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ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients

  • Original Article
  • Published:
Tumor Biology

Abstract

Despite one third of breast (BC) and colorectal cancer (CRC) cases having a hereditary component, only a small proportion can be explained by germline mutations. The aim of this study was to identify potential genomic alterations related to cancer predisposition. Copy number variations (CNVs) were interrogated in 113 unrelated cases fulfilling the criteria for hereditary BC/CRC and presenting non-pathogenic mutations in BRCA1, BRCA2, MLH1, MSH2, TP53, and CHEK2 genes. An identical germline deep intronic deletion of ROBO1 was identified in three index patients using two microarray platforms (Agilent 4x180K and Affymetrix CytoScan HD). The ROBO1 deletion was confirmed by quantitative PCR (qPCR). Six relatives were also evaluated by CytoScan HD Array. Genomic analysis confirmed a co-segregation of the ROBO1 deletion with the occurrence of cancer in two families. Direct sequencing revealed no pathogenic ROBO1 point mutations. Transcriptomic analysis (HTA 2.0, Affymetrix) in two breast carcinomas from a single patient revealed ROBO1 down-expression with no splicing events near the intronic deletion. Deeper in silico analysis showed several enhancer regions and a histone methylation mark in the deleted region. The ROBO1 deletion in a putative transcriptional regulatory region, its down-expression in tumor samples, and the results of the co-segregation analysis revealing the presence of the alteration in affected individuals suggest a pathogenic effect of the ROBO1 in cancer predisposition.

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Acknowledgments

The authors would like to thank the Nucleic Acid Bank and Department of Pathology of A.C. Camargo Cancer Center (São Paulo, Brazil) and the patients and families who agreed to participate in this study. We are also grateful for the assistance with the sequencing data given by Dr. Felipe C. da Silva.

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    Authors and Affiliations

    1. International Research Center (CIPE), A.C. Camargo Cancer Center, Rua Taguá 440, São Paulo, CEP: 01508-010, SP, Brazil

      Rolando A. R. Villacis, Francine B. Abreu, Priscila M. Miranda, Dirce M. Carraro & Silvia R. Rogatto

    2. Department of Pathology, Faculty of Medicine, University of São Paulo State (UNESP), Botucatu, SP, Brazil

      Maria A. C. Domingues

    3. Oncology Center, Sírio-Libanês Hospital, São Paulo, SP, Brazil

      Erika M. M. Santos & Benedito M. Rossi

    4. Department of Pathology, A.C. Camargo Cancer Center, São Paulo, SP, Brazil

      Victor P. Andrade

    5. Department of Oncogenetics, A.C. Camargo Cancer Center, São Paulo, SP, Brazil

      Maria I. Achatz

    6. Department of Urology, Faculty of Medicine, University of São Paulo State (UNESP), CEP: 18618-970, Botucatu, SP, Brazil

      Silvia R. Rogatto

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    1. Rolando A. R. Villacis
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    Correspondence to Silvia R. Rogatto.

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    Funding

    This study was supported by grants from the National Institute of Science and Technology in Oncogenomics (INCITO FAPESP 2008/57887-9 and CNPq 573589/08-9), FAPESP (2010/15901-5 and 2011/07742-7).

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    Rolando A. R. Villacis and Francine B. Abreu contributed equally to this work.

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    Supplemental Fig. 1

    Electropherograms showing the silent ROBO1 mutations identified in all relatives tested of patient P2 (P2-2, P2-3, P2-4 and P2-5) (1), in P2-2 and P2-3 only (2) and in P2-4 and P2-5 only (3). (GIF 32 kb)

    High resolution image (TIFF 384 kb)

    Supplemental Fig. 2

    Hierarchical clustering analysis using the 795 differentially expressed coding transcripts between normal breast and tumor samples. In the heatmap red represents over-expression and blue down-expression. (GIF 28 kb)

    High resolution image (TIFF 195 kb)

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    Villacis, R.A.R., Abreu, F.B., Miranda, P.M. et al. ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients. Tumor Biol. 37, 3145–3153 (2016). https://doi.org/10.1007/s13277-015-4145-0

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    • DOI: https://doi.org/10.1007/s13277-015-4145-0

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