exon 1 of the GLA gene in a female Fabry disease patient. Bioinformatic predictions and biochemical analyses revealed that this variant significantly reduced the stability and activity of α-galactosidase A, confirming its pathogenicity. Using an adenine base-editing technique, the mutation was corrected in vitro. The study not only identified a novel variant but also demonstrated the potential of the CRISPR/Cas9 system.

Background: Fabry disease is a rare disorder caused by mutations in the GLA gene, leading to reduced α-galactosidase A activity. It is an X-linked lysosomal storage disorder with an estimated prevalence of 1 in 100,000. Approximately 0.12% of patients develop end-stage renal disease. The primary pathogenic mechanism involves mutations in the GLA gene, which play a critical role in the disease process and are important for developing treatment strategies.
Clinically, patients experience multi-organ damage, with severe cases resulting in cardiovascular complications and end-stage renal disease. The disease often begins in childhood, progresses with age, and significantly reduces quality of life. As a primary treatment, enzyme replacement therapy (ERT) with agalsidase α or β has been effective for many patients. However, its high cost and the need for lifelong injections reduce its feasibility. Long-term ERT can also lead to the development of anti-ERT antibodies.
Recently, genome-editing tools like CRISPR-Cas9 have revolutionized biomedical research, showing great promise for genetic diseases. Base editors, containing Cas9 nickase and cytosine or adenine deaminases, enable base changes and are particularly useful for genetic diseases caused by point mutations.
Objective and methods: The study used the patented Therapeutic Performance Mapping System (TPHS), which integrates systems biology, machine learning, and pattern recognition techniques. A comprehensive literature review was conducted through PubMed and Medline to identify candidate proteins. Additionally, gene expression data were gathered from the NCBI Gene Expression Omnibus, ArrayExpress, and Omics Discovery Index databases. Statistically significant genes were identified and mapped to candidate proteins. Findings were mathematically modeled using the TPHS system. Candidate proteins were filtered using various statistical methods, and the final candidates were analyzed using the Human Protein Atlas database to identify proteins measurable in urine or blood.

Material and Methods: In this study, the c.146G>A mutation was identified in a female patient. The pathogenicity of the mutation was determined using bioinformatics and biochemical methods. For the base-editing strategy, a transgenic HEK293T cell line carrying the c.146G>A mutation was developed. Adenine base editors (ABEs) were then created and optimized to minimize unwanted off-target edits. Among the designed editors, ABE8e-947DPAPAP-C successfully corrected the mutation.

Results: This study clinically identified the c.146G>A mutation and demonstrated its correction through an in vitro genome-editing strategy, highlighting the potential of these tools. The research underscores the potential of tailored base editors for treating genetic diseases like Fabry disease. Future research could further develop these therapeutic strategies for clinical applications. The CRISPR/Cas9 technology holds transformative potential for treating genetic diseases. Findings from this study demonstrate the importance of this technology in advancing gene editing and therapeutic strategies.

Prepared by Hamza Umut Karakurt, Gene2Info Senior R&D Engineer