Abstract
DICER1 is an endoribonuclease responsible for the production of mature microRNAs which are small, single-stranded RNA molecules that regulate gene expression post-transcriptionally by binding to mRNA and repressing the expression of target genes. Germ-line mutations in DICER1 are responsible for a rare cancer syndrome, including tumors that can co-occur with multinodular goiter (MNG). Using Sanger sequencing, we screened all DICER1 exons and intron boundaries in 20 suspected mutation carriers: nine with ovarian sex cord-stromal tumors (including Sertoli–Leydig cell tumors (SLCTs)), five with pleuropulmonary blastoma, one with cystic nephroma, one with nasal chondromesenchymal hamartoma and four with more than one manifestation suggestive of a germ-line DICER1 mutation. All were negative for any apparently deleterious variants. We developed a Multiplex Ligation-based Probe Amplification assay for DICER1 to screen for large deletions or duplications. Synthetic oligonucleotides were designed to cover all exons in three probe-mixes. In a child with a SLCT and MNG, and in her mother and brother (both diagnosed with MNG), we identified a heterozygous germ-line deletion of approximately 3 kilobases that eliminates exon 21 of DICER1 and two-thirds of intron 21, accompanied by an insertion of a G nucleotide at the 3′ end of the deletion (c.3270-6_4051-1280delinsG). This allele is expressed in the patient’s cDNA, creating an out-of-frame deletion predicted to result in a truncated protein (r.3270_4050del; p.Tyr1091Ser*28). Our novel finding of a disease-causing large deletion in DICER1 emphasizes the need to include assays that can detect rearrangements, duplications and deletions in any DICER1 screening protocol.
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Acknowledgements
We thank Dr John R Priest for his continued support of our work and all those who provided samples, especially Tomasz Dzik, MD, Ph.D. We would like to thank Jonathan Jarry, M.Sc. and George Chong, Ph.D. for providing input and help with the implementation of the MLPA assay. We also thank the technical support team from PREMIER Biosoft and Jelger van der Meer Ph.D. (MRC-Holland) for advice in devising the probe-mixes and MLPA assays, respectively. The study was financially supported by the Mendon F Schutt Foundation and the Jewish General Hospital Foundation.
AUTHOR CONTRIBUTIONS
NS developed the MLPA assay, performed the validation in genomic DNA and wrote the manuscript; AS prepared the RNA and cDNA and performed the cDNA experiments; NH oversaw the laboratory work and edited the manuscript; FP helped to recruit the cases; MM and MN provided samples and key clinical information; WDF wrote the paper with NS and led the project. All authors read and approved the final submitted manuscript.
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Sabbaghian, N., Srivastava, A., Hamel, N. et al. Germ-line deletion in DICER1 revealed by a novel MLPA assay using synthetic oligonucleotides. Eur J Hum Genet 22, 564–567 (2014). https://doi.org/10.1038/ejhg.2013.215
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DOI: https://doi.org/10.1038/ejhg.2013.215
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