Germ-line deletion in DICER1 revealed by a novel MLPA assay using synthetic oligonucleotides.
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.
1] Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada [2] Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montreal, Quebec, Canada.
Nelly
N
Sabbaghian
1] Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada [2] Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montreal, Quebec, Canada.
Archana
A
Srivastava
1] Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada [2] The Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.
Nancy
N
Hamel
1] Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada [2] Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montreal, Quebec, Canada.
François
F
Plourde
Department of Nuclear Medicine and Out-Patient Pediatric Endocrine Clinic, Children's Memorial Health Institute, Warsaw, Poland.
Malgorzata
M
Gajtko-Metera
Department of Pediatric Endocrinology and Rheumatology, Poznan University of Medical Sciences, Poznan, Poland.
Marek
M
Niedziela
1] Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada [2] Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montreal, Quebec, Canada [3] The Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.
William D
WD
Foulkes
D000483:Alleles; D002648:Child; D053487:DEAD-box RNA Helicases / Q000235:genetics*; D005091:Exons; D005260:Female; D016368:Frameshift Mutation; D015321:Gene Rearrangement; D005854:Germ Cells / Q000737:chemistry*; D018095:Germ-Line Mutation; D006044:Goiter, Nodular / Q000150:complications / Q000235:genetics; D006801:Humans; D007438:Introns; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D010051:Ovarian Neoplasms / Q000150:complications / Q000235:genetics; D010375:Pedigree; D043244:Ribonuclease III / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D009841:Oligonucleotides; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases