TUBEROUS SCLEROSIS, LYMPHANGIOMYOMATOSIS,
AND POLYCYSTIC KIDNEY DISEASE

Our laboratory studies three inter-related disorders: tuberous sclerosis complex (TSC), lymphangiomyomatosis (LAM), and autosomal dominant polycystic kidney disease (ADPKD). Tuberous sclerosis is an autosomal dominantly inherited disease with an incidence of approximately one in 10,000. TSC is characterized by seizures, mental retardation, autism, and benign tumors or "hamartomas." These tumors can occur in virtually any organ system, particularly brain, kidney, heart, and skin. Malignant tumors can also occur in tuberous sclerosis, particularly in the kidney, although they are less frequent than benign tumors. There are two genes that cause TSC, TSC1 and TSC2. We have been interested in understanding how mutations in these genes lead to human disease.

LAM, which affects women almost exclusively, is a rare disease of pulmonary smooth muscle proliferation. LAM shares two features with TSC: a distinctive pulmonary infiltrate and benign renal angiomyolipomas. Because of these similarities, we hypothesized that LAM and TSC have a common genetic basis. However, LAM is not genetically transmitted. Microscopically, there is cystic distortion of the normal pulmonary architecture by an infiltration of smooth muscle. Most patients have a slowly declining clinical course. Lung transplantation is the only effective therapy for patients with end-stage disease. LAM can occur without other disease or in association with TSC.

ADPKD is one of the most common hereditary disorders in the general population, accounting for 8 to 10% of all cases of end-stage renal disease. In ADPKD, the kidney becomes replaced by hundreds of cysts, ranging in size from a few millimeters to several centimeters, leading to renal failure. Hepatic cysts and cerebral vascular aneurysms also occur in ADPKD. Two genes, PKD1 and PDK2, have been implicated in the genesis of ADPKD. We are interested in determining whether mutations in the PDK1 gene cause ADPKD by a gain-of-function or loss-of-function mechanism.

The product of TSC2, tuberin, appears to be localized to the Golgi apparatus and may function in vesicular transport. The function of hamartin, the product of TSC1, is not known. We have demonstrated an interaction between hamartin and tuberin, which is detectable at endogenous protein levels. Hamartin is present in a cell line derived from the Eker rat that lacks functional tuberin, indicating that the stability of hamartin is not dependent on its interaction with tuberin. Hamartin is localized to the membrane/particulate (P100) fraction of cultured cells. The P100 localization is unchanged in the Eker cells. Finally, we have shown that at endogenous expression levels hamartin has a punctate pattern of immunofluorescence in the cytoplasm. Taken together, hamartin's presence in the membrane/particulate fraction and its pattern of cytoplasmic staining suggest that it is localized to cytoplasmic vesicles. If altered vesicular trafficking leads to tumorigenesis in tuberous sclerosis, TSC1 and TSC2 may have a novel mechanism of tumor suppression.

By Western blot analysis, hamartin is strongly expressed in brain, kidney, and heart, all of which are frequently affected in TSC. Using immunohistochemistry with an antibody that we have developed to the carboxy terminus of hamartin, the expression pattern of hamartin in normal human tissues was found to be nearly identical to that of tuberin. This observation is consistent with the recent finding that tuberin and hamartin interact and with clinical similarity between TSC1- and TSC2-linked disease. Strong hamartin expression was seen in cortical neurons, renal tubular epithelial cells, pancreatic islet cells, bronchial epithelial cells, and pulmonary macrophages. Hamartin was also expressed in endocrine tissues, including islet cells of the pancreas, follicular cells of the thyroid, and the zona reticularis of the adrenal cortex. In eight angiomyolipomas from a TSC1-linked patient, no hamartin expression was detected, while tuberin, the product of the TSC2 gene, was expressed. In 19 angiomyolipomas from a TSC2-linked patient, in whose angiomyolipomas we have previously shown loss of tuberin expression, hamartin expression was present. These data suggest that tuberin and hamartin immunoreactivity can distinguish tumors with underlying TSC1 mutations from those with TSC2 mutations, which may have diagnostic implications.

The types of malignancy that occur in TSC have not been fully characterized. We have studied eight malignant tumors from six TSC patients ranging in age from 22 months to 21 years old. Six tumors were renal, one was from the inguinal region, and one was from the brain. The tumors were analyzed for loss of heterozygosity (LOH) in the chromosomal regions of the TSC1, TSC2, and VHL genes. Three patients (ages 7, 8, and 20) had renal cell carcinomas (RCCs). Two of these patients had multifocal RCCs. All three patients with RCC also had prominent multifocal dysplasia of renal cyst epithelium. Two patients (ages 20 and 21) had malignant angiomyolipomas, one renal and one inguinal. One patient (age 22 months) had a grade 4 giant cell astrocytoma (glioblastoma multiforme). LOH in the region of the TSC2 gene was found, either in the malignant tumor or in benign tumors, in all five patients whose DNA could be analyzed. We conclude that children with TSC, as well as adults with the disease, are at risk for developing malignant tumors. Two types of renal malignancy occur in TSC: renal cell carcinoma, which appears to arise from dysplastic renal cyst epithelial cells, and malignant angiomyolipoma. Tumors cytologically similar to malignant angiomyolipomas may also occur at extra-renal sites. LOH analyses suggest that most patients with TSC who develop malignant tumors have germline TSC2, rather than TSC1 gene mutations.

LAM, which affects women almost exclusively, is a rare disease of unknown etiology that was first described 60 years ago. LOH in the chromosomal regions of the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas from 13 women with sporadic LAM for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven of the angiomyolipomas (54%). We also found TSC2 LOH in four of five lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, LAM is not genetically transmitted. Women with LAM may have germline TSC2 mutations with limited expression or they may be mosaic containing TSC2 gene mutations in lung and kidney, but not in brain or skin.

ADPKD is caused by mutations in one of two genes: PKD1 on chromosome 16p13 and PKD2 on chromosome 4. The PKD1 gene is immediately adjacent to TSC2. To determine whether mutations in the PKD1 gene causes ADPKD by an activating (gain-of-function) or an inactivating (loss-of-function) model, we analyzed DNA from cyst epithelial cells for LOH in the PKD1 region of chromosome 16p13 using microsatellite markers. Twenty-nine cysts from four patients were studied. Five cysts from three patients had chromosome 16p13 LOH (Brasier and Henske, J. Clin. Invest. 99:194, 1997). Four of the cysts had loss of two chromosome 16p13 markers that flank the PKD1 gene. In two patients, microsatellite analysis of family members was consistent with loss of the wild type copy of PKD1 in the cysts. In the third patient, 16p13 LOH was detected in three separate cysts, all of which showed loss of the same alleles. Chromosome 3p21 LOH was detected in one cyst. No LOH was detected in four other genomic regions. These results demonstrate that some renal cyst epithelial cells exhibit clonal chromosomal abnormalities with loss of the wild- type copy of PKD1. This supports a LOH model for ADPKD, with a germline mutation inactivating one copy of PKD1 and somatic mutation or deletion inactivating the remaining wild type copy.

AL-SALEEM, T., WESSNER, L.L., SCHEITHAUER, B.W., PATTERSON, K., ROACH, E.S., DREYER, S.J., FUJIKAWA, K., BJORNSSON, J., BERNSTEIN, J., HENSKE, E.P. Malignant tumors of the kidney, brain, and soft tissues in children and young adults with tuberous sclerosis. Cancer 83:208-2216, 1998.

HENSKE, E.P., AO, X., SHORT, M.P., GREENBERG, R., NEUMANN, H.P.H., KWIATKOWSKI, D.J., RUSSO, I. Frequent progesterone receptor immunoreactivity in tuberous sclerosis-associated renal angiomyolipomas. Mod. Pathol. 11:665-668, 1998.

HENSKE, E.P., KWIATKOWSKI, D.J. Human microsatellite repeat markers and their application to analysis of clonality and allelic loss in tumors. In Human Genome Methods, edited by K.W. Adolph. CRC Press, Boca Raton, Florida, pp. 3-21, 1998.

HENSKE, E.P., THORNER, P., PATTERSON, K., ZHUANG, Z., BERNSTEIN, J. Renal cell carcinoma in children with diffuse cystic hyperplasia of the kidneys. Pediatr. Dev. Pathol. (in press).

KWIATKOWSKA, J., JOZWIAK, S., HALL, F., HENSKE, E.P., HAINES, J.H., McNAMARA, P., BRASIER, J., WIGOWSKA-SOWINSKA, J., KASPRZYK-OBARA, J., SHORT, M.P., KWIATKOWSKI, D.J. Comprehensive mutational analysis of the TSC1 gene: observations on frequency of mutation, associated features, and nonpenetrance. Ann. Hum. Genet. 62:277-285, 1998.

NEUMANN, H.P.H., SCHWARZKOPF, G., HENSKE, E.P. Renal angiomyolipomas, cysts, and cancer in tuberous sclerosis complex. Semin. Pediatr. Neurol. 5(4):269-275, 1998.

PLANK, T.L., HENSKE, E.P. Tumor suppressor genes. In Basic Science of Cancer, edited by G.D. Kruh and K. Tew. Current Medicine, Inc. (in press).

PLANK, T.L., LOGGINIDOU, E., KLEIN-SZANTO, A., HENSKE, E.P. The expression of hamartin, the product of the TSC1 gene, in normal human tissues and in TSC1- and TSC2-linked angiomyolipomas. Mod. Pathol. (in press).

PLANK, T.L., YEUNG, R.S., HENSKE, E.P. Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res. 58:4766-4770, 1998.

SMOLAREK, T.A., WESSNER, L.L., McCORMACK, F.X., MYLET, J.C., MENON, A.G., HENSKE, E.P. Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis. Am. J. Hum. Genet. 62:810-815, 1998.

BJORNSSON, J., HENSKE, E.P., BERNSTEIN, J. Renal manifestations. In The Tuberous Sclerosis Complex, Third Edition. Oxford University Press (in press).

^§   Fox Chase researcher

^a   R.S. Yeung: University of Washington, Seattle, WA 98195

^b   J. Bernstein: William Beaumont Research Institute, Royal Oak, MI 48073

^c    F.X. McCormack, A. Menon, T. Smoralek: University of Cincinnati, Cincinnati, OH 45267

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998