Analysis of the ABCA4 gene by next-generation sequencing

Area of Science:

• Genetics
• Ophthalmology
• Molecular Biology

Background:

• ABCA4 gene mutations are linked to inherited retinal diseases like Stargardt disease and cone-rod dystrophy.
• Comprehensive variant identification is crucial for accurate diagnosis and genetic counseling.

Purpose of the Study:

• To identify all potential disease-associated variants within the coding regions of the ABCA4 gene in a large patient cohort.
• To evaluate the diagnostic utility of next-generation sequencing (NGS) for ABCA4-associated conditions.

Main Methods:

• A cohort of 168 patients with suspected ABCA4-associated diseases underwent initial screening using an ABCA4 microarray.
• Patients with identified or suspected mutations were further analyzed using NGS to sequence the entire ABCA4 coding region and splice sites.
• Novel variants were validated via Sanger sequencing and assessed for pathogenicity using in silico tools and segregation analysis.

Main Results:

• NGS successfully sequenced 159 patients, identifying the second disease-associated allele in approximately 48% of patients with one known mutation.
• In patients with no initial mutations, NGS detected both disease-associated alleles in 4 patients and one mutation in 10 patients.
• A total of 57 previously unknown variants were discovered, including missense, nonsense, deletion, and splice-site altering types, with 55 deemed pathogenic.

Conclusions:

• A significant number of ABCA4 gene mutations remain undiscovered, potentially located in noncoding regions.
• While ABCA4 microarrays serve as a useful initial screening tool, NGS offers a more time- and cost-effective approach for large-scale cohort analysis.
• NGS significantly enhances the detection rate of pathogenic ABCA4 variants, improving diagnostic capabilities for associated retinal diseases.