Danielle Bodmer

Heritable somatic methylation and inactivation of MSH2 in families with Lynch syndrome due to deletion of the 3' exons of TACSTD1.

Lynch syndrome patients are susceptible to colorectal and endometrial cancers owing to inactivating germline mutations in mismatch repair genes, including MSH2 (ref. 1). Here we describe patients from Dutch and Chinese families with MSH2-deficient tumors carrying heterozygous germline deletions of the last exons of TACSTD1, a gene directly upstream of MSH2 encoding Ep-CAM. Due to these deletions, transcription of TACSTD1 extends into MSH2. The MSH2 promoter in cis with the deletion is methylated in Ep-CAM positive but not in Ep-CAM negative normal tissues, thus revealing a correlation between activity of the mutated TACSTD1 allele and epigenetic inactivation of the corresponding MSH2 allele. Gene silencing by transcriptional read-through of a neighboring gene in either sense, as demonstrated here, or antisense direction, could represent a general mutational mechanism. Depending on the expression pattern of the neighboring gene that lacks its normal polyadenylation signal, this may cause either generalized or mosaic patterns of epigenetic inactivation.

Risk of urothelial bladder cancer in Lynch syndrome is increased, in particular among MSH2 mutation carriers

Background Colorectal, endometrial and upper urinary tract tumours are characteristic for Lynch syndrome (hereditary non-polyposis colon carcinoma, HNPCC). The aim of the present study was to establish whether carriers of mutations in mismatch repair genes MLH1, MSH2 or MSH6 are at increased risk of urinary bladder cancer. Methods Carriers and first degree relatives of 95 families with a germline mutation in the MLH1 (n=26), MSH2 (n=43), or MSH6 (n=26) gene were systematically questioned about the occurrence of carcinoma. The cumulative risk of cancer occurring before the age of 70 years (CR70) was compared to the CR70 of the general Dutch population. Microsatellite instability (MSI) testing and/or immunohistochemistry (IHC) for mismatch repair proteins was performed on bladder tumour tissue. Results Bladder cancer was diagnosed in 21 patients (90% men) from 19 Lynch syndrome families (2 MLH1, 15 MSH2, and 4 MSH6). CR70 for bladder cancer was 7.5% (95% CI 3.1% to 11.9%) for men and 1.0% (95% CI 0% to 2.4%) for women, resulting in relative risks for mutation carriers and first degree relatives of 4.2 (95% CI 2.2 to 7.2) for men and 2.2 (95% CI 0.3 to 8.0) for women. Men carrying an MSH2 mutation and their first degree relatives were at highest risks: CR70 for bladder and upper urinary tract cancer being 12.3% (95% CI 4.3% to 20.3%) and 5.9% (95% CI 0.7% to 11.1%). Bladder cancer tissue was MSI positive in 6/7 tumours and loss of IHC staining was found in 14/17 tumours, indicating Lynch syndrome aetiology. Conclusion Patients with Lynch syndrome carrying an MSH2 mutation are at increased risk of urinary tract cancer including bladder cancer. In these cases surveillance should be considered.

Recurrence and variability of germline EPCAM deletions in Lynch syndrome

Recently, we identified 3′ end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele‐specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch‐like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2‐deficient families from multiple geographical origins with varying deletions all encompassing the 3′ end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu‐repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3′ end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics. Hum Mutat 32:1–8, 2011.

An alternative route for multistep tumorigenesis in a novel case of hereditary renal cell cancer and a t(2;3)(q35;q21) chromosome translocation.

Through allele-segregation and loss-of-heterozygosity analyses, we demonstrated loss of the translocation-derivative chromosome 3 in five independent renal cell tumors of the clear-cell type, obtained from three members of a family in which a constitutional t(2;3)(q35;q21) was encountered. In addition, analysis of the von Hippel-Lindau gene, VHL, revealed distinct insertion, deletion, and substitution mutations in four of the five tumors tested. On the basis of these results, we conclude that, in this familial case, an alternative route for renal cell carcinoma development is implied. In contrast to the first hit in the generally accepted two-hit tumor-suppressor model proposed by Knudson, the familial translocation in this case may act as a primary oncogenic event leading to (nondisjunctional) loss of the der(3) chromosome harboring the VHL tumor-suppressor gene. The risk of developing renal cell cancer may be correlated directly with the extent of somatic (kidney) mosaicism resulting from this loss.

Patients with an unexplained microsatellite instable tumour have a low risk of familial cancer

The cancer risk is unknown for those families in which a microsatellite instable tumour is neither explained by MLH1 promoter methylation nor by a germline mutation in a mismatch repair (MMR) gene. Such information is essential for genetic counselling. Families suspected of Lynch syndrome (n=614) were analysed for microsatellite instability, MLH1 promoter methylation and/or germline mutations in MLH1, MSH2, MSH6, and PMS2. Characteristics of the 76 families with a germline mutation (24 MLH1, 2 PMS2, 32 MSH2, and 18 MSH6) were compared with those of 18 families with an unexplained microsatellite instable tumour. The mean age at diagnosis of the index patients in both groups was comparable at 44 years. Immunohistochemistry confirmed the loss of an MMR protein. Together this suggests germline inactivation of a known gene. The Amsterdam II criteria were fulfilled in 50/75 families (66%) that carried a germline mutation in an MMR gene and in only 2/18 families (11%) with an unexplained microsatellite instable tumour (P<0.0001). Current diagnostic strategies can detect almost all highly penetrant MMR gene mutations. Patients with an as yet unexplained microsatellite instable tumour likely carry a different type of mutation that confers a lower risk of cancer for relatives.

Molecular analysis of a familial case of renal cell cancer and a t(3;6)(q12;q15).

We identified a novel familial case of clear‐cell renal cancer and a t(3;6)(q12;q15). Subsequent cytogenetic and molecular analyses showed the presence of several abnormalities within tumour samples obtained from different patients. Loss of the der(3) chromosome was noted in some, but not all, of the samples. A concomitant VHL gene mutation was found in one of the samples. In addition, cytogenetic and molecular evidence for heterogeneity was obtained through analysis of several biopsy samples from one of the tumours. Based on these results and those reported in the literature, we conclude that loss of der(3) and subsequent VHL gene mutation may represent critical steps in the development of renal cell cancers in persons carrying the chromosome 3 translocation. Moreover, preliminary data suggest that other (epi)genetic changes may be related to tumour initiation.

Disruption of a novel gene, DIRC3, and expression of DIRC3-HSPBAP1 fusion transcripts in a case of familial renal cell cancer and t(2;3)(q35;q21)

Previously, we identified a family with renal cell cancer and a t(2;3)(q35;q21). Positional cloning of the chromosome 3 breakpoint led to the identification of a novel gene, DIRC2, that spans this breakpoint. Here we have characterized the chromosome 2 breakpoint in detail and found that another novel gene, designated DIRC3, spans this breakpoint. In addition, we found that the first two exons of DIRC3 can splice to the second exon of HSPBAP1, a JmjC‐Hsp27 domain gene that maps proximal to the breakpoint on chromosome 3. This splice results in the formation of DIRC3‐HSPBAP1 fusion transcripts. We propose that these fusion transcripts may affect normal HSPBAP1 function and concomitant chromatin remodeling and/or stress response signals within t(2;3)(q35;q21)‐positive kidney cells. As a consequence, familial renal cell cancer may develop.

Chromosome 3 Translocations and Familial Renal Cell Cancer

Renal cell carcinomas (RCCs) occur in both sporadic and familial forms. In a subset of families the occurrence of RCCs co-segregates with the presence of constitutional chromosome 3 translocations. Previously, such co-segregation phenomena have been widely employed to identify candidate genes in various hereditary (cancer) syndromes. Here we survey the translocation 3-positive RCC families that have been reported to date and the subsequent identification of its respective candidate genes using positional cloning strategies. Based on allele segregation, loss of heterozygosity and mutation analyses of the tumors, a multi-step model for familial RCC development has been generated. This model is relevant for (i) understanding familial tumorigenesis and (ii) rational patient management. In addition, a high throughput microarray-based strategy is presented that will enable the rapid identification of novel positional candidate genes via a single step procedure. The functional consequences of the (fusion) genes that have been identified so far, the multi-step model and its consequences for clinical diagnosis, the identification of persons at risk and genetic counseling in RCC families are discussed.

Cytogenetic and molecular analysis of early stage renal cell carcinomas in a family with a translocation (2;3)(q35;q21).

Previously, we described a family with renal cell carcinoma (RCC) and a constitutional balanced t(2;3) (q35;q21). Based on loss of heterozygosity and von Hippel-Lindau (VHL) gene mutation analyses in five tumor biopsies from three patients in this family, we proposed a multistep model for RCC development in which the familial translocation may act as a primary oncogenic event leading to (nondisjunctional) loss of the translocation-derived chromosome 3, and somatic mutation of the VHL gene as a secondary event related to tumor progression. Here, we describe the cytogenetic and molecular analysis of three novel tumors at early stages of development in two members of this family. Again, loss of derivative chromosome 3 was found in two of these tumors and a VHL mutation in one of them. In the third tumor, however, none of these abnormalities could be detected. These results underline our previous notion that loss of derivative chromosome 3 and VHL gene mutation play critical roles in familial RCC. In addition, they show that both anomalies may occur at relatively early stages of tumor development.

Young age and a positive family history of colorectal cancer are complementary selection criteria for the identification of Lynch syndrome

Families at high risk for Lynch syndrome can effectively be recognised by microsatellite instability (MSI) testing. The aim of the present study is to compare the effectiveness of a MSI test for the identification of Lynch syndrome in patients selected by a pathologist mainly based on young age at diagnosis (MSI-testing-indicated-by-a-Pathologist; MIPA), with that of patients selected by a clinical geneticist mainly based on family history (MSI-testing-indicated-by-Family-History; MIFH). Patients with a Lynch syndrome associated tumour were selected using MIPA (n=362) or MIFH (n=887). Germline DNA mutation testing was performed in 171 out of 215 patients (80%) with a MSI positive tumour. MSI was tested positive in 20% of the MIPA-group group compared to 16% in the MIFH-group (P=0.291). In 91 of 171 patients with MSI positive tumours tested for germline mutations were identified as Lynch syndrome patients: 42% in the MIPA-group and 56% in the MIFH-group (P=0.066). Colorectal cancer (CRC) or endometrial cancer (EC) presenting at an age below 50 years would have led to the diagnosis of Lynch syndrome in 89% of these families (CRC below 50 years: 88% and EC below 50 years: 12%). Families detected by MIPA were characterised more often by extracolonic Lynch syndrome associated malignancies, especially EC (P<0.001). Our results indicate that recognition of Lynch syndrome by CRC or EC below 50 years is as effective as a positive family history. Families from patients selected by individual criteria more often harbour extracolonic Lynch syndrome associated malignancies.