CommentaryNailing Down a Link between Tuberin and RenalCysts

David M. HockenberyFrom the Division of Clinical Research, Fred Hutchinson Cancer

Research Center, Seattle, Washington

Transgenic technology, despite its power to analyze andcreate animal models for gene function, sometimescomes up short of answers. In this issue of The AmericanJournal of Pathology, Cai and colleagues1 demonstratethat astute analysis of mutant strains can also deliver thegoods. Tuberous sclerosis complex (TSC) is inherited asan autosomal dominant trait, with two genes, TSC1(hamartin) and TSC2 (tuberin), each responsible for�50% of familial cases.2 The disease presents similarly,albeit with a high index of variability, with both mutations,excepting the occurrence of renal cystic disease.3 Renalcysts occur almost exclusively in TSC2 patients, with asubset developing a severe form similar to polycystickidney disease (PKD), with early progression to renalfailure. Many of these patients have deletions involvingboth TSC2 and PKD1, which are contiguous genes lo-cated at chromosome 16p13.3.4 Despite this convincingexplanation, some questions remained. Autosomal dom-inant PKD differs in both natural history and pathologyfrom the TSC2/PKD1 contiguous gene deletion patients,and although PKD involves somatic loss of the normalPKD1 allele, this has not been demonstrated to occur inrenal cysts from TSC2 patients.

A dominant pattern of inheritance for a single gene traitdoes not indicate whether the mutant allele has dominantor recessive effects in the cell. For recessive mutants,inheritance of a mutant allele from each parent is only oneoption. Studies of cancer genetics focused attention on asecond mechanism, somatic mutation or deletion (loss ofheterozygosity) of the remaining normal allele of a tumorsuppressor gene.5 Generation of cells with both copiesinactivated by this second-hit mechanism provides a se-lective advantage for clonal expansion and evolution.Reduction to homozygosity may also occur by mitoticrecombination, or chromosome nondisjunction, yieldingtwo copies of the maternal or paternal allele bearing themutation (uniparental disomy). As proposed by Hall,6 ifpostzygotic reduction to homozygosity occurs sufficientlyearly in development, mosaicism may result in an exag-gerated phenotype with segmental patterns.

In this issue, Cai and colleagues1 perform geneticanalysis on three Eker rats with variant, severe PKD phe-notypes and demonstrate mosaicism attributed to unipa-rental disomy of a mutant TSC2 allele. Reidar Eker7 firstdescribed autosomal inheritance of renal adenomas andcarcinomas as a spontaneous mutant in Wistar rats in1954. Renal neoplasms in the Eker rat arise from proximaland collecting duct epithelial cells on a background ofcystic tubular dilation. Yeung and colleagues8 in 1993reported the mapping of the Eker renal tumor suscepti-bility gene to a region on rat chromosome 10 syntenicwith human chromosome 16p13. In the same year, ahuman gene responsible for tuberous sclerosis, TSC2,was identified in the 16p13.3 region.9 Cloning of the ratTSC2 followed, and sequencing of the Eker TSC2 locusidentified insertion of a retrotransposon at one allele,disrupting the open reading frame.10 Final confirmation ofthe role of TSC2 was obtained by demonstrating that awild-type TSC2 transgene completely suppressed theEker phenotype.11 The cellular recessive nature of theTSC2 phenotype was demonstrated by showing loss ofheterozygosity or point mutations in the remaining normalTSC2 allele within Eker renal tumors, but not uninvolvedtissues.12

The identification of germline mutations in TSC2 asresponsible for the Eker phenotype was surprising be-cause of the notable differences between human and ratdisease phenotypes. Renal tumors are the only lesion inthe Eker rat model observed with 100% penetrance. Uter-ine leiomyomas, splenic hemangiomas, and pituitary ad-enomas occur in decreasing frequency in the Eker rat.13

Although renal disease (most commonly renal angiomyo-lipomas) occurs in the majority of TSC patients, renal cellcarcinoma is diagnosed in �1% of patients.14 Skin le-sions (hypomelanotic macules and angiofibromas), car-diac rhabdomyomas, and pulmonary hamartomas occurcommonly in TSC, but are not reported in Eker rats.Central nervous system tumors are the leading cause ofmorbidity and mortality in TSC. Cortical tubers (hence thename tuberous sclerosis) are distinctly uncommon in

Accepted for publication November 20, 2002.

Address reprint requests to David M. Hockenberry, Division of ClinicalResearch, Fred Hutchinson Cancer Research Center, 1100 Fairview Av-enue North, Seattle, Washington 98109-1024. E-mail: dhocken@fhcrc.org.

American Journal of Pathology, Vol. 162, No. 2, February 2003

Copyright © American Society for Investigative Pathology

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Eker rats. One possible explanation for the species-spe-cific phenotypes may be that the loss of the wild-typecopy through somatic deletion occurs at an earlier stageof development and/or in different cell lineages in thehuman disease. Attempts to analyze Tsc2 null animalshave failed because of embryonic lethality in both gene-targeted mice and rats with two germline copies of theEker TSC2 allele.15,16

Cai and colleagues1 collected three Eker rats withsevere, early bilateral kidney disease from two differentanimal colonies. Pathological examination found diffusePKD. These lesions resembled the typical Eker kidneylesions only in the histological appearance of papillaryadenomas and carcinomas forming within a portion of thecysts. A single nucleotide polymorphism between EkerTSC2 and the allele from the congenic Long-Evans strainbackground was discriminated using polymerase chainreaction-denaturing high performance liquid chromatog-raphy (dHPLC) and confirmed by restriction fragmentlength polymorphism and allele-specific polymerasechain reaction. Using an epithelial cell line derived fromone of the polycystic kidneys and DNA from normal andaffected organs, the group demonstrated loss of the wild-type TSC2 allele and the adjacent PKD1 allele and dupli-cation of the Eker allele. These results, combined withkaryotypes showing two normal copies of chromosome10 in affected cells, confirm mosaicism for uniparentalisodisomy, probably because of postzygotic chromosomalnondisjunction. Somatic recombination is also a possiblemechanism for the mosaicism described, although the re-ciprocal mosaic genotype, including two wild-type TSC2alleles, was not identified.

The pattern of mosaicism in the Eker rats with PKDindicates that uniparental disomy occurred near the timeof condensation of intermediate and lateral plate meso-derm in early organogenesis. Intriguingly, the loss ofTSC2 function earlier in development did not recapitulatethe TSC phenotype in humans. In particular, hamartoma-tous lesions such as angiomyolipomas and oncocytomasinvolving multiple organs are not observed. However,there is accumulating evidence that the cellular conse-quences of the loss of tuberin are conserved in rodentsand humans. Multiple angiogenic growth factors are ac-tivated in angiofibromas from tuberous sclerosis patientsand TSC2(�/�) rat embryonic fibroblasts support endo-thelial cell proliferation.17 Eker rat uterine leiomyomasand TSC pulmonary hamartomas and lymphangio-leiomyomatosis express high levels of the chromosomalprotein HMG2, normally restricted to embryonic tis-sues.18

A number of hypotheses for tuberin function have beenput forward. Based on sequence similarity with a GTPase-activating protein for Rap1 (GAP3), two groups reportedthat tuberin has GAP activity for both Rap1a andRab5.19,20 Identification of the Drosophila melanogasterlocus, gigas, as a TSC2 homolog has led to importantinsights into intracellular signaling pathways involving tu-berin. Mutant alleles for gigas cause enlarged eyes andwings because of cellular hypertrophy and hyperpla-sia.21 Crossing gigas flies with other mutant backgroundsestablished an epistasis relationship between dTsc2 and

insulin-receptor signaling components, with cell growthphenotypes in insulin receptor, dPTEN and dPKB mutantsdependent on dTsc2 function. Conversely, a downstreamtarget of insulin receptor signaling, ribosomal protein S6kinase, acted epistatically dominant to dTsc2. Studies inmammalian cells have determined that TSC2 is phos-phorylated by PKB and that TSC2 function is required forregulation of S6 kinase by nutrients.22,23 Under condi-tions of amino acid deprivation, S6 kinase is inactivated,leading to suppression of ribosomal protein synthesis. InTSC2 mutant backgrounds, ribosomal protein synthesisis insensitive to amino acid supply. Understanding tu-berin as an inhibitor of S6K, the loss of regulation ofribosomal protein synthesis may explain the cellular phe-notypes of increased proliferation and growth in tuberin-deficient cells.

The clear implication of the phenotype of the mosaicEker rats is that the consequences of tuberin deficiencyare limited to renal tubular epithelium in the rat kidney,despite the opportunity for earlier developmental pathol-ogy affecting multiple cell lineages. The link betweentuberous sclerosis and renal tubular cysts was first ob-served in patients with the contiguous gene deletion syn-drome involving TSC2 and PKD1, presenting as earlyonset, severe renal cystic disease. A recent study byKleymenova and colleagues24 uncovered another rela-tionship between tuberin and polycystin-1. They demon-strated that functional localization of polycystin-1 at thebasolateral membrane was defective in TSC2 mutantcells, thus presenting the possibility that tuberin defi-ciency can mimic a PKD1 null genotype, manifesting asPKD. The mosaic Eker rats described by Cai and col-leagues1 with UPD for the TSC2 mutant and two normalPKD1 alleles are entirely consistent with this hypothesis.

Although mosaicism is rarely reported in inherited can-cer predisposition syndromes, it seems to be a commonmechanism in other types of autosomal dominant, cellularrecessive diseases.25 Inherited skin diseases such asneurofibromatosis 1, cutaneous leiomyomatosis, and dis-seminated superficial actinic porokeratosis frequentlypresent with a pattern of pronounced segmental lesionssuperimposed on the usual nonsegmental phenotype.This phenotype, termed type 2 segmental involvement byHapple,26 has been proposed to result from genetic mo-saicism with reduction to homozygosity or hemizygosityof an inherited mutation. As shown in Cai and col-leagues,1 recognition of additional examples of mosa-icism in human disease and corresponding animal mod-els may yield surprising and novel insights into genefunction.

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