BAP1 missense mutation c.2054 A\>T (p.E685V) completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line

Our plain-English summary of BAP1 missense mutation c.2054 A>T (p.E685V) completely disrupts normal splicing through creation of a novel 5' splice site in a human mesothelioma cell line, by A. Morrison, Y. Chekaluk, R. Bacares, et al (PloS One, 2015).

The study

• Authors: A. Morrison, Y. Chekaluk, R. Bacares, M. Ladanyi, L. Zhang
• Journal: PloS One
• Published: 2015;10(4):e0119224
• Cited by: 10 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

BAP1 is a tumor suppressor gene that is lost or deleted in diverse cancers, including uveal mela¬noma, malignant pleural mesothelioma (MPM), clear cell renal carcinoma, and cholangiocarcinoma. Recently, BAP1 germline mutations have been reported in families with combinations of these same cancers. A particular challenge for mutation screening is the classification of non-truncating BAP1 sequence variants because it is not known whether these subtle changes can affect the protein function sufficiently to predispose to cancer development. Here we report mRNA splicing analysis on a homozygous substitution mutation, BAP1 c. 2054 A&T (p.Glu685Val), identified in an MPM cell line derived from a mesothelioma patient. The mutation occurred at the 3rd nucleotide from the 3' end of exon 16. RT-PCR, cloning and subsequent sequencing revealed several aberrant splicing products not observed in the controls: 1) a 4 bp deletion at the end of exon 16 in all clones derived from the major splicing product. The BAP1 c. 2054 A&T mutation introduced a new 5' splice site (GU), which resulted in the deletion of 4 base pairs and presumably protein truncation; 2) a variety of alternative splicing products that led to retention of different introns: introns 14-16; introns 15-16; intron 14 and intron 16; 3) partial intron 14 and 15 retentions caused by activation of alternative 3' splice acceptor sites (AG) in the introns. Taken together, we were unable to detect any correctly spliced mRNA transcripts in this cell line. These results suggest that aberrant splicing caused by this mutation is quite efficient as it completely abolishes normal splicing through creation of a novel 5' splice site and activation of cryptic splice sites. These data support the conclusion that BAP1 c.2054 A&T (p.E685V) variant is a pathogenic mutation and contributes to MPM through disruption of normal splicing.

In plain English

This laboratory study examined one human mesothelioma cell line, HMeso01A, derived from a patient with malignant pleural mesothelioma. Gene sequencing showed the cell line was homozygous for a change in the BAP1 gene called c.2054 A>T (p.Glu685Val). The article does not discuss asbestos. The authors asked whether this single-letter change, at the third letter from the end of exon 16, affects how the gene's RNA message is spliced. Computer programs predicted the change to be damaging and to create a strong new splice site 4 base pairs upstream of the normal one. The cell line's RNA showed abnormal splicing products not seen in four control individuals without the change. Of 30 clones sequenced, all 19 from the main product had a 4 base pair deletion. The authors said this presumably leads to a shortened protein of 689 amino acids instead of 729. Other products retained whole or partial introns. No correctly spliced message was found. The authors concluded that changes outside the core splice sites may disrupt normal splicing. They noted that other abnormal products outside the tested region may have gone undetected.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory molecular study of a single cell line: computer (in silico) predictions, then RT-PCR, cloning and sequencing of BAP1 RNA compared with controls
• Who was studied: One human mesothelioma cell line, HMeso01A, derived from a malignant pleural mesothelioma patient, compared with RNA from four control individuals seen at Memorial Sloan Kettering Cancer Center who do not carry the mutation
• Exposure: No asbestos or other environmental exposure was studied. The factor examined was a genetic change: a homozygous BAP1 c.2054 A>T (p.Glu685Val) substitution in exon 16.
• Main results:
  • Three prediction programs rated the mutation as damaging: PolyPhen 'probably damaging', SIFT 'damaging', MutationTaster 'disease causing'
  • A splice prediction program (NNSplice) predicted that the mutation creates a strong new 5' splice site 4 base pairs upstream of the original one
  • The HMeso01A cell line showed abnormal splicing products that were not seen in the four wild type controls
  • Of 30 clones sequenced, all 19 clones from the bottom band in HMeso01A had a 4 base pair deletion
  • The authors stated that the 4 base pair deletion presumably leads to a stop signal at amino acid position 690, giving a shortened protein (689 instead of 729 amino acids). This was presumed, not directly measured.
  • Other splicing products in HMeso01A retained different introns, either whole or in part
  • No correctly spliced BAP1 mRNA was detected in the HMeso01A cell line
• The authors' conclusion: The authors concluded that mutations outside the highly conserved GU/AG splice sites may disrupt normal splicing rather than simply changing one amino acid. They also stated that this BAP1 variant contributes to malignant pleural mesothelioma through disruption of normal splicing.
• Limitations the authors state:
  • The largest product, with introns 14, 15 and 16 retained, might have come from traces of genomic DNA contaminating the RNA
  • Because the primers covered only exons 14 to 17, other abnormal splicing products outside this region may have gone undetected

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: BAP1, Mesothelioma, Tumor Suppressor Gene, Pleural Mesothelioma, Carcinoma, Germline Mutation.

Related studies

• Malignant cells from pleural fluids in malignant mesothelioma patients reveal novel mutations — S. Sneddon, I. Dick, et al. (2018)
• Splicing modulation as novel therapeutic strategy against diffuse malignant peritoneal mesothelioma — R. Sciarrillo, A. Wojtuszkiewicz, et al. (2019)
• Novel LRRK2 mutations and other rare, non-BAP1-related candidate tumor predisposition gene variants in high-risk cancer families with mesothelioma and other tumors — M. Cheung, Y. Kadariya, et al. (2021)
• Intrahepatic cholangiocarcinoma development in a patient with a novel BAP1 germline mutation and low exposure to asbestos — G. Brandi, M. Deserti, et al. (2020)
• A population-based analysis of germline BAP1 mutations in melanoma — S.J. O'Shea, C.D. Robles-Espinoza, et al. (2017)
• Loss of BAP1 expression is associated with genetic mutation and can predict outcomes in gallbladder cancer — T. Hirosawa, M. Ishida, et al. (2018)

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