Members of the Tenerife families carrying the hMSH2(M688R)
mutation may develop a single cancer during their lifetime, while other members developed multiple cancers and often in a short period of time. In addition, some family members developed multiple tumors prior to the age of 30 years, including unusual childhood tumors such as glioblastoma and lymphoma. In recent years, several case reports have described children with Turcot or CMMR-D syndromes with compound heterozygous or homozygous MMR gene defects (22–26
). These patients developed tumors at early ages and sometimes combined with characteristics of neurofibromatosis type 1. While LS/HNPCC-related tumors are clearly observed, lymphoma, hematological and CNS tumors are most common in these patients (22–25
). Turcot and CMMR-D were ruled out with our 6-year-old glioblastoma carrier where we identified a heterozygous hMSH2(M688R)
mutation and no other mutations in the hMSH2, hMSH6 or hMLH1 genes. However, the age of onset and the likelihood that another 5-year-old patient with lymphoma also was a hMSH2(M688R)
carrier suggested a potentially more aggressive genetic phenotype than simple LS/HNPCC.
The collection of tumor pathology samples in conditions adequate for IHC has only recently been structured on the Canary Islands that include Tenerife. Despite this technical difficulty, we were able to collect 18 tumor samples from hMSH2(M688R) carriers that were amenable to IHC analysis. All of these tumors were MSI (data not shown). Sixteen of the 18 samples, including all of the classic LS/HNPCC type tumors, showed loss of hMSH2 and hMSH6 expression with normal expression of hMLH1. Two samples (11%) displayed expression of hMSH2 and hMSH6 proteins within the tumor. These was a metachronous bladder tumor from a patient who developed a second bladder tumor, which paradoxically showed loss of hMSH2 and hMSH6 expression. The other was a brain tumor from a patient who originally developed a leiomyosarcoma at 18 years that showed loss of hMSH2 and hMSH6 expression. The leiomyosarcoma patient also developed a lung metastasis that displayed loss of hMSH2 and hMSH6 expression while the brain tumor showed expression of both hMSH2 and hMSH6. These results suggest that the brain tumor was a metachronous MSI tumor that expressed the hMSH2–hMSH6 complex although an unusual metastasis cannot be entirely ruled out. Nevertheless, the expression of hMSH2 in this subset of MSI tumors suggested a molecular mechanism of tumor promotion where an MMR defect was present irrespective of MMR protein expression.
Because of the location of the hMSH2(M688) residue, we considered a dominant negative function as one possible explanation for tumor promotion activity in the presence of protein expression. Since a number of studies have implicated the hMSH2(M688I)
alteration as causative for LS/HNPCC (19–21
), we purified both the hMSH2(M688I)–hMSH6 and hMSH2(M688R)–hMSH6 proteins. Biochemical analysis of the hMSH2(M688I)–hMSH6 heterodimer suggested only modest differences compared with the WT. We conclude that the hMSH2(M688I)
alteration is likely to be a non-causative polymorphism. In contrast, the hMSH2(M688R)–hMSH6 heterodimer binds mismatched nucleotides but is completely incapable of processing ATP normally. Moreover, the hMSH2(M688R)–hMSH6 protein significantly interferes with mismatch-dependent excision gap formation at a 1:1 ratio with the WT hMSH2–hMSH6 and to a lesser extent the complete MMR reaction catalyzed by an hMSH2-deficient extract complemented by the WT hMSH2–hMSH6. These studies are consistent with the conclusion that hMSH2(M688R)–hMSH6 may function as a dominant negative for MMR in vitro
Can the hMSH2(M688R)
function as a dominant negative in vivo
? The ability of the hMSH2(M688R)–hMSH6 heterodimer to function as a dominant negative will depend on its cellular expression. Moreover, a complete inhibition of MMR function would require an excess of mutant protein expression compared to the WT allele. However, it is clear that even a partial inhibition may result in modestly elevated mutation rates and increased drug resistance, which could ultimately lead to the selection of a complete MMR defect and enhanced drug resistance (45
The IHC results suggest that the mutant protein may be expressed in some tissues but not in others. These observations are consistent with the hypothesis that the hMSH2(M688R)
mutation may function as a tissue-dependent dominant negative. In such a case, its effectiveness as a dominant negative would depend on its tissue expression. Based on the IHC results, one might predict that the hMSH2(M688R)–hMSH6 heterodimer may be expressed in brain and lymphoid tissues. Such a tissue-dependent dominant negative could explain the relatively high incidence of CNS and early-onset tumors that appear similar to Turcot and CMMR-D syndromes. It is interesting to note that reports of dominant negative MMR mutations suggest it is likely to be extremely rare and often controversial (47–50
). Continued surveillance of the Tenerife families and acquisition of new early-onset tumors amenable to IHC will ultimately confirm whether the hypothesis that the hMSH2(M688R)
mutation functions as a tissue-dependent dominant negatve.