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Background <p>Missense variants are frequently classified as variants of uncertain significance (VUS) according to the guidelines of the American College of Medical Genetics and Genomics and the Association of Molecular Pathology (ACMG/AMP). Consequently, disease relevance remains elusive, impeding molecular genetic diagnostics, patients` and family genetic counseling, and identification of patients eligible for clinical trials. Functional studies are critical for resolving the clinical significance of VUS. <i>CNGA1</i> encodes the main subunit of the rod cyclic nucleotide-gated (CNG) channel, a vital component of the phototransduction cascade. Variants in <i>CNGA1</i> are a rare cause of autosomal recessive retinitis pigmentosa and a phase I/II gene augmentation trial (NCT06291935) is currently ongoing highlighting the necessity to differentiate benign from pathogenic variants.</p> Methods <p><i>CNGA1</i> missense variants compiled from retinal disease patient cohorts, public databases and literature were functionally investigated using a medium-throughput aequorin-based assay and in vitro minigene splice assays for predicted exonic spliceogenic variants. Functional data were correlated with the in silico prediction of five variant effect predictors (VEPs) and applied to support or revise variants’ ACMG/AMP classification.</p> Results <p>Data mining revealed 86 missense <i>CNGA1</i> variants – including three novel – most of them lacking functional data; 65.1% of the variants were initially classified as VUS. The aequorin-based assay showed that 72.1% of tested variants significantly impaired CNG channel function and were classified as functionally abnormal, while 23.3% were functionally normal and 5% remained functionally uncertain. Correlation of the functional data with in silico predictions identified AlphaMissense and CPT-1 to be the most suitable tools for assessing <i>CNGA1</i> missense variants. Using in vitro minigene splice assays, two putative missense variants were shown to induce missplicing. Based on the functional findings, 62.1% of the variants initially classified as VUS were re-categorized as likely pathogenic or likely benign. Furthermore, 93.3% of the variants initially classified as likely pathogenic showed an effect on CNGA1 channel function, confirming their disease relevance and supporting their reclassification as pathogenic.</p> Conclusion <p>This study represents the first comprehensive functional assessment of disease-associated <i>CNGA1</i> missense variants, thus significantly advancing the understanding of their disease relevance and improving molecular genetic diagnostics in patients.</p>

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Systematic functional evaluation of CNGA1 missense variants associated with retinitis pigmentosa

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月27日

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  • Peggy Reuter,
  • Jennifer Schroeder,
  • Marc Sturm,
  • Mathieu Quinodoz,
  • Veronika Vaclavik,
  • Miriam Bauwens,
  • Marieke De Bruyne,
  • Bart Leroy,
  • Joseph van Aerschot,
  • Katarina Stingl,
  • Susanne Kohl

摘要

Background

Missense variants are frequently classified as variants of uncertain significance (VUS) according to the guidelines of the American College of Medical Genetics and Genomics and the Association of Molecular Pathology (ACMG/AMP). Consequently, disease relevance remains elusive, impeding molecular genetic diagnostics, patients` and family genetic counseling, and identification of patients eligible for clinical trials. Functional studies are critical for resolving the clinical significance of VUS. CNGA1 encodes the main subunit of the rod cyclic nucleotide-gated (CNG) channel, a vital component of the phototransduction cascade. Variants in CNGA1 are a rare cause of autosomal recessive retinitis pigmentosa and a phase I/II gene augmentation trial (NCT06291935) is currently ongoing highlighting the necessity to differentiate benign from pathogenic variants.

Methods

CNGA1 missense variants compiled from retinal disease patient cohorts, public databases and literature were functionally investigated using a medium-throughput aequorin-based assay and in vitro minigene splice assays for predicted exonic spliceogenic variants. Functional data were correlated with the in silico prediction of five variant effect predictors (VEPs) and applied to support or revise variants’ ACMG/AMP classification.

Results

Data mining revealed 86 missense CNGA1 variants – including three novel – most of them lacking functional data; 65.1% of the variants were initially classified as VUS. The aequorin-based assay showed that 72.1% of tested variants significantly impaired CNG channel function and were classified as functionally abnormal, while 23.3% were functionally normal and 5% remained functionally uncertain. Correlation of the functional data with in silico predictions identified AlphaMissense and CPT-1 to be the most suitable tools for assessing CNGA1 missense variants. Using in vitro minigene splice assays, two putative missense variants were shown to induce missplicing. Based on the functional findings, 62.1% of the variants initially classified as VUS were re-categorized as likely pathogenic or likely benign. Furthermore, 93.3% of the variants initially classified as likely pathogenic showed an effect on CNGA1 channel function, confirming their disease relevance and supporting their reclassification as pathogenic.

Conclusion

This study represents the first comprehensive functional assessment of disease-associated CNGA1 missense variants, thus significantly advancing the understanding of their disease relevance and improving molecular genetic diagnostics in patients.