Functional analysis and clinical classification of 462 germline BRCA2 missense variants affecting the DNA binding domain.

Variants of uncertain significance (VUSs) in BRCA2 are a common result of hereditary cancer genetic testing. While more than 4,000 unique VUSs, comprised of missense or intronic variants, have been identified in BRCA2, the few missense variants now classified clinically as pathogenic or likely pathogenic are predominantly located in the region encoding the C-terminal DNA binding domain (DBD). We report on functional evaluation of the influence of 462 BRCA2 missense variants affecting the DBD on DNA repair activity of BRCA2 using a homology-directed DNA double-strand break repair assay. Of these, 137 were functionally abnormal, 313 were functionally normal, and 12 demonstrated intermediate function. Comparisons with other functional studies of BRCA2 missense variants yielded strong correlations. Sequence-based in silico prediction models had high sensitivity, but limited specificity, relative to the homology-directed repair assay. Combining the functional results with clinical and genetic data in an American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP)-like variant classification framework from a clinical testing laboratory, after excluding known splicing variants and functionally intermediate variants, classified 431 of 442 (97.5%) missense variants (129 as pathogenic/likely pathogenic and 302 as benign/likely benign). Functionally abnormal variants classified as pathogenic by ACMG/AMP rules were associated with a slightly lower risk of breast cancer (odds ratio [OR] 5.15, 95% confidence interval [CI] 3.43-7.83) than BRCA2 DBD protein truncating variants (OR 8.56, 95% CI 6.03-12.36). Overall, functional studies of BRCA2 variants using validated assays substantially improved the variant classification yield from ACMG/AMP models and are expected to improve clinical management of many individuals found to harbor germline BRCA2 missense VUS.

Evidence-based recommendations for gene-specific ACMG/AMP variant classification from the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel.

The ENIGMA research consortium develops and applies methods to determine clinical significance of variants in hereditary breast and ovarian cancer genes. An ENIGMA BRCA1/2 classification sub-group, formed in 2015 as a ClinGen external expert panel, evolved into a ClinGen internal Variant Curation Expert Panel (VCEP) to align with Food and Drug Administration recognized processes for ClinVar contributions. The VCEP reviewed American College of Medical Genetics and Genomics/Association of Molecular Pathology (ACMG/AMP) classification criteria for relevance to interpreting BRCA1 and BRCA2 variants. Statistical methods were used to calibrate evidence strength for different data types. Pilot specifications were tested on 40 variants and documentation revised for clarity and ease of use. The original criterion descriptions for 13 evidence codes were considered non-applicable or overlapping with other criteria. Scenario of use was extended or re-purposed for eight codes. Extensive analysis and/or data review informed specification descriptions and weights for all codes. Specifications were applied to pilot variants with pre-existing ClinVar classification as follows: 13 uncertain significance or conflicting, 14 pathogenic and/or likely pathogenic, and 13 benign and/or likely benign. Review resolved classification for 11/13 uncertain significance or conflicting variants and retained or improved confidence in classification for the remaining variants. Alignment of pre-existing ENIGMA research classification processes with ACMG/AMP classification guidelines highlighted several gaps in the research processes and the baseline ACMG/AMP criteria. Calibration of evidence strength was key to justify utility and strength of different data types for gene-specific application. The gene-specific criteria demonstrated value for improving ACMG/AMP-aligned classification of BRCA1 and BRCA2 variants.

Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup.

The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) framework for classifying variants uses six evidence categories related to the splicing potential of variants: PVS1, PS3, PP3, BS3, BP4, and BP7. However, the lack of guidance on how to apply such codes has contributed to variation in the specifications developed by different Clinical Genome Resource (ClinGen) Variant Curation Expert Panels. The ClinGen Sequence Variant Interpretation Splicing Subgroup was established to refine recommendations for applying ACMG/AMP codes relating to splicing data and computational predictions. We utilized empirically derived splicing evidence to (1) determine the evidence weighting of splicing-related data and appropriate criteria code selection for general use, (2) outline a process for integrating splicing-related considerations when developing a gene-specific PVS1 decision tree, and (3) exemplify methodology to calibrate splice prediction tools. We propose repurposing the PVS1_Strength code to capture splicing assay data that provide experimental evidence for variants resulting in RNA transcript(s) with loss of function. Conversely, BP7 may be used to capture RNA results demonstrating no splicing impact for intronic and synonymous variants. We propose that the PS3/BS3 codes are applied only for well-established assays that measure functional impact not directly captured by RNA-splicing assays. We recommend the application of PS1 based on similarity of predicted RNA-splicing effects for a variant under assessment in comparison with a known pathogenic variant. The recommendations and approaches for consideration and evaluation of RNA-assay evidence described aim to help standardize variant pathogenicity classification processes when interpreting splicing-based evidence.

Closing the gap: Systematic integration of multiplexed functional data resolves variants of uncertain significance in BRCA1, TP53, and PTEN.

Clinical interpretation of missense variants is challenging because the majority identified by genetic testing are rare and their functional effects are unknown. Consequently, most variants are of uncertain significance and cannot be used for clinical diagnosis or management. Although not much can be done to ameliorate variant rarity, multiplexed assays of variant effect (MAVEs), where thousands of single-nucleotide variant effects are simultaneously measured experimentally, provide functional evidence that can help resolve variants of unknown significance (VUSs). However, a rigorous assessment of the clinical value of multiplexed functional data for variant interpretation is lacking. Thus, we systematically combined previously published BRCA1, TP53, and PTEN multiplexed functional data with phenotype and family history data for 324 VUSs identified by a single diagnostic testing laboratory. We curated 49,281 variant functional scores from MAVEs for these three genes and integrated four different TP53 multiplexed functional datasets into a single functional prediction for each variant by using machine learning. We then determined the strength of evidence provided by each multiplexed functional dataset and reevaluated 324 VUSs. Multiplexed functional data were effective in driving variant reclassification when combined with clinical data, eliminating 49% of VUSs for BRCA1, 69% for TP53, and 15% for PTEN. Thus, multiplexed functional data, which are being generated for numerous genes, are poised to have a major impact on clinical variant interpretation.

Simplified and more sensitive criteria for identifying individuals with pathogenic CDH1 variants.

BACKGROUND: Hereditary diffuse gastric cancer (HDGC) is an autosomal-dominant syndrome most often caused by pathogenic variants in CDH1. The International Gastric Cancer Linkage Consortium (IGCLC) recently updated its criteria for genetic testing. The purpose of this study was to estimate the sensitivity of IGCLC's 2020 criteria for identifying carriers of CDH1 pathogenic variants and to formulate a new set of criteria that is simpler and more sensitive. METHODS: Medical histories of 112 CDH1 mutation carriers, identified predominantly by multigene panel testing, and their 649 family members were reviewed. The percentage of subjects fulfilling the IGCLC 2015 and 2020 criteria was calculated, once without making any assumptions about unavailable pathology, and once assuming gastric cancer to be diffuse when pathology was unavailable. For comparison, we calculated the percentage of subjects who fulfilled our proposed criteria. RESULTS: When making no assumptions about missing pathology, a small (19%) and equal percentage of CDH1 mutation carriers fulfilled the IGCLC 2015 and 2020 criteria. When assuming unspecified gastric cancer to be diffuse, 45 out of 112 (40%) subjects met the 2015 criteria and 53 out of 112 (47%) met the 2020 criteria. Eighty-seven per cent (97/112) fulfilled our proposed criteria. CONCLUSION: In consecutive cases, mostly unselected for clinical criteria of HDGC, the IGCLC 2020 criteria are, at best, marginally more sensitive than previous iterations, but they are also more cumbersome. Unavailable cancer pathology reports are a real-world obstacle to their proper application. Our proposed Yale criteria both address this issue and offer significantly greater sensitivity than the IGCLC 2020 criteria.

Optimising clinical care through CDH1-specific germline variant curation: improvement of clinical assertions and updated curation guidelines.

BACKGROUND: Germline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications. METHODS: CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories. RESULTS: Updated CDH1-specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing. CONCLUSIONS: The development and evolution of CDH1-specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations.

Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach.

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.

NUBPL mitochondrial disease: new patients and review of the genetic and clinical spectrum.

BACKGROUND: The nucleotide binding protein-like (NUBPL) gene was first reported as a cause of mitochondrial complex I deficiency (MIM 613621, 618242) in 2010. To date, only eight patients have been reported with this mitochondrial disorder. Five other patients were recently reported to have NUBPL disease but their clinical picture was different from the first eight patients. Here, we report clinical and genetic findings in five additional patients (four families). METHODS: Whole exome sequencing was used to identify patients with compound heterozygous NUBPL variants. Functional studies included RNA-Seq transcript analyses, missense variant biochemical analyses in a yeast model (Yarrowia lipolytica) and mitochondrial respiration experiments on patient fibroblasts. RESULTS: The previously reported c.815-27T>C branch-site mutation was found in all four families. In prior patients, c.166G>A [p.G56R] was always found in cis with c.815-27T>C, but only two of four families had both variants. The second variant found in trans with c.815-27T>C in each family was: c.311T>C [p.L104P] in three patients, c.693+1G>A in one patient and c.545T>C [p.V182A] in one patient. Complex I function in the yeast model was impacted by p.L104P but not p.V182A. Clinical features include onset of neurological symptoms at 3-18 months, global developmental delay, cerebellar dysfunction (including ataxia, dysarthria, nystagmus and tremor) and spasticity. Brain MRI showed cerebellar atrophy. Mitochondrial function studies on patient fibroblasts showed significantly reduced spare respiratory capacity. CONCLUSION: We report on five new patients with NUBPL disease, adding to the number and phenotypic variability of patients diagnosed worldwide, and review prior reported patients with pathogenic NUBPL variants.

Clinical features and cancer risk in families with pathogenic CDH1 variants irrespective of clinical criteria.

BACKGROUND: The clinical phenotype of CDH1 pathogenic variant carriers has mostly been studied in families that fulfil criteria of hereditary diffuse gastric cancer (HDGC). We aimed at determining cancer phenotype and cancer risk estimation among families with CDH1 pathogenic variants not selected by HDGC clinical criteria. METHODS: Patients were all consecutively identified CDH1 pathogenic variant carriers from a clinical laboratory tested with multigene panel testing and from an academic cancer genetics programme. Clinical and demographic features, cancer phenotypes and genotype-phenotype correlations were determined among CDH1 families. Age-specific cumulative cancer risks (penetrance) were calculated based on 38 families with available pedigrees. RESULTS: Within the 113 CDH1 pathogenic variant probands and 476 relatives, 113 had gastric cancer, 177 breast cancer and 196 other cancers. Mean age at diagnosis was 47 for gastric and 54 for breast cancer. Forty-six per cent fulfilled criteria of HDGC. While 36% of families had both gastric and breast cancers, 36% had breast but no gastric cancers and 16% had gastric but not breast cancers. Cumulative risk of cancer by age 80 was 37.2% for gastric and 42.9% for breast cancer. CONCLUSION: In unselected CDH1 pathogenic variant carrier families, gastric cancer risks were lower and age at diagnosis higher than previously reported in families pre-selected for HDGC criteria. A substantial proportion of families did not present with any gastric cancers and their cancers were limited to breast. Thus, clinical criteria for CDH1 testing should be widened, including breast cancer families only, and a consideration for delayed prophylactic gastrectomy/surveillance should be evaluated.

Alternative splicing and ACMG-AMP-2015-based classification of PALB2 genetic variants: an ENIGMA report.

BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.

Correction: DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

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DNA breakpoint assay reveals a majority of gross duplications occur in tandem reducing VUS classifications in breast cancer predisposition genes.

PURPOSE: Gross duplications are ambiguous in terms of clinical interpretation due to the limitations of the detection methods that cannot infer their context, namely, whether they occur in tandem or are duplicated and inserted elsewhere in the genome. We investigated the proportion of gross duplications occurring in tandem in breast cancer predisposition genes with the intent of informing their classifications. METHODS: The DNA breakpoint assay (DBA) is a custom, paired-end, next-generation sequencing (NGS) method designed to capture and detect deep-intronic DNA breakpoints in gross duplications in BRCA1, BRCA2, ATM, CDH1, PALB2, and CHEK2. RESULTS: DBA allowed us to ascertain breakpoints for 44 unique gross duplications from 147 probands. We determined that the duplications occurred in tandem in 114 (78%) carriers from this cohort, while the remainder have unknown tandem status. Among the tandem gross duplications that were eligible for reclassification, 95% of them were upgraded to pathogenic. CONCLUSION: DBA is a novel, high-throughput, NGS-based method that informs the tandem status, and thereby the classification of, gross duplications. This method revealed that most gross duplications in the investigated genes occurred in tandem and resulted in a pathogenic classification, which helps to secure the necessary treatment options for their carriers.

Detection of structural variation using target captured next-generation sequencing data for genetic diagnostic testing.

PURPOSE: Structural variation (SV) is associated with inherited diseases. Next-generation sequencing (NGS) is an efficient method for SV detection because of its high-throughput, low cost, and base-pair resolution. However, due to lack of standard NGS protocols and a limited number of clinical samples with pathogenic SVs, comprehensive standards for SV detection, interpretation, and reporting are to be established. METHODS: We performed SV assessment on 60,000 clinical samples tested with hereditary cancer NGS panels spanning 48 genes. To evaluate NGS results, NGS and orthogonal methods were used separately in a blinded fashion for SV detection in all samples. RESULTS: A total of 1,037 SVs in coding sequence (CDS) or untranslated regions (UTRs) and 30,847 SVs in introns were detected and validated. Across all variant types, NGS shows 100% sensitivity and 99.9% specificity. Overall, 64% of CDS/UTR SVs were classified as pathogenic/likely pathogenic, and five deletions/duplications were reclassified as pathogenic using breakpoint information from NGS. CONCLUSION: The SVs presented here can be used as a valuable resource for clinical research and diagnostics. The data illustrate NGS as a powerful tool for SV detection. Application of NGS and confirmation technologies in genetic testing ensures delivering accurate and reliable results for diagnosis and patient care.

RNA homeostasis governed by cell type-specific and branched feedback loops acting on NMD.

Nonsense-mediated mRNA decay (NMD) is a conserved RNA decay pathway that degrades aberrant mRNAs and directly regulates many normal mRNAs. This dual role for NMD raises the possibility that its magnitude is buffered to prevent the potentially catastrophic alterations in gene expression that would otherwise occur if NMD were perturbed by environmental or genetic insults. In support of this, here we report the existence of a negative feedback regulatory network that directly acts on seven NMD factors. Feedback regulation is conferred by different branches of the NMD pathway in a cell type-specific and developmentally regulated manner. We identify feedback-regulated NMD factors that are rate limiting for NMD and demonstrate that reversal of feedback regulation in response to NMD perturbation is crucial for maintaining NMD. Together, our results suggest the existence of an intricate feedback network that maintains both RNA surveillance and the homeostasis of normal gene expression in mammalian cells.

Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay.

Nonsense-mediated decay (NMD) degrades both normal and aberrant transcripts harboring stop codons in particular contexts. Mutations that perturb NMD cause neurological disorders in humans, suggesting that NMD has roles in the brain. Here, we identify a brain-specific microRNA-miR-128-that represses NMD and thereby controls batteries of transcripts in neural cells. miR-128 represses NMD by targeting the RNA helicase UPF1 and the exon-junction complex core component MLN51. The ability of miR-128 to regulate NMD is a conserved response occurring in frogs, chickens, and mammals. miR-128 levels are dramatically increased in differentiating neuronal cells and during brain development, leading to repressed NMD and upregulation of mRNAs normally targeted for decay by NMD; overrepresented are those encoding proteins controlling neuron development and function. Together, these results suggest the existence of a conserved RNA circuit linking the microRNA and NMD pathways that induces cell type-specific transcripts during development.

Hereditary lobular breast cancer with an emphasis on E-cadherin genetic defect.

Recent studies have reported germline CDH1 mutations in cases of lobular breast cancer (LBC) not associated with the classical hereditary diffuse gastric cancer syndrome. A multidisciplinary workgroup discussed genetic susceptibility, pathophysiology and clinical management of hereditary LBC (HLBC). The team has established the clinical criteria for CDH1 screening and results' interpretation, and created consensus guidelines regarding genetic counselling, breast surveillance and imaging techniques, clinicopathological findings, psychological and decisional support, as well as prophylactic surgery and plastic reconstruction. Based on a review of current evidence for the identification of HLBC cases/families, CDH1 genetic testing is recommended in patients fulfilling the following criteria: (A) bilateral LBC with or without family history of LBC, with age at onset <50 years, and (B) unilateral LBC with family history of LBC, with age at onset <45 years. In CDH1 asymptomatic mutant carriers, breast surveillance with clinical examination, yearly mammography, contrast-enhanced breast MRI and breast ultrasonography (US) with 6-month interval between the US and the MRI should be implemented as a first approach. In selected cases with personal history, family history of LBC and CDH1 mutations, prophylactic mastectomy could be discussed with an integrative group of clinical experts. Psychodecisional support also plays a pivotal role in the management of individuals with or without CDH1 germline alterations. Ultimately, the definition of a specific protocol for CDH1 genetic screening and ongoing coordinated management of patients with HLBC is crucial for the effective surveillance and early detection of LBC.

Gene-specific criteria for PTEN variant curation: Recommendations from the ClinGen PTEN Expert Panel.

The ClinGen PTEN Expert Panel was organized by the ClinGen Hereditary Cancer Clinical Domain Working Group to assemble clinicians, researchers, and molecular diagnosticians with PTEN expertise to develop specifications to the 2015 ACMG/AMP Sequence Variant Interpretation Guidelines for PTEN variant interpretation. We describe finalized PTEN-specific variant classification criteria and outcomes from pilot testing of 42 variants with benign/likely benign (BEN/LBEN), pathogenic/likely pathogenic (PATH/LPATH), uncertain significance (VUS), and conflicting (CONF) ClinVar assertions. Utilizing these rules, classifications concordant with ClinVar assertions were achieved for 14/15 (93.3%) BEN/LBEN and 16/16 (100%) PATH/LPATH ClinVar consensus variants for an overall concordance of 96.8% (30/31). The variant where agreement was not reached was a synonymous variant near a splice donor with noncanonical sequence for which in silico models cannot predict the native site. Applying these rules to six VUS and five CONF variants, adding shared internal laboratory data enabled one VUS to be classified as LBEN and two CONF variants to be as classified as PATH and LPATH. This study highlights the benefit of gene-specific criteria and the value of sharing internal laboratory data for variant interpretation. Our PTEN-specific criteria and expertly reviewed assertions should prove helpful for laboratories and others curating PTEN variants.

Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline CDH1 sequence variants.

The variant curation guidelines published in 2015 by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provided the genetics community with a framework to assess variant pathogenicity; however, these rules are not gene specific. Germline pathogenic variants in the CDH1 gene cause hereditary diffuse gastric cancer and lobular breast cancer, a clinically challenging cancer predisposition syndrome that often requires a multidisciplinary team of experts to be properly managed. Given this challenge, the Clinical Genome Resource (ClinGen) Hereditary Cancer Domain prioritized the development of the CDH1 variant curation expert panel (VCEP) to develop and implement rules for CDH1 variant classifications. Here, we describe the CDH1 specifications of the ACMG/AMP guidelines, which were developed and validated after a systematic evaluation of variants obtained from a cohort of clinical laboratory data encompassing ∼827,000 CDH1 sequenced alleles. Comparing previously reported germline variants that were classified using the 2015 ACMG/AMP guidelines to the CDH1 VCEP recommendations resulted in reduced variants of uncertain significance and facilitated resolution of variants with conflicted assertions in ClinVar. The ClinGen CDH1 VCEP recommends the use of these CDH1-specific guidelines for the assessment and classification of variants identified in this clinically actionable gene.

The unfolded protein response is shaped by the NMD pathway.

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD's ability to regulate normal mRNAs.