DICER1 mutations in familial multinodular goiter with and without ovarian Sertoli-Leydig cell tumors

Abstract

Context: Nontoxic multinodular goiter (MNG) is frequently observed in the general population, but little is known about the underlying genetic susceptibility to this disease. Familial cases of MNG have been reported, and published reports describe 5 families that also contain at least 1 individual with a Sertoli-Leydig cell tumor of the ovary (SLCT). Germline mutations in DICER1, a gene that codes for an RNase III endoribonuclease, have been identified in families affected by pleuropulmonary blastoma (PPB), some of whom include cases of MNG and gonadal tumors such as SLCTs.

Objective: To determine whether familial MNG with or without SLCT in the absence of PPB was associated with mutations in DICER1.

Design, setting, and patients: From September 2009 to September 2010, we screened 53 individuals from 2 MNG and 3 MNG/SLCT families at McGill University for mutations in DICER1. We investigated blood lymphocytes and MNG and SLCT tissue from family members for loss of the wild-type DICER1 allele (loss of heterozygosity), DICER1 expression, and microRNA (miRNA) dysregulation.

Main outcome measure: Detection of germline DICER1 gene mutations in familial MNG with and without SLCT.

Results: We identified and characterized germline DICER1 mutations in 37 individuals from 5 families. Two mutations were predicted to be protein truncating, 2 resulted in in-frame deletions, and 1 was a missense mutation. Molecular analysis of the 3 SLCTs showed no loss of heterozygosity of DICER1, and immunohistochemical analysis in 2 samples showed strong expression of DICER1 in Sertoli cells but weak staining of Leydig cells. miRNA profiling of RNA from lymphoblastoid cell lines from both affected and unaffected members of the familial MNG cases revealed miRNA perturbations in DICER1 mutation carriers.

Conclusions: DICER1 mutations are associated with both familial MNG and MNG with SLCT, independent of PPB. These germline DICER1 mutations are associated with dysregulation of miRNA expression patterns.

Figure 1. MNG/SCLT and familial MNG pedigrees

A–E, pedigrees and electrophoretograms of probands with DICER1 mutations and familial MNG/SCLTO (A–C) or familial MNG (D–E). The genomic reference sequence used is refseq NG_016311.1. The vertical line in electrophoretogram E represents the intron/exon boundary. Symbols in black indicate MNG; “+” or “−” denotes mutation status (+ = mutation). SLCT = Sertoli-Leydig Cell Tumor, CRC = colorectal cancer, FM = fibrolamellar meningioma, RMS = rhabdomyosarcoma.

Figure 2. DICER1 mutations and their effect on mRNA

A: mutant mRNAs from families A and C are targeted and degraded by the nonsense-mediated mRNA decay. LCLs from carriers of the c.871_874delAAAG (left) or c.5018-5021delTCAA (right) mutations from family A and C, respectively and from a non-carrier were treated or not treated with cycloheximide (CHX), a potent inhibitor of the NMD. Amplification of the 7–8 exon junction (left) or part of exon 23 (right) in treated and non-treated LCLS indicated that in presence of CHX the amount of mutant mRNA was increased in both carriers. No mutant mRNA was detected in the controls. B: amplification of the exon 15–16 junction of mRNA derived from LCLs from one individual from family B indicated that mutant mRNA produced is insensitive to NMD (left). The upper band seen in this individual is a non-specific heteroduplex. Sequencing of the PCR products revealed that the g.49351C>G mutation leads to the creation of a de novo splice site at position 22 of exon 16 and the in-frame deletion of the first 21 bases of exon 16 (right). C: Splice mutation in family E. Primers in DICER1 exons 17 and 19 amplify a heterozygous 183bp deletion in c.2805-1G>T mutation carrier cDNA (left). Sequence analysis of the 2 amplified products in G/T carrier (right) shows perfect excision of exon 18 in the smaller fragment (middle trace) and wild-type sequence in the larger fragment (upper and lower traces).

Figure 3. Loss of Heterozygosity and Immunohistochemistry studies

A: LOH analysis of Family B, no loss of heterozygosity in proband affected tissue. I and III = gDNA from lymphocytes; II = gDNA from goiter; IV = gDNA from SLCT. Arrow indicates mutation. B: Immunohistochemistry of MNG and SLCT. Left: representative staining of DICER1 in MNG from a non-hereditary case (top) and from a mutation carrier from family D (bottom). DICER1 staining is detected in cells bordering the vesicles and is heterogeneous, being partly dependent on the amount of cytoplasm, which is reduced when surrounding large vesicles. No difference in staining is evident when comparing hereditary and non-hereditary cases of MNG. Right: representative staining of DICER1 in normal ovary (top) compared to a Sertoli-Leydig cell tumor. This tumor is composed of prominent cords and tubules of immature Sertoli cells with discrete clusters of Leydig cells. The Leydig cells contained a round nucleus with prominent nucleolus and an abundant pale and eosinophilic cytoplasm. The immature Sertoli cells have small round nuclei and scanty cytoplasm. No staining was detected in normal ovary. Cytoplasm of Sertoli cells (S) was strongly stained and Leydig cells (L) show very weak staining.

Figure 4. miRNA microarray studies

A: Differentially expressed miRNAs between carriers and non-carriers of mutations in DICER1 from family D and E. Unsupervised hierarchical clustering showed clustering of the samples according their mutation status. The Cluster 3.0 software was used, wherein miRNA expression values were median-centered and clustered using Pearson uncentered correlation distance and average linkage. B: Quantification of expression levels of miRNAs miR-345 and let-7a by real-time PCR in frozen tissues. miRNA-345 and let-7a are downregulated in goiter tissue from individual IV-4 from family E compared to normal thyroid and follicular thyroid cancer tissues from non-carriers of DICER1 mutations.

Figure 4. miRNA microarray studies

A: Differentially expressed miRNAs between carriers and non-carriers of mutations in DICER1 from family D and E. Unsupervised hierarchical clustering showed clustering of the samples according their mutation status. The Cluster 3.0 software was used, wherein miRNA expression values were median-centered and clustered using Pearson uncentered correlation distance and average linkage. B: Quantification of expression levels of miRNAs miR-345 and let-7a by real-time PCR in frozen tissues. miRNA-345 and let-7a are downregulated in goiter tissue from individual IV-4 from family E compared to normal thyroid and follicular thyroid cancer tissues from non-carriers of DICER1 mutations.