HNPCC: Six new pathogenic mutations

Background Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disease with a high risk for colorectal and endometrial cancer caused by germline mutations in DNA mismatch-repair genes (MMR). HNPCC accounts for approximately 2 to 5% of all colorectal cancers. Here we present 6 novel mutations in the DNA mismatch-repair genes MLH1, MSH2 and MSH6. Methods Patients with clinical diagnosis of HNPCC were counselled. Tumor specimen were analysed for microsatellite instability and immunohistochemistry for MLH1, MSH2 and MSH6 protein was performed. If one of these proteins was not detectable in the tumor mutation analysis of the corresponding gene was carried out. Results We identified 6 frameshift mutations (2 in MLH1, 3 in MSH2, 1 in MSH6) resulting in a premature stop: two mutations in MLH1 (c.2198_2199insAACA [p.N733fsX745], c.2076_2077delTG [p.G693fsX702]), three mutations in MSH2 (c.810_811delGT [p.C271fsX282], c.763_766delAGTGinsTT [p.F255fsX282], c.873_876delGACT [p.L292fsX298]) and one mutation in MSH6 (c.1421_1422dupTG [p.C475fsX480]). All six tumors tested for microsatellite instability showed high levels of microsatellite instability (MSI-H). Conclusions HNPCC in families with MSH6 germline mutations may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations.


Background
Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by very high risk for colorectal cancer [1]. In addition, endometrial and ovary cancer risk as well as risk for tumors of the ureter, renal pelvic and small intestine is increased. Germline mutations of MLH1 and MSH2 account for 70% of all HNPCC cases [2]. Approximately 5-10% of HNPCC families carry a germline mutation in the MSH6 gene [3][4][5]. Here we report six germline mutations in the mismatch-repair genes MLH1, MSH2 and MSH6, that to our best knowledge have not been described (Medline and HNPCC mutation database http:/ /www.nfdht.nl.

Methods
This study was approved by the ethical committee of the Medical Faculty of the Ruhr-University Bochum. All patients underwent interdisciplinary counselling by a geneticist, clinician and psychologist. Patients included in this study fulfilled the Amsterdam or Bethesda criteria [1]. Substantial pedigree information moreover including the revised Bethesda criteria [6] is given in table 1. After given informed consent blood samples were drawn for genetic testing. Genomic DNA was extracted using standard protocol [7].
Formalin fixed and paraffin embedded tumor tissues were obtained from different primary pathologists and sent upon request to the local reference pathology of the Familial Colorectal Cancer Center of the Ruhr University Bochum. Tumors were reexamined for histomorphological HNPCC features, immunohistochemical MMR repair protein expression and microsatellite instability.

Microsatellite Analysis
Tumor and surrounding normal tissue were microdissected by a skilled pathologist. Tumor cell cellularity was at least 70%. DNA was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Microsatellite mark-ers BAT-25, BAT-26, D5S346, D17S250, D2S123 and BAT-40 were amplified. The markers included the NIH reference panel according to the international guidelines for the evaluation of MSI in colorectal cancer (Boland et al., 1998). Primer sequences are available on request. Tumors were classified as having high grade microsatellite instability (MSI-H) if at least 30% of the markers showed instabilities. Microsatellite-PCR reactions were performed in 96-well microtiter plates, in 20 mmol/L Tris-HCl, pH 8.4, 5 mmol/L KCl, 1.5 mmol/L MgCl2, 100 ng of each primer, 200 mmol/L dNTPs, 60 mmol/L TMAC (Sigma, Taufkirchen, Germany), 1.5% formamide, 2 ml DNA template (tumor or normal tissue), and 1.5 units Taq DNA polymerase (Gibco BRL, Karlsruhe, Germany), in a final volume of 15 ml. Reactions were performed in a Hybaid Touchdown temperature cycler (MWG-Biotech, Ebersberg, Germany), for 40 cycles of 94°C for 15 seconds, different annealing temperatures for each marker for 30 seconds, and 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. BAT-25, BAT-26, BAT-40 and D2S123 were amplified with an annealing temperature of 60°C, whereas D5S346 was performed at 58°C and D17S250 at 53°C. PCR products were separated on 6% polyacrylamide, 8 mol/L urea gels and DNA fragments were visualized by silver staining.

Results and discussion
All tumor specimens of the investigated patients showed MSI in at least 2 of five tested loci and therefore were classified as MSI-H. Tumor tissue of all patients was immunohistochemically stained (    Patient 0531-X had tubulovillous adenoma at the age of 39. Colonoscopy was performed annually and the patient developed seven adenomas within a two years follow-up period. The sister presented with an adenoma of unknown histology at the age of 41. The 40 year old grandmother died of colorectal cancer. So far no female developed endometrial cancer. This pedigree fulfilled criterion 7 of the Bethesda guidelines [1]. Criterion 1 of the revised Bethesda guidelines is met. Tumor tissue of patient 0531-X revealed MSI-H. Firstly, immunohistochemistry of MLH1 and MSH2 was performed only and revealed normal expression of MLH1 and MSH2 protein in the tumor tissue. In addition, immunohistochemistry of MSH6 was done and showed loss of MSH6 protein expression. The frameshift mutation c.1421_1422dupTG [p.C475fsX480] in the MSH6 gene was detected. The father of the index patient was not affected by HNPCCassociated cancers and is currently aged 65. Although the published data supports the notion of a later onset of disease in HNPCC families carrying an MSH6 mutation, this was not the case in our family. The index patient and her sister started to develop adenomas by the age of 39 and 41 years respectively. Moreover the paternal grandmother died of colorectal cancer at age 40. These findings are comparable to data for MLH1 and MSH2 mutation positive individuals with HNPCC.

Conclusions
Our phenotype data imply that HNPCC associated with MSH6 gene mutations may show an age of onset that is MSH2 staining Figure 2 MSH2 staining Patient 0660-6: Mucinous adenocarcinoma of the colon with a negative staining of the tumor cells for MSH2. As an internal positive control, lymphocytes (arrowheads) in an adjacent lymph follicle show a positive nuclear staining for MSH2 comparable to this of patients with MLH1 and MSH2 mutations. Therefore, screening recommendations should not be loosened for families with MSH6 gene mutations.