Tuberous sclerosis 2 gene and uses thereof

Details Tuberous sclerosis 2 gene and uses thereof Tuberous sclerosis 2 gene and uses thereof

1996-10-01

2001-05-15

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023500, C536S024310, C536S024330, C536S024300, C435S006120, C435S320100, C435S325000

Reexamination Certificate

active

06232452

ABSTRACT:

The present invention relates to the tuberous sclerosis 2 (TSC2) gene, mutations thereof in patients having TSC2-associated disorders, the protein encoded by the TSC2 gene, and their uses in diagnosis and therapy.
BACKGROUND TO THE INVENTION
All references mentioned hereinbelow are listed at the end of this description and are herein incorporated by reference in their entirety.
Tuberous sclerosis (TSC) is an autosomal dominant disorder, classified as a phakomatosis (van der Hoeve, 1933) and characterised by the widespread development of growths, usually described as hamartomata, in many tissues and organs. The unpredictable distribution of these lesions, particularly within the brain, eyes, skin, kidneys, heart, lungs and skeleton results in a wide variety of signs, symptoms and complications (Gomez, 1988). Although most frequently diagnosed as a result of neurological or dermatological manifestations, renal disease was found to be the leading cause of mortality ({fraction (11/40)} deaths) in the largest series of TSC deaths reported so far. Renal complications including haemorrhage, hypertension and end stage renal disease (ESRD) are associated with the development of cysts and hamartomatous growths (angiomyolipomata) in the kidneys. Angiomyolipomata probably arise due to coexistent inactivating constitutional and somatic mutations, consistent with the TSC genes functioning as tumour or growth suppressor genes (Green et al (1994) and Green et al (in press)). Therefore the frequency of diagnosed cases is likely to under-represent true prevalence which may be as high as 1 in 5,800 (Osborne et al., 1991). The pathogenesis of TSC is poorly understood and efforts to establish the primary underlying defect have focused on positional cloning of the causative gene(s).
Linkage studies have established locus heterogeneity (Sampson et al., 1989a & 1992, Haines et al., 1991a&b, Janssen et al., 1991, Povey et al.,1991, Northrup et al., 1992) with disease determining genes on chromosomes 9 (Fryer et al., 1987) and 16 (Kandt et al., 1992) leading to apparently indistinguishable phenotypes. In most, if not all, affected multigeneration families the disease can be accounted for by the gene at one or other of these loci (Kwiatkowski et al., 1993). The Genome Database Nomenclature Committee recently agreed that the loci on chromosomes 9 and 16 should be termed TSC1 and TSC2 respectively. Analysis of meiotic recombination events in TSC families has refined the positions of TSC1 and TSC2 to small regions in the telomeric chromosomal bands 9q34.3 and 16p13.3. The candidate region at 16p13.3 extends between the markers MS205.2 (D16S309) and 16AC2.5 (D16S291) (Kwiatkowski et al., 1993), representing an estimated 1.5 megabases of DNA.
Loss of heterozygosity for alleles at 16p has been observed in hamartomata from TSC patients (Green and Yates, 1993; Smith et al., 1993), indicating that a second somatic mutation may be required to produce the TSC phenotype at a cellular level. This observation is consistent with the chromosome 16 TSC gene acting as a tumour suppressor, a feature shared by genes causing the other phakomatoses, neurofibromatosis type 1 (NF1) (Legius et al., 1993) and type 2 (NF2) (Trofatter et al., 1993), and von Hippel-Lindau disease (VHL) (Latif et al., 1993). If a two-hit mechanism, as proposed by Knudson (1971), does apply to TSC, then inactivating constitutional mutations would be anticipated. TSC has not been noted in individuals with the chromosome 16 &agr;-thalassaemia/mental retardation syndrome (ATR-16) and terminal deletions of 16p which extend into the distal part of the candidate region (Wilkie et al., 1990). The inventors of the present invention therefore investigated the proximal part of the candidate region for deletions.
Some 60% of TSC cases appear to represent new mutations (Sampson et al., 1989b) and the inventors reasoned that a proportion of these might be large deletions. Such deletions, detectable by pulsed field gel electrophoresis (PFGE), would greatly facilitate identification of the gene, as has been demonstrated in NF1, NF2 and VHL (Viskochil et al., 1990; Trofatter et al., 1993; Latif et al., 1993). The inventors have now identified 5 TSC associated constitutional interstitial deletions of between 30 and 75 kb in the proximal part of the candidate region. These have been mapped to a 120kb segment from which the inventors have identified a number of genes, one of which was disrupted by all the deletions. Mutation analysis and expression studies provide strong evidence that this gene, which we term TSC2, is the chromosome 16 tuberous sclerosis determining gene.
SUMMARY OF THE INVENTION
Accordingly, in one aspect this invention provides an isolated, purified or recombinant nucleic acid sequence comprising:
(a) a TSC2 gene or its complementary strand,
(b) a sequence substantially homologous to, or capable of hybridising to, a substantial portion of a molecule defined in (a) above,
(e) a fragment of a molecule defined in (a) or (b) above.
In particular, there is provided a DNA molecule having a sequence corresponding to all or a portion of the nucleotide sequence of
FIG. 3
[SEQ ID NO: 1], or a complementary sequence, or a sequence which hybridises to any of the above sequences.
In another aspect this invention provides a purified DNA molecule characterised as follows:
(i) it is present in the telomeric chromosomal band 16p 13.3,
(ii) it is mutated in TSC patients,
(iii) it lies between markers GGG
1
and 16AC2.
The sequence is preferably contained in cosmids ZDS-5 and LADS-4. The DNA may be genomic but it is preferred for it to be a cDNA.
The TSC2 gene described herein is a gene found on human chromosone 16, and the results of familial studies described herein form the basis for concluding that this TSC2 gene encodes a protein called TSC2 protein or tuberin which has a role in the prevention or suppression of TSC. The TSC2 gene therefore includes the DNA sequences shown in
FIG. 3
[SEQ ID NO: 1], and all functional equivalents. The gene furthermore includes regulatory regions which control the expression of the TSC2 coding sequence, including promotor, enhancer and terminator regions. Other DNA sequences such as introns spliced from the end-product TSC2 RNA transcript are also encompassed. Although work has been carried out in relation to the human gene, the corresponding genetic and functional sequences present in lower animals are also encompassed.
The present invention therefore further provides a TSC2 gene or its complementary strand having the sequence according to
FIG. 3
[SEQ ID NO: 1]. In particular, it provides a TSC2 gene or its complementary strand having the sequence of
FIG. 3
[SEQ ID NO: 1]which gene or strand is mutated in some TSC patients (more specifically, TSC2 patients).
The invention further provides a nucleic acid sequence comprising a mutant TSC2 gene, especially one selected from a sequence comprising a sequence according to
FIG. 3
[SEQ ID NO: 1]when:
(a) [WS-13] about 32 kb are deleted flanked by CW13 and CW9;
(b) [WS-9) about 46 kb are deleted with breakpoints in SM9 and CW12;
(c) [WS-211] about 75 kb are deleted with breakpoints between CW9 and CW15 distally, and between CW23 and CW21 proximally;
(d) [WS-97] about 75 kb are deleted between BFS2 and SM9 distally, and within CW20 proximally;
(e) [WS-53] about 35 kb are deleted between, distally, CW23 next to JH1 and, proximally, such that 0.6 kb of TSC2 is deleted, the deletion lying proximally between SH6 and JH13;
(f) (WS212] about 75 kb are deleted between SM9-CW9 distally and the TSC2 3′UTR proximally as shown in
FIG. 8
;
(g) [WS-215] about 160 kb are deleted between CW20 and CW10-CW36 as shown in
FIG. 8
;
(h) [WS-227] about 50 kb are deleted between CW20 and JH11 as shown in
FIG. 8
;
(i) [WS-219] about 27 kb are deleted between JH1 and JH6 as shown in
FIG. 8
; and
(j) [WS-250] about 160 kb are deleted in CW20 as shown in FIG.

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