Analysis of CCM1 expression uncovers novel minor-form exons and variable splicing patterns

Abstract

Cerebral cavernous malformations (CCM) are vascular lesions, which occur sporadically or following an autosomal dominant inheritance pattern with variable expression and incomplete penetrance. Three genes have been associated with the disease (CCM1, CCM2 and CCM3). CCM1 has been reported to express atypical transcripts in addition to alternative splicing of non-coding exons. Here, we report the identification of novel alternative splicing events in the CCM1 gene. 5′RACE analysis revealed several transcription start sites, novel exons located in introns 2 and 7, and a larger exon 13. The 5′UTR CCM1 region showed at least eight splicing variants which were differentially transcribed. The results shown here expand our knowledge of CCM1 gene expression, which seems to be more complex than previously reported. The novel minor-form exons herein described should be considered in molecular diagnosis of CCM. These findings support new functional transcript sequences that could be considered in the pathogenesis and variable clinical penetrance of Krit1-linked CCMs.

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Acknowledgments

This work has been supported by grants CP10/00526 (Instituto de Salud Carlos III, Spain) and P07-CVI-02790 (Junta de Andalucía, Spain). RM received a grant for Rio Hortega specialised healthcare post-training contract (Instituto de Salud Carlos III). MD received a fellowship of Asociación Neuroinvest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Correspondence to Miguel Lucas.

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The authors declare that they have no conflict of interest.

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This research adhered to the tenets of the Declaration of Helsinki. A written consent was obtained from each of the adult individuals tested. This study was approved by the ‘‘Committee of Ethics and Clinical Investigation’’ from Hospital Universitario Virgen Macarena.

Electronic supplementary material

Amplification of exon 13L using different 5′ primers showed bands of variable intensity. The products were separated in agarose gel as described in “Materials and methods” section. The bands refer to fragments of exonic sequences; 1-12, 1-13L, 5-13L, 7-13L and 11-13L. Mw: 100-bp molecular-weight marker.

CCM patients included in this study

5′RACE methodology

Primers used for RT-qPCR

Efficiency of quantitative amplification

Location of transcriptional start sites of the CCM1 gene

Amplification of exon 13L with upstream exons

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Mondejar, R., Delgado, M., Solano, F. et al. Analysis of CCM1 expression uncovers novel minor-form exons and variable splicing patterns. Genes Genom 38, 879–889 (2016). https://doi.org/10.1007/s13258-016-0435-1

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Keywords

  • Cerebral cavernous malformations (CCMs)
  • CCM1
  • Atypical splicing
  • 5′UTR exons
  • Transcription start site (TSS)
  • Minor-form exons