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Tuberous sclerosis seems to fit well into this category o f inherited cancers - it is dom inantly inherited with a variable phenotype (the clinical signs found in each patient could depend on w hich cells in the developing em bryo receive a second h it on a TSC gene and at w hat stage of development). X -inactivation studies (Green et al. 1996) show that ham artom as are clonal; i.e. that despite being a m ixture o f cell types, they have arisen from one ancestral cell. The num ber o f renal tum ours in the Eker heterozygote rat induced by ionising radiation is linearly dependent on the dose o f

radiation received (Hino et al. 1993), suggesting that only one som atic m utation (in addition to the E ker germ line mutation) is neccessary for tum our form ation. In support of the idea o f the tuberous sclerosis genes as tum our suppressors, som atic second hits have been observed at both hum an loci and at the rat rTsc2 locus in several studies, sum m arised in table 1.1.

As expected, no patient has shown LOH at both loci, so that finding LO H can point to w hich locus a germ line m utation will be found in. D ifferent lesions from the same patient have been show n to have different extents of LO H (but at the sam e locus), suggesting that different ham artom as have arisen as a result o f independent somatic events.

In som e lesions w here LO H is not observed, point m utations in T S C l (van Slegtenhorst

et al. 1997) or rTsc2 (K obayashi et al. 1997) have been found, confirm ing the idea that

it is usually the loss of both copies of either T S C l or TSC 2 w hich causes ham artom a form ation. H ow ever K obayashi et al. (1997) reported several renal cell carcinom as w here neither LO H or a somatic point m utation could be found in rTsc2 despite extensive SSC P analysis. It is not clear whether the failure to find a second hit in these lesions was due to technical problem s (contam ination w ith non-tum our cells or inefficient SSC P), that the second hit could not have been found by this analysis (perhaps in the prom oter region or in an intron) or that it really was not present and that the lesion occurred despite the presence of a normal copy o f rTsc2.

Several observations have been made by different investigators about their LO H data w hich hold true w ith com bined data. Loss of heterozygosity seem s m ore frequent at the TSC2 locus than at T S C l. This is likely to be because m ost o f the lesions studied com e from sporadic patients, m ore of whom seem to have TSC 2 germ line m utations than T S C l m utations (see chapter 6). It is also possible that the difference reflects a different m ode o f pathology o f the two loci - perhaps the second hit o f T S C l is often a small m utation rather than a large genom ic deletion (m atching the germ line m utation spectrum ) and is therefore harder to detect, or perhaps a germ line m utation in T S C l has a haploinsufficiency effect so that a second hit is unnecessary for form ation o f a

hamartoma. Identification o f the germline mutation in the patients studied for LOH would help to elucidate this. These questions are discussed further in chapter 7.

W w ... , s 16pl3 LOH 9q34 LOH Reference / A ll LOH

giant cell astrocytoma 3/9 2/9 Sepp eta l. (1996)

3/7 1/7 Carbonara et al. ( 1996)

1/1 van Slegtenhorst et al.

(1997)

1/11 0/11 Henske et al. (1996)

cortical tuber 2/5 1/5 Sepp et al. (1996)

0/14 0/13 Henske et al. (1996)

0/1 0/1 Carbonara et al. (1996)

Subtotal brain 9/47(19% ) 5/47(11% ) 14/47 (30% )

angiomyolipoma 6/17 1/17 Sepp et al. (1996)

28/49 4/49 Henske et al. (1996)

6/9 0/9 Carbonara et al. (1996)

renal cell carcinoma 0/1 1/1 Sepp et al. (1996)

Subtotal kidney 40/76 (53%) 6/77 (8%) 46/77 (60%)

cardiac rhabdomyoma 1/1 0/1 Sepp et al. (1996)

2/6 0/6 Henske et al. (1996)

0/3 0/3 Carbonara et al. ( 1996)

Subtotal heart 3/10(30% ) 0/10 (0%) 3/1 0(3 0% )

fibrom a 3/8 0/8 Sepp eta l. (1996)

shagreen patch 1/1 0/1 Sepp et al. ( 1996)

0/1 0/1 Henske et al. (1996)

angiofibrom a 0/2 0/2 Henske et al. (1996)

Subtotal skin 4/12 (33%) 0/12 (0% ) 4/12 (33% )

Total 56/145 (39%) 11/146 (8%) 67/146 (46% )

LOH in non-TSC lesions 1

non-TSC AML 3/29(10% ) 0/29 (0%) Henske et al. (1995)

LOH in the Eker rat ratchr. 10

preneoplastic renal tubules 4/19 - Kubo g /a /. (1995)

renal carcinoma 4/5 - YQwnget al. (1993)

renal tumours and cell lines 21/29 - Y eung et al. (1994)

renal carcinoma 11/25 - Y eung et al. (1995)

renal carcinom a derived cell lines

22/31 - Y eung et al. (1995)

uterine leiomyoma 4/6 - Yeung et al. (1995)

leiomyom a or

leiomyosarcom a derived

cell lines

5/7 Yeung et al. (1995)

splenic hem angiosarcom a 0/8 - Y eung e ra /. (1995)

unspecified 6/10 - Kubo e ta l. (1994)

pituitary tumours 1/5 - Kubo e ta l. (1994)

Eker rat total 78/145 (54%) -

Table 1.1 Somatic second hits reported in tuberous sclerosis esions

It has also been suggested that LO H is m ore frequent in angiom yolipom as than in brain lesions (H enske et al. 1996). This could reflect a haploinsufficiency effect in brain, rather than a tw o-hit m odel of pathogenesis but it may also reflect sm aller second hit m utations in brain, or that kidney lesions are m ore well defined than brain lesions m aking it easier to find LO H w ithout interference from infiltrating norm al cells. If H en sk e’s data are excluded from this analysis, there is little difference betw een the two tissues; the total LO H in brain is 13/23 lesions (57%) and in kidney 15/28 (54% ). A lack o f LO H in Eker rat splenic hem angiosarcom as has also been observed (Y eung et al.

1995) probably for sim ilar reasons to those m entioned above for hum an brain lesions.

O ne lesion, a giant cell astrocytom a, also showed LO H in a small region o f 9p21 (including D 9S104 but not the nearby IFNA locus or M LM tum our suppressor gene) (C arbonara et al. 1994). Interestingly, studies of Eker rat renal carcinom a cell lines suggest that there is a locus involved in the progression o f the carcinom as on rat chrom osom e 5q31-q33, near the Infa (a-interferon) locus (H ino et al. 1993) and the Ink4B and Ink4A loci (Hino et al. 1995). At least two genes on chrom osom e 9p are com m only lost in various tum ours (Coleman et al. 1994) and it is possible that one or both of these genes is som etim es involved in ham artom a form ation or progression in both hum ans and rats.

Cell lines from Eker rat tum ours (shown to lack a norm al rTsc2 gene) had their tum origenicity reduced and contact inhibition restored by réintroduction o f the full length rTsc2 or ju st the C-term inus (amino acids 1049-1809 o f rat tuberin - Jin et al.

1996, O rim oto et al. 1996). Renal carcinom as can be prevented in Eker heterozygote rats by the addition o f a rTsc2 transgene (Kobayashi et al. 1997), further supporting the idea o f TSC 2 as a tum our suppressor gene.