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Clinical significance of mRNA nonstop decay in rare disease diagnosis

Laboratory Genetics and Genomics
  • Primary Categories:
    • Laboratory Genetics
  • Secondary Categories:
    • Laboratory Genetics
Introduction:
Nonstop decay (NSD) is a cellular surveillance mechanism that targets and degrades mRNAs lacking stop codons, preventing the translation of potentially harmful truncated proteins. Stop-loss variants, such as stop codon single nucleotide variants or frameshift variants upstream of the natural stop codon, can trigger NSD if no downstream compensatory stop codon is present before the poly(A) tail. Despite the well-established functional importance of NSD, variants that activate the NSD pathway are currently not included in the American College of Medical Genetics and Genomics (ACMG) / Association for Molecular Pathology (AMP) variant interpretation guidelines or the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) Workgroup's PVS1 criterion recommendations, potentially leading to the underestimation of pathogenic variants in the diagnosis of rare diseases.

Methods:
We developed a computational pipeline to predict whether the stop-loss variants will undergo the nonstop decay pathway in human genes. This pipeline was applied to analyze 663 genes relevant to carrier screening, prenatal sequencing, and hereditary cancer testing, and the results were compared to manual analysis. We are extending this pipeline broadly to the human genome to identify additional NSD-susceptible genes that are related to human disease. We also used this pipeline to analyze genome sequencing (GS) data from our patient cohort to determine the prevalence of NSD variants in the disease population.

Results:
The comparision of the computational pipeline with manual analysis demonstrated full concordance across the 663 genes, confirming its reliability. Our analysis revealed that 44 genes (6.64%) are susceptible to NSD in at least one of the three reading frames when stop-loss variants occur. In our WGS database, we identified a significant number of NSD variants, some contributing directly to rare disease diagnoses. Notably, we present here several GS cases in which NSD variants were identified in the disease-causing genes that are consistent with the clinical phenotypes of the patients. For example, a homozygous NSD variant, NM_005101.4:c.463dup (p.R155Pfs*?), was identified in the ISG15 gene in an individual with clinical phenotypes that align with autosomal recessive Immunodeficiency 38 (OMIM: 616126). These features include skin rash, recurrent skin infections, dry skin, and mucosal ulcers. In another case, an individual carrying an NSD variant in the NDUFS7 gene, NM_024407.5:c.610del (p.E204Sfs*?), in trans with a pathogenic variant c.364G>A (p.V122M), presents with clinical phenotypes consistent with Mitochondrial Complex I Deficiency, Nuclear Type 3 (OMIM: 618224). This individual exhibits respiratory failure, abnormal MRI findings with extensive non-enhancing confluent brainstem lesions and diffuse upper cervical spine signals, motor developmental delay, hypotonia, lethargy, and respiratory arrest. These NSD variants would have been classified as variants of uncertain significance under the current ACMG/AMP variant interpretation guidelines, underscoring the importance of recognizing mRNA nonstop decay in rare disease diagnosis.

Conclusion:
NSD variants are clinically significant in rare disease diagnosis and are prevalent across genes associated with human diseases. These results support applying the PVS1 criterion in the ACMG/AMP variant interpretation guidelines to stop-loss variants in identified NSD susceptible genes to improve diagnostic accuracy and enhance patient care.

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