Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

Abstract

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

Data availability

All data generated or analysed during this study are included in the manuscript, supporting files, and as Source data and Source code.

Article and author information

Author details

  1. Hirokazu Kimura

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  2. Raymond M Paranal

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  3. Neha Nanda

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  4. Laura D Wood

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  5. James R Eshleman

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  6. Ralph H Hruban

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    Ralph H Hruban, has the right to receive royalty payments from Thrive Earlier Diagnosis for the GNAS in pancreatic cysts invention in a relationship overseen by Johns Hopkins University..
  7. Michael G Goggins

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  8. Alison P Klein

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  9. Nicholas Jason Roberts

    The Johns Hopkins University, Baltimore, United States
    For correspondence
    nrobert8@jhmi.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8709-0664

Funding

The Sol Goldman Pancreatic Cancer Research Center (Pilot Grant)

  • Nicholas Jason Roberts

National Institutes of Health (S10 OD026859)

  • Nicholas Jason Roberts

The Rolfe Pancreatic Cancer Foundation

  • Nicholas Jason Roberts

National Institutes of Health (P50 CA622924)

  • Alison P Klein
  • Nicholas Jason Roberts

The Japanese Society of Gastroenterology

  • Hirokazu Kimura

The Japan Society for Promotion of Science

  • Hirokazu Kimura

The Joseph C Monastra Foundation

  • Nicholas Jason Roberts

The Geral O Mann Foundation

  • Nicholas Jason Roberts

Art Creates Cures Foundation

  • Nicholas Jason Roberts

Susan Wojcicki and Denis Troper

  • Nicholas Jason Roberts

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2022, Kimura et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Hirokazu Kimura
  2. Raymond M Paranal
  3. Neha Nanda
  4. Laura D Wood
  5. James R Eshleman
  6. Ralph H Hruban
  7. Michael G Goggins
  8. Alison P Klein
  9. Nicholas Jason Roberts
(2022)
Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance
eLife 11:e71137.
https://doi.org/10.7554/eLife.71137

Share this article

https://doi.org/10.7554/eLife.71137

Further reading

    1. Cancer Biology
    Zhenhui Chen, Lu Yu ... Yi Ding
    Research Article

    The prevalence and mortality rates of colorectal cancer (CRC) are increasing worldwide. Radiation resistance hinders radiotherapy, a standard treatment for advanced CRC, leading to local recurrence and metastasis. Elucidating the molecular mechanisms underlying radioresistance in CRC is critical to enhance therapeutic efficacy and patient outcomes. Bioinformatic analysis and tumour tissue examination were conducted to investigate the CPT1A mRNA and protein levels in CRC and their correlation with radiotherapy efficacy. Furthermore, lentiviral overexpression and CRISPR/Cas9 lentiviral vectors, along with in vitro and in vivo radiation experiments, were used to explore the effect of CPT1A on radiosensitivity. Additionally, transcriptomic sequencing, molecular biology experiments, and bioinformatic analyses were employed to elucidate the molecular mechanisms by which CPT1A regulates radiosensitivity. CPT1A was significantly downregulated in CRC and negatively correlated with responsiveness to neoadjuvant radiotherapy. Functional studies suggested that CPT1A mediates radiosensitivity, influencing reactive oxygen species (ROS) scavenging and DNA damage response. Transcriptomic and molecular analyses highlighted the involvement of the peroxisomal pathway. Mechanistic exploration revealed that CPT1A downregulates the FOXM1-SOD1/SOD2/CAT axis, moderating cellular ROS levels after irradiation and enhancing radiosensitivity. CPT1A downregulation contributes to radioresistance in CRC by augmenting the FOXM1-mediated antioxidant response. Thus, CPT1A is a potential biomarker of radiosensitivity and a novel target for overcoming radioresistance, offering a future direction to enhance CRC radiotherapy.

    1. Cancer Biology
    2. Evolutionary Biology
    Arman Angaji, Michel Owusu ... Johannes Berg
    Research Article

    In growing cell populations such as tumours, mutations can serve as markers that allow tracking the past evolution from current samples. The genomic analyses of bulk samples and samples from multiple regions have shed light on the evolutionary forces acting on tumours. However, little is known empirically on the spatio-temporal dynamics of tumour evolution. Here, we leverage published data from resected hepatocellular carcinomas, each with several hundred samples taken in two and three dimensions. Using spatial metrics of evolution, we find that tumour cells grow predominantly uniformly within the tumour volume instead of at the surface. We determine how mutations and cells are dispersed throughout the tumour and how cell death contributes to the overall tumour growth. Our methods shed light on the early evolution of tumours in vivo and can be applied to high-resolution data in the emerging field of spatial biology.