Article Text

Germline DICER1 mutations and familial cystic nephroma
  1. Amin Bahubeshi1,2,
  2. Nebil Bal3,
  3. Thomas Rio Frio1,4,
  4. Nancy Hamel1,4,
  5. Carly Pouchet1,2,
  6. Ahmet Yilmaz1,2,4,
  7. Dorothée Bouron-Dal Soglio5,
  8. Gretchen M Williams6,
  9. Marc Tischkowitz1,2,
  10. John R Priest6,
  11. William D Foulkes1,2,4
  1. 1Program in Cancer Genetics, Departments of Oncology and Human Genetics, McGill University, Montreal, Quebec, Canada
  2. 2Lady Davis Institute, Segal Cancer Centre, Jewish General Hospital, Montreal, Quebec, Canada
  3. 3Department of Pathology, Baskent University Faculty of Medicine, Ankara, Turkey
  4. 4The Research Institute, McGill University Health Centre, Montreal, Quebec, Canada
  5. 5Department of Pathology, CHU Sainte Justine, Montréal, Québec, Canada
  6. 6The International Pleuropulmonary Blastoma Registry, St Paul, Minnesota, USA
  1. Correspondence to Dr William D Foulkes, Lady Davis Institute and Segal Cancer Centre, Jewish General Hospital, 3755 Côte St-Catherine, Montreal QC H3T 1E2, Canada; william.foulkes{at}mcgill.ca

Abstract

Background Multilocular cystic nephroma (CN) is a benign kidney tumour and is part of a family of kidney neoplasms including cystic partially differentiated nephroblastoma and Wilms tumour (WT). CN is rarely familial or bilateral, but it occurs in about 10% of families where pleuropulmonary blastoma (PPB) is present. Recently, germline mutations in DICER1 were found in familial PPB.

Objective To search for DICER1 mutations in two families with familial CN; PPB was present in one family. Additionally, to test germline DNA from 50 children with sporadic WT for DICER1 mutations.

Results Both families with multiple CN were found to have mutations in DICER1 leading to premature stop codons, predicted to result in loss of the ribonuclease and dsRNA binding domains. These domains are essential to the function of DICER1. No germline mutations were found in any of the 50 children who had developed WT.

Conclusion It has been established that DICER1 mutations cause familial CN and may be implicated in bilateral CN. No germline mutations were found in the patients with WT, suggesting that DICER1 mutations are unlikely to have a major role in the aetiology of sporadic WT. These results provide further evidence implicating miRNA dysregulation in tumourigenesis.

  • Kidney
  • Wilms tumour
  • hereditary
  • miRNA
  • pleuropulmonary blastoma
  • clinical genetics
  • molecular genetics
  • screening
  • renal medicine
  • genetic epidemiology

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Introduction

Cystic nephroma (CN), or multilocular cystic nephroma, is a rare kidney tumour that lies at the benign end of a spectrum of kidney neoplasms which include cystic partially differentiated nephroblastoma (CPDN) and Wilms tumour (WT). CN and CPDN must be differentiated from each other by histological analysis as imaging is often inconclusive. Although CN and CPDN are almost identical conditions, a delineating factor is the differentiated septa without blastemal elements in CN.1 In CPDN, septa contain blastema or other partially undifferentiated cells, yet it can remain benign, unlike WT which is overtly malignant.1 2

CN most commonly occurs in individuals below 2 years of age, with a preponderance of male subjects being affected. There is, however, a second incidence peak which occurs after the age of 40 years, where women are predominantly affected.3 4 In a review of 58 cases of CN, none were familial and only three were bilateral, suggesting that CN is usually sporadic and that bilateral CN is rare.4 Notably, a recent report described two siblings with CN, one of whom had bilateral CN.5

Although little is known about the aetiology of CN, it can be a manifestation of the distinctive familial disease complex associated with pleuropulmonary blastoma (PPB), a rare malignant tumour of pleura and lung occurring in children 6 years of age or less.6–12 In approximately 35% of families in which a child has PPB, the patient or a family member manifests one or more additional conditions from an unusual array of dysontogenetic-dysplastic and malignant conditions, known as the PPB family tumour and dysplasia syndrome (PPB FTDS) (OMIM #601200). CN, including familial,6 8 and bilateral CN, are found in 9–10% of family members affected by PPB.7 The first description of familial CN and PPB was reported by Delahunt et al in 1993 and described two siblings with CN who had a sister with PPB.8 In the study reported here we have analysed DICER1 in two other previously reported families, family S, first described by Bal et al, where a 13-month-old boy was found to have CN while his older sister had previously presented with both CN and PPB6 and family K, a case of familial CN without PPB more recently reported by Ashley and Reinberg, where one of the two affected children had bilateral CN.5 The pedigrees of both families are presented in figure 1A.

Figure 1

Family pedigrees, mutation traces and their predicted effect on DICER1 and immunohistochemistry of mutant DICER1. (A) Pedigrees of the two mutation carrier families studied. Individuals tested for DICER1 mutations are indicated with + (mutation present) or − (mutation absent). Other individuals in the families were not tested. ‘Bilateral’ (individual II-1, family K) indicates the cystic nephroma (CN) was bilateral; ‘PPB’ (pleuropulmonary blastoma) indicates the deceased individual (III-1, family S) who was diagnosed with both CN and PPB. Multiple cases of goitre occurred on both sides of the extended family S, several of whom had to have operations to remove the goitres: I:2—operation at 50 years, II:3—operation at 24 years, II:4—operation at 27 years. Note the younger age at operation for II:3 and II:4 on the maternal side of the family where the mutation segregates. Unlike the CN and PPB cases in the families, we do not have pathological confirmation of a diagnosis of thyroid disease. (B) Electrophoretograms showing the predicted c.4309_4312delGACT mutation in exon 23 in family K and the predicted c.5477C→A mutation in exon 25 in family S. (C) Graphical illustration of the relative positions of the D1437Mfs (family K) and S1826X (family S) amino acid changes along the DICER1 protein structure. (D) Immunohistochemistry of DICER1 in kidney from an unaffected individual (top) and in a cystic nephroma from family S, individual III-2 (bottom). In the normal kidney, DICER1 staining is strong in tubules and absent in the glomerulus. Analysis of the CN reveals weak staining in the tubules.

The families described by Delahunt and by Bal were included in a larger review by the International PPB Registry,7 which summarised cases of CN occurring in patients with PPB or their family members. A total of four instances of familial CN have been reported in the literature (table 1), three of which occurred in the context of PPB.

Table 1

Published cases of familial cystic nephroma (CN) with and without pleuropulmonary blastoma (PPB)

In 2009, Hill et al screened the DICER1gene in affected probands from families with PPB and other diagnoses associated with PPB, including CN,13 and identified mutations in all 11 cases analysed. With childhood CN firmly established as a disease associated with PPB, we set out to determine whether CN in families both with6 and without PPB5 is associated with germline DICER1 mutations. As WT is closely related to CN and also occurs occasionally with PPB, we also screened lymphocyte DNA isolated from 50 children diagnosed with WT for DICER1 mutations.

Methods

All families in this study gave IRB approved consent. The WT cases were diagnosed at several hospitals in eastern Canada between 1970 and 1998 (median age at diagnosis 34 months, range 8.5 months to 15 years).

DNA extracted from peripheral blood lymphocytes or saliva from affected individuals and their immediate relatives (figure 1) was sequenced for the gene DICER1. All exons and their flanking intronic regions were sequenced primarily using previously published primers.13 The DNA from the patients with WT were screened through high-resolution melt analysis using the LightScanner instrument (Idaho Technologies Inc, Utah, USA) and samples with altered melting curves were directly sequenced. High-resolution melt could not be adequately optimised for exons 13, 20, 22 and 23 so these were directly sequenced for all samples. DNA was extracted from paraffin-embedded CN tissue from patient III:2 (family S) using the Qiagen FFPE DNA extraction system (Qiagen, Mississauga, Canada) and was amplified by PCR, along with DNA extracted from lymphocytes, using the following primers: forward 5′-AGGAGATCTGAGGAGGATGAAG-3′ and reverse 5′-CCGCATCATGGGATAGTACA-3′. The 150 bp PCR products were analysed by direct sequencing and the relative intensity of the peaks at the position of the c.5477C→A mutation was compared between lymphocyte and CN DNA to determine loss of heterozygosity (LOH) status.

For immunohistochemistry, deparaffinised 5 μm tissue sections were incubated with anti-DICER antibody (1:50, ab14601, Abcam, Massachusetts, USA). Staining was completed using Dako Envision+ system-HRP (Dako, Denmark).

Statistical analysis on expected mutation frequency of WT cases was performed using the single-proportion test and the exact 95% CI was calculated by the Clopper–Pearson method. The penetrance of CN within 282 PPB FTDS kindred (containing 298 PPB cases), was calculated using a person-years approach. We counted all individuals within these kindred as contributing person-years if they were affected with a condition likely to be part of the PPB FDTS spectrum (see supplementary web-only file) or were unaffected, but in a lineage that linked them to another affected individual (n=423). The numerator consisted of the 21 cases of CN and four cases of unbiopsied renal cysts. Person-year counts were truncated at age 4, because no cases of CN have been known to occur after this age in PPB FTDS.

Results

We identified a heterozygous 4 bp deletion in exon 23 in family K and a heterozygous nonsense mutation in exon 25 of family S (figure 1A,B). The predicted c.4309_4312delGACT mutation in family K found in the two brothers with CN (II-1 and II-2) would lead to a D1437Mfs amino acid substitution and result in a stop codon at amino acid 1453 (figure 1A–C). The mutation was also found in the unaffected mother (I-3) and her unaffected child (II-3), who had a normal chest CT and renal ultrasound in June 2010. The predicted c.5477C→A mutation in family S is a nonsense mutation that would create a S1826X amino acid change. The mutation was found in the DNA extracted from paraffin-embedded CN from III-1, who died from PPB, in lymphocyte DNA from III-2 (affected with CN), in the unaffected mother (II-3) and two unaffected sisters, III-3 and III-4 (figure 1A–C). LOH studies using the CN from III-2 showed no allelic loss, with both the mutant and wild-type alleles still present in equal proportions (data not shown), and immunohistochemical staining of this CN revealed very reduced staining of DICER1 in the tubules (figure 1D, bottom image) compared with a normal kidney control (figure 1D, top image).

No exonic mutations or variants close to the intron–exon boundaries were found in the constitutional DNA from the 50 patients with WT. Using the single-proportion test, the exact Clopper–Pearson 95% CI upper limit is 0.071. In other words, it is very unlikely that more than three out of 50 children with WT will carry a germline DICER1 mutation.

We attempted to calculate a minimum estimate of the risk for CN within PPB FTDS kindred. Based on 298 PPB cases enrolled into the International Pleuropulmonary Blastoma Registry, we identified 423 individuals who were affected with a PPB-related disorder or were obligate carriers of putative genetic lesions, such as DICER1 mutations, which are associated with these conditions. These individuals contributed 1624.5 person-years of risk for CN. Twenty-five children had a CN or renal cysts on imaging that were consistent with a diagnosis of CN. Therefore the annual rate of CN in these kindred was 1539 per 100 000 person-years. The overall risk for CN is about 6% (25/423). In the Registry, there are three cases of WT that are definitely part of the PPB FTDS spectrum, so the risk for WT is 0.71% in the same kindred, and the annual rate is 185 per 100 000 person-years. In a comparison of proportions, the difference between the incidence of CN and WT is highly significant (p<0.0001).

Discussion

The DICER1 mutations in these two families with familial CN, one with and one without concomitant PPB, strongly implicate this mutation as the main genetic determinant of familial CN. Both mutations identified are predicted to result in truncated proteins which exclude or impede functionally important domains, including the double-stranded RNA binding domain and one of the ribonuclease domains (figure 1C). The double-stranded RNA binding domain is essential for pre-miRNA to attach to DICER1 and the ribonuclease domain conducts the primary activity of the protein.

Both increased and decreased DICER1 expression levels have been linked to several cancers, including oesophageal, breast and ovarian carcinoma,14–17 but the PPB FTDS is the first disease complex in which germline DICER1 mutations have been linked to human disease.13 Notably, in the linkage analysis that was a prelude to the identification of DICER1 mutations in children with PPB, individuals with lung cysts, CN and rhabdomyosarcoma were counted as affected.13 The phenotype of DICER1 mutations is highly variable, but patterns can be discerned. Taken with previous studies, our findings demonstrate that DICER1 mutations can present in families with PPB alone, CN alone, PPB and CN together and are found in individuals with no obvious phenotype. Only a detailed phenotypic analysis of all individuals with DICER1 mutations will provide insight into the genotype–phenotype relationship and perhaps also into the effect of individual mutations themselves. Current data tabulated by the Registry include 21 Registry and 10 literature cases of CN in the context of a PPB kindred; in addition, three cases of WT have occurred in Registry PPB families.12 Interestingly, one child in a family with PPB had renal medullary cysts with intralobar nephroblastomatosis, which is a potential precursor of WT. All CN associated with PPB have been diagnosed in children under 4 years of age and all have survived (unpublished Registry data). In two children, bilateral CN has been proliferative and required multiple resections with one of these needing bilateral nephrectomies and a renal transplant.18 Among the 31 cases of CN associated with PPB are four individuals with bilateral CN, all of which occurred in patients with PPB (unpublished Registry data). Preliminary data in a group of patients with both sporadic and familial PPB suggest that DICER1 mutations are identified in 50–60% of PPB cases,19 and it will be important to determine the frequency of DICER1 mutations in unselected cases of CN.

DICER1 is a key component of the miRNA biogenesis pathway. It is responsible for cutting pre-miRNA into its final 20–22 nt strands which become incorporated into the RNA-induced silencing complex. Current estimates suggest that miRNA, all of which must be processed by DICER1, modifies expression of up to 30% of all mRNA transcripts.20 It is unknown why disabling one copy of a universally important gene like DICER1 would have effects as specific as those presented here, and as is the case for most tumour suppressor genes, the genotype–phenotype relationship for DICER1 is not easily explained. Recent mouse knockout models have shown that losing a single copy of DICER1 from tumours results in impaired miRNA processing and reduced survival, suggesting that DICER1 is a haploinsufficient tumour suppressor gene21 and that mice with biallelic loss of DICER1 are non-viable.22 Loss of the wild-type allele in tumours is a typical mechanism by which tumour suppressor genes modify cell behaviour, but no LOH was seen in PPB tumours previously analysed,13 and our own results from CN tissue support this pattern. Immunohistochemical analysis showed that the DICER1 protein was significantly reduced or absent in the one DICER1-related CN that was available to us. As this is the only CN that has been studied thus far, it is currently not possible to draw any conclusions about pathogenetic mechanisms of CN in individuals with germline DICER1 mutations, but our findings conflict with previous results in PPB, wherein the tumour showed staining for DICER1 protein but the overlying (apparently benign) epithelium did not express detectable DICER1 protein.

Our calculations suggest that about 6% of individuals within PPB kindred will develop a CN. In contrast, the risk for WT seems to be <1%. These figures are only a rough estimate, and clearly as more genetic testing for DICER1 mutations is performed, the numbers will change.

The lack of tumours in adults carrying germline DICER1 mutations both here and in other studies is notable. Considering that one of the primary functions of miRNA appears to be facilitating the differentiation of stem cells,23 24 it might be postulated that a lack of fully functioning DICER1 on both alleles is less consequential in adults, where most of the cells with broad development potential have already become terminally differentiated.

Hemizygous loss of DICER1 has been found in malignant tissue from a wide variety of human cancers such as breast and pancreatic carcinoma, but homozygous deletions or truncations have never been reported in humans.21 It is interesting to note that at least one other member of the miRNA pathway, TARBP2, has been implicated in tumour growth. This codes for a stabilising cofactor of DICER1,25 and somatic mutations were found in endometrial and colorectal cancer cell lines, both of which showed diminished DICER1 concentrations. It is therefore possible that other members and cofactors of the miRNA pathway may also be candidate cancer predisposition genes. Drosha and Pasha (also known as DGCR8), which handle pri-miRNA processing in the nucleus before it is transported to DICER1 in the cytoplasm as pre-miRNA, have been screened for somatic mutations in various cancers, and while expression levels were found to be correlated with prognosis, deleterious somatic or germline mutations have not been reported.16 25

We did not identify germline DICER1 mutations in 50 WT cases. The upper limit of 95% CI for this observation suggests that at most, seven of 100 children with WT will carry DICER1 mutations. Given, therefore, the known familial occurrences of CN and PPB and of WT and PPB, it is possible that mutation analysis of a much larger series of children with WT may reveal the presence of germline DICER1 mutations. WT occurring within a PPB kindred may be predicted to be more likely attributable to DICER1 mutations than are sporadic WT, or WT in other syndromes predisposing to WT.

Conclusions

Here we show that DICER1 mutations can cause familial and bilateral CN, in families with and without PPB. In contrast, DICER1 mutations are not commonly seen in sporadic cases of WT. From a clinical perspective, the importance of these findings is that DICER1 mutation testing should be considered in all cases of familial and/or bilateral CN and may also be warranted in sporadic unilateral tumours, especially if there is a family history of other unusual paediatric malignancies. Moreover, if a DICER1 mutation is found in a patient with CN, they and their family may be susceptible to other conditions associated with PPB, some of which warrant early detection.12

Note added in proof: Further investigation of a CN from Family K (individual II-1) revealed no evidence of loss of heterozygosity at the DICER1 locus. Immunohistochemical staining of this tumor with DICER1 antibodies revealed a pattern of staining that was identical to that seen in individual III-2 (Figure 1D, lower panel).

Acknowledgments

We would like to thank the families who took part in this study and Dr Y Reinberg for his help. We thank Jean-Sébastien Brunet for statistical advice.

References

Supplementary materials

Footnotes

  • Funding AB is supported by the CIHR/FRSQ training grant in cancer research FRN53888 of the McGill Integrated Cancer Research Training Program. WDF and MT are grateful to the Turner Cancer Research Fund for support. WDF holds a Fonds de la Recherche en Santé du Québec (FRSQ) national scientist award and MT holds a FRSQ clinician-scientist award. TRF was funded by the Research Institute of the McGill University Health Centre and by the Henry R. Shibata Fellowship of the Cedars Cancer Institute. JRP is supported by the Pine Tree Apple Tennis Classic and the Theodora H. Lang Charitable Trust.

  • Competing interests John R. Priest is named in a patent registration involving genetic sequencing methodology of DICER1 gene.

  • Ethics approval This study was conducted with the approval of McGill University in Montreal, Canada and Bashkent University in Ankara, Turkey.

  • Provenance and peer review Not commissioned; externally peer reviewed.