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, KoreaCorrespondence 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
Copyright © The Korean Society for Laboratory Medicine.
The American College of Medical Genetics (ACMG) and the Association for Molecular Pathology (AMP) established standards and guidelines for the interpretation of sequence variants . 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 .
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.  published a recommendation for applying the PS3/BS3 criterion to provide a more structured approach for evaluating functional evidence. Furthermore, Kanavy et al.  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 (
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 .
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 .
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 . 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 .
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) .
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 . 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 . Migration is the directed movement of cells on a 2D surface without an obstructive fiber network . Invasion requires adhesion, proteolysis of extracellular matrix components, and migration . 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 . 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 .
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
We selected four genes causing hereditary cancer syndromes, each with different molecular roles:
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 . In the reviewed study , 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 . However, in other contexts, wild-type p53 allows DNA repair via cell-cycle arrest and prevents cell death from unresolved DNA damage ; 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 .
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 . Among these, one study used the latter and the split-GFP reassembly method . 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 . 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 . 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 . 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  and a gamma-tubulin adapter protein , thus preventing centrosome reduplication during the same cell cycle . 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
The cadherin 1 (
Germline mutations in
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 . 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.  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.
The phosphatase and TENsin homolog (
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
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 (
Conflicts of Interest
- 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. Genet Med 2015;17:405-24.
- Brnich SE, Abou Tayoun AN, Couch FJ, Cutting GR, Greenblatt MS, Heinen CD, et al. Recommendations for application of the functional evidence PS3/BS3 criterion using the ACMG/AMP sequence variant interpretation framework. Genome Med 2019;12:3.
- Kanavy DM, McNulty SM, Jairath MK, Brnich SE, Bizon C, Powell BC, et al. Comparative analysis of functional assay evidence use by ClinGen Variant Curation Expert Panels. Genome Med 2019;11:77.
- Fortuno C, Lee K, Olivier M, Pesaran T, Mai PL, de Andrade KC, et al. Specifications of the ACMG/AMP variant interpretation guidelines for germline TP53 variants. Hum Mutat 2021;42:223-36.
- Lee K, Krempely K, Roberts ME, Anderson MJ, Carneiro F, Chao E, et al. Specifications of the ACMG/AMP variant curation guidelines for the analysis of germline
CDH1sequence variants. Hum Mutat 2018;39:1553-68.
- Mester JL, Ghosh R, Pesaran T, Huether R, Karam R, Hruska KS, et al. Gene-specific criteria for PTEN variant curation: Recommendations from the ClinGen
PTENExpert Panel. Hum Mutat 2018;39:1581-92.
- Starita LM, Machida Y, Sankaran S, Elias JE, Griffin K, Schlegel BP, et al. BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number. Mol Cell Biol 2004;24:8457-66.
- Ko JL, Chiao MC, Chang SL, Lin P, Lin JC, Sheu GT, et al. A novel
p53mutant retained functional activity in lung carcinomas. DNA Repair (Amst) 2002;1:755-62.
- Gilbert DF, Friedrich O. Cell viability assays. New York, NY: Springer, 2017.
- Doffe F, Carbonnier V, Tissier M, Leroy B, Martins I, Mattsson JSM, et al. Identification and functional characterization of new missense SNPs in the coding region of the
TP53gene. Cell Death Differ 2021;28:1477-92.
- Lang V, Pallara C, Zabala A, Lobato-Gil S, Lopitz-Otsoa F, Farrás R, et al. Tetramerization-defects of p53 result in aberrant ubiquitylation and transcriptional activity. Mol Oncol 2014;8:1026-42.
- Fidler IJ. Critical determinants of metastasis. Semin Cancer Biol 2002;12:89-96.
- Steeg PS. Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006;12:895-904.
- Debruyne D, Boterberg T, Bracke ME. Cell aggregation assays. Methods Mol Biol 2014;1070:77-92.
- Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschläger M, et al. In vitro cell migration and invasion assays. Mutat Res 2013;752:10-24.
- Friedl P, Wolf K. Plasticity of cell migration: a multiscale tuning model. J Cell Biol 2010;188:11-9.
- Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV.
In vitrocell migration and invasion assays. J Vis Exp :51046.
- Zhang Y, Feng Y, Justus CR, Jiang W, Li Z, Lu JQ, et al. Comparative study of 3D morphology and functions on genetically engineered mouse melanoma cells. Integr Biol (Camb) 2012;4:1428-36.
- Spinelli L, Leslie NR. Assays to measure PTEN lipid phosphatase activity in vitro from purified enzyme or immunoprecipitates. Methods Mol Biol 2016;1447:95-105.
- el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. Definition of a consensus binding site for p53. Nat Genet 1992;1:45-9.
- Zilfou JT, Lowe SW. Tumor suppressive functions of p53. Cold Spring Harb Perspect Biol 2009;1:a001883.
- Riley T, Sontag E, Chen P, Levine A. Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 2008;9:402-12.
- Cho Y, Gorina S, Jeffrey PD, Pavletich NP. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 1994;265:346-55.
- Clore GM, Omichinski JG, Sakaguchi K, Zambrano N, Sakamoto H, Appella E, et al. High-resolution structure of the oligomerization domain of p53 by multidimensional NMR. Science 1994;265:386-91.
- Jeffrey PD, Gorina S, Pavletich NP. Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms. Science 1995;267:1498-502.
- Kato S, Han SY, Liu W, Otsuka K, Shibata H, Kanamaru R, et al. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proc Natl Acad Sci U S A 2003;100:8424-9.
- Giacomelli AO, Yang X, Lintner RE, McFarland JM, Duby M, Kim J, et al. Mutational processes shape the landscape of
TP53mutations in human cancer. Nat Genet 2018;50:1381-7.
- Kharaziha P, Ceder S, Axell O, Krall M, Fotouhi O, Böhm S, et al. Functional characterization of novel germline
TP53variants in Swedish families. Clin Genet 2019;96:216-25.
- Yamada H, Shinmura K, Okudela K, Goto M, Suzuki M, Kuriki K, et al. Identification and characterization of a novel germ line
p53mutation in familial gastric cancer in the Japanese population. Carcinogenesis 2007;28:2013-8.
- Li J, Yang L, Gaur S, Zhang K, Wu X, Yuan YC, et al. Mutants TP53 p.R273H and p.R273C but not p.R273G enhance cancer cell malignancy. Hum Mutat 2014;35:575-84.
- van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsper-ger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998;31:1-9.
- Willis A, Jung EJ, Wakefield T, Chen X. Mutant p53 exerts a dominant negative effect by preventing wild-type p53 from binding to the promoter of its target genes. Oncogene 2004;23:2330-8.
- Brosh R, Rotter V. When mutants gain new powers: news from the mutant p53 field. Nat Rev Cancer 2009;9:701-13.
- Pietenpol JA, Tokino T, Thiagalingam S, el-Deiry WS, Kinzler KW, Vogelstein B. Sequence-specific transcriptional activation is essential for growth suppression by p53. Proc Natl Acad Sci U S A 1994;91:1998-2002.
- Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004;303:844-8.
- Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML, et al. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 1993;362:849-52.
- Lukin DJ, Carvajal LA, Liu WJ, Resnick-Silverman L, Manfredi JJ. p53 Promotes cell survival due to the reversibility of its cell-cycle checkpoints. Mol Cancer Res 2015;13:16-28.
- Takaoka M, Miki Y.
BRCA1gene: function and deficiency. Int J Clin Oncol 2018;23:36-44.
- Hashizume R, Fukuda M, Maeda I, Nishikawa H, Oyake D, Yabuki Y, et al. The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation. J Biol Chem 2001;276:14537-40.
- Wang B, Matsuoka S, Ballif BA, Zhang D, Smogorzewska A, Gygi SP, et al. Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 2007;316:1194-8.
- Yu X, Wu LC, Bowcock AM, Aronheim A, Baer R. The C-terminal (BRCT) domains of
BRCA1interact in vivowith CtIP, a protein implicated in the CtBP pathway of transcriptional repression. J Biol Chem 1998;273:25388-92.
- Cantor SB, Bell DW, Ganesan S, Kass EM, Drapkin R, Grossman S, et al. BACH1, a novel helicase-like protein, interacts directly with
BRCA1and contributes to its DNA repair function. Cell 2001;105:149-60.
- Humphrey JS, Salim A, Erdos MR, Collins FS, Brody LC, Klausner RD. Human BRCA1 inhibits growth in yeast: potential use in diagnostic testing. Proc Natl Acad Sci U S A 1997;94:5820-5.
- Bork P, Hofmann K, Bucher P, Neuwald AF, Altschul SF, Koonin EV. A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J 1997;11:68-76.
- Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science 1996;274:1664-72.
- Caligo MA, Bonatti F, Guidugli L, Aretini P, Galli A. A yeast recombination assay to characterize human
BRCA1missense variants of unknown pathological significance. Hum Mutat 2009;30:123-33.
- Millot GA, Berger A, Lejour V, Boulé JB, Bobo C, Cullin C, et al. Assessment of human Nter and Cter
BRCA1mutations using growth and localization assays in yeast. Hum Mutat 2011;32:1470-80.
- Drost R, Bouwman P, Rottenberg S, Boon U, Schut E, Klarenbeek S, et al. BRCA1 RING function is essential for tumor suppression but dispensable for therapy resistance. Cancer Cell 2011;20:797-809.
- Ransburgh DJ, Chiba N, Ishioka C, Toland AE, Parvin JD. Identification of breast tumor mutations in
BRCA1that abolish its function in homologous DNA recombination. Cancer Res 2010;70:988-95.
- Stynen B, Tournu H, Tavernier J, Van Dijck P. Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. Microbiol Mol Biol Rev 2012;76:331-82.
- Sarkar M, Magliery TJ. Re-engineering a split-GFP reassembly screen to examine RING-domain interactions between BARD1 and BRCA1 mutants observed in cancer patients. Mol Biosyst 2008;4:599-605.
- Starita LM, Young DL, Islam M, Kitzman JO, Gullingsrud J, Hause RJ, et al. Massively parallel functional analysis of BRCA1 RING domain variants. Genetics 2015;200:413-22.
- Lodovichi S, Vitello M, Cervelli T, Galli A. Expression of cancer related
BRCA1missense variants decreases MMS-induced recombination in Saccharomyces cerevisiaewithout altering its nuclear localization. Cell Cycle 2016;15:2723-31.
- Coupier I, Baldeyron C, Rousseau A, Mosseri V, Pages-Berhouet S, Caux-Moncoutier V, et al. Fidelity of DNA double-strand break repair in heterozygous cell lines harbouring
BRCA1missense mutations. Oncogene 2004;23:914-9.
- Cervelli T, Lodovichi S, Bellè F, Galli A. Yeast-based assays for the functional characterization of cancer-associated variants of human DNA repair genes. Microb Cell 2020;7:162-74.
- Phelan CM, Dapic V, Tice B, Favis R, Kwan E, Barany F, et al. Classification of
BRCA1missense variants of unknown clinical significance. J Med Genet 2005;42:138-46.
- Hayes F, Cayanan C, Barillà D, Monteiro AN. Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation. Cancer Res 2000;60:2411-8.
- Di Cecco L, Melissari E, Mariotti V, Iofrida C, Galli A, Guidugli L, et al. Characterisation of gene expression profiles of yeast cells expressing
BRCA1missense variants. Eur J Cancer 2009;45:2187-96.
- Monteiro AN, August A, Hanafusa H. Evidence for a transcriptional activation function of BRCA1 C-terminal region. Proc Natl Acad Sci U S A 1996;93:13595-9.
- Ostrow KL, McGuire V, Whittemore AS, DiCioccio RA. The effects of
BRCA1missense variants V1804D and M1628T on transcriptional activity. Cancer Genet Cytogenet 2004;153:177-80.
- Sankaran S, Crone DE, Palazzo RE, Parvin JD. BRCA1 regulates gamma-tubulin binding to centrosomes. Cancer Biol Ther 2007;6:1853-7.
- Kais Z, Parvin JD. Regulation of centrosomes by the BRCA1-dependent ubiquitin ligase. Cancer Biol Ther 2008;7:1540-3.
- Lovelock PK, Healey S, Au W, Sum EY, Tesoriero A, Wong EM, et al. Genetic, functional, and histopathological evaluation of two C-terminal
BRCA1missense variants. J Med Genet 2006;43:74-83.
- Kais Z, Chiba N, Ishioka C, Parvin JD. Functional differences among
BRCA1missense mutations in the control of centrosome duplication. Oncogene 2012;31:799-804.
- Norton JA, Ham CM, Van Dam J, Jeffrey RB, Longacre TA, Huntsman DG, et al.
CDH1truncating mutations in the E-cadherin gene: an indication for total gastrectomy to treat hereditary diffuse gastric cancer. Ann Surg 2007;245:873-9.
- Shore EM, Nelson WJ. Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells. J Biol Chem 1991;266:19672-80.
- van Roy F, Berx G. The cell-cell adhesion molecule E-cadherin. Cell Mol Life Sci 2008;65:3756-88.
- Shenoy S.
CDH1(E-cadherin) mutation and gastric cancer: genetics, molecular mechanisms and guidelines for management. Cancer Manag Res 2019;11:10477-86.
- Guilford P, Hopkins J, Harraway J, McLeod M, McLeod N, Harawira P, et al. E-cadherin germline mutations in familial gastric cancer. Nature 1998;392:402-5.
- Corso G, Intra M, Trentin C, Veronesi P, Galimberti V.
CDH1germline mutations and hereditary lobular breast cancer. Fam Cancer 2016;15:215-9.
- Pharoah PD, Guilford P, Caldas C. Incidence of gastric cancer and breast cancer in
CDH1(E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology 2001;121:1348-53.
- Barber M, Murrell A, Ito Y, Maia AT, Hyland S, Oliveira C, et al. Mechanisms and sequelae of E-cadherin silencing in hereditary diffuse gastric cancer. J Pathol 2008;216:295-306.
- Melo S, Figueiredo J, Fernandes MS, Gonçalves M, Morais-de-Sá E, Sanches JM, et al. Predicting the functional impact of
CDH1missense mutations in hereditary diffuse gastric cancer. Int J Mol Sci 2017;18:2687.
- Gottardi CJ, Wong E, Gumbiner BM. E-cadherin suppresses cellular transformation by inhibiting beta-catenin signaling in an adhesion-independent manner. J Cell Biol 2001;153:1049-60.
- Jeanes A, Gottardi CJ, Yap AS. Cadherins and cancer: how does cadherin dysfunction promote tumor progression? Oncogene 2008;27:6920-9.
- Bruner HC, Derksen PWB. Loss of E-cadherin-dependent cell-cell adhesion and the development and progression of cancer. Cold Spring Harb Perspect Biol 2018;10:a029330.
- Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res 2008;68:3645-54.
- Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell 2008;14:818-29.
- Berx G, van Roy F. Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb Perspect Biol 2009;1:a003129.
- Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V. Cell-cell adhesion and cytoskeleton tension oppose each other in regulating tumor cell aggregation. Cancer Res 2015;75:2426-33.
- Ozawa M, Ringwald M, Kemler R. Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. Proc Natl Acad Sci U S A 1990;87:4246-50.
- Takeichi M. Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 1993;5:806-11.
- Christofori G, Semb H. The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends Biochem Sci 1999;24:73-6.
- Suriano G, Oliveira C, Ferreira P, Machado JC, Bordin MC, De Wever O, et al. Identification of
CDH1germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands. Hum Mol Genet 2003;12:575-82.
- Corso G, Pedrazzani C, Pinheiro H, Fernandes E, Marrelli D, Rinnovati A, et al. E-cadherin genetic screening and clinico-pathologic characteristics of early onset gastric cancer. Eur J Cancer 2011;47:631-9.
- Brooks-Wilson AR, Kaurah P, Suriano G, Leach S, Senz J, Grehan N, et al. Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria. J Med Genet 2004;41:508-17.
- Jonkman JE, Cathcart JA, Xu F, Bartolini ME, Amon JE, Stevens KM, et al. An introduction to the wound healing assay using live-cell microscopy. Cell Adh Migr 2014;8:440-51.
- Suriano G, Oliveira MJ, Huntsman D, Mateus AR, Ferreira P, Casares F, et al. E-cadherin germline missense mutations and cell phenotype: evidence for the independence of cell invasion on the motile capabilities of the cells. Hum Mol Genet 2003;12:3007-16.
- Worby CA, Dixon JE. PTEN. Annu Rev Biochem 2014;83:641-69.
- Weng LP, Brown JL, Baker KM, Ostrowski MC, Eng C. PTEN blocks insulin-mediated ETS-2 phosphorylation through MAP kinase, independently of the phosphoinositide 3-kinase pathway. Hum Mol Genet 2002;11:1687-96.
- Weng LP, Brown JL, Eng C. PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model. Hum Mol Genet 2001;10:599-604.
- Planchon SM, Waite KA, Eng C. The nuclear affairs of PTEN. J Cell Sci 2008;121:249-53.
- Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor
PTEN. Cell 1998;95:29-39.
- Milella M, Falcone I, Conciatori F, Cesta Incani U, Del Curatolo A, Inzerilli N, et al. PTEN: multiple functions in human malignant tumors. Front Oncol 2015;5:24.
- Sansal I, Sellers WR. The biology and clinical relevance of the
PTENtumor suppressor pathway. J Clin Oncol 2004;22:2954-63.
- Hlobilkova A, Guldberg P, Thullberg M, Zeuthen J, Lukas J, Bartek J. Cell cycle arrest by the
PTENtumor suppressor is target cell specific and may require protein phosphatase activity. Exp Cell Res 2000;256:571-7.
- Eng C.
PTEN: one gene, many syndromes. Hum Mutat 2003;22:183-98.
- Nieuwenhuis MH, Kets CM, Murphy-Ryan M, Yntema HG, Evans DG, Colas C, et al. Cancer risk and genotype-phenotype correlations in
PTENhamartoma tumor syndrome. Fam Cancer 2014;13:57-63.
- Kwabi-Addo B, Giri D, Schmidt K, Podsypanina K, Parsons R, Greenberg N, et al. Haploinsufficiency of the
PTENtumor suppressor gene promotes prostate cancer progression. Proc Natl Acad Sci U S A 2001;98:11563-8.
- Álvarez-Garcia V, Tawil Y, Wise HM, Leslie NR. Mechanisms of PTEN loss in cancer: It's all about diversity. Semin Cancer Biol 2019;59:66-79.
- Han SY, Kato H, Kato S, Suzuki T, Shibata H, Ishii S, et al. Functional evaluation of
PTENmissense mutations using in vitro phosphoinositide phosphatase assay. Cancer Res 2000;60:3147-51.
- Mighell TL, Evans-Dutson S, O'Roak BJ. A saturation mutagenesis approach to understanding PTEN lipid phosphatase activity and genotype-phenotype relationships. Am J Hum Genet 2018;102:943-55.
- Sun H, Lesche R, Li DM, Liliental J, Zhang H, Gao J, et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci U S A 1999;96:6199-204.
- Tan MH, Mester J, Peterson C, Yang Y, Chen JL, Rybicki LA, et al. A clinical scoring system for selection of patients for
PTENmutation testing is proposed on the basis of a prospective study of 3042 probands. Am J Hum Genet 2011;88:42-56.
- Spinelli L, Black FM, Berg JN, Eickholt BJ, Leslie NR. Functionally distinct groups of inherited
PTENmutations in autism and tumour syndromes. J Med Genet 2015;52:128-34.