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Functional analysis of PTEN variants of unknown significance from PHTS patients unveils complex patterns of PTEN biological activity in disease

European Journal of Human Genetics volume 31, pages 568–577 (2023)Cite this article

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Abstract

Heterozygous germline mutations in PTEN gene predispose to hamartomas and tumors in different tissues, as well as to neurodevelopmental disorders, and define at genetic level the PTEN Hamartoma Tumor Syndrome (PHTS). The major physiologic role of PTEN protein is the dephosphorylation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), counteracting the pro-oncogenic function of phosphatidylinositol 3-kinase (PI3K), and PTEN mutations in PHTS patients frequently abrogate PTEN PIP3 catalytic activity. PTEN also displays non-canonical PIP3-independent functions, but their involvement in PHTS pathogeny is less understood. We have previously identified and described, at clinical and genetic level, novel PTEN variants of unknown functional significance in PHTS patients. Here, we have performed an extensive functional characterization of these PTEN variants (c.77 C > T, p.(Thr26Ile), T26I; c.284 C > G, p.(Pro95Arg), P95R; c.529 T > A, p.(Tyr177Asn), Y177N; c.781 C > G, p.(Gln261Glu), Q261E; c.829 A > G, p.(Thr277Ala), T277A; and c.929 A > G, p.(Asp310Gly), D310G), including cell expression levels and protein stability, PIP3-phosphatase activity, and subcellular localization. In addition, caspase-3 cleavage analysis in cells has been assessed using a C2-domain caspase-3 cleavage-specific anti-PTEN antibody. We have found complex patterns of functional activity on PTEN variants, ranging from loss of PIP3-phosphatase activity, diminished protein expression and stability, and altered nuclear/cytoplasmic localization, to intact functional properties, when compared with PTEN wild type. Furthermore, we have found that PTEN cleavage at the C2-domain by the pro-apoptotic protease caspase-3 is diminished in specific PTEN PHTS variants. Our findings illustrate the multifaceted molecular features of pathogenic PTEN protein variants, which could account for the complexity in the genotype/phenotype manifestations of PHTS patients.

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Fig. 1: Depiction of PTEN 3D structure (accession ID5R) and localization of the amino acids targeted by mutations which were analysed in this study (Thr26, Pro95, Tyr177, Gln261, Thr277, and Asp310).
Fig. 2: Steady-state expression and stability of PTEN PHTS variants.
Fig. 3: PIP3-phosphatase functional activity of PTEN PHTS variants in cells.
Fig. 4: Subcellular localization of PTEN PHTS variants.
Fig. 5: Cleavage of PTEN PHTS variants at Asp301 as determined using the anti-PTEN SP227 mAb.

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Data availability

The datasets generated during and/or analysed during the current study are available in the LOVD gene variant database (http://www.lovd.nl/3.0/home), with the following accession ID: 0000352584 (T26I), 0000878862 (P95R), 0000878883 (Y177N), 0000878885 (Q261E), 0000878886 (T277A), 0000878890 (D310G).

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Acknowledgements

We thank Gustavo Pérez‐Nanclares and Ana Belén de la Hoz (Genetics-Genomics Core facility, Biocruces Bizkaia Health Research Institute) for their expert assistance with DNA sequencing., and Javier Díez Garcı́a (Microscopy core facility, Biocruces Bizkaia Health Research Institute) for expert microscopy technical support.

Funding

This work has been supported in part by grant BBH-19-001 (to RP) from PTEN Research Foundation (United Kingdom); grants SAF2016-79847-R (to RP and JIL), and PID2019-105342GB-I00 (to VJC and MM) from Ministerio de Economía y Competitividad (Spain and The European Regional Development Fund); and grant S2017/BMD‐3691(InGEMICS‐CM) from Comunidad de Madrid and European Structural and Investment Funds (to VJC and MM). LT has been the recipient of a predoctoral fellowship from Asociación Española Contra el Cáncer (AECC, Junta Provincial de Bizkaia, Spain). JM has been the recipient of a predoctoral fellowship (PRE_2014_1_285) from Gobierno Vasco, Departamento de Educación (Basque Country, Spain). CN-X is the recipient of a Miguel Servet Research Contract from Instituto de Salud Carlos III (grant number CP20/00008).

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Authors and Affiliations

  1. Biocruces Bizkaia Health Research Institute, Barakaldo, Spain

    Leire Torices, Janire Mingo, Sandra Luna, Caroline E. Nunes-Xavier, José I. López & Rafael Pulido

  2. Departamento de Microbiología y Parasitología, Facultad de Farmacia, UCM & Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain

    Isabel Rodríguez-Escudero, Teresa Fernández-Acero, María Molina & Víctor J. Cid

  3. Institute for Cancer Research, Oslo University Hospital, Oslo, Norway

    Caroline E. Nunes-Xavier

  4. Department of Pathology, Cruces University Hospital, Barakaldo, Spain

    José I. López

  5. Familial Cancer Clinical Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

    Fátima Mercadillo, María Currás & Miguel Urioste

  6. Ikerbasque, The Basque Foundation for Science, Bilbao, Spain

    Rafael Pulido

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  1. Leire Torices
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Contributions

LT, JM, IR-E, TF-A, and SL designed and performed experiments, and performed data analysis. CEN-X designed experiments, supervised the work and performed data analysis. JIL and MM supervised the work. FM, MC, and MU shared information and provided feedback. VJC and RP designed experiments, performed data analysis and wrote the manuscript. All authors revised the manuscript.

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Correspondence to Rafael Pulido.

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Torices, L., Mingo, J., Rodríguez-Escudero, I. et al. Functional analysis of PTEN variants of unknown significance from PHTS patients unveils complex patterns of PTEN biological activity in disease. Eur J Hum Genet 31, 568–577 (2023). https://doi.org/10.1038/s41431-022-01265-w

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