Mutations in the TSC1 gene account for a minority of patients with tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by tumour-like malformations (hamartomas) of the brain, skin, and other organs, often associated with seizures and learning disability. There is genetic heterogeneity with loci for TSC on chromosomes 9q34 (TSC1) and 16p13.3 (TSC2). The recently cloned TSC1 gene has 23 exons spanning some 40 kb of genomic DNA with an 8.6 kb transcript. We now report the results of mutation screening by SSCP and heteroduplex analysis of genomic DNA for all 21 coding exons of TSC1 in 83 unrelated cases of tuberous sclerosis. TSC1 gene mutations were found in 16 of the 83 cases (19%). These comprised base substitutions, small insertions, or small deletions giving rise to six nonsense mutations, eight frameshifts, and two splice site mutations, all of which would be expected to result in a truncated or absent protein. In the 10 cases predicted to have TSC1 mutations by linkage analysis or loss of heterozygosity studies, the mutation was identified in eight (80%). In the remaining 73 unassigned cases, only eight mutations were found (11%). From these data we estimate that TSC1 mutations accounted for 24% of the cases in this sample (and an estimated 22% of all TSC cases). This contrasts with data from linkage studies suggesting that TSC1 and TSC2 mutations account for approximately equal numbers of families.

Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT).

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.

Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

STAT1 pathway is involved in activation of caprine arthritis-encephalitis virus long terminal repeat in monocytes.

The caprine arthritis-encephalitis virus (CAEV) long terminal repeat (LTR) is activated by gamma interferon (IFN-gamma) in promonocytic cells. We have previously shown that a 70-bp element is necessary and sufficient for the response of the CAEV LTR to this cytokine. At the 5' end, this 70-bp IFN-gamma response element contains sequence similarity to the gamma activated site (GAS). Here we demonstrate that the putative GAS element in the CAEV LTR binds specifically to a cellular factor induced by IFN-gamma in promonocytic cells. Substitution mutations in this consensus sequence eliminate binding of the inducible factor. The GAS element from the 70-bp motif is sufficient to confer responsiveness to IFN-gamma using a heterologous minimal promoter. Consistent with the binding data, the same mutations in the GAS element eliminate responsiveness to IFN-gamma in the context of both a functional CAEV LTR and a heterologous promoter. The cellular factor that binds to the GAS element is present from 5 min to 14 h after stimulation with IFN-gamma. Binding of the nuclear factor to the GAS element in the CAEV LTR is inhibited by antibody directed against STAT1 (p91/84). Thus, the GAS sequence in the CAEV LTR is essential for the response to IFN-gamma and a STAT1-like factor binds to this site. The STAT-1 signaling pathway provides at least one mechanism for activation of the CAEV LTR by IFN-gamma in monocytes. These data are the first demonstration of a role for a STAT family member in the regulation of a viral promoter.

Female germline mosaicism in tuberous sclerosis confirmed by molecular genetic analysis.

We have investigated a family in which three siblings with the autosomal dominant disorder tuberous sclerosis had unaffected parents. The family were typed for polymorphic markers spanning the two genes known to cause tuberous sclerosis located at 9q34 (TSC1) and 16p13.3 (TSC2). TSC1 markers showed different maternal and paternal haplotypes in affected children, excluding a mutation in TSC1 as the cause of the disease. For the TSC2 markers all the affected children had the same maternal and paternal haplotypes, as did three of their unaffected siblings. Mutation screening by RT-PCR and direct sequencing of the TSC2 gene identified a 4 bp insertion TACT following nucleotide 2077 in exon 18 which was present in the three affected children but not in five unaffected siblings or the parents. This mutation would cause a frameshift and premature termination at codon 703. Absence of the mutation in lymphocyte DNA from the parents was consistent with germline mosaicism and this was confirmed by our finding of identical chromosome 16 haplotypes in affected and unaffected siblings, providing unequivocal evidence of two different cell lines in the gametes. Molecular analysis of the TSC2 alleles present in the affected subjects showed that the mutation had been inherited from the mother. This is the first case of germline mosaicism in tuberous sclerosis proven by molecular genetic analysis and also the first example of female germline mosaicism for a characterized autosomal dominant gene mutation apparently not associated with somatic mosaicism.

Loss of heterozygosity in tuberous sclerosis hamartomas.

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.

Clonality of tuberous sclerosis harmatomas shown by non-random X-chromosome inactivation.

Tuberous sclerosis (TSC) is an autosomal dominant condition characterised by tumour-like malformations (hamartomas) in the brain and other organs. A proportion of hamartomas from patients with TSC show loss of heterozygosity (LOH) for DNA markers in the region of either the TSC1 gene on chromosome 9q34 or the TSC2 gene on 16p13.3. This implies that these lesions are clonal. We have studied X-chromosome inactivation, as a marker of clonality, in 13 hamartomas from females with TSC. The hamartomas comprised five renal angiomyolipomas, three fibromas and seven other lesions. In previous studies, four of the lesions showed LOH. A polymerase chain reaction assay was used to analyse differential methylation of an HpaII restriction site adjacent to the androgen-receptor triplet-repeat polymorphism on Xq11-12. In 12 of the lesions, there was a skewed inactivation pattern with one X chromosome being fully methylated and the other unmethylated. Normal tissue showed a random pattern of inactivation. These data confirm that most TSC hamartomas are clonal in origin. This is an intriguing finding, since these lesions are composed of more than one cell type.

Activation of caprine arthritis-encephalitis virus long terminal repeat by gamma interferon.

Caprine arthritis-encephalitis virus (CAEV) is a lymphotropic lentivirus whose replication increases during monocyte maturation. We examined gene expression directed by the CAEV long terminal repeat (LTR) in a promonocytic cell line stimulated with several agents. Our results demonstrate that the CAEV LTR is activated by treatment of immature monocytes with gamma interferon (IFN-gamma) or a phorbol ester but not with tumor necrosis factor alpha or lipopolysaccharide. The cis-acting element in the LTR for the IFN-gamma response localizes to a duplicated 70-bp motif that contains an IFN-gamma response element, the gamma-activated site. One copy of the motif is necessary and sufficient for the response to IFN-gamma. Multiple copies contribute to basal transcriptional activity in the context of a heterologous promoter. This IFN-gamma response element in the CAEV LTR differs from the element required for the response to phorbol esters. Thus, activation of the CAEV LTR in monocytes that are stimulated by IFN-gamma, a cytokine that is secreted in response to viral infections, could contribute to conversion from latent to high-level viral replication in infected hosts.

Giardiavirus-resistant Giardia lamblia lacks a virus receptor on the cell membrane surface.

Giardia lamblia virus (GLV) is a small nonenveloped double-stranded RNA virus that infects specifically the parasitic protozoan G. lamblia. Among the many collected strains of G. lamblia, a few turn out to be highly resistant to the virus infection. Two of these strains, Ac and JH, were subjected to electroporation with the RNA from GLV-infected G. lamblia WB strain. Subsequent studies indicated the presence of GLV double-stranded RNA and GLV protein in the electroporated and propagated cells. Virus particles, released by the transfected cells into the culture medium, were capable of infecting the virus-sensitive G. lamblia WB strain. When the WB cells were incubated with GLV at 4 degrees C and treated with the bifunctional cross-linking reagent disuccinimidyl suberate, little GLV protein was detectable inside the cells by immunofluorescent staining. However, patches of fluorescent granules were found on the membrane surface of the cells, suggesting cross-linking of the viruses with a certain membrane component(s). Similar treatment of the resistant strains Ac and JH showed no fluorescence either inside or outside of the cells. Two other closely related parasitic protozoa, Tritrichomonas foetus and Trichomonas vaginalis, cannot be infected by GLV via either viral infection or RNA transfection. The [35S]cysteine-labeled protein profiles in Triton X-114 extracts of G. lamblia WB, Ac, and JH were compared. The profile of the WB strain differs clearly from that of Ac and JH. It remains to be seen, however, whether this difference is related at all to the different susceptibilities to GLV infection.

Investigations of virus-protozoa relationships in the model of the free-living ciliate Tetrahymena pyriformis and adenovirus type 3.

Cultures of the free-living ciliate, Tetrahymena pyriformis, strain GL were experimentally infected with adenovirus type 3 (Ad3). The course of interaction was followed over a five-day period and in four successively passaged subcultures in virus-free medium. Possible presence of viral genomic DNA in the ciliates was assayed by DNA-DNA hybridization, allowing the detection of 100 pg of homologous DNA. The presence of viral protein was tested by time-resolved fluoroimmunoassay (TR FIA) enabling to detect less than 5 ng of Ad3 hexon antigen per ml. In ciliate culture supernatants the presence of virus was tested by TR FIA and by estimation of viral titers (PFU tests). Viral genome was detected in the ciliates over the first two days post-infection and not later. Antigen concentration in the supernatant was negative by the fourth day of the experiment and on the tenth day no virus was detected by PFU tests. During passages in virus-free medium, viral genome was detected only in the ciliates of the first subculture, the antigen concentration was negative after the second passage. In addition, no viral titer could be detected after this time. Our data do not support the hypothesis that Ad3-replication takes place in T. pyriformis.

Novel ribonucleic acid species in Eimeria nieschulzi are associated with RNA-dependent RNA polymerase activity.

Sporulated oocysts of Eimeria nieschulzi, E. tenella and E. acervulina were screened for the presence of putative viral nucleic acids. An RNA-dependent RNA polymerase activity in E. nieschulzi correlated with the presence of unknown nucleic acid species that were absent in the two other species of Eimeria. The novel nucleic acid species also served as templates for RNA-polymerase activity in in vitro synthesis of full-length labelled transcripts. These nucleic acid species were shown to be RNAse-sensitive and were suspected to represent the genomic RNA of a putative virus.