Quantification of mutant E-cadherin using bioimaging analysis of in situ fluorescence microscopy. A new approach to CDH1 missense variants.
Missense mutations result in full-length proteins containing an amino acid substitution that can be neutral or deleterious, interfering with the normal conformation, localization, and function of a protein. A striking example is the presence of CDH1 (E-cadherin gene) germline missense variants in hereditary diffuse gastric cancer (HDGC), which represent a clinical burden for genetic counseling and surveillance of mutation carriers and their families. CDH1 missense variants can compromise not only the function of E-cadherin but also its expression pattern. Here, we propose a novel method to characterize E-cadherin signature in order to identify cases with E-cadherin deregulation and functional impairment. The strategy includes a bioimaging pipeline to quantify the expression level and characterize the distribution of the protein from in situ immunofluorescence images. The algorithm computes 1D (dimension intensity) radial and internuclear fluorescence profiles to generate expression outlines and 2D virtual cells representing a typical cell within the populations analyzed. Using this new approach, we verify that cells expressing mutant forms of E-cadherin display fluorescence profiles distinct from those of the wild-type cells. Mutant proteins showed a significantly decrease of fluorescence intensity at the membrane and often abnormal expression peaks in the cytoplasm, reflecting the underlying molecular mechanism of trafficking deregulation. Our results suggest employing this methodology as a complementary approach to evaluate the pathogenicity of E-cadherin missense variants. Moreover, it can be applied to a wide range of proteins and, more importantly, to diseases characterized by aberrant protein expression or trafficking deregulation.
Institute for Systems and Robotics and Department of Bioengineering from the Instituto Superior Técnico, Technical University of Lisbon, Lisbon, Portugal.
João Miguel
JM
Sanches
Department of Cancer Genetics, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Joana
J
Figueiredo
Institute for Systems and Robotics and Department of Bioengineering from the Instituto Superior Técnico, Technical University of Lisbon, Lisbon, Portugal.
Martina
M
Fonseca
Department of Cancer Genetics, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Cecília
C
Durães
Department of Cancer Genetics, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
Soraia
S
Melo
Institute for Systems and Robotics and Department of Bioengineering from the Instituto Superior Técnico, Technical University of Lisbon, Lisbon, Portugal.
Sofia
S
Esménio
1] Department of Cancer Genetics, IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal [2] Faculty of Medicine of the University of Porto, Porto, Portugal.
Raquel
R
Seruca
D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D005817:Genetic Counseling; D020022:Genetic Predisposition to Disease; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D020125:Mutation, Missense; D013274:Stomach Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology
D015820:Cadherins