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유전성 암 유전자에서 검출된 염기 변이의 해석을 위한 기능 연구 근거의 적용
Applying Functional Assay Evidence to Interpret Sequence Variants Identified in Hereditary Cancer Genes
연세대학교 의과대학 진단검사의학교실
Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea
Correspondence to:This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Lab Med Online 2022; 12(3): 145-158
Published July 1, 2022 https://doi.org/10.47429/lmo.2022.12.3.145
Copyright © The Korean Society for Laboratory Medicine.
Abstract
Keywords
INTRODUCTION
The American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) established standards and guidelines for the interpretation of sequence variants [1]. Criteria for classifying pathogenic or benign variants have been developed. However, some evidence, such as PS3/BS3, is somewhat ambiguous, and many laboratories have difficulty applying the criteria in variant interpretation. According to the ACMG/AMP guideline, PS3 and BS3 are defined by “well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product” and “well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing,” respectively [1].
Although functional studies can provide evidence to interpret a variant’s effect on protein function, leading to the reclassification of variants of uncertain significance (VUS), detailed guidance on how functional evidence can be evaluated and applied has not been provided by the original ACMG/AMP guidelines. Brnich et al. [2] published a recommendation for applying the PS3/BS3 criterion to provide a more structured approach for evaluating functional evidence. Furthermore, Kanavy et al. [3] evaluated the comparative analysis of PS3/BS3 of six Variant Curation Expert Panels (
Nevertheless, clinical laboratory practitioners are often unfamiliar with the various experimental procedures used for functional validation. We selected four genes (
-
Table 1 Summary of the general functional assays introduced in Part I
Mechanism Endpoint Example Expected result in affected cell-lines Gene expression and protein turnover mRNA/protein level PCR
Western blotGenetic material is not amplified.
Protein band is absent.Transactivation Reporter gene expression level Fluorescent reporter proteins, luciferase assays Fluorescence is not detected. Cell viability Indicator of cell life or death Colony formation assay
Apoptosis assayColonies grow despite lack of cell anchorage.
Cells are resistant to apoptosis.Binding Interaction between two molecules Tetramerization assay Protein band is detected at a different location on western blot. Cell motility Indicator of cell movement Cell aggregation assay, cell invasion assay, wound closure assay Cell adhesion loss and increased cell motility Enzyme activity Indicator of enzyme activity involved in a common pathway Phosphatase assay Varies depending on enzyme kinetics and inhibi- tion in certain pathways.
-
Table 2 PS3/BS3 interpretation suggested by ClinGen
Gene Criteria Specification TP53 *PS3_Strong Transactivation assays in yeast (IARC classification based on data from Kato et al.) that demonstrate a low functioning allele ( < 20% activity) AND:
-Evidence of a dominant-negative effect (DNE)+evidence of a LOF from Giacomelli et al. data
OR
-There is a second assay showing low function (colony formation assays, apoptosis assays, tetramer assays, knock-in mouse models, and growth suppression assays).PS3_Moderate A)Transactivation assays in yeast (IARC classification based on data from Kato et al.) that demonstrate a partially functioning allele ( > 20% and ≤ 75% activity) AND:
-Evidence of a DNE+evidence of a LOF from Giacomelli et al. data.
OR
-There is a second assay showing low function.Do not use code with conflicting evidence.
B)No transactivation assays (IARC classification based on data Kato et al.) available BUT:
-Evidence of a DNE+evidence of a LOF from Giacomelli et al. data.
AND
-There is a second assay showing low function.Do not use code with conflicting evidence. BS3_Strong Transactivation assays in yeast (IARC classification based on data from Kato et al.) that show retained function (76–140% activity) or super- transactivation function AND:
-No evidence of a DNE+no evidence of a LOF from Giacomelli et al. data.
OR
-There is a second assay, including colony formation assays, apoptosis assays, tetramer assays, growth suppression, and knock-in mouse models demonstrating retained function.BS3_Supporting Transactivation assays in yeast (IARC classification based on data from Kato et al.) that demonstrate a partially functioning allele ( > 20% and ≤ 75% activity) AND:
-No evidence of a DNE+no evidence of a LOF from Giacomelli et al. data.
OR
-There is a second assay demonstrating retained function.Do not use code with conflicting evidence. CDH1 †PS3_Strong RNA assay demonstrating abnormal out-of-frame transcripts.
This rule can only be applied to demonstrate splicing defects.PS3_Supporting RNA assay demonstrating abnormal in-frame transcripts.
This rule can only be applied to demonstrate splicing defects.BS3_Strong Functional RNA studies demonstrating no impact on transcript composition
This rule can only be used to demonstrate a lack of splicing and can only be applied to synonymous, intronic, or non-coding variants. BS3 may be downgraded based on data quality.PTEN ‡PS3_Strong Disease-Specific
Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.
-Phosphatase activity < 50% of wild-type
-RNA, mini-gene, or other assays show impact on splicingPS3_Supporting Disease-Specific; Strength Modified
Abnormal in vitro cellular assay or transgenic model with a phenotype different from the wild-type that does not meet PS3.BS3_Strong Disease-Specific
Well-established in vitro or in vivo functional studies show no damaging effect on protein function. To be applied for missense variants with both lipid phosphatase activity AND results from a second assay appropriate to the protein domain demonstrating no statistically significant difference from the wild-type. For intronic or synonymous variants, RNA, mini-gene, or other splicing assays demonstrate no splicing impact.BS3_Supporting Disease-Specific; Strength Modified
In vitro or in vivo functional study or studies showing no damaging effect on protein function but BS3 not met.*
TP53 PS3/BS3 interpretation is suggested by Fortuno et al.; †CDH1 PS3/BS3 interpretation is suggested by Lee et al.; ‡PTEN PS3/BS3 interpretation is suggested by Mester et al.
PART I
1. Gene expression and protein turnover assay
Several conditions must be met to assess the impact of a variant on the function of a gene. First, the protein encoded by the gene must be produced, as stated by the central dogma, and carried to the correct subcellular location. Finally, it must not be degraded before it can perform its function. Researchers carrying out functional studies must ensure that these conditions are met before making any hasty interpretations.
Several experimental methods can be used for that purpose proposed. As the central dogma states, a gene must undergo transcription to produce its corresponding mRNA, which in turn must be translated to generate its corresponding protein. The polymerase chain reaction (PCR) can measure the transcription step of the central dogma, while western blotting can prove translation. The correct subcellular localization can be visualized via immunofluorescence. Flow cytometry can also be used if the target organelle is the plasma membrane. Lastly, double fluorescence can be used to verify whether the protein is ubiquitylated and becomes prone to proteasomal degradation [7].
2. Transactivation assay
In the context of gene regulation, transactivation describes the increased expression of specific target genes through an intermediate transactivator protein binding to a response element (RE) located within the promoter or enhancer region. Therefore, transactivation assays can be used to evaluate transcription factor gene variants. They require REs of target genes upstream of either the target genes themselves or reporter genes, such as the green fluorescence protein (GFP), in addition to the transcription factor gene [8].
3. Cell viability assay
Cell viability assays gauge how well or poorly cells proliferate by measuring an indicator of cell life or death. They can assess the physiological, structural, and functional aspects of cultured cells [9]. Cell life indicators include cell number, ATP content, DNA content, dehydrogenase activity, and membrane integrity. Cell death indicators include caspase activity, chromatin condensation, and phospholipid redistribution. Cell proliferation, colony formation, growth suppression, and apoptosis assays are examples of cell viability assays [10].
4. Binding assay
Binding assays are used to quantify interactions between two molecules, such as small molecule-proteins, protein–protein, and protein–DNA. Examples of binding assays include ATP-binding assays (small molecule-protein) and tetramerization assays (protein–protein) [11].
5. Cell motility assay
Motility is an essential cell feature. Thus, methods to study cell migratory behavior are valuable tools to observe cell characteristics, especially in cancer research, which includes migration and invasion through the extracellular matrix, intravasation into blood circulation, attachment to a distant site, and extravasation to form distant foci [12, 13]. Cell aggregation, cell invasion, and wound closure assays are well-known methods for observing cell motility. Cell aggregation assays have frequently been used to test cells’ E-cadherin-dependent cell-cell adhesions, and assess the functionality of the complex in epithelioid cells [14]. A cell aggregation assay is a useful tool for distinguishing between invasive and noninvasive cell types. Cell invasion assays are different from cell migration assays in the field of experimental biology. Invasion is the movement of a cell through a 3D matrix that modifies the cell shape and interacts with the extracellular matrix [15]. Migration is the directed movement of cells on a 2D surface without an obstructive fiber network [15]. Invasion requires adhesion, proteolysis of extracellular matrix components, and migration [16]. Therefore, cell invasion assays help observe how invasive cells penetrate a barrier in response to chemoattractants or inhibiting compounds. The wound closure assay is the simplest method for determining the migration ability of collective cell migration [17]. In the wound closure assay, the migration of cells was measured as a closed distance over time and compared to a control. Observing single-cell lamellipodium formation, tail retraction, and directional movement may reveal any impaired migratory phenotypes [18].
6. Enzyme activity assay
Enzyme assays for the study of enzyme kinetics and enzyme inhibition help measure enzymatic activity. Assays to measure phosphatase activity are a type of enzyme assay. Phosphatase assays can be employed to study the catalytic activity of
PART II
We selected four genes causing hereditary cancer syndromes, each with different molecular roles:
1. TP53
The tumor suppressor gene
1) Transactivation assay
Transactivation assays are used to investigate the effects of variants on the transactivation function of transcription factors. In functional studies that employed transactivation assays, transfected cell-lines were used to evaluate how efficiently
2) Colony formation assay
The colony formation assay is based on the fact that normal cells are prevented from anchorage-independent growth due to anoikis (a type of apoptosis triggered specifically by a lack of cell anchorage), while transformed cells are capable of proliferating without binding to a substrate. In this assay, cells are grown in a soft agar layer mixed with a cell culture medium resting on another layer containing a higher agar concentration. Studies employing this assay used the p53-null non-small-cell carcinoma cell line, H1299, to determine whether transfection with wild-type or mutant
3) Apoptosis assay
Apoptosis assays are based on the fact that apoptotic cells have reduced DNA content and undergo morphological changes making them distinguishable from viable cells via flow cytometry. In particular, the appearance of phosphatidylserine in the outer plasma membrane of early apoptotic cells due to a loss of plasma membrane asymmetry distinguishes early and late apoptotic cells [31]. In the reviewed study [10], H1299 cells co-transfected with a range of
4) Tetramerization assay
A tetramerization assay evaluated missense variants within the oligomerization domain (residues 323–356). As p53 needs to form a homotetramer to function as a transcription factor, pathogenic variants that prevent tetramer formation or promote the formation of heterotetramers with p53 can exert a dominant-negative effect or act as gain-of-function mutations [32, 33]. H1299 and U2OS cell-lines transfected with either wild-type
5) Growth suppression assay
Growth suppression assays aim to verify whether the mutated tumor suppressor genes confer resistance to small molecules and certain drugs, such as nutlin-3 and etoposide. In one study using such an assay, cell cultures at 50% confluence were transfected with either wild-type
Etoposide is a DNA double-strand break-inducing agent that activates p53 and induces apoptosis in mouse thymocytes [36]. However, in other contexts, wild-type p53 allows DNA repair via cell-cycle arrest and prevents cell death from unresolved DNA damage [37]; indeed, the authors found that wild-type p53 expression in p53-null cells prevented cell death upon etoposide treatment, whereas mutant p53 expression had no effect. Variant frequencies were measured after 12 days of incubation, and Z-scores were calculated for each variant. Evidence of a dominant negative effect (DNE) and LOF as defined by the ClinGen expert panel saw Z-scores of ≥0.61 and ≤-0.21 for p53-wild-type nutlin-3 and etoposide, respectively. Evidence of no DNE and no LOF was defined by Z-scores of <0.61 and >-0.21 for p53-wild-type nutlin-3 and etoposide, respectively [4].
2. BRCA1
1) SCP assay
The SCP assay is based on the observation that
2) Protein binding assay
Hetero-dimerization of BRCA1 with BARD1 via its RING domain is crucial for homologous recombination-mediated DNA repair. RING variants that disrupt dimerization result in the loss of tumor suppression [48, 49]. There are several ways to study protein–protein interactions, including co-immunoprecipitation and TAP-tag, protein arrays, mass spectrometry, yeast two-hybrid analysis, and split protein complementation assays [50]. Among these, one study used the latter and the split-GFP reassembly method [51]. Folding-reporter GFP (frGFP) was generated from the 5´ fragment (for residues 1–84) of EGFP and the 3´ fragment (residues 85–238) of GFPuv. These were fused with
Another study used yeast two-hybrid analysis, where a yeast transcription factor was split into two fragments instead of a fluorescence protein [52]. In this study, the DNA-binding domain of Gal4 was fused to
3) E3 ubiquitin ligase assay
The BRCA1/BARD1 complex functions as an E3 ubiquitin ligase [39]. Therefore, E3 ubiquitin ligase activity may also be a BRCA1 functional assay endpoint. In one study using such a functional assay, a fusion protein of BARD1 (residues 26–126) and BRCA1 (residues 2–304) capable of auto-ubiquitination in vitro was used in a phage display assay [52]. BARD1-BRCA1 fusion proteins with different variants of BRCA1 were expressed at the C-terminus of the bacteriophage T7 coat protein. The multiple phage strains displaying BRCA1 variants were incubated in ubiquitination reactions (containing E1, E2, FLAG-tagged ubiquitin, and ATP). Under such conditions, phages carrying active
4) Recombination assay
The aforementioned assays can only evaluate missense variants in either the BRCT or RING domains. Thus, assays capable of investigating the pathogenicity of variants located throughout
5) Transactivation assay
Although homologous recombination is the main function of BRCA1, it also functions as a transcription factor. Therefore, transactivation assays, which have been used to study
6) Centrosome amplification assay
In addition to its numerous nuclear functions, BRCA1 also has cytoplasmic roles. Having exactly two centrosomes is crucial for the proper segregation of chromosomes in dividing cells. BRCA1 regulates centrosome amplification through its E3 ubiquitin ligase activity by ubiquitylating gamma-tubulin [7] and a gamma-tubulin adapter protein [61], thus preventing centrosome reduplication during the same cell cycle [62]. Functional studies using centrosome amplification assays used GFP-tagged centrins or anti-pericentrin antibodies to visualize centrosomes. Subsequently, the proportion of cells with abnormal numbers of centrosomes in strains transfected with a range of
3. CDH1
The cadherin 1 (
Germline mutations in
LOF through
1) Aggregation/Invasion assay
Cell aggregation depends on cell-cell adhesion, and the cadherin-catenin complex is necessary between epithelial cells [14, 80]. Downregulation of the complex is often observed in tumor cells during tumor progression. It is associated with high tumor infiltrative and metastatic abilities due to cell adhesion loss and increased cell motility [81-83]. Aggregation and collagen invasion assays can be used to evaluate
Chinese hamster ovary (CHO) cells are often utilized because they do not express
2) Wound closure assay
A wound closure assay, also called wound healing assay, is a simple method to test cell motility. Removing the cells from an area through mechanical, thermal, or chemical damage creates a cell-free area in a confluent monolayer [87]. This assay is usually performed under conditions of suppressed cell proliferation. Introducing a cell-free area next to the cell monolayer induces cell migration into the gap. Suriano et al. [88] used an in vitro wound closure assay to test p.Ala617Thr (p.A617T), p.Thr340Ala (p.T340A), p.Ala634Val (p.A634V), and p.Val832Met (p.V832M) mutations compared to wild-type and mock cells. According to the results, p.Ala617Thr (p.A617T) and wild-type cells showed similar cell motility. p.Thr340Ala (p.T340A) and p.Ala634Val (p.A634V) showed high cell motility. p.Val832Met (p.V832M) and mock cells showed very low cell motility, failing to migrate unidirectionally due to low polarization. However, these results caused the destabilization of the E-cadherin adhesion complex, implying that motile capability is neither necessary nor sufficient for cells to invade.
4. PTEN
The phosphatase and TENsin homolog (
The
Furthermore,
1) Phosphatase activity
Most missense mutations are clustered around the phosphatase domain. Therefore, an assay to measure phosphatase activity is useful for the functional analysis of
2) PTEN/pAKT expression level
PTEN dephosphorylates PIP3, preventing the downstream pathway of AKT phosphorylation. Therefore, cells with
CONCLUSION
This study is intended to help clinical laboratories apply functional evidence criteria when interpreting the sequence variants found in clinical genetic testing. For this purpose, four cancer susceptibility genes with different mechanisms of action (
-
Table 3 Literature on functional assays used in each gene
Gene Transactivation assay Cell viability assay Binding assay Cell motility assay Enzyme activity assay HDR Centrosome TP53 7878469, 30224644, 31081129, 17690113, 24816189 24816189, 12509279, 24677579, 19693097, 8479523 17690113 PTEN 26504226 26504226 10866302, 29706350, 21828076, 26504226, 19915616, 25875300, 28263967, 25527629 CDH1 12588804, 21106365, 15235021, 14500541, 22470475, 18772194, 12944922, 16924464 BRCA1 32656256, 15689452, 10811118, 19493677, 8942979, 18087219 11301010, 18680205 23628597, 18493658 18493658 8939848, 22172724, 25823446, 27484786 18087219, 18927495, 15923272 Literatures are represented using PMID number.
Abbreviation: HDR, homology directed repair.
Conflicts of Interest
None declared.
Acknowledgements
This study was supported by the Research Fund of the Quality Control Committee of the Korean Society for Laboratory Medicine (KSLM Research Project 2021-05-012).
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