Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework

Brnich, Sarah E.; Tayoun, Ahmad Abou; Couch, Fergus J.; Cutting, Garry R.; Greenblatt, Marc S.; Heinen, Christopher D.; Kanavy, Dona M.; Luo, Xi; McNulty, Shannon M.; Tavtigian, Sean V.; Wright, Mathew W.; Harrison, Steven M.; Biesecker, Leslie G.; Berg, Jonathan S.

doi:10.1101/709428

Cited by 98 publications

(194 citation statements)

References 29 publications

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“…We demonstrate that massively parallel functional assays can accurately measure the impacts of variants in the key Lynch Syndrome gene MSH2. This functional effect map, if rationally combined 54,91 with other lines of clinical evidence 9 , will enable more accurate classification of Lynch Syndrome variants.…”

Section: Discussionmentioning

confidence: 99%

“…Bioinformatic tools are frequently used to interpret clinical variants when other forms of evidence, such as co-segregation with disease, functional studies, or population variant allele frequency, are unavailable or uninformative54 . We compared the classification performance of our LOF scores with that of six computational predictors, including three specific to MMR genes: MAPP-MMR 55 , FoldX 56 thermostability predictions from Nielsen et al38 and PON-MMR57 , and three general-purpose tools REVEL 35 , CADD58 , and PolyPhen2 34 .…”

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confidence: 99%

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Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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Introduction 3The lack of functional evidence for the majority of missense variants limits their clinical interpretability, 4 and poses a key barrier to the broad utility of carrier screening. In Lynch Syndrome (LS), one of the most 5 highly prevalent cancer syndromes, nearly 90% of clinically observed missense variants are deemed 6 'variants of uncertain significance' (VUS). To systematically resolve their functional status, we performed 7 a massively parallel screen in human cells to identify loss-of-function missense variants in the key DNA 8 mismatch repair factor MSH2. The resulting sequence-function map is substantially complete, covering 9 94% of the 17,746 possible variants, and is highly concordant (≥96%) with existing functional data and 10 expert clinicians' interpretations. The large majority (89%) of missense variants were functionally neutral, 11 perhaps unexpectedly in view of its evolutionary conservation. These data provide ready-to-use 12 functional evidence to resolve the ~1,300 extant missense VUSs in MSH2, and may facilitate the 13 prospective classification of newly discovered variants in the clinic. 14 15 Main text 16 17 Lynch Syndrome (LS, OMIM:120435), the first discovered familial cancer syndrome 1 , confers 18 predisposition to colorectal and endometrial cancers due to the loss of DNA mismatch repair (MMR) and 19 resulting mutagenic burden. Most affected individuals inherit a single loss-of-function variant in one of 20 four MMR factors (MSH2, MLH1, 21 MSH6, PMS2), followed by a somatic 22 'second hit' inactivating the remaining 23 functional copy. Due to high 24 population prevalence (≥1:300) 2,3 , 25 clear genetic etiology, and potential 26 for prevention through intensified 27 surveillance, MMR gene variants are 28 considered clinically actionable 4 . 29 30 Missense changes together comprise 31 20-30% of LS variants 5 , and are 32 particularly challenging to interpret: 33 their functional impacts may range 34 from minimal to profound, most are 35 individually rare, and they exhibit 36 incomplete penetrance 6 . 37 Consequently, the overwhelming 38 majority of LS gene missense variants 39 listed in ClinVar (4,762/5,473, 87.0%) 40 are deemed 'variants of uncertain 41 significance', or VUS 42( Supplementary Fig.1), and cannot 43 be used to guide diagnosis. 45To address these challenges, we 46 performed a deep mutational scan 7 to 47 systematically measure the functional 48 impact of missense variants in the 49 major Lynch Syndrome gene MSH2. 50We established a human cell system 51 to model MSH2 variant function using 52 the mismatch repair proficient 8 cell 53 line HAP1 (Fig. 1a,c). First, to disrupt MMR, we derived clonal MSH2 knockout cells bearing a 19-bp frameshift deletion in MSH2 exon 6, and 55 verified that it lacked detectable MSH2 protein expression ( Supplementary Fig. 2). To restore MMR, we 56 stably reintroduced into cells either wild-type MSH2 cDNA (KO+WT) or a pathogenic founder allele 57 (KO+A636P) on inducible lentiviral constructs. After inducing expression with doxycycl...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Jia

Burugula

Chen

et al. 2020

Preprint

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“…A crucial aspect for a proper evaluation of the sequence variants is represented by the choice of appropriate functional studies. Although recommendations have been recently issued to provide a detailed guidance on the evaluation of functional data [143,144], for the large genes discussed here, we do not have a general agreement on the assays providing sufficient evidence. Moreover, the large size of the coding region is a considerable issue for specific applications (e.g.…”

Section: Final Considerations and Future Perspectivesmentioning

confidence: 80%

Is Gene-Size an Issue for the Diagnosis of Skeletal Muscle Disorders?

Savarese

Välipakka

Johari

et al. 2020

JND

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Human genes have a variable length. Those having a coding sequence of extraordinary length and a high number of exons were almost impossible to sequence using the traditional Sanger-based gene-by-gene approach. High-throughput sequencing has partly overcome the size-related technical issues, enabling a straightforward, rapid and relatively inexpensive analysis of large genes. Several large genes (e.g. TTN, NEB, RYR1, DMD) are recognized as disease-causing in patients with skeletal muscle diseases. However, because of their sheer size, the clinical interpretation of variants in these genes is probably the most challenging aspect of the high-throughput genetic investigation in the field of skeletal muscle diseases. The main aim of this review is to discuss the technical and interpretative issues related to the diagnostic investigation of large genes and to reflect upon the current state of the art and the future advancements in the field.

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“…The advantage of in vitro studies is that the impact of different variants can be compared without the need to consider the genetic background of the assay. Indeed, recent guidelines on assigning functional evidence scores (PS3/BS3) in the ACMG classification framework treat these two lines of evidence differently and recommend using PP4 score for patient‐derived data, while assigning PS3/BS3 for evidence obtained from validated in vitro functional assays (Brnich et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay

et al. 2020

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Improving the accuracy of variant interpretation during diagnostic sequencing is a major goal for genomic medicine. To explore an often‐overlooked splicing effect of missense variants, we developed the functional assay (“minigene”) for the majority of exons of CAPN3, the gene responsible for limb girdle muscular dystrophy. By systematically screening 21 missense variants distributed along the gene, we found that eight clinically relevant missense variants located at a certain distance from the exon–intron borders (deep exonic missense variants) disrupted normal splicing of CAPN3 exons. Several recent machine learning‐based computational tools failed to predict splicing impact for the majority of these deep exonic missense variants, highlighting the importance of including variants of this type in the training sets during the future algorithm development. Overall, 24 variants in CAPN3 gene were explored, leading to the change in the American College of Medical Genetics and Genomics classification of seven of them when results of the “minigene” functional assay were considered. Our findings reveal previously unknown splicing impact of several clinically important variants in CAPN3 and draw attention to the existence of deep exonic variants with a disruptive effect on gene splicing that could be overlooked by the current approaches in clinical genetics.

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Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework

Cited by 98 publications

References 29 publications

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Massively parallel functional testing ofMSH2missense variants conferring Lynch Syndrome risk

Is Gene-Size an Issue for the Diagnosis of Skeletal Muscle Disorders?

Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay

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