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Figure 80.5 Variegated translocation mosaicism in primary fibroblasts from a Werner syndrome patient. The figure is a spectral (or SKY) karyotype of the chromosomal complement of a skin fibroblast from a male WS patient (reported in Melcher et al., 2000). Note the many stable chromosomal changes including reciprocal translocations (e.g., involving chromosomes 1 and 8), together with translocations that are not obviously reciprocal in nature and may be accompanied by deletions (e.g., the translocation of material from chromosome 1 to chromosomes 7 and 17). This original spectral karyotype was kindly provided by Dr. Holger Hoehn, University of Wùrzburg, Wùrzburg, Germany. See the color plate section.

a hyper-recombination syndrome—and thus of particular conceptual value.

The model depicted in Figure 80.4 provides a useful way to integrate molecular, biochemical, and cytologic data on WS, and begins to explain mechanistic links among recombination, cell viability, and mutagenesis in WS cells. It also provides a useful way to further explore mechanistic aspects of WRN function. One example of this is illustrated by recent work to determine whether one or both of the WRN catalytic activities must be lost in order to generate the HR repair and cell survival defects characteristically seen in cells from WS patients. These experiments used single amino acid substitutions to disrupt the WRN exonuclease or helicase activities, together with the reexpression of mutant protein in WS cell lines. We found that both the WRN exonuclease and helicase activities needed to be lost to reveal the WRN recombination defect. However, and in contrast to WRN-deficient cells, the expression of either single missense mutant supported high cell viability after DNA crosslink damage (Swanson et al., 2004).

These results indicate that the spectrum of WRN mutations identified in WS patients reflects the need to lose both WRN catalytic activities from cells in order to generate the cellular and clinical defects characteristic of WS. Our results also raise the intriguing possibility that WRN missense mutations that selectively affect helicase or exonuclease activity may be segregating in the human population, and could be associated with disease pheno-types in addition to WS that resulted from a selective loss of WRN-mediated HR repair.

WRN Function and Disease Pathogenesis in Cell Lineages

The elucidation of molecular aspects of WRN function, and of how the loss of WRN function generates WS

cellular phenotypes, together begin to suggest how the WS clinical phenotype may originate and progress. The postulated role for WRN in insuring successful, high-fidelity DNA replication, HR repair, and telomere maintenance, as outlined earlier (see Figure 80.4), indicates that a loss of function will be accompanied by genomic instability and reduced cell viability in many cell lineages during and after development. These two cellular consequences are likely to be intermediate phenotypes that lead to mutation accumulation and cell loss, that together drive the development of cell type-, cell lineage-, or tissue-specific defects in WS patients.

This idea of pathogenesis is shown in Figure 80.6. Further rounds of mutation accumulation, cell dysfunction, and cell loss can occur in continuously or conditionally replicating cell lineages, with the eventual compromise of tissue or organ structure and function and, in some tissues, the emergence of mutation-dependent neoplastic proliferation. This model of clinical progression indicates that the appearance of the first features of WS during adolescence is a reflection of the progressive accumulation of cellular defects during development and over the first decade of life, rather than something that is driven by the endocrine and physiologic changes that accompany puberty. Thus puberty reveals, rather than generates, the WS phenotype in affected individuals.

This view is consistent with the clinical and mechanistic view of WS as outlined earlier, and emphasizes the importance of genomic instability and replicative senescence in the generation of the WS phenotype. If this picture is accurate, however, why are dividing cell lineages not selectively affected during adult life by the loss of WRN function? And why are some organs such as the CNS spared the consequences of loss of WRN function? One likely explanation is the following. All cell lineages are generated by mitotic division during development and thus have the potential to be affected by mutation

progeroid cell loss ~ / features

Figure 80.6 Model for pathogenesis of disease in the absence of WRN function. WRN loss as a result of inherited germline mutations (see Figure 80.3) leads from the beginning of development to genetic instability and cell loss in many or all cell lineages. These changes, following the completion of development, can be perpetuated or amplified in specific cell lineages or tissues where division potential is retained. How the intermediate consequences or phenotypes of the absence of WRN function, i.e., mutation accumulation, cell dysfunction and cell loss, affect specific lineages or tissues to lead to the emergence of either neoplastic, atrophic, or progeroid outcomes is heavily conditioned by normal lineage biology (see text for additional discussion). Two different time lines above the figure indicate the progressive nature of cell and cell lineage defects, and their origins during development.

accumulation or cell loss in the absence of WRN function. Continuously dividing lineages during adult life such as skin, gut, and bone marrow may be tolerant of the loss of WRN function by virtue of mutation expansion-limiting lineage architecture, normally stringent cell editing by a combination of apoptosis and terminal differentiation, and large reserves of stem cells or lineage repopulating cells.

Conversely, in the absence of WRN function, those cell lineages or tissues that are largely postmitotic following the completion of development (e.g., many CNS cell lineages) may take advantage of normal developmental regulatory mechanisms such as compensatory cell proliferation and cell editing by programmed cell death to compensate for increased cell loss or dysfunction to insure the completion of development with normal structure and function. Key variables that determine the eventual outcome are the number of cell divisions required to generate a mature lineage; how much cell editing via apoptotic cell death occurs during and after development; and what functional redundancy is present to compensate for cell loss or dysfunction.

The fibroblast lineage and other mesenchymal or mesodermally derived cell lineages may be selectively affected by the loss of WRN function for several of these reasons, including the persistence of conditional cell division throughout life; the ability to accumulate genetic variation, and perhaps genetic damage, in conjunction with a comparative resistance to damage-induced apop-tosis; and the absence of a compartmentalized tissue architecture that could effectively suppress the prolifera-tive defects that result in neoplasia. In the absence of WRN function this combination of features may predispose mesenchymal lineages to the progressive accumulation of mutant and dysfunctional or senescent cells, together with progressive disruption of trophic or regulatory interactions with adjacent epithelial or stromal cells (reviewed in Campisi, 2005).

This line of pathogenetic reasoning leads to two important conclusions. First, we need to know more about the normal biology of specific human cell lineages before we will be able to understand and predict in vivo consequences of a loss or absence of WRN function. A second important conclusion is that we clearly need experimentally tractable animal models in which to study cell lineage-specific functions of WRN.

Animal Models of WS

Animal models are clearly important if we are to study WRN function and WS pathogenesis at the levels of cell lineage and the whole organism. The only mammalian models of WS that have been developed thus far have been in the mouse. Three different types of mouse model have been published: a complete knockout or null of murine Wrn leading to a loss of Wrn protein expression in all tissues (Lombard et al., 2000); an in-frame deletion of the helicase domain of murine Wrn, leading to a truncated protein that retains exonuclease activity though lacks helicase activity (Lebel and Leder, 1998); and transgenic expression of a human K577M WRN variant protein that lacks helicase activity against a background of normal murine Wrn expression (Wang et al., 2000).

Of these three models, the only one that faithfully recapitulates the genetic and biochemical defect observed in WS patients is the knockout that does not express Wrn protein. However, despite having faithfully recapitulated the biochemical defect observed in WS patients, the Wrn knockout mouse model does not have an obvious aging, genetic instability, or cancer phenotype. Several reasons have been suggested to explain the apparent absence of an obvious organismal phenotype in Wrn knockout mice: that mice simply do not live long enough to develop the changes first observed in WS patients beginning in the second and third decades of life; that mice may have different, or more robust, ways to compensate for the loss of Wrn function; or that humans may represent for reasons of lack of redundancy, long life span or environmental exposures, the equivalent of a sensitized background for revealing WRN function at the cell and organismal levels. Three caveats in interpreting these results and arguments are that the phenotyping of the Wrn knockout mouse model has been very modest to date; there has not as yet been a careful aging cohort study taken to completion; and knockout mice have not been systematically challenged with DNA damaging agents such as cross-linkers that may reveal defects in the murine Wrn functional pathway that parallel those observed in human cells.

One obvious way to test these ideas and further develop the murine model of WS is to generate sensitized mouse genetic backgrounds by altering the genetic constitution of Wrn knockout mice to place additional stress on the replication, HR repair or telomere maintenance pathways where Wrn is likely to function. One example of this approach was taken by two collaborating groups that made use of a murine telomerase RNA template-deficient (or Terc-deficient) mouse in conjunction with the loss of Wrn (Chang et al., 2004), or of Wrn and Blm (Du et al., 2004), function to reveal the progressive appearance of changes observed in WS patients.

Changes observed in these double or triple mutants include the graying and loss of hair, osteoporosis, diabetes mellitus, and cataracts. These changes appear to depend on critically short telomeres as a primary driver of the phenotype in the absence of Wrn and Blm. This provides an explanation as to why changes are observed in only a portion of mice, and in those affected only in late-generation Terc-deficiency where telomere erosion is substantial. These results are encouraging and have begun to provide mouse models in which to investigate Wrn function while identifying telomeres as a potential substrate for Wrn function in vivo.

Conclusion

Werner syndrome is one of a growing number of human diseases that have revealed the importance of genetic instability, replicative senescence, and cell death as intermediate phenotypes that can shape the risk of human disease or modify disease pathogenesis. WS is further distinguished as one of the growing number of human cancer predispositions that appear to result from a defect in homologous recombination function. One prominent role for WRN function in vivo is as part of one or more resolution complexes that act on common nucleic acid substrates that are generated during DNA replication or recombination, or that are part of the structure or metabolism of telomeres. This resolution function of WRN insures the successful completion of replication, recombination, or telomere maintenance, and thus high cell viability and genetic stability in all human cell lineages during development. WRN may also play comparable roles in cell lineages that retain the ability to divide during adult life.

Much work remains to be done to improve our understanding of WS as a biological and clinical human disease state. WRN function clearly modulates normal biology and physiology, and thus disease risk or pathogenesis in many human tissues or cell lineages. There are also tantalizing suggestions that the importance of WRN function in human biology will extend well beyond the small number of individuals affected with classic WS, as a modulator of both neoplastic and non-neoplastic disease risk in the general population. These hints suggest that additional work on the biology and medicine of WS will be challenging though richly rewarding.

Recommended Resources PRINT RESOURCES

Epstein, C.J., Martin, G.M., Schultz, A.L., and Motulsky, A.G. (1966). Werner's syndrome: A review of its symptomatology, natural history, pathologic features, genetics and relationship to the natural aging process. Medicine 45, 177-221. (A modern classic detailing the clinical, pathological, a and formal genetic analysis of contemporary and 122 reported WS cases.)

Salk, D., Fujiwara, Y., and Martin, G.M. (Eds.) (1985). Werner's Syndrome and Human Aging, Advances in Experimental Medicine and Biology, Vol. 190. New York: Plenum Press. (Proceedings of the Kobe, Japan meeting in 1982 that contains edited reprints of important primary references, a translation of Otto Werner's thesis of 1904, and many useful chapters detailing clinical, pathologic, and biological aspects of WS.)

Tollefsbol, T.O. and Cohen, H.J. (1984). Werner's syndrome: An underdiagnosed disorder resembling premature aging. Age 7, 75-88. (Useful compilation of clinical and biological information on WS gleaned from literature reports.) Goto, M. and Miller, R.W. (2001). From Premature Gray Hair to Helicase-Werner Syndrome: Implications for Aging and Cancer. Gann Monograph Cancer Res. 49. (Relatively recent compilation of reviews covering historical, clinical, and biological features of WS.) Monnat, R. J. Jr. and Saintigny, Y. (2004). The Werner syndrome protein: Unwinding function to explain disease. SAGE-KE. http://sageke.sciencemag.org/cgi/ reprint/2004/13/re3. (Recent summary of biological and clinical aspects of WS that provides a detailed discussion of information supporting the model of WRN function shown in Figure 80.4.)

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