Clinical significance of mRNA nonstop decay in rare disease diagnosis and recommendations for its application in variant classification

Clinical significance of mRNA nonstop decay in rare disease diagnosis and recommendations for its application in variant classification | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Clinical significance of mRNA nonstop decay in rare disease diagnosis and recommendations for its application in variant classification Yue Zhou, Dandan He, Nikita Mehta, Christian C. Taborda, Robert Rigobello, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9126009/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background : Nonsense-mediated decay (NMD) is routinely considered during clinical interpretation of predicted loss-of-function (LoF) variants, whereas mRNA nonstop decay (NSD)—a translation-coupled surveillance pathway that degrades transcripts lacking an in-frame termination codon—is rarely considered. Variants that abolish the canonical stop codon or remove the final stop-containing exon(s) can create nonstop transcripts, but current American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant classification guidelines and the Clinical Genome Resource (ClinGen) PVS1 recommendations do not explicitly address NSD, creating a potential mechanism-specific gap in variant classification. Methods : We developed a computational workflow to identify disease-associated genes and transcript regions in which variants are predicted to generate nonstop transcripts in any of the three reading frames. Disease genes were obtained from OMIM and transcript annotations were retrieved from Ensembl. NSD-potential genes were defined as those lacking an in-frame stop codon in the 3′ UTR prior to the annotated polyadenylation site for at least one reading frame. We then screened 2,439 clinical genome sequencing (GS) variant call format (VCF) files for variants overlapping NSD-susceptible regions and performed case-level review. Results : Among 4,954 OMIM disease-associated protein-coding genes, 333 (6.72%) had NSD potential in ≥1 reading frame, yielding 546 genomic NSD-susceptible loci spanning 34,744 bp (~0.0011% of the human genome). Screening of 2,439 GS cases identified 359 candidate NSD variants; six variants in six probands were consistent with the individuals’ phenotypes and the genes’ disease mechanism. In these cases, incorporation of NSD as a transcript-null mechanism would increase the weight of LoF evidence and support reclassification from variant of uncertain significance to likely pathogenic or pathogenic when combined with additional clinical and genetic evidence. Conclusions : NSD-predicted variants are not ultra-rare among disease genes and can be clinically relevant in rare disease diagnosis. We propose practical recommendations for recognizing NSD-susceptible variants and incorporating NSD into gene-/disease-specific LoF frameworks (e.g., PVS1) to improve consistency and diagnostic yield. nonstop decay mRNA surveillance variant classification PVS1 loss-of-function stop-loss rare disease genome sequencing Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Messenger RNA (mRNA) quality control pathways maintain proteostasis and transcriptome integrity by recognizing and degrading aberrant RNAs that would otherwise produce potentially toxic proteins. Among the best-characterized translation-coupled pathways are nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD). NMD targets transcripts harboring premature termination codons (PTCs), typically when translation terminates upstream of exon–exon junction complexes or other features that signal premature termination [1–3]. NSD, in contrast, is triggered when translation reaches the 3′ end of an mRNA in the absence of an in-frame stop codon, leading to ribosome stalling at the poly(A) tail and recruitment of factors that couple ribosome rescue with 3′→5′ mRNA degradation by the SKI complex and the cytoplasmic RNA exosome [2–4] (Figure 1). In clinical genomic variant classification, prediction of transcript-level consequences is central to interpreting variants that reduce or abolish protein production. The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework includes the PVS1 criterion for variants predicted to cause loss of function (LoF) in genes where LoF is a known disease mechanism [5]. Subsequently, the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) working group recommendations provide a decision tree for assigning PVS1 strength (Very Strong/Strong/Moderate/Supporting) based on variant type, location, and predicted impact (e.g., NMD-competence, critical domains, and percentage of protein truncation) [6]. However, the current ACMG/AMP and SVI PVS1 guidance does not explicitly address NSD as a mechanism by which 3′-terminal frameshift, stop-loss, or terminal exon splice or deletion variants can generate a functional null allele. Consequently, variants expected to yield nonstop transcripts are frequently treated as C-terminal protein alterations with limited predicted impact, even when the true molecular outcome is transcript degradation through NSD. We hypothesized that NSD-susceptible variation is more prevalent among disease genes than generally appreciated and that explicit recognition of NSD would improve the consistency and accuracy of LoF evidence assignment in variant classification. Here, we (i) systematically identified disease-associated genes with transcript architectures susceptible to NSD across reading frames, (ii) defined the genomic regions in which variants could generate nonstop transcripts, (iii) assessed the prevalence of candidate NSD variants in a clinical genome sequencing (GS) cohort, and (iv) propose practical recommendations for incorporating NSD into LoF variant classification, with an exemplar implementation in DDX41 -related hematologic malignancy predisposition. Methods Identification of disease-associated gene set. Disease-associated genes were obtained from the Online Mendelian Inheritance in Man (OMIM) database (genemap2.txt; accessed January 3, 2025). Genes were filtered to retain entries with phenotype annotations, approved HGNC gene symbols, and Ensembl gene identifiers. In total, 4,954 disease-associated protein-coding genes were included. Disease-associated but non–protein-coding genes (n = 24) were excluded from downstream analyses. Transcript assembly and annotation. For each gene, transcript annotations were retrieved from Ensembl using the Ensembl REST API, including exon coordinates and transcript biotypes. For each gene, exon sequences for the canonical transcript were retrieved and validated for completeness; full-length transcript sequences were assembled by concatenating exon sequences in genomic order and transcriptional orientation. Coding sequence (CDS) boundaries and the terminal exon were annotated by alignment to Ensembl GRCh38 CDS FASTA references. Polyadenylation site selection. NSD depends on whether a compensatory stop codon is present before the poly(A) tail. Because tissue-specific alternative polyadenylation may alter the effective 3′ end of transcripts, we used polyadenylation sites corresponding to RefSeq transcripts where feasible, prioritizing those with the longest annotated 3′ UTR among mapped candidates. Ensembl–RefSeq mappings were obtained via BioMart; manual review was performed when automated mappings were unavailable. Identification of NSD-potential genes and NSD-susceptible regions. For each assembled transcript, we scanned the 3′ UTR (from the canonical stop codon to the poly(A) site) in all three reading frames for in-frame stop codons. Transcripts lacking an in-frame stop codon within the 3′ UTR prior to the poly(A) site in at least one reading frame were classified as NSD-potential. For NSD-potential genes, we defined the transcript intervals in which a variant could generate a nonstop transcript for each reading frame (Figure 2). In the +0 frame, only disruption of the canonical stop codon (3 nucleotides) yields an in-frame nonstop transcript. For +1 and +2 frames, we simulated a frameshifted transcript and defined the NSD-susceptible region as the interval between the last available stop codon in that shifted frame and the canonical stop codon. Transcript-relative coordinates were converted to genomic coordinates to create a unified NSD coordinate reference set. Clinical GS patient cohort A retrospective analysis of GS results from 2,439 patients at Baylor Genetics was performed. These individuals underwent GS either as proband-only, duo, or trio-based to investigate suspected genetic etiologies. This diagnostic laboratory is certified by the College of American Pathologists (CAP) and complies with the Clinical Laboratory Improvement Amendments. The analysis of deidentified data was approved by the Institutional Review Board at Baylor College of Medicine (IRB: H-41191) Genome sequencing Peripheral blood samples and buccal samples were provided in most cases, although other sources of DNA were also accepted. Library preparation was performed using a PCR-free approach following the manufacturer’s protocols (Illumina, San Diego, US). The GS protocol also incorporated either a coding single-nucleotide polymorphism array procedure or a spike-in plasmid tracer for quality control and sample tracking. Each library underwent sequencing with paired-end 150bp reads to achieve an average nuclear genome coverage ≥40x. Variant detection and classification Variant calling was conducted using in-house and proprietary bioinformatics pipelines. The scope of variant detection included single nucleotide variants (SNV), small insertions and deletions (<50bp, indels), copy-number variants (CNV), absence of heterozygosity, uniparental disomy, tandem repeat expansions, and mitochondrial variants. For clinical reporting, the genomic findings were classified into five tiers (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign, and Benign) according to Baylor Genetics’ classification scheme, which incorporates the guidelines recommended by the ACMG/AMP [5]. Variant screening We screened the 2,439 GS VCFs for variants overlapping NSD-susceptible regions (Figure 3). Variants were annotated for overlapping position, predicted reading frame, and variant consequence. Candidate NSD variants underwent post-screening review to determine (i) whether LoF is an established disease mechanism for the gene, (ii) whether zygosity and inheritance were consistent with the gene–disease association, and (iii) whether the individual’s phenotype was compatible with the associated disorder. This study is descriptive; no hypothesis-testing statistics were applied. Results Genome-wide landscape of NSD susceptibility among disease genes Among 4,954 OMIM disease-associated protein-coding genes, 333 (6.72%) were classified as NSD-potential in at least one reading frame. Across these 333 genes, we identified 546 distinct genomic regions capable of harboring variants that generate nonstop transcripts (Supplemental Table 1). In aggregate, NSD-susceptible loci spanned 34,744 nucleotides—approximately 0.0011% of the total chromosomal sequence—indicating that while the genomic footprint is small, the number of disease genes with NSD potential is substantial (Figure 4). Prevalence of candidate NSD variants in a clinical GS cohort Screening of 2,439 GS cases identified 359 candidate NSD variants across NSD-susceptible loci. Most variants were observed in blood group–related genes (n = 332) and there were three genes which LoF is not the established disease mechanism (n = 3). Eighteen candidate NSD variants were found in genes associated with autosomal recessive disease; in the absence of a second reportable variant in trans and compatible phenotypes, these findings were not clinically reportable but may be relevant in the context of reproductive testing (e.g., carrier screening). Six probands harbored NSD-predicted variants in disease-causing genes that were consistent with their clinical phenotypes and inheritance expectations (Table 1). These variants would typically receive only PVS1_Moderate or PVS1_Strong under existing PVS1 decision trees because they occur near the 3′ end and are predicted to alter only a small C-terminal portion of the protein. Incorporating NSD as a transcript-null mechanism supports treating these variants as functional null alleles (subject to gene- and transcript-specific considerations) and can increase the overall weight of pathogenic evidence when combined with additional data (e.g., de novo occurrence, phenotype specificity, segregation and allelic data, in silico predictions). Case vignettes illustrating NSD-relevant reclassification NDUFS7 (autosomal recessive mitochondrial complex I deficiency, nuclear type 3, OMIM: 618224). Compound heterozygous variants were identified in the NDUFS7 gene: a paternal inherited frameshift variant NM_024407.5:c.610del (p.Glu204Serfs*?) predicted to abolish the canonical stop codon and generate a nonstop transcript, and a known pathogenic missense variant NM_024407.5:c.364G>A (p.Val122Met). Without NSD consideration, the frameshift might be interpreted as a near-terminal truncation removing nine amino acids and receive only PVS1_Moderate; with NSD incorporation, it is consistent with a null allele acting in trans with a pathogenic variant and a highly concordant phenotype with mitochondrial complex I deficiency, supporting pathogenic classification. ISG15 (autosomal recessive immunodeficiency 38, OMIM: 616126). A homozygous NM_005101.4:c.463dup (p.Arg155Profs*?) frameshift variant predicted to create a nonstop transcript was identified in the ISG15 gene. Because the frameshift occurs near the 3′ end with only ten animo acids downstream, this variant would typically receive PVS1_Moderate; NSD provides a mechanistic rationale for transcript degradation and supports pathogenic classification in the context of a compatible phenotype with immunodeficiency and recessive inheritance in this patient. HCN2 (autosomal dominant HCN2 -related epilepsy, OMIM: 602477). A heterozygous de novo frameshift NM_001194.4:c.2328_2334dup (p.Ser779Profs*?) variant predicted to generate a nonstop transcript was detected in the HCN2 gene. This variant disrupts normal transcript termination and generates a nonstop mRNA predicted to be degraded through the NSD pathway. This variant had a PVS1_Strong criteria code since the altered 3’ coding region of protein is more than 10% and was also reclassified from variant of uncertain significance (VUS) to likely pathogenic after the incorporation of NSD rules in variant classification which is consistent with the clinical diagnosis of epilepsy. HNRNPD (autosomal dominant HNRNPD -related neurodevelopmental disorder, CCID: 009030). A de novo canonical splice acceptor variant in the last coding exon of the HNRNPD gene (NM_031369.3:c.944-1G>A) was identified. For this gene, LoF/haploinsufficiency is an established disease mechanism. Current splicing recommendations often assign PVS1_Moderate for the last-exon splice site variants in the absence of evidence for NMD [7]; NSD provides an additional pathway by which last-exon splice disruption can lead to transcript loss when the termination codon is removed and no compensatory stop codon exists prior to polyadenylation, supporting a higher PVS1 strength when transcript context is consistent. SHANK2 (autosomal dominant SHANK2 -related neurodevelopmental disorder, CCID: 006134). A heterozygous frameshift variant was detected in the SHANK2 gene (NM_012309.5:c.5302_5305del; p.Leu1768Glnfs*?). In genes where haploinsufficiency is the disease mechanism, NSD prediction is consistent with a null allele and supports upgrading LoF evidence from PVS1_Moderate to PVS1. This variant was also reclassified from VUS to likely pathogenic which is consistent with the neurodevelopmental phenotype in this patient. TYMP (autosomal recessive mitochondrial DNA depletion syndrome type 1 (MNGIE), OMIM: 603041). Compound heterozygous variants were detected in the TYMP gene including a frameshift duplication variant NM_001953.5:c.865_889dup (p.Ala297Glyfs*?) predicted to abolish the native stop codon and induce NSD, in trans with a likely pathogenic in-frame deletion NM_001953.5:c.1198_1203del (p.Val400_Leu401del). Considering LoF as the disease mechanism, NSD supports treating the frameshift as a null allele consistent with molecular and clinical diagnosis of mitochondrial DNA depletion syndrome type 1 (MNGIE) in this patient. Exemplar implementation in gene-/disease-specific variant classification guidance: DDX41 ClinGen Variant Curation Expert Panels (VCEPs) develop gene-/disease-specific ACMG/AMP specifications to improve consistency and accuracy in variant classification. The DDX41 gene is associated with DDX41 -related hematologic malignancy predisposition syndrome (CCID: 004638). The condition is primarily characterized by adult-onset acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Malignant transformation is driven by biallelic inactivation of DDX41 with the second hit frequently being one of several highly specific somatic hotspot variants [8,9]. Given DDX41 functions as a tumor suppressor gene, null variants are typically identified in patients at risk for this condition. While many pathogenic DDX41 variants are truncating, a subset of terminal frameshift variants is predicted to generate nonstop transcripts depending on the location of compensatory stop codons relative to the poly(A) tail. We summarize published and database-reported individuals who carry DDX41 frameshift variants consistent with NSD prediction (Table 2). In several reported cases, coexistence of a hotspot somatic variant provides phenotype/genotype coherence that can strengthen pathogenicity assessment when NSD is recognized as a functional null mechanism. For DDX41 (NM_016222.4), the annotated polyadenylation signal and insertion site imply that +1 and −1 frameshift variants in the terminal coding region can place the next in-frame stop codon downstream of the poly(A) tail, producing nonstop transcripts. In this setting, treating such variants as LoF is mechanistically and clinically reasonable, contingent on transcript selection and gene mechanism considerations. This illustrates how NSD criteria can be incorporated into ClinGen VCEP specifications alongside established PVS1 decision trees. Recommendations for incorporating NSD into variant classification Recommendation 1: Treat NSD as a transcript-null mechanism analogous to NMD when LoF is an established disease mechanism. NSD is expected to reduce transcript abundance through translation-coupled degradation when an mRNA lacks an in-frame termination codon and translation reaches the poly(A) tail [2–4]. For genes in which haploinsufficiency or biallelic LoF causes disease, variants predicted to trigger NSD should generally be evaluated under LoF frameworks (e.g., PVS1), rather than being discounted as minor C-terminal alterations. Recommendation 2: Define NSD susceptibility on a transcript-specific basis. NSD prediction depends on (i) the clinically relevant transcript (preferably MANE Select and/or MANE Plus Clinical where available), (ii) the location of the poly(A) site (including alternative polyadenylation), and (iii) the presence of compensatory stop codons in the relevant reading frame between the disrupted canonical stop codon and the poly(A) tail. Laboratories should document the transcript used and the stop-codon landscape in the relevant frame as part of the interpretation rationale. Recommendation 3: Recognize the variant classes most likely to trigger NSD. These include: (a) stop-loss variants that abolish the canonical stop codon without the alteration of upstream coding sequence; (b) frameshift variants in the terminal coding region that eliminate the canonical stop codon; (c) splice acceptor/donor or other variants that cause skipping of the last (or penultimate) coding exon containing the termination codon or cause a frameshift into a NSD-susceptible reading frame; and (d) copy-number or structural variants that delete the final stop-containing exon(s). Recommendation 4: Calibrate strength of evidence using existing PVS1 logic, with explicit NSD checkpoints. We recommend adding an NSD checkpoint to the PVS1 decision tree [6] for variants near the 3′ end that would otherwise be downgraded due to limited predicted truncation. If the variant is predicted to generate a nonstop transcript on the clinically relevant transcript and LoF is a known disease mechanism, applying PVS1 at the same strength as other null variants of comparable confidence is appropriate (Table 3). In semi-quantitative adaptations of ACMG/AMP, Very Strong pathogenic evidence corresponds to 8 points [10,11], and an NSD-triggering variant in a LoF gene can reasonably contribute at this level when prediction confidence is high. (Notably, an ACMG/AMP/CAP/ClinGen SVC v4.0 standard is in development; laboratories should align NSD implementation with the finalized framework when released.) Recommendation 5: Distinguish canonical NSD from long 3′-tail/termination inefficiency mechanisms. Some transcripts may terminate far downstream of the canonical stop due to readthrough or creation of a distant stop codon; evidence suggests that unusually long 3′ UTR translation can trigger decay mechanisms distinct from classic NSD [12]. When the next stop codon is present but far downstream (e.g., >150 nt beyond the canonical termination site), we suggest considering a reduced-strength LoF contribution (PVS1_Moderate) unless additional experimental evidence demonstrates transcript loss (Table 3). This approach parallels existing guidance to modulate PVS1 strength based on predicted molecular outcome confidence [6]. Operational checklist. When evaluating a candidate NSD variant, we recommend documenting: (1) gene–disease mechanism (LoF established?); (2) transcript selection (MANE Select/ MANE Plus Clinical/ clinically curated transcript); (3) whether the variant removes the canonical stop codon or stop-containing exon(s); (4) whether an in-frame compensatory stop codon exists prior to polyadenylation in the relevant reading frame; (5) whether alternative transcripts or polyadenylation could rescue termination; and (6) any corroborating evidence (RNA studies, segregation, de novo status, phenotype specificity). Discussion This study highlights NSD as an under-recognized but potentially important mechanism in clinical variant classification. Although the genomic regions capable of generating nonstop transcripts are small in aggregate, we found that ~7% of OMIM disease-associated genes have transcript architectures that permit NSD in at least one reading frame. This observation challenges the perception that NSD-relevant variants are “ultra-rare” and suggests that NSD should be considered routinely when interpreting stop-loss, terminal frameshift, or last-exon splice or deletion variants in LoF-mediated conditions. Our case series illustrates a recurring interpretive pitfall: near-terminal variants are often downgraded because they appear to remove only a short C-terminal segment. This logic is appropriate when translation is expected to terminate soon after the variant and produce a largely intact protein, but it can be misleading when the true outcome is loss of the termination codon and degradation of the transcript. In most examples here, NSD provides a mechanistically coherent explanation for functional null alleles that would otherwise be categorized as VUS due to reduced PVS1 strength. Importantly, NSD should not be applied indiscriminately: prediction requires transcript-specific assessment of compensatory stop codons and polyadenylation sites, and clinical classification must still integrate additional evidence (inheritance, segregation, de novo status, phenotype specificity, population frequency, in silico prediction, experimental evidence) per ACMG/AMP/ClinGen guidelines [5,6]. The DDX41 exemplar underscores how NSD can be operationalized within ClinGen VCEP specifications. In tumor suppressor genes with characteristic second-hit somatic hotspots, recognizing NSD as a LoF mechanism can align molecular predictions with observed genotype patterns. More broadly, NSD-aware LoF assessment may improve cross-laboratory consistency by reducing reliance on variable, ad hoc interpretations of terminal frameshift or stop-loss variants. Limitations. First, NSD susceptibility was inferred computationally from reference transcript annotations and selected polyadenylation sites; alternative splicing and alternative polyadenylation could mitigate or exacerbate NSD in a tissue-specific manner. Second, we did not systematically quantify transcript abundance changes (e.g., RNA sequencing) for candidate variants; future work integrating RNA data would help calibrate prediction confidence and PVS1 strength. Third, our GS cohort screening reflects a clinical testing population and is enriched for disease-causing gene and variants which may limit generalizability of raw prevalence estimates. Finally, interpretation in oncology predisposition genes may require additional somatic context and careful distinction between germline and somatic events. Conclusion Variants predicted to trigger mRNA nonstop decay are present across a non-trivial fraction of disease-associated genes and can contribute directly to rare disease molecular diagnoses. Incorporating NSD into LoF variant classification—using transcript-specific checks and gene mechanism awareness—can improve the consistency and accuracy of PVS1 strength assignment and reduce the number of clinically relevant variants remaining as VUS. We recommend that NSD checkpoints be incorporated into gene-/disease-specific ACMG/AMP/ClinGen specifications and, as consensus frameworks evolve, into general variant classification guidance. Abbreviations ACMG: American College of Medical Genetics and Genomics; AML: Acute Myeloid Leukemia; AMP: Association for Molecular Pathology; CAP: College of American Pathologists; CCID: ClinGen Curation Identification; CDS: coding sequence; ClinGen: Clinical Genome Resource; GS: genome sequencing; LoF: loss of function; MANE: Matched Annotation from NCBI and EMBL-EBI; MDS: Myelodysplastic Syndromes; NGD: no-go decay; NMD: nonsense-mediated decay; NSD: nonstop decay; PTC: premature termination codon; SVI: Sequence Variant Interpretation; VCEP: Variant Curation Expert Panel; VCF: variant call format; VUS: variant of uncertain significance. Declarations Ethics approval and consent to participate This study was conducted in accordance with protocol (H-41191) approved by the Institutional Review Board at Baylor College of Medicine. The study was also conducted in accordance with the Declaration of Helsinki. Consent for publication Consent was waived for the individuals included in this study per Institutional Review Board protocol. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests Y.Z., C.C.T., R.R., M.D., J.L., N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are employed by Baylor Genetics. Baylor College of Medicine and Miraca Holdings Inc. have formed a joint venture with shared ownership and governance of Baylor Genetics, which performs genetic testing and derives revenue. N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are also employees of Baylor College of Medicine and derive support through a professional services agreement with Baylor Genetics. Fundings No funding was received for the current study. Authors' contributions Study conception: Y.Z., X.L. Study design: Y.Z., X.L. Data curation: Y.Z., D.H., N.M., C.C.T., J.L., X.L. Data analysis: Y.Z., D.H., N.M., J.L., X.L. Data interpretation: Y.Z., D.H., N.M., J.L., N.L., Y.W., X.L. Writing-original draft: Y.Z., D.H., N.M., R.R., M.D., N.L., X.L. Writing-review and editing: Y.Z., D.H., N.M., C.C.T., R.R., M.D., J.L., N.L., Y.W., D.W., L.A.G., L.V., X.Z., L.M., C.M.E., F.X., and X.L. Acknowledgements Not applicable. References Wolin SL, Maquat LE. Cellular RNA surveillance in health and disease. Science (1979). American Association for the Advancement of Science; 2019. p. 822–7. https://doi.org/10.1126/science.aax2957 Monaghan L, Longman D, Cáceres JF. Translation‐coupled mRNA quality control mechanisms. EMBO J. Springer Science and Business Media LLC; 2023;42. https://doi.org/10.15252/embj.2023114378 Powers KT, Szeto JYA, Schaffitzel C. New insights into no-go, non-stop and nonsense-mediated mRNA decay complexes. Curr. Opin. Struct. Biol. Elsevier Ltd; 2020. p. 110–8. https://doi.org/10.1016/j.sbi.2020.06.011 Klauer AA, van Hoof A. Degradation of mRNAs that lack a stop codon: A decade of nonstop progress. Wiley Interdiscip. Rev. RNA. 2012. p. 649–60. https://doi.org/10.1002/wrna.1124 Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine. Nature Publishing Group; 2015;17:405–24. https://doi.org/10.1038/gim.2015.30 Abou Tayoun AN, Pesaran T, DiStefano MT, Oza A, Rehm HL, Biesecker LG, et al. Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum Mutat. John Wiley and Sons Inc.; 2018;39:1517–24. https://doi.org/10.1002/humu.23626 Walker LC, Hoya M de la, Wiggins GAR, Lindy A, Vincent LM, Parsons MT, et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup. Am J Hum Genet. Cell Press; 2023;110:1046–67. https://doi.org/10.1016/j.ajhg.2023.06.002 Polprasert C, Schulze I, Sekeres MA, Makishima H, Przychodzen B, Hosono N, et al. Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms. Cancer Cell. Cell Press; 2015;27:658–70. https://doi.org/10.1016/j.ccell.2015.03.017 Kadono M, Kanai A, Nagamachi A, Shinriki S, Kawata J, Iwato K, et al. Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia. Exp Hematol. Elsevier Inc.; 2016;44:745-754.e4. https://doi.org/10.1016/j.exphem.2016.04.017 Tavtigian S V., Greenblatt MS, Harrison SM, Nussbaum RL, Prabhu SA, Boucher KM, et al. Modeling the ACMG/AMP variant classification guidelines as a Bayesian classification framework. Genetics in Medicine. Nature Publishing Group; 2018;20:1054–60. https://doi.org/10.1038/gim.2017.210 Tavtigian S V., Harrison SM, Boucher KM, Biesecker LG. Fitting a naturally scaled point system to the ACMG/AMP variant classification guidelines. Hum Mutat. John Wiley and Sons Inc; 2020;41:1734–7. https://doi.org/10.1002/humu.24088 Takata A, Hamanaka K, Matsumoto N. Refinement of the clinical variant interpretation framework by statistical evidence and machine learning. Med. Cell Press; 2021;2:611-632.e9. https://doi.org/10.1016/j.medj.2021.02.003 Tables Table 1. Positive GS cases with NSD variants identified in disease-causing genes consistent with clinical phenotypes. Gene Disease Inheritance/ Zygosity Variant Protein Full Length PVS1 criteria without or with NSD incorporation ACMG/AMP/ClinGen Classification without or with NSD incorporation NDUFS7 Mitochondrial Complex I Deficiency, Nuclear Type 3 (OMIM: 618224). AR/ Compound Heterozygous NM_024407.5: c..610del (p.E204Sfs*?); c.364G>A (p.V122M) 213 PVS1_Moderate → PVS1 VUS → Pathogenic ISG15 Immunodeficiency 38 (OMIM: 616126) AR/ Homozygous NM_005101.4: c.463dup (p.R155Pfs*?) 165 PVS1_Moderate → PVS1 VUS → Pathogenic HCN2 HCN2 -related epilepsy (OMIM: 602477) AD/ De Novo NM_001194.4: c.2328_2334dup, (p.S779Pfs*?) 889 PVS1_Strong → PVS1 VUS → Likely Pathogenic HNRNPD HNRNPD -related neurodevelopmental disorder (CCID: 009030) AD/ De Novo NM_031369.3:c.944-1G>A 355 PVS1_Moderate → PVS1 VUS → Pathogenic SHANK2 SHANK2 -related neurodevelopmental disorder (CCID: 006134) AD/ Heterozygous NM_012309.5: c.5302_5305del (p.Leu1768Glnfs*?) 1800 PVS1_Moderate → PVS1 VUS → Likely Pathogenic TYMP Mitochondrial DNA depletion syndrome type 1 (MNGIE) (OMIM: 603041) AR/ Compound Heterozygous NM_001953.5:c.865_889dup (p.Ala297Glyfs*?); c.1198_1203del (p.Val400_Leu401del) 482 PVS1_Strong → PVS1 Likely Pathogenic → Pathogenic Table 2. DDX41 frameshift variants reported in literature or public databases that are predicted to be subject to non-stop decay. Germline cDNA Germline protein Germline VAF Somatic cDNA Somatic protein Somatic VAF Origin confirmed? Age Diagnosis Reference c.1791_1792del p.(Lys597Asnfs*?) >40% c.1574G>A p.Arg525His 10% No 66 AML [1,2] c.1811_1812insCATATGTGCTAT p.(Lys604Asnfs*?) 52% c.1574G>A p.Arg525His 23% No N/A Myeloid neoplasm [3], ClinVar: 3371864 c.1836_1837del p.(Asp613LeufsTer?) 35-40% N/A N/A N/A No 60-65 None [4], gnomAD: 5-177511822-TCC-T None None N/A c.1857dup c.1589G>A p.Met620Hisfs*? p.Gly530Asp 34.8% 30.5% Yes 72 Myeloid neoplasm [5] c.1814del p.(Gln605ArgfsTer?) 41.9% c.1574G>A p.Arg525His 5.3% Yes N/A MDS [6] c.1721del p.(Leu574Argfs*?) N/A N/A N/A N/A N/A N/A MDS, AML ClinVar:1338572 c.1773del p.(Ile592Serfs*?) 45-50% N/A N/A N/A No N/A N/A gnomAD: 5-177511886-TC-T c.1843del p.(Leu615TrpfsTer?) 45-50% N/A N/A N/A No 40-45 N/A gnomAD: 5-177511816-AG-A Table 3. Conceptual mapping of termination-related mRNA surveillance mechanisms and proposed evidence strength in variant classification. Scenario Description Pathway Triggered LoF evidence criteria Premature stop codon (early termination) Nonsense-Mediated Decay (NMD) Upstream exon–exon junction → decay triggered PVS1; +8 points No stop codon → ribosome reaches poly(A) Non-stop Decay (NSD) Ribosome stalls at poly(A), mRNA degraded → decay triggered PVS1; +8 points Stop codon far downstream (>150 nt past normal site) Extended nonstop-like surveillance Ribosome reads through long 3′ region → decay may be triggered PVS1_Moderate; +2 points References 1. Ebert MS´, Passet M, Raimbault A, Rahmé R, Rahmé R, Raffoux E, et al. Germline DDX41 mutations define a significant entity within adult MDS/AML patients. Blood. 2019. 2. Duployez N, Largeaud L. Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study. Blood. American Society of Hematology; 2020;136:2125–32. https://doi.org/10.1182/BLOOD.2019000962 3. Aguilera-Diaz A, Larrayoz MJ, Palomino-Echeverría S, Vazquez I, Ariceta B, Mañú A, et al. Strategy for identification of a potential inherited leukemia predisposition in a 299 patient’s cohort with tumor-only sequencing data. Leuk Res. Elsevier Ltd; 2020;95. https://doi.org/10.1016/j.leukres.2020.106386 4. Kovilakam SC, Gu M, Dunn WG, Marando L, Barcena C, England G, et al. Prevalence and significance of DDX41 gene variants in the general population. 5. Qu S, Li B, Qin T, Xu Z, Pan L, Hu N, et al. Molecular and clinical features of myeloid neoplasms with somatic DDX41 mutations. Br J Haematol. Blackwell Publishing Ltd; 2021;192:1006–10. https://doi.org/10.1111/bjh.16668 6. Maierhofer A, Mehta N, Chisholm RA, Hutter S, Baer C, Nadarajah N, et al. The clinical and genomic landscape of patients with DDX41 variants identified during diagnostic sequencing. Blood Adv. American Society of Hematology; 2023;7:7346–57. https://doi.org/10.1182/bloodadvances.2023011389 Additional Declarations Competing interest reported. Y.Z., C.C.T., R.R., M.D., J.L., N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are employed by Baylor Genetics. Baylor College of Medicine and Miraca Holdings Inc. have formed a joint venture with shared ownership and governance of Baylor Genetics, which performs genetic testing and derives revenue. N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are also employees of Baylor College of Medicine and derive support through a professional services agreement with Baylor Genetics. Supplementary Files NSDSupplementalTable1.csv Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 10 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 24 Mar, 2026 Editor assigned by journal 23 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 14 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9126009","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611628733,"identity":"ca5151c6-9ef9-400a-9ecc-afa171b8755d","order_by":0,"name":"Yue Zhou","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhou","suffix":""},{"id":611628735,"identity":"b0e31c23-b2f9-4c9c-870c-b6119b657af0","order_by":1,"name":"Dandan He","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"He","suffix":""},{"id":611628738,"identity":"e49ed0ef-6372-4dce-ace5-5cf1d9f912b4","order_by":2,"name":"Nikita Mehta","email":"","orcid":"","institution":"Memorial Sloan Kettering Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Nikita","middleName":"","lastName":"Mehta","suffix":""},{"id":611628741,"identity":"193a5a32-7b78-4f8c-9b15-a151624aa18e","order_by":3,"name":"Christian C. Taborda","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"C.","lastName":"Taborda","suffix":""},{"id":611628745,"identity":"2be95995-11de-4634-a42b-a77e06b40aea","order_by":4,"name":"Robert Rigobello","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Rigobello","suffix":""},{"id":611628747,"identity":"cd5f2f97-3fec-4b9a-853b-e4a7ac2b212d","order_by":5,"name":"Morgan Driver","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Morgan","middleName":"","lastName":"Driver","suffix":""},{"id":611628748,"identity":"001141ff-5546-4c28-9055-cd1d1bf3971a","order_by":6,"name":"John Lattier","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"Lattier","suffix":""},{"id":611628753,"identity":"fb2d0a4e-d8f1-4ba9-886e-326c7e4e54b8","order_by":7,"name":"Ning Liu","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Ning","middleName":"","lastName":"Liu","suffix":""},{"id":611628756,"identity":"3dc1221b-d765-412e-bb7f-1a33a274e269","order_by":8,"name":"Yue Wang","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wang","suffix":""},{"id":611628762,"identity":"3a7d2535-163a-4278-8cb9-5dd679d67996","order_by":9,"name":"David Wu","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Wu","suffix":""},{"id":611628765,"identity":"53664e22-d7d0-4209-a8dd-520a375d483e","order_by":10,"name":"Lucy A. Godley","email":"","orcid":"","institution":"Northwestern University","correspondingAuthor":false,"prefix":"","firstName":"Lucy","middleName":"A.","lastName":"Godley","suffix":""},{"id":611628766,"identity":"3037818a-3164-4b28-8f23-5c93e650197f","order_by":11,"name":"Liesbeth Vossaert","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Liesbeth","middleName":"","lastName":"Vossaert","suffix":""},{"id":611628768,"identity":"2e9eb69e-9e6f-4e43-9e4a-827159842de5","order_by":12,"name":"Xiaonan Zhao","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Xiaonan","middleName":"","lastName":"Zhao","suffix":""},{"id":611628769,"identity":"731f1f74-8991-440c-a58c-2ada99bf8c82","order_by":13,"name":"Linyan Meng","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Linyan","middleName":"","lastName":"Meng","suffix":""},{"id":611628770,"identity":"279f8609-f235-4035-8831-1591d3b39679","order_by":14,"name":"Christine M. Eng","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"M.","lastName":"Eng","suffix":""},{"id":611628772,"identity":"17c163b4-a061-4436-ad96-2e7c1cf607ae","order_by":15,"name":"Fan Xia","email":"","orcid":"","institution":"Baylor Genetics","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Xia","suffix":""},{"id":611628774,"identity":"d7a9005c-c382-447d-b892-f7544f2239e0","order_by":16,"name":"Xi Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApUlEQVRIiWNgGAWjYDACZgbGAwwMNglgDg+RWhiAWtJI0cIA1nKYBC0Gx4HqeSrO5/FPO8D44G0bEVokm0FaztwulridwGw4lxgt/EC/HOZtu524QTqBTZqXGC1sEC3nQFrYfxOlBWrLAbAtzERpkWxmbDg450wy0C+JzZJzzhGhxeD84YMP3lTY5fHPTj744U0ZEVoYGBgb0BmjYBSMglEwCigGAB2VMoAYFoupAAAAAElFTkSuQmCC","orcid":"","institution":"Baylor Genetics","correspondingAuthor":true,"prefix":"","firstName":"Xi","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2026-03-15 03:38:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9126009/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9126009/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105566577,"identity":"9b2c5f35-1469-4deb-a574-88c38ed2f0a7","added_by":"auto","created_at":"2026-03-27 12:56:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":426292,"visible":true,"origin":"","legend":"\u003cp\u003eNonstop decay mechanism. Translation of an mRNA lacking a stop codon leads to ribosome stalling at the poly(A) tail and recruitment of factors that couple ribosome rescue with 3′→5′ mRNA degradation (SKI complex and cytoplasmic exosome) and nascent peptide handling via the proteasome.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126009/v1/a748320f6288732f0bea6f0f.jpg"},{"id":105566070,"identity":"b2e040a8-69f4-4348-b764-15ec01085aee","added_by":"auto","created_at":"2026-03-27 12:55:13","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":652244,"visible":true,"origin":"","legend":"\u003cp\u003eSequence analysis workflow to identify NSD-susceptible genomic regions. For each gene, the mRNA is analyzed in three reading frames. For each frame, the last in-frame stop codon and the canonical stop codon are highlighted in red and transcript intervals in which variants could produce nonstop transcripts are defined in blue.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126009/v1/f05a5206fb9fae16782928b4.jpg"},{"id":105751970,"identity":"9fabaaa0-6b88-483d-9caa-d3196f86cf47","added_by":"auto","created_at":"2026-03-30 15:52:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":549935,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of clinical GS screening for NSD candidate variants.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126009/v1/9208f64e46b6167719e51827.jpg"},{"id":105497423,"identity":"0dccfa61-3afe-4811-818b-b0f5a4491602","added_by":"auto","created_at":"2026-03-26 16:45:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":385134,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of NSD-capable loci throughout the genome.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9126009/v1/38c82a2b62d8f3eb73678517.jpg"},{"id":105752511,"identity":"b16fb1a9-dae8-41f7-823e-d0db899f6bc7","added_by":"auto","created_at":"2026-03-30 16:02:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3050579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9126009/v1/17c071d2-ae8d-44c4-b6b7-402c208c466c.pdf"},{"id":105497421,"identity":"849e1eb3-345f-4ab8-aa9c-fb368739ae9b","added_by":"auto","created_at":"2026-03-26 16:45:05","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":61569,"visible":true,"origin":"","legend":"","description":"","filename":"NSDSupplementalTable1.csv","url":"https://assets-eu.researchsquare.com/files/rs-9126009/v1/16f9180b90153f452fd07ff9.csv"}],"financialInterests":"Competing interest reported. Y.Z., C.C.T., R.R., M.D., J.L., N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are employed by Baylor Genetics. Baylor College of Medicine and Miraca Holdings Inc. have formed a joint venture with shared ownership and governance of Baylor Genetics, which performs genetic testing and derives revenue. N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are also employees of Baylor College of Medicine and derive support through a professional services agreement with Baylor Genetics.","formattedTitle":"Clinical significance of mRNA nonstop decay in rare disease diagnosis and recommendations for its application in variant classification","fulltext":[{"header":"Background","content":"\u003cp\u003eMessenger RNA (mRNA) quality control pathways maintain proteostasis and transcriptome integrity by recognizing and degrading aberrant RNAs that would otherwise produce potentially toxic proteins. Among the best-characterized translation-coupled pathways are nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop decay (NSD). NMD targets transcripts harboring premature termination codons (PTCs), typically when translation terminates upstream of exon–exon junction complexes or other features that signal premature termination [1–3]. NSD, in contrast, is triggered when translation reaches the 3′ end of an mRNA in the absence of an in-frame stop codon, leading to ribosome stalling at the poly(A) tail and recruitment of factors that couple ribosome rescue with 3′→5′ mRNA degradation by the SKI complex and the cytoplasmic RNA exosome [2–4] (Figure 1).\u003c/p\u003e\n\u003cp\u003eIn clinical genomic variant classification, prediction of transcript-level consequences is central to interpreting variants that reduce or abolish protein production. The American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework includes the PVS1 criterion for variants predicted to cause loss of function (LoF) in genes where LoF is a known disease mechanism [5]. Subsequently, the Clinical Genome Resource (ClinGen) Sequence Variant Interpretation (SVI) working group recommendations provide a decision tree for assigning PVS1 strength (Very Strong/Strong/Moderate/Supporting) based on variant type, location, and predicted impact (e.g., NMD-competence, critical domains, and percentage of protein truncation) [6]. However, the current ACMG/AMP and SVI PVS1 guidance does not explicitly address NSD as a mechanism by which 3′-terminal frameshift, stop-loss, or terminal exon splice or deletion variants can generate a functional null allele. Consequently, variants expected to yield nonstop transcripts are frequently treated as C-terminal protein alterations with limited predicted impact, even when the true molecular outcome is transcript degradation through NSD.\u003c/p\u003e\n\u003cp\u003eWe hypothesized that NSD-susceptible variation is more prevalent among disease genes than generally appreciated and that explicit recognition of NSD would improve the consistency and accuracy of LoF evidence assignment in variant classification. Here, we (i) systematically identified disease-associated genes with transcript architectures susceptible to NSD across reading frames, (ii) defined the genomic regions in which variants could generate nonstop transcripts, (iii) assessed the prevalence of candidate NSD variants in a clinical genome sequencing (GS) cohort, and (iv) propose practical recommendations for incorporating NSD into LoF variant classification, with an exemplar implementation in \u003cem\u003eDDX41\u003c/em\u003e-related hematologic malignancy predisposition.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eIdentification of disease-associated gene set.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDisease-associated genes were obtained from the Online Mendelian Inheritance in Man (OMIM) database (genemap2.txt; accessed January 3, 2025). Genes were filtered to retain entries with phenotype annotations, approved HGNC gene symbols, and Ensembl gene identifiers. In total, 4,954 disease-associated protein-coding genes were included. Disease-associated but non–protein-coding genes (n = 24) were excluded from downstream analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscript assembly and annotation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each gene, transcript annotations were retrieved from Ensembl using the Ensembl REST API, including exon coordinates and transcript biotypes. For each gene, exon sequences for the canonical transcript were retrieved and validated for completeness; full-length transcript sequences were assembled by concatenating exon sequences in genomic order and transcriptional orientation. Coding sequence (CDS) boundaries and the terminal exon were annotated by alignment to Ensembl GRCh38 CDS FASTA references.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolyadenylation site selection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNSD depends on whether a compensatory stop codon is present before the poly(A) tail. Because tissue-specific alternative polyadenylation may alter the effective 3′ end of transcripts, we used polyadenylation sites corresponding to RefSeq transcripts where feasible, prioritizing those with the longest annotated 3′ UTR among mapped candidates. Ensembl–RefSeq mappings were obtained via BioMart; manual review was performed when automated mappings were unavailable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of NSD-potential genes and NSD-susceptible regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each assembled transcript, we scanned the 3′ UTR (from the canonical stop codon to the poly(A) site) in all three reading frames for in-frame stop codons. Transcripts lacking an in-frame stop codon within the 3′ UTR prior to the poly(A) site in at least one reading frame were classified as NSD-potential. For NSD-potential genes, we defined the transcript intervals in which a variant could generate a nonstop transcript for each reading frame (Figure 2). In the +0 frame, only disruption of the canonical stop codon (3 nucleotides) yields an in-frame nonstop transcript. For +1 and +2 frames, we simulated a frameshifted transcript and defined the NSD-susceptible region as the interval between the last available stop codon in that shifted frame and the canonical stop codon. Transcript-relative coordinates were converted to genomic coordinates to create a unified NSD coordinate reference set.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical GS patient cohort\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA retrospective analysis of GS results from 2,439 patients at Baylor Genetics was performed. These individuals underwent GS either as proband-only, duo, or trio-based to investigate suspected genetic etiologies. This diagnostic laboratory is certified by the College of American Pathologists (CAP) and complies with the Clinical Laboratory Improvement Amendments. The analysis of deidentified data was approved by the Institutional Review Board at Baylor College of Medicine (IRB: H-41191)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood samples and buccal samples were provided in most cases, although other sources of DNA were also accepted. Library preparation was performed using a PCR-free approach following the manufacturer’s protocols (Illumina, San Diego, US). The GS protocol also incorporated either a coding single-nucleotide polymorphism array procedure or a spike-in plasmid tracer for quality control and sample tracking. Each library underwent sequencing with paired-end 150bp reads to achieve an average nuclear genome coverage ≥40x.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVariant detection and classification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVariant calling was conducted using in-house and proprietary bioinformatics pipelines. The scope of variant detection included single nucleotide variants (SNV), small insertions and deletions (\u0026lt;50bp, indels), copy-number variants (CNV), absence of heterozygosity, uniparental disomy, tandem repeat expansions, and mitochondrial variants. For clinical reporting, the genomic findings were classified into five tiers (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign, and Benign) according to Baylor Genetics’ classification scheme, which incorporates the guidelines recommended by the ACMG/AMP [5].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVariant screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe screened the 2,439 GS VCFs for variants overlapping NSD-susceptible regions (Figure 3). Variants were annotated for overlapping position, predicted reading frame, and variant consequence. Candidate NSD variants underwent post-screening review to determine (i) whether LoF is an established disease mechanism for the gene, (ii) whether zygosity and inheritance were consistent with the gene–disease association, and (iii) whether the individual’s phenotype was compatible with the associated disorder. This study is descriptive; no hypothesis-testing statistics were applied.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eGenome-wide landscape of NSD susceptibility among disease genes\u003c/h2\u003e\n\u003cp\u003eAmong 4,954 OMIM disease-associated protein-coding genes, 333 (6.72%) were classified as NSD-potential in at least one reading frame. Across these 333 genes, we identified 546 distinct genomic regions capable of harboring variants that generate nonstop transcripts (Supplemental Table 1). In aggregate, NSD-susceptible loci spanned 34,744 nucleotides—approximately 0.0011% of the total chromosomal sequence—indicating that while the genomic footprint is small, the number of disease genes with NSD potential is substantial (Figure 4).\u003c/p\u003e\n\u003ch2\u003ePrevalence of candidate NSD variants in a clinical GS cohort\u003c/h2\u003e\n\u003cp\u003eScreening of 2,439 GS cases identified 359 candidate NSD variants across NSD-susceptible loci. Most variants were observed in blood group–related genes (n = 332) and there were three genes which LoF is not the established disease mechanism (n = 3). Eighteen candidate NSD variants were found in genes associated with autosomal recessive disease; in the absence of a second reportable variant in trans and compatible phenotypes, these findings were not clinically reportable but may be relevant in the context of reproductive testing (e.g., carrier screening).\u003c/p\u003e\n\u003cp\u003eSix probands harbored NSD-predicted variants in disease-causing genes that were consistent with their clinical phenotypes and inheritance expectations (Table 1). These variants would typically receive only PVS1_Moderate or PVS1_Strong under existing PVS1 decision trees because they occur near the 3′ end and are predicted to alter only a small C-terminal portion of the protein. Incorporating NSD as a transcript-null mechanism supports treating these variants as functional null alleles (subject to gene- and transcript-specific considerations) and can increase the overall weight of pathogenic evidence when combined with additional data (e.g., de novo occurrence, phenotype specificity, segregation and allelic data, in silico predictions).\u003c/p\u003e\n\u003ch2\u003eCase vignettes illustrating NSD-relevant reclassification\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eNDUFS7\u003c/em\u003e (autosomal recessive mitochondrial complex I deficiency, nuclear type 3, OMIM: 618224). Compound heterozygous variants were identified in the \u003cem\u003eNDUFS7\u003c/em\u003e gene: a paternal inherited frameshift variant NM_024407.5:c.610del (p.Glu204Serfs*?) predicted to abolish the canonical stop codon and generate a nonstop transcript, and a known pathogenic missense variant NM_024407.5:c.364G\u0026gt;A (p.Val122Met). Without NSD consideration, the frameshift might be interpreted as a near-terminal truncation removing nine amino acids and receive only PVS1_Moderate; with NSD incorporation, it is consistent with a null allele acting in trans with a pathogenic variant and a highly concordant phenotype with mitochondrial complex I deficiency, supporting pathogenic classification.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eISG15\u003c/em\u003e (autosomal recessive immunodeficiency 38, OMIM: 616126). A homozygous NM_005101.4:c.463dup (p.Arg155Profs*?) frameshift variant predicted to create a nonstop transcript was identified in the \u003cem\u003eISG15\u003c/em\u003e gene. Because the frameshift occurs near the 3′ end with only ten animo acids downstream, this variant would typically receive PVS1_Moderate; NSD provides a mechanistic rationale for transcript degradation and supports pathogenic classification in the context of a compatible phenotype with immunodeficiency and recessive inheritance in this patient.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHCN2\u003c/em\u003e (autosomal dominant \u003cem\u003eHCN2\u003c/em\u003e-related epilepsy, OMIM: 602477). A heterozygous de novo frameshift NM_001194.4:c.2328_2334dup (p.Ser779Profs*?) variant predicted to generate a nonstop transcript was detected in the \u003cem\u003eHCN2\u003c/em\u003e gene. This variant disrupts normal transcript termination and generates a nonstop mRNA predicted to be degraded through the NSD pathway. This variant had a PVS1_Strong criteria code since the altered 3’ coding region of protein is more than 10% and was also reclassified from variant of uncertain significance (VUS) to likely pathogenic after the incorporation of NSD rules in variant classification which is consistent with the clinical diagnosis of epilepsy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHNRNPD\u003c/em\u003e (autosomal dominant \u003cem\u003eHNRNPD\u003c/em\u003e-related neurodevelopmental disorder, CCID: 009030). A de novo canonical splice acceptor variant in the last coding exon of the \u003cem\u003eHNRNPD\u003c/em\u003e gene (NM_031369.3:c.944-1G\u0026gt;A) was identified. For this gene, LoF/haploinsufficiency is an established disease mechanism. Current splicing recommendations often assign PVS1_Moderate for the last-exon splice site variants in the absence of evidence for NMD [7]; NSD provides an additional pathway by which last-exon splice disruption can lead to transcript loss when the termination codon is removed and no compensatory stop codon exists prior to polyadenylation, supporting a higher PVS1 strength when transcript context is consistent.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSHANK2\u003c/em\u003e (autosomal dominant \u003cem\u003eSHANK2\u003c/em\u003e-related neurodevelopmental disorder, CCID: 006134). A heterozygous frameshift variant was detected in the \u003cem\u003eSHANK2\u003c/em\u003e gene (NM_012309.5:c.5302_5305del; p.Leu1768Glnfs*?). In genes where haploinsufficiency is the disease mechanism, NSD prediction is consistent with a null allele and supports upgrading LoF evidence from PVS1_Moderate to PVS1. This variant was also reclassified from VUS to likely pathogenic which is consistent with the neurodevelopmental phenotype in this patient.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTYMP\u003c/em\u003e (autosomal recessive mitochondrial DNA depletion syndrome type 1 (MNGIE), OMIM: 603041). Compound heterozygous variants were detected in the \u003cem\u003eTYMP\u003c/em\u003e gene including a frameshift duplication variant NM_001953.5:c.865_889dup (p.Ala297Glyfs*?) predicted to abolish the native stop codon and induce NSD, in trans with a likely pathogenic in-frame deletion NM_001953.5:c.1198_1203del (p.Val400_Leu401del). Considering LoF as the disease mechanism, NSD supports treating the frameshift as a null allele consistent with molecular and clinical diagnosis of mitochondrial DNA depletion syndrome type 1 (MNGIE) in this patient.\u003c/p\u003e\n\u003ch2\u003eExemplar implementation in gene-/disease-specific variant classification guidance: \u003cem\u003eDDX41\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eClinGen Variant Curation Expert Panels (VCEPs) develop gene-/disease-specific ACMG/AMP specifications to improve consistency and accuracy in variant classification. The \u003cem\u003eDDX41\u003c/em\u003e gene is associated with \u003cem\u003eDDX41\u003c/em\u003e-related hematologic malignancy predisposition syndrome (CCID: 004638). The condition is primarily characterized by adult-onset acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Malignant transformation is driven by biallelic inactivation of \u003cem\u003eDDX41\u003c/em\u003e with the second hit frequently being one of several highly specific somatic hotspot variants [8,9]. Given \u003cem\u003eDDX41\u003c/em\u003e functions as a tumor suppressor gene, null variants are typically identified in patients at risk for this condition. While many pathogenic \u003cem\u003eDDX41\u003c/em\u003e variants are truncating, a subset of terminal frameshift variants is predicted to generate nonstop transcripts depending on the location of compensatory stop codons relative to the poly(A) tail. We summarize published and database-reported individuals who carry \u003cem\u003eDDX41\u003c/em\u003e frameshift variants consistent with NSD prediction (Table 2). In several reported cases, coexistence of a hotspot somatic variant provides phenotype/genotype coherence that can strengthen pathogenicity assessment when NSD is recognized as a functional null mechanism.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eDDX41\u003c/em\u003e (NM_016222.4), the annotated polyadenylation signal and insertion site imply that +1 and −1 frameshift variants in the terminal coding region can place the next in-frame stop codon downstream of the poly(A) tail, producing nonstop transcripts. In this setting, treating such variants as LoF is mechanistically and clinically reasonable, contingent on transcript selection and gene mechanism considerations. This illustrates how NSD criteria can be incorporated into ClinGen VCEP specifications alongside established PVS1 decision trees.\u003c/p\u003e\n\u003ch1\u003eRecommendations for incorporating NSD into variant classification\u003c/h1\u003e\n\u003cp\u003eRecommendation 1: Treat NSD as a transcript-null mechanism analogous to NMD when LoF is an established disease mechanism. NSD is expected to reduce transcript abundance through translation-coupled degradation when an mRNA lacks an in-frame termination codon and translation reaches the poly(A) tail [2–4]. For genes in which haploinsufficiency or biallelic LoF causes disease, variants predicted to trigger NSD should generally be evaluated under LoF frameworks (e.g., PVS1), rather than being discounted as minor C-terminal alterations.\u003c/p\u003e\n\u003cp\u003eRecommendation 2: Define NSD susceptibility on a transcript-specific basis. NSD prediction depends on (i) the clinically relevant transcript (preferably MANE Select and/or MANE Plus Clinical where available), (ii) the location of the poly(A) site (including alternative polyadenylation), and (iii) the presence of compensatory stop codons in the relevant reading frame between the disrupted canonical stop codon and the poly(A) tail. Laboratories should document the transcript used and the stop-codon landscape in the relevant frame as part of the interpretation rationale.\u003c/p\u003e\n\u003cp\u003eRecommendation 3: Recognize the variant classes most likely to trigger NSD. These include: (a) stop-loss variants that abolish the canonical stop codon without the alteration of upstream coding sequence; (b) frameshift variants in the terminal coding region that eliminate the canonical stop codon; (c) splice acceptor/donor or other variants that cause skipping of the last (or penultimate) coding exon containing the termination codon or cause a frameshift into a NSD-susceptible reading frame; and (d) copy-number or structural variants that delete the final stop-containing exon(s).\u003c/p\u003e\n\u003cp\u003eRecommendation 4: Calibrate strength of evidence using existing PVS1 logic, with explicit NSD checkpoints. We recommend adding an NSD checkpoint to the PVS1 decision tree [6] for variants near the 3′ end that would otherwise be downgraded due to limited predicted truncation. If the variant is predicted to generate a nonstop transcript on the clinically relevant transcript and LoF is a known disease mechanism, applying PVS1 at the same strength as other null variants of comparable confidence is appropriate (Table 3). In semi-quantitative adaptations of ACMG/AMP, Very Strong pathogenic evidence corresponds to 8 points [10,11], and an NSD-triggering variant in a LoF gene can reasonably contribute at this level when prediction confidence is high. (Notably, an ACMG/AMP/CAP/ClinGen SVC v4.0 standard is in development; laboratories should align NSD implementation with the finalized framework when released.)\u003c/p\u003e\n\u003cp\u003eRecommendation 5: Distinguish canonical NSD from long 3′-tail/termination inefficiency mechanisms. Some transcripts may terminate far downstream of the canonical stop due to readthrough or creation of a distant stop codon; evidence suggests that unusually long 3′ UTR translation can trigger decay mechanisms distinct from classic NSD [12]. When the next stop codon is present but far downstream (e.g., \u0026gt;150 nt beyond the canonical termination site), we suggest considering a reduced-strength LoF contribution (PVS1_Moderate) unless additional experimental evidence demonstrates transcript loss (Table 3). This approach parallels existing guidance to modulate PVS1 strength based on predicted molecular outcome confidence [6].\u003c/p\u003e\n\u003cp\u003eOperational checklist. When evaluating a candidate NSD variant, we recommend documenting: (1) gene–disease mechanism (LoF established?); (2) transcript selection (MANE Select/ MANE Plus Clinical/ clinically curated transcript); (3) whether the variant removes the canonical stop codon or stop-containing exon(s); (4) whether an in-frame compensatory stop codon exists prior to polyadenylation in the relevant reading frame; (5) whether alternative transcripts or polyadenylation could rescue termination; and (6) any corroborating evidence (RNA studies, segregation, de novo status, phenotype specificity).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights NSD as an under-recognized but potentially important mechanism in clinical variant classification. Although the genomic regions capable of generating nonstop transcripts are small in aggregate, we found that ~7% of OMIM disease-associated genes have transcript architectures that permit NSD in at least one reading frame. This observation challenges the perception that NSD-relevant variants are “ultra-rare” and suggests that NSD should be considered routinely when interpreting stop-loss, terminal frameshift, or last-exon splice or deletion variants in LoF-mediated conditions.\u003c/p\u003e\n\u003cp\u003eOur case series illustrates a recurring interpretive pitfall: near-terminal variants are often downgraded because they appear to remove only a short C-terminal segment. This logic is appropriate when translation is expected to terminate soon after the variant and produce a largely intact protein, but it can be misleading when the true outcome is loss of the termination codon and degradation of the transcript. In most examples here, NSD provides a mechanistically coherent explanation for functional null alleles that would otherwise be categorized as VUS due to reduced PVS1 strength. Importantly, NSD should not be applied indiscriminately: prediction requires transcript-specific assessment of compensatory stop codons and polyadenylation sites, and clinical classification must still integrate additional evidence (inheritance, segregation, de novo status, phenotype specificity, population frequency, in silico prediction, experimental evidence) per ACMG/AMP/ClinGen guidelines [5,6].\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eDDX41\u003c/em\u003e exemplar underscores how NSD can be operationalized within ClinGen VCEP specifications. In tumor suppressor genes with characteristic second-hit somatic hotspots, recognizing NSD as a LoF mechanism can align molecular predictions with observed genotype patterns. More broadly, NSD-aware LoF assessment may improve cross-laboratory consistency by reducing reliance on variable, ad hoc interpretations of terminal frameshift or stop-loss variants.\u003c/p\u003e\n\u003cp\u003eLimitations. First, NSD susceptibility was inferred computationally from reference transcript annotations and selected polyadenylation sites; alternative splicing and alternative polyadenylation could mitigate or exacerbate NSD in a tissue-specific manner. Second, we did not systematically quantify transcript abundance changes (e.g., RNA sequencing) for candidate variants; future work integrating RNA data would help calibrate prediction confidence and PVS1 strength. Third, our GS cohort screening reflects a clinical testing population and is enriched for disease-causing gene and variants which may limit generalizability of raw prevalence estimates. Finally, interpretation in oncology predisposition genes may require additional somatic context and careful distinction between germline and somatic events.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eVariants predicted to trigger mRNA nonstop decay are present across a non-trivial fraction of disease-associated genes and can contribute directly to rare disease molecular diagnoses. Incorporating NSD into LoF variant classification—using transcript-specific checks and gene mechanism awareness—can improve the consistency and accuracy of PVS1 strength assignment and reduce the number of clinically relevant variants remaining as VUS. We recommend that NSD checkpoints be incorporated into gene-/disease-specific ACMG/AMP/ClinGen specifications and, as consensus frameworks evolve, into general variant classification guidance.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACMG: American College of Medical Genetics and Genomics; AML: Acute Myeloid Leukemia; AMP: Association for Molecular Pathology; CAP: College of American Pathologists; CCID: ClinGen Curation Identification; CDS: coding sequence; ClinGen: Clinical Genome Resource; GS: genome sequencing; LoF: loss of function; MANE: Matched Annotation from NCBI and EMBL-EBI; MDS: Myelodysplastic Syndromes; NGD: no-go decay; NMD: nonsense-mediated decay; NSD: nonstop decay; PTC: premature termination codon; SVI: Sequence Variant Interpretation; VCEP: Variant Curation Expert Panel; VCF: variant call format; VUS: variant of uncertain significance.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThis study was conducted in accordance with protocol (H-41191) approved by the Institutional Review Board at Baylor College of Medicine. The study was also conducted in accordance with the Declaration of Helsinki.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eConsent was waived for the individuals included in this study per Institutional Review Board protocol.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eY.Z., C.C.T., R.R., M.D., J.L., N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are employed by Baylor Genetics. Baylor College of Medicine and Miraca Holdings Inc. have formed a joint venture with shared ownership and governance of Baylor Genetics, which performs genetic testing and derives revenue. N.L., Y.W., L.V., X.Z., L.M., C.M.E., F.X., and X.L. are also employees of Baylor College of Medicine and derive support through a professional services agreement with Baylor Genetics. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFundings\u003c/h2\u003e\n\u003cp\u003eNo funding was received for the current study.\u003c/p\u003e\n\u003ch2\u003eAuthors' contributions\u003c/h2\u003e\n\u003cp\u003eStudy conception: Y.Z., X.L. Study design: Y.Z., X.L. Data curation: Y.Z., D.H., N.M., C.C.T., J.L., X.L. Data analysis: Y.Z., D.H., N.M., J.L., X.L. Data interpretation: Y.Z., D.H., N.M., J.L., N.L., Y.W., X.L. Writing-original draft: Y.Z., D.H., N.M., R.R., M.D., N.L., X.L. Writing-review and editing: Y.Z., D.H., N.M., C.C.T., R.R., M.D., J.L., N.L., Y.W., D.W., L.A.G., L.V., X.Z., L.M., C.M.E., F.X., and X.L.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWolin SL, Maquat LE. Cellular RNA surveillance in health and disease. Science (1979). American Association for the Advancement of Science; 2019. p. 822\u0026ndash;7. https://doi.org/10.1126/science.aax2957\u003c/li\u003e\n\u003cli\u003eMonaghan L, Longman D, C\u0026aacute;ceres JF. Translation‐coupled mRNA quality control mechanisms. EMBO J. Springer Science and Business Media LLC; 2023;42. https://doi.org/10.15252/embj.2023114378\u003c/li\u003e\n\u003cli\u003ePowers KT, Szeto JYA, Schaffitzel C. New insights into no-go, non-stop and nonsense-mediated mRNA decay complexes. Curr. Opin. Struct. Biol. Elsevier Ltd; 2020. p. 110\u0026ndash;8. https://doi.org/10.1016/j.sbi.2020.06.011\u003c/li\u003e\n\u003cli\u003eKlauer AA, van Hoof A. Degradation of mRNAs that lack a stop codon: A decade of nonstop progress. Wiley Interdiscip. Rev. RNA. 2012. p. 649\u0026ndash;60. https://doi.org/10.1002/wrna.1124\u003c/li\u003e\n\u003cli\u003eRichards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine. Nature Publishing Group; 2015;17:405\u0026ndash;24. https://doi.org/10.1038/gim.2015.30\u003c/li\u003e\n\u003cli\u003eAbou Tayoun AN, Pesaran T, DiStefano MT, Oza A, Rehm HL, Biesecker LG, et al. Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. Hum Mutat. John Wiley and Sons Inc.; 2018;39:1517\u0026ndash;24. https://doi.org/10.1002/humu.23626\u003c/li\u003e\n\u003cli\u003eWalker LC, Hoya M de la, Wiggins GAR, Lindy A, Vincent LM, Parsons MT, et al. Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup. Am J Hum Genet. Cell Press; 2023;110:1046\u0026ndash;67. https://doi.org/10.1016/j.ajhg.2023.06.002\u003c/li\u003e\n\u003cli\u003ePolprasert C, Schulze I, Sekeres MA, Makishima H, Przychodzen B, Hosono N, et al. Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms. Cancer Cell. Cell Press; 2015;27:658\u0026ndash;70. https://doi.org/10.1016/j.ccell.2015.03.017\u003c/li\u003e\n\u003cli\u003eKadono M, Kanai A, Nagamachi A, Shinriki S, Kawata J, Iwato K, et al. Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia. Exp Hematol. Elsevier Inc.; 2016;44:745-754.e4. https://doi.org/10.1016/j.exphem.2016.04.017\u003c/li\u003e\n\u003cli\u003eTavtigian S V., Greenblatt MS, Harrison SM, Nussbaum RL, Prabhu SA, Boucher KM, et al. Modeling the ACMG/AMP variant classification guidelines as a Bayesian classification framework. Genetics in Medicine. Nature Publishing Group; 2018;20:1054\u0026ndash;60. https://doi.org/10.1038/gim.2017.210\u003c/li\u003e\n\u003cli\u003eTavtigian S V., Harrison SM, Boucher KM, Biesecker LG. Fitting a naturally scaled point system to the ACMG/AMP variant classification guidelines. Hum Mutat. John Wiley and Sons Inc; 2020;41:1734\u0026ndash;7. https://doi.org/10.1002/humu.24088\u003c/li\u003e\n\u003cli\u003eTakata A, Hamanaka K, Matsumoto N. Refinement of the clinical variant interpretation framework by statistical evidence and machine learning. Med. Cell Press; 2021;2:611-632.e9. https://doi.org/10.1016/j.medj.2021.02.003\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Positive GS cases with NSD variants identified in disease-causing genes consistent with clinical phenotypes.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"864\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003eDisease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eInheritance/\u003c/p\u003e\n \u003cp\u003eZygosity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eVariant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eProtein Full Length\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1 criteria without or with NSD incorporation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eACMG/AMP/ClinGen Classification without or with NSD incorporation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eNDUFS7\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003eMitochondrial Complex I Deficiency, Nuclear Type 3 (OMIM: 618224).\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAR/\u003c/p\u003e\n \u003cp\u003eCompound Heterozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eNM_024407.5: c..610del (p.E204Sfs*?);\u003c/p\u003e\n \u003cp\u003ec.364G\u0026gt;A (p.V122M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1_Moderate \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePVS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eVUS \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eISG15\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003eImmunodeficiency 38\u003c/p\u003e\n \u003cp\u003e(OMIM: 616126)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAR/\u003cbr\u003e\u0026nbsp;Homozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eNM_005101.4: c.463dup (p.R155Pfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1_Moderate \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePVS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eVUS \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eHCN2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cem\u003eHCN2\u003c/em\u003e-related\u003cbr\u003e\u0026nbsp;epilepsy\u003c/p\u003e\n \u003cp\u003e(OMIM: 602477)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAD/\u003cbr\u003e\u0026nbsp;De Novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eNM_001194.4: c.2328_2334dup,\u003cbr\u003e\u0026nbsp;(p.S779Pfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1_Strong \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePVS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eVUS \u0026rarr;\u003c/p\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eHNRNPD\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cem\u003eHNRNPD\u003c/em\u003e-related neurodevelopmental disorder\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(CCID: 009030)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAD/\u003cbr\u003e\u0026nbsp;De Novo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eNM_031369.3:c.944-1G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e355\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1_Moderate \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePVS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eVUS \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eSHANK2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cem\u003eSHANK2\u003c/em\u003e-related neurodevelopmental disorder\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(CCID: 006134)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAD/\u003cbr\u003e\u0026nbsp;Heterozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eNM_012309.5: c.5302_5305del (p.Leu1768Glnfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e1800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1_Moderate \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePVS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eVUS \u0026rarr;\u003c/p\u003e\n \u003cp\u003eLikely Pathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cem\u003eTYMP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003eMitochondrial DNA depletion syndrome type 1 (MNGIE)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(OMIM: 603041)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAR/\u003cbr\u003e\u0026nbsp;Compound Heterozygous\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 180px;\"\u003e\n \u003cp\u003eNM_001953.5:c.865_889dup (p.Ala297Glyfs*?);\u003c/p\u003e\n \u003cp\u003ec.1198_1203del (p.Val400_Leu401del)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e482\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 140px;\"\u003e\n \u003cp\u003ePVS1_Strong \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePVS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 160px;\"\u003e\n \u003cp\u003eLikely Pathogenic \u0026rarr;\u003c/p\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. \u003cem\u003eDDX41\u003c/em\u003e frameshift variants reported in literature or public databases that are predicted to be subject to non-stop decay.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eGermline cDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eGermline protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eGermline VAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eSomatic cDNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eSomatic protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eSomatic VAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eOrigin confirmed?\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eDiagnosis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1791_1792del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Lys597Asnfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e\u0026gt;40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003ec.1574G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003ep.Arg525His\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eAML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e[1,2]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1811_1812insCATATGTGCTAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Lys604Asnfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e52%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003ec.1574G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003ep.Arg525His\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e23%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eMyeloid neoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e[3], ClinVar: 3371864\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1836_1837del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Asp613LeufsTer?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e35-40%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e60-65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e[4], gnomAD: 5-177511822-TCC-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003ec.1857dup\u003cbr\u003e\u0026nbsp;c.1589G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003ep.Met620Hisfs*?\u003cbr\u003e\u0026nbsp;p.Gly530Asp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e34.8%\u003cbr\u003e\u0026nbsp;30.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eMyeloid neoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e[5]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1814del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Gln605ArgfsTer?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e41.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003ec.1574G\u0026gt;A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003ep.Arg525His\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e5.3%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eMDS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e[6]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1721del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Leu574Argfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eMDS,\u0026nbsp;AML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eClinVar:1338572\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1773del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Ile592Serfs*?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e45-50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003egnomAD: 5-177511886-TC-T\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003ec.1843del\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 144px;\"\u003e\n \u003cp\u003ep.(Leu615TrpfsTer?)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e45-50%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e40-45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003egnomAD: 5-177511816-AG-A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Conceptual mapping of termination-related mRNA surveillance mechanisms and proposed evidence strength in variant classification.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"864\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eScenario\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eDescription\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003ePathway Triggered\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 210px;\"\u003e\n \u003cp\u003eLoF evidence criteria\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003ePremature stop codon (early termination)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eNonsense-Mediated Decay (NMD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003eUpstream exon\u0026ndash;exon junction \u0026rarr; decay triggered\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePVS1; +8 points\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eNo stop codon \u0026rarr; ribosome reaches poly(A)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eNon-stop Decay (NSD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003eRibosome stalls at poly(A), mRNA degraded \u0026rarr; decay triggered\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePVS1; +8 points\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 186px;\"\u003e\n \u003cp\u003eStop codon far downstream (\u0026gt;150 nt past normal site)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 162px;\"\u003e\n \u003cp\u003eExtended nonstop-like surveillance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 306px;\"\u003e\n \u003cp\u003eRibosome reads through long 3\u0026prime; region \u0026rarr; decay may be triggered\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 210px;\"\u003e\n \u003cp\u003ePVS1_Moderate; +2 points\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e1. Ebert MS\u0026acute;, Passet M, Raimbault A, Rahm\u0026eacute; R, Rahm\u0026eacute; R, Raffoux E, et al. Germline DDX41 mutations define a significant entity within adult MDS/AML patients. Blood. 2019.\u0026nbsp;\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e2. Duployez N, Largeaud L. Prognostic impact of DDX41 germline mutations in intensively treated acute myeloid leukemia patients: an ALFA-FILO study. Blood. American Society of Hematology; 2020;136:2125\u0026ndash;32. https://doi.org/10.1182/BLOOD.2019000962\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e3. Aguilera-Diaz A, Larrayoz MJ, Palomino-Echeverr\u0026iacute;a S, Vazquez I, Ariceta B, Ma\u0026ntilde;\u0026uacute; A, et al. Strategy for identification of a potential inherited leukemia predisposition in a 299 patient\u0026rsquo;s cohort with tumor-only sequencing data. Leuk Res. Elsevier Ltd; 2020;95. https://doi.org/10.1016/j.leukres.2020.106386\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e4. Kovilakam SC, Gu M, Dunn WG, Marando L, Barcena C, England G, et al. Prevalence and significance of DDX41 gene variants in the general population.\u0026nbsp;\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e5. Qu S, Li B, Qin T, Xu Z, Pan L, Hu N, et al. Molecular and clinical features of myeloid neoplasms with somatic DDX41 mutations. Br J Haematol. Blackwell Publishing Ltd; 2021;192:1006\u0026ndash;10. https://doi.org/10.1111/bjh.16668\u003c/p\u003e\n\u003cp class=\"MsoNormal\"\u003e6. Maierhofer A, Mehta N, Chisholm RA, Hutter S, Baer C, Nadarajah N, et al. The clinical and genomic landscape of patients with DDX41 variants identified during diagnostic sequencing. Blood Adv. American Society of Hematology; 2023;7:7346\u0026ndash;57. https://doi.org/10.1182/bloodadvances.2023011389\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genome-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Genome Medicine](https://genomemedicine.biomedcentral.com/)","snPcode":"13073","submissionUrl":"https://submission.springernature.com/new-submission/13073/3","title":"Genome Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"nonstop decay, mRNA surveillance, variant classification, PVS1, loss-of-function, stop-loss, rare disease, genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-9126009/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9126009/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Nonsense-mediated decay (NMD) is routinely considered during clinical interpretation of predicted loss-of-function (LoF) variants, whereas mRNA nonstop decay (NSD)—a translation-coupled surveillance pathway that degrades transcripts lacking an in-frame termination codon—is rarely considered. Variants that abolish the canonical stop codon or remove the final stop-containing exon(s) can create nonstop transcripts, but current American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant classification guidelines and the Clinical Genome Resource (ClinGen) PVS1 recommendations do not explicitly address NSD, creating a potential mechanism-specific gap in variant classification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We developed a computational workflow to identify disease-associated genes and transcript regions in which variants are predicted to generate nonstop transcripts in any of the three reading frames. Disease genes were obtained from OMIM and transcript annotations were retrieved from Ensembl. NSD-potential genes were defined as those lacking an in-frame stop codon in the 3′ UTR prior to the annotated polyadenylation site for at least one reading frame. We then screened 2,439 clinical genome sequencing (GS) variant call format (VCF) files for variants overlapping NSD-susceptible regions and performed case-level review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Among 4,954 OMIM disease-associated protein-coding genes, 333 (6.72%) had NSD potential in ≥1 reading frame, yielding 546 genomic NSD-susceptible loci spanning 34,744 bp (~0.0011% of the human genome). Screening of 2,439 GS cases identified 359 candidate NSD variants; six variants in six probands were consistent with the individuals’ phenotypes and the genes’ disease mechanism. In these cases, incorporation of NSD as a transcript-null mechanism would increase the weight of LoF evidence and support reclassification from variant of uncertain significance to likely pathogenic or pathogenic when combined with additional clinical and genetic evidence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: NSD-predicted variants are not ultra-rare among disease genes and can be clinically relevant in rare disease diagnosis. We propose practical recommendations for recognizing NSD-susceptible variants and incorporating NSD into gene-/disease-specific LoF frameworks (e.g., PVS1) to improve consistency and diagnostic yield.\u003c/p\u003e","manuscriptTitle":"Clinical significance of mRNA nonstop decay in rare disease diagnosis and recommendations for its application in variant classification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-26 16:45:01","doi":"10.21203/rs.3.rs-9126009/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"123819104942639184498517080348160216233","date":"2026-05-11T00:40:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93480434162609711851377879893965424548","date":"2026-05-08T05:22:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55771874564895432018732533252619579957","date":"2026-04-23T05:38:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-24T13:49:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-23T05:00:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T11:42:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genome Medicine","date":"2026-03-15T03:32:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genome-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Genome Medicine](https://genomemedicine.biomedcentral.com/)","snPcode":"13073","submissionUrl":"https://submission.springernature.com/new-submission/13073/3","title":"Genome Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54238db5-3053-4e00-8fd8-b957986e4069","owner":[],"postedDate":"March 26th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"123819104942639184498517080348160216233","date":"2026-05-11T00:40:26+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"93480434162609711851377879893965424548","date":"2026-05-08T05:22:17+00:00","index":34,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-26T16:45:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-26 16:45:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9126009","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9126009","identity":"rs-9126009","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}