DICER1 RNase IIIb domain mutations are infrequent in testicular germ cell tumours

Abstract

Background: Testicular Germ Cell Tumours (TGCT) are the most frequently occurring malignancy in males from 15-45 years of age. They are derived from germ cells unable to undergo physiological maturation, although the genetic basis for this is poorly understood. A recent report showed that mutations in the RNase IIIb domain of DICER1, a micro-RNA (miRNA) processing enzyme, are common in non-epithelial ovarian cancers. DICER1 mutations were found in 60% of Sertoli-Leydig cell tumours, clustering in four codons encoding metal-binding sites. Additional analysis of 14 TGCT DNA samples identified one case that also contained a mutation at one of these sites.

Findings: A number of previous studies have shown that DICER1 mutations are found in <1% of most cancers. To provide a more accurate estimate of the frequency of such mutations in TGCTs, we have analysed 96 TGCT samples using high resolution melting curve analysis for sequence variants in these four codons. Although we did not detect any mutations in any of these sites, we did identify a novel mutation (c.1725 R>Q) within the RNase IIIb domain in one TGCT sample, which was predicted to disturb DICER1 function.

Conclusion: Overall our findings suggest a mutation frequency in TGCTs of ~1%. We conclude therefore that hot-spot mutations, frequently seen in Sertoli-Leydig cell tumours, are not common in TGCTs.

Figure 1

HRM analysis detection of previously identified mutations within the DICER1 RNase IIIb domain. Nucleotide and amino acid numbering are based on DICER1 reference sequence [NCBI:NM_177438]. 1A. Aberrant HRM curves resulting from four different heterozygous mutations, affecting amino acids 1705 and 1709 of DICER1. #1 = c.5113G>A (p.E1705K); #2 = c.5125G>A (p.D1709N); #3 = c.5126A>G(p.D1709G); #4 = c.5127T>A (p.D1709E); Ref = reference sequence. 1B. Aberrant HRM curves resulting from two different heterozygous mutations, affecting amino acids 1810 and 1813 of DICER1. #1 = c.5428G>T (p.D1810Y); #2 = c.5437G>C (E1813Q); Ref = reference sequence.

Figure 2

A novel mutation identified within the DICER1 RNase IIIb domain in a single seminoma sample. Amino acid numbering is based on DICER1 reference sequence [GenBank:NM_177438]. 2A. The aberrant HRM curve corresponding to the DICER1 mutation. The curves on the baseline represent samples without a sequence variant (this was confirmed with Sanger sequencing in one sample). 2B. A Sanger sequencing trace from the sample that showed the aberrant HRM curve in 2A. The heterozygous sequence variant is indicated by an arrow. This mutation is predicted to change an Arginine to a Glutamine at position 1725 of DICER1. 2C. The RNase IIIb domain in DICER1, containing amino acids 1666–1824. The location of the mutation (R1725Q) identified in this study is shown by the red bar. The locations of the four metal-binding residues (all acidic amino acids) frequently mutated in Sertoli-Leydig cell tumours are shown by black bars. The region of 100% conservation across at least 42 species at the amino acid level (residues 1705–1741) is indicated by the horizontal black bar.