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RNASEL mutations in hereditary prostate cancer
  1. H Chen1,
  2. A R Griffin1,
  3. Y-Q Wu1,
  4. L P Tomsho1,
  5. K A Zuhlke1,
  6. E M Lange5,
  7. S B Gruber1,3,
  8. K A Cooney1,2,4
  1. 1Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
  2. 2Department of Urology, University of Michigan Medical School, Ann Arbor, MI, USA
  3. 3Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, MI, USA
  4. 4Ann Arbor Department of Veteran’s Affairs, Ann Arbor, MI, USA
  5. 5Section of Biostatistics, Department of Public Health Sciences, Wake Forest University School of Medicine, Winston-Salem, NC, USA
  1. Correspondence to:
 Dr K A Cooney, 7310 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0946, USA; 
 kcooney{at}umich.edu

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Prostate cancer linkage to a broad region on chromosome 1q24-25 was first described by Smith et al1 in 1996. In this initial report, analysis of 91 multiplex prostate cancer families from North America and Sweden showed significant evidence for prostate cancer linkage surrounding marker D1S158 with a peak multipoint lod score assuming heterogeneity of 5.43 at marker D1S422. Although approximately one-third of prostate cancer families were estimated to be linked to this locus in this first set of families, additional studies suggest that the percentage of families with prostate cancer attributable to mutations in HPC1 may be significantly less (<5%).2

Recently, Carpten et al3 provided evidence that the RNASEL gene may be a candidate gene for HPC1. This gene is in the previously described 1q linkage peak, and deleterious mutations were identified in two of eight families with evidence for linkage to 1q24-25 markers. Additional functional data in support of the hypothesis that RNASEL mutations contribute to hereditary prostate cancer include: (1) evidence of loss of heterozygosity (LOH) of the wild type allele in prostate cancer from a patient harbouring a germline truncating mutation (E265X), and (2) absent RNASEL expression in prostate cancer tissue from a patient with the E265X mutation.

The University of Michigan Prostate Cancer Genetics Project (PCGP) was established in 1995 with the major goal of characterising the molecular basis of prostate cancer susceptibility. Fifty-nine families from the PCGP were used in the first confirmatory report of prostate cancer linkage to chromosome 1q24-25 markers.4 In this present report, we set out to determine the contribution of RNASEL mutations to prostate cancer susceptibility in a set of PCGP families.

MATERIAL AND METHODS

Patient selection

Men with prostate cancer selected for this study are participants in the University of Michigan PCGP. Current enrolment criteria for this family based study of inherited prostate cancer susceptibility include families with two or more living members with prostate cancer. Confirmation of the diagnosis is obtained via review of medical records and/or death certificates. In some cases, the diagnosis of prostate cancer in dead subjects is achieved through verbal confirmation by two independent family members. Genomic DNA was isolated from whole blood using a commercial kit (Puregene, Gentra Systems, Inc, Plymouth, MN).

Ninety-five men with prostate cancer were selected from 75 PCGP families for direct sequence analysis of the RNASEL gene. Genotype data using two markers that surround the gene, D1S466 and D1S158, were available from affected men in 39 of the 75 families. Nineteen of the 39 families with genotype data were considered to have high evidence of linkage because the family non-parametric linkage (NPL) score was >1 (table 1). The 11 families with moderate evidence of linkage consisted of families with NPL scores ≤1 but ≥−1. The remaining nine families with low evidence of linkage consisted of families who had NPL scores <−1. In the 11 families with moderate evidence for linkage to 1q24-25 markers, genomic DNA samples from all affected men in these families were evaluated by direct sequencing (28 subjects). DNA samples from all affected men from three additional families without available linkage data were also sequenced (six men). In the remaining 61 families, which included 19 families with strong evidence and nine families with little evidence for linkage using markers D1S466 and D1S158, genomic DNA from only one affected man from each family was sequenced.

Table 1

Characteristics of the 75 PCGP families analysed for RNASEL mutations. Criteria used to describe evidence for prostate cancer linkage to 1q24-25 markers are described in the Material and methods

Key points

  • Mutations in the RNASEL gene have been identified in some prostate cancer families leading to the hypothesis that RNASEL is the prostate cancer susceptibility gene HPC1 on chromosome 1q.

  • To test this hypothesis, we tested genomic DNA from 95 affected men in 75 multiplex prostate cancer families, including 19 families with evidence of linkage to 1q24-25 markers, from the University of Michigan Prostate Cancer Genetics Project for RNASEL mutations.

  • Direct sequence analysis of the coding region showed four rare missense/nonsense mutations (G59S, I97L, V247M, and E265X) occurring in four unrelated families. None of four reported rare mutations (M1I, G59S, V247M, and E265X) were observed in a panel of 75 control DNA samples or in any of the 103 cases and 323 control samples from a population based study of prostate cancer in African Americans.

  • Additional studies are needed to elucidate more clearly the role of RNASEL mutations in hereditary prostate cancer.

Control samples

To estimate allele frequencies in both African Americans and whites, control samples were obtained from two sources. The Flint Men’s Health Study (FMHS) is a continuing population based case-control study of prostate cancer in African American men conducted in Genesee County, Michigan.5 For this report, DNA samples from 103 men with prostate cancer and 323 male controls were used. The average age at diagnosis for cases was 62.8 ± 8.3 years. All control men were screened for prostate cancer with serum PSA and digital rectal exam per protocol. The average age of the controls at the time of consent was 56.0 ± 10.2 years. Genomic DNA samples from 27 men and 48 women were also obtained from a continuing study called Genes, Environment and Melanoma (GEM) at the University of Michigan. The 75 DNA samples were obtained from spouses of study participants with melanoma. These subjects are white and range between 20 and 85 years of age.

For participants in all three studies (PCGP, FMHS, and GEM), written consent was obtained and all protocols were approved by the Institutional Review Board at the University of Michigan Medical School.

DNA sequencing

PCR was used to amplify 20-40 ng of genomic DNA using primer sequences from Carpten et al.3 PCR primers were M13 tailed and purchased from GIBCO BRL (Invitrogen, Carlsbad, CA). Each reaction contained 2.5 mmol/l MgCl2, 0.2 mmol/l dNTPs, 0.55 μmol/l for both forward and reverse primers, 1 unit of Platinum™ Taq (Invitrogen, Carlsbad, CA), and 1 unit of AmpliTaq Gold™ (Applied Biosystems, Foster City, CA) DNA polymerases. Amplification was carried out as follows: denaturing (95°C/14 minutes), annealing (35 cycles of 95°C/30 seconds, 56°C/30 seconds, and 72°C/45 seconds), and final extension (72°C/10 minutes). PCR products were purified using Microcon™ PCR centrifugal filter devices (Millipore Corporation, Bedford, MA). Sequencing of PCR products was performed by the University of Michigan DNA Sequencing Core using ABI PRISM™ 3700 Genetic Analyzers (Applied Biosystems, Foster City, CA). M13 forward and reverse primers were used to obtain both strand sequences. Sample sequences were aligned with RNASEL DNA sequence using the NCBI BLAST program to identify changes. Only changes clearly present in both strands were further evaluated.

Allele specific oligonucleotide (ASO) hybridisation and restriction fragment length polymorphism (RFLP) analysis

ASO experiments were performed as previously described.6 Control DNA samples for the M1I substitution were kindly provided by W Issacs. For RFLP analysis, PCR products were generated using the appropriate RNASEL primers, incubated at 36°C in the presence of BseRI or 65°C in the presence of BsrDI for one hour, and analysed on 2% agarose gels.

RESULTS

Sixteen of the 75 families (17%) included in this report were African American and the remainder of the families were white (table 1). The average number of confirmed affected men in the families was 4.7 ± 2.4. The clinical features of the 95 men with prostate cancer who were sequenced for RNASEL mutations are shown in table 2.

Table 2

Clinical features of 95 men with prostate cancer from 75 PCGP families

Sequence analysis of the RNASEL gene in 95 men with prostate cancer selected from 75 PCGP families showed three rare missense substitutions and one nonsense mutation (table 3). One of the missense substitutions, I97L, was identified in two unrelated affected men. Sequence analysis of the other affected men in each family from which DNA was available (family 345: brother and maternal cousin; family 392: second cousin) failed to identify this mutation. These results were also confirmed using RFLP analysis, since this mutation creates a novel BseRI site in the PCR product created using primer RNASEL2 (data not shown). This I97L substitution has been identified in men with prostate cancer in two separate reports with a frequency of ≤1%.3,7

Table 3

Summary of missense and nonsense mutations identified through sequence analysis of 95 men with prostate cancer from 75 PCGP families. All mutations were identified through sequence analysis, and five of the substitutions were previously reported by Carpten et al3

A novel missense mutation was identified in subject 168-01, which results in the substitution of methionine for valine at amino acid position 247 (table 3). The presence of this mutation in the proband was confirmed by RFLP analysis (data not shown). We also identified the E265X nonsense mutation previously described by Carpten et al3 in one (subject 1115-02) of the 95 men with prostate cancer who were selected for the initial mutation screen (table 3). Sequence analysis confirmed the presence of the stop codon in one of two affected brothers but also in one of two unaffected brothers in this same family (fig 1C). All three men who were heterozygous for the E265X substitution were also heterozygous for the G59S substitution.

Figure 1

PCGP family with RNASEL E265X mutation. The pedigree structure is provided for family 1115 in which men with prostate cancer are illustrated by filled boxes, and the age at diagnosis is listed below each subject. The arrow indicates the participant who was selected first for sequence analysis. Mutation status for E265X is also indicated for family members that have provided a DNA sample for laboratory analysis. The same family members that are heterozygous for the E265X mutation are also heterozygous for the G59S mutation.

In the report of Carpten et al,3 the E265X mutation was identified in 0.54% of unaffected white men (n=186) from a prostate cancer screening study and 0.52% of CEPH parents (n=96). Over 50% of the participants in the PCGP receive their care at the University of Michigan Medical Center. To determine the frequency of this mutation in the patient population at our medical centre, we analysed DNA from 75 spouses of patients with melanoma (GEM study) for the E265X mutation as well as the G59S and V247M substitutions (table 4). No heterozygous or homozygous carriers of any of these mutations were identified in this panel of controls. We also tested genomic DNA samples from a population based prostate cancer case-control study that focuses solely on African American men for the M1I mutation. This substitution of isoleucine for methionine at codon 1 was one of the two deleterious mutations described in the report of Carpten et al3 and it was identified in a single African American family. None of the case or control men was shown to be a carrier of the M1I mutation or the G59S, V247M, or E265X mutations.

Table 4

Results of ASO hybridisation experiments to determine frequency of M1I, G59S, G247S, and E265X mutations. DNA samples from 75 controls from the GEM study were studied for the presence of each of four previously reported RNASEL mutations. 103 men with prostate cancer and 323 unaffected controls from the FMHS, which is a population based case-control study of prostate cancer in African American men, were also analysed. In each assay, several samples were not amplified sufficiently for this analysis and were removed from the total number of samples assayed

DISCUSSION

There are several factors that make RNASEL a plausible candidate for the prostate cancer susceptibility gene HPC1. First, although the candidate region remains large, linkage peaks and recombinant data often indicate that the most likely region for HPC1 is near marker D1S158.3 The gene encodes an endoribonuclease which is a member of the interferon regulated 2-5A system. In vitro experiments suggest that the RNASEL gene may function as a tumour suppressor gene.8 RNase L -/- murine thymocytes have suppressed apoptosis,9 although similar studies have not been completed with prostate cells in which the RNASEL gene has been inactivated. Finally, the identification of deleterious mutations in two of eight multiplex prostate cancer families linked to 1q24-25 suggest that RNASEL requires further study as a prostate cancer susceptibility gene.

Our data provide limited support for the hypothesis that the RNASEL gene is HPC1. Analysis of one or more probands from 75 large (average >4 affected men/family) prostate cancer families has shown four rare missense/nonsense mutations among four unrelated families. Only two of 19 families with strong evidence of HPC1 linkage were found to have RNASEL mutations (families 168 and 392). In family 168, DNA samples from two affected brothers are not available for RNASEL mutation testing. In family 392, however, only one of two affected family members harboured the I97L mutation (see below). The other RNASEL mutations detected in our study (G59S, I97L, and E265X) did not completely cosegregate with affected status in three families.

Prostate cancer genetic studies are complicated by several factors including the variable participation in early detection or screening tests and the occasional diagnosis of latent or clinically insignificant prostate cancer within a family. The E265X nonsense mutation was first identified in a family with four affected brothers and each brother was heterozygous for the allele with the stop codon.3 Clinical information was available from three of the four brothers; all three men had clinically significant prostate cancer with Gleason scores in each case ≥7. However, in our report, only two of three affected brothers in family 1115 were discovered to be heterozygous carriers of the E265X mutation (fig 1). Review of clinical records shows no significant differences in clinical stage at presentation between the brothers with and the one without the E265X mutation. There are also two unaffected brothers in family 1115 who are participating in the PCGP research project; both brothers have had serum PSA tests with values <2.5 ng/ml within the last 12 months. Thus, it is unlikely that the unaffected brother harbouring the E265X mutation (1115-07), who was 57 at the time of study enrolment, has undetected prostate cancer. Rokman et al10 recently reported the identification of the RNASEL E265X mutation in five index patients from 116 Finnish hereditary prostate cancer families. Similar to our results, segregation of the E265X mutation with disease status was observed in only one of the five families. In our study, the E265X mutation was found to be in complete linkage disequilibrium with the G59S mutation. This observation is consistent with the findings of others10 and suggests the occurrence of a rare (allele frequency <1%) founder allele in the US white population.

Previous studies by us and others have shown rare cases of allelic loss in prostate cancer tumour tissue using markers that map to the HPC1 candidate interval. The frequency of 1q24-25 LOH appears to be ∼10% in both sporadic (C Bettis, L Huang, K A Cooney, unpublished data)11,12 and hereditary12–14 prostate cancer. Previous investigations in our laboratory using tumour tissue from subject 392-01 showed evidence of allelic loss using two chromosome 1q markers (D1S158 and D1S518, data not shown). Examination of the linkage data from subject 392-01 and his affected second degree cousin (392-02) showed that the non-shared, presumably normal, allele was deleted consistent with the hypothesis that HPC1 may be acting as a tumour suppressor gene in this family. This observation led us to include both 392-01 and his affected second cousin 392-02 in our sequence analysis of the RNASEL gene. However, the I97L RNASEL missense mutation was identified only in subject 392-02 and presumably on the non-shared allele. Taken together, the findings do not provide evidence to support the hypothesis that the RNASEL gene is a tumour suppressor gene causing hereditary prostate cancer in this family.

Review of published linkage studies provides reasonable evidence to support the existence of a prostate cancer susceptibility gene at 1q24-25. Mutations in this HPC1 gene are likely to be relatively rare and may preferentially lead to aggressive prostate cancer.15RNASEL mutations may contribute to prostate cancer in some families, as suggested by our results, as well as those contained in two recent publications.7,16 Wang et al7 screened 326 men with prostate cancer from 163 unrelated families for RNASEL mutations. These investigators failed to identify any clearly deleterious mutations (including E265X); however, a common polymorphism, namely Arg462Gln, was associated with familial prostate cancer compared to either sporadic prostate cancer or compared to control men. Rennert et al16 identified a founder frameshift mutation in RNASEL (471delAAAG) in Ashkenazi Jews that was found in higher frequency among men with prostate cancer (6.9%) compared to either elderly male or young women controls (2.4% and 4.0%, respectively). Additional studies, including a meta-analysis, are needed to elucidate more clearly the role of RNASEL mutations in hereditary prostate cancer.

Acknowledgments

This work was supported by USPHS Grants P50-CA69568 (SPORE) and R01-CA79596. We gratefully acknowledge Caroline Mohai, Gina Claeys, Kay Doerr, and the Flint Men’s Health Study Research Team for assistance with patient recruitment and data collection.

REFERENCES

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