Development of a versatile HPLC-based method to evaluate the activation status of small GTPases

Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood.     

SNP identification,haplotype analysis,and parental origin of mutations in TSC2

Inactivating mutations in the TSC2 gene, consisting of 41coding exons in 40 kb on 16p13, cause the hamartoma syndrome tuberous sclerosis. During TSC2 mutational analysis we identified ten SNPs that occur within or close to exon boundaries at minor allele frequencies greater than 5%. We determined the haplotypes for six of these SNPs and the microsatellite marker kg8 in the 3' region of TSC2 in a set of 40 parent-child trios. The most common haplotypes accounted for 53%, 11%, 6%, and 5% of chromosomes. Thirty-eight TSC2 mutation-bearing haplotypes had a similar distribution, indicating that there was no haplotype that predisposed to mutation in this region of TSC2. Family analysis was possible in 12 sporadic cases, and indicated that the mother was the parent of origin in 7 cases (3 point mutations, 2 small deletions, 2 large deletions), while the father was in 5 cases (2 point mutations, 3 small deletions). We conclude that TSC2 mutations occur at substantial frequency on both the maternally and paternally derived TSC2 alleles, in contrast to many other genetic diseases including NF1. The observations have implications for genetic counseling in TSC.     

Human TSC-associated renal angiomyolipoma cells are hypersensitive to ER stress

Tuberous sclerosis complex (TSC), an inherited tumor predisposition syndrome associated with mutations in TSC1 or TSC2, affects ～1 in 6,000 individuals. Eighty percent of TSC patients develop renal angiomyolipomas, and renal involvement is a major contributor to patient morbidity and mortality. Recent work has shown that mammalian target of rapamycin complex 1 (mTORC1) inhibition caused angiomyolipoma shrinkage but that this treatment may cause cytostatic not a cytotoxic effect. Endoplasmic reticulum (ER) stress can develop in TSC-associated cells due to mTORC1-driven protein translation. We hypothesized that renal angiomyolipoma cells experience ER stress that can be leveraged to result in targeted cytotoxicity. We used immortalized human angiomyolipoma cells stably transfected with empty vector or TSC2 (encoding tuberin). Using cell number quantification and cell death assays, we found that mTORC1 inhibition with RAD001 suppressed angiomyolipoma cell proliferation in a cytostatic manner. Angiomyolipoma cells exhibited enhanced sensitivity to proteasome inhibitor-induced ER stress compared with TSC2-rescued cells. After proteasome inhibition with MG-132, Western blot analyses showed greater induction of C/EBP-homologous protein (CHOP) and more poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage, supporting ER stress-induced apoptosis. Live cell numbers also were decreased and cell death increased by MG-132 in angiomyolipoma cells compared with TSC2 rescued. Intriguingly, while pretreatment of angiomyolipoma cells with RAD001 attenuated CHOP and BiP induction, apoptotic markers cleaved PARP and caspase-3 and eukaryotic translation initiation factor 2α phosphorylation were increased, along with evidence of increased autophagy. These results suggest that human angiomyolipoma cells are uniquely susceptible to agents that exacerbate ER stress and that additional synergy may be achievable with targeted combination therapy.     

Germ-line mutational analysis of the TSC2 gene in 90 tuberous-sclerosis patients.

Ninety patients with tuberous-sclerosis complex (TSC) were tested for subtle mutations in the TSC2 gene, by means of single-strand conformational analysis (SSCA) of genomic DNA. Patients included 56 sporadic cases and 34 familial probands. For all patients, SSCA was performed for each of the 41 exons of the TSC2 gene. We identified 32 SSCA changes, 22 disease-causing mutations, and 10 polymorphic variants. Interestingly, we detected mutations at a much higher frequency in the sporadic cases (32%) than in the multiplex families (9%). Among the eight families for which linkage to the TSC2 region had been determined, only one mutation was found. Mutations were distributed equally across the gene; they included 5 deletions, 3 insertions, 10 missense mutations, 2 nonsense mutations, and 2 tandem duplications. We did not detect an increase in mutations either in the GTPase-activating protein (GAP)-related domains of TSC2 or in the activating domains that have been identified in rat tuberin. We did not detect any mutations in the exons (25 and 31) that are spliced out in the isoforms. There was no evidence for correspondence between variability of phenotype and type of mutation (missense versus early termination). Diagnostic testing will be difficult because of the genetic heterogeneity of TSC (which has at least two causative genes: TSC1 and TSC2), the large size of the TSC2 gene, and the variety of mutations. More than half of the mutations that we identified (missense, small in-frame deletion, and tandem duplication) are not amenable to the mutation-detection methods, such as protein-truncation testing, that are commonly employed for genes that encode proteins with tumor-suppressor function.     

Estradiol and tamoxifen stimulate LAM-associated angiomyolipoma cell growth and activate both genomic and nongenomic signaling pathways

Lymphangioleiomyomatosis (LAM) is a progressive lung disease affecting almost exclusively women. The reasons for this strong gender predisposition are poorly understood. Renal angiomyolipomas occur in 50-60% of sporadic LAM patients. The smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly indistinguishable morphologically. Here, we report the first successful cell culture of a LAM-associated renal angiomyolipoma. The cells carried inactivating mutations in both alleles of the TSC2 gene and expressed estrogen receptor , estrogen receptor , and androgen receptor. To elucidate the cellular pathways through which steroid hormones influence LAM pathogenesis, we treated the cells with both estradiol and tamoxifen. Cell growth was stimulated by estradiol, associated with phosphorylation of p44/42 MAPK at 5 min and an increase in c-myc expression at 4 h. Tamoxifen citrate also stimulated cell growth, associated with increased phosphorylation of p44/42 MAPK and expression of c-myc, indicating that tamoxifen has agonist effects on angiomyolipoma cells. This response to tamoxifen in human angiomyolipoma cells differs from prior studies of Eker rat leiomyoma cells, possibly reflecting cell type or species differences in cells lacking tuberin. Our data provide the first evidence that estradiol stimulates the growth of angiomyolipoma cells, that tamoxifen has agonist effects in angiomyolipoma cells, and that estradiol and tamoxifen impact both genomic and nongenomic signaling pathways in angiomyolipoma cells. The responsiveness of angiomyolipoma cells to estradiol may be related to the underlying reasons that LAM affects primarily women.     

Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for 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 (54%) of the angiomyolipomas. We also found TSC2 LOH in four 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, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.     

Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.     

Analysis of renal lesions in Chinese tuberous sclerosis complex patients with different types of TSC gene mutations

We sought to explore the relationship between renal lesion features and genetic mutations in tuberous sclerosis complex (TSC) patients. TSC patients with renal lesions were subjected to TSC1/2 gene next-generation sequencing (NGS). TSC1/2 mutation types and imaging examinations were screened for combined analysis of genetic and clinical features. Seventy-three probands among TSC patients with renal lesions were included. Twenty affected relatives were also included. In total, 93 patients were included. Eighty patients (86.0%) had bilateral renal angiomyolipomas (AMLs), and one had epithelioid AML. Two patients had polycystic kidney disease, one had renal cell carcinoma, and one had Wilms tumor. Among the 73 probands, four had TSC1 mutations, 53 had TSC2 mutations, and 16 had no mutations identified (NMI). There was no statistically significant difference between TSC1 mutation, TSC2 mutation and NMI group (P= 0.309), or between familial and sporadic groups (P= 0.775) when considering AML size. There was no statistically significant difference between pathogenic/likely pathogenic and benign/likely benign/NMI groups (P= 0.363) or among patients with different mutation types of TSC2 (P= 0.906). The relationship between the conditions of TSC gene mutations and the severity of renal lesions still needs more analysis. Patients with NMI, particularly those with familial disease, need more attention because the pathogenesis remains unknown.     

Analysis of PKD1 for genomic deletion by multiplex ligation-dependent probe assay: absence of hot spots

Autosomal dominant polycystic kidney disease is largely due to mutations in PKD1. PKD1 has an unusual genomic structure, including a 2.5-kb polypyrimidine sequence in intron 21, which has been postulated to lead to a high rate of spontaneous genomic mutation events. In addition, the majority of the gene is duplicated three to six times at 97-99% identity elsewhere in the genome. To identify genomic mutations in PKD1, we developed a multiplex ligation-dependent probe assay (MLPA) in which sites of variation between PKD1 and its copies were positioned at the ligation sites of the MLPA probe sets. Thirteen probe sets covered PKD1 exons 2 through 46, at an average spacing of 2.5 kb. Analysis of 27 independent PKD patient samples showed no evidence for genomic deletions confined to PKD1. Analysis of 15 tuberous sclerosis patient samples in which deletions in TSC2 extended into PKD1 showed no evidence of clustering of breakpoints near the polypyrimidine tract.     

Germ-line mosaicism in tuberous sclerosis: how common?

Two-thirds of cases of tuberous sclerosis complex (TSC) are sporadic and usually are attributed to new mutations, but unaffected parents sometimes have more than one affected child. We sought to determine how many of these cases represent germ-line mosaicism, as has been reported for other genetic diseases. In our sample of 120 families with TSC, 7 families had two affected children and clinically unaffected parents. These families were tested for mutations in the TSC1 and TSC2 genes, by Southern blotting and by single-strand conformational analysis. Unique variants were detected in six families. Each variant was present and identical in both affected children of a family but was absent in both parents and the unaffected siblings. Sequencing of the variants yielded two frameshift mutations, one missense mutation, and two nonsense mutations in TSC2 and one nonsense mutation in TSC1. To determine which parent contributed the affected gametes, the families were analyzed for linkage to TSC1 and TSC2, by construction of haplotypes with markers flanking the two genes. Linkage analysis and loss-of-heterozygosity studies indicated maternal origin in three families, paternal origin in one family, and either being possible in two families. To evaluate the possibility of low-level somatic mosaicism for TSC, DNA from lymphocytes of members of the six families were tested by allele-specific PCR. In all the families, the mutant allele was detected only in the known affected individuals. We conclude that germ-line mosaicism was present in five families with mutations in the TSC2 gene and in one family with the causative mutation in the TSC1 gene. The results have implications for genetic counseling of families with seemingly sporadic TSC.     

Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.     

Mosaic and Intronic Mutations in TSC1/TSC2 Explain the Majority of TSC Patients with No Mutation Identified by Conventional Testing

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor gene syndrome due to germline mutations in either TSC1 or TSC2. 10–15% of TSC individuals have no mutation identified (NMI) after thorough conventional molecular diagnostic assessment. 53 TSC subjects who were NMI were studied using next generation sequencing to search for mutations in these genes. Blood/saliva DNA including parental samples were available from all subjects, and skin tumor biopsy DNA was available from six subjects. We identified mutations in 45 of 53 subjects (85%). Mosaicism was observed in the majority (26 of 45, 58%), and intronic mutations were also unusually common, seen in 18 of 45 subjects (40%). Seventeen (38%) mutations were seen at an allele frequency < 5%, five at an allele frequency < 1%, and two were identified in skin tumor biopsies only, and were not seen at appreciable frequency in blood or saliva DNA. These findings illuminate the extent of mosaicism in TSC, indicate the importance of full gene coverage and next generation sequencing for mutation detection, show that analysis of TSC-related tumors can increase the mutation detection rate, indicate that it is not likely that a third TSC gene exists, and enable provision of genetic counseling to the substantial population of TSC individuals who are currently NMI.     

Multicenter phase 2 trial of sirolimus for tuberous sclerosis: kidney angiomyolipomas and other tumors regress and VEGF- D levels decrease

Background

Tuberous sclerosis (TSC) related tumors are characterized by constitutively activated mTOR signaling due to mutations in TSC1 or TSC2.

Methods

We completed a phase 2 multicenter trial to evaluate the efficacy and tolerability of the mTOR inhibitor, sirolimus, for the treatment of kidney angiomyolipomas.

Results

36 adults with TSC or TSC/LAM were enrolled and started on daily sirolimus. The overall response rate was 44.4% (95% confidence intervals [CI] 28 to 61); 16/36 had a partial response. The remainder had stable disease (47.2%, 17/36), or were unevaluable (8.3%, 3/36). The mean decrease in kidney tumor size (sum of the longest diameters [sum LD]) was 29.9% (95% CI, 22 to 37; n = 28 at week 52). Drug related grade 1–2 toxicities that occurred with a frequency of >20% included: stomatitis, hypertriglyceridemia, hypercholesterolemia, bone marrow suppression (anemia, mild neutropenia, leucopenia), proteinuria, and joint pain. There were three drug related grade 3 events: lymphopenia, headache, weight gain. Kidney angiomyolipomas regrew when sirolimus was discontinued but responses tended to persist if treatment was continued after week 52. We observed regression of brain tumors (SEGAs) in 7/11 cases (26% mean decrease in diameter), regression of liver angiomyolipomas in 4/5 cases (32.1% mean decrease in longest diameter), subjective improvement in facial angiofibromas in 57%, and stable lung function in women with TSC/LAM (n = 15). A correlative biomarker study showed that serum VEGF-D levels are elevated at baseline, decrease with sirolimus treatment, and correlate with kidney angiomyolipoma size (Spearman correlation coefficient 0.54, p = 0.001, at baseline).

Conclusions

Sirolimus treatment for 52 weeks induced regression of kidney angiomyolipomas, SEGAs, and liver angiomyolipomas. Serum VEGF-D may be a useful biomarker for monitoring kidney angiomyolipoma size. Future studies are needed to determine benefits and risks of longer duration treatment in adults and children with TSC.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00126672","term_id":"NCT00126672"}}NCT00126672     

Analysis of the COL1A1 and COL1A2 genes by PCR amplification and scanning by conformation-sensitive gel electrophoresis identifies only COL1A1 mutations in 15 patients with osteogenesis imperfecta type I: identification of common sequences of null-allele mutations.

Although >90% of patients with osteogenesis imperfecta (OI) have been estimated to have mutations in the COL1A1 and COL1A2 genes for type I procollagen, mutations have been difficult to detect in all patients with the mildest forms of the disease (i.e., type I). In this study, we first searched for mutations in type I procollagen by analyses of protein and mRNA in fibroblasts from 10 patients with mild OI; no evidence of a mutation was found in 2 of the patients by the protein analyses, and no evidence of a mutation was found in 5 of the patients by the RNA analyses. We then searched for mutations in the original 10 patients and in 5 additional patients with mild OI, by analysis of genomic DNA. To assay the genomic DNA, we established a consensus sequence for the first 12 kb of the COL1A1 gene and for 30 kb of new sequences of the 38-kb COL1A2 gene. The sequences were then used to develop primers for PCR for the 103 exons and exon boundaries of the two genes. The PCR products were first scanned for heteroduplexes by conformation-sensitive gel electrophoresis, and then products containing heteroduplexes were sequenced. The results detected disease-causing mutations in 13 of the 15 patients and detected two additional probable disease-causing mutations in the remaining 2 patients. Analysis of the data developed in this study and elsewhere revealed common sequences for mutations causing null alleles.     

Ultra deep sequencing detects a low rate of mosaic mutations in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by mutations in TSC1 and TSC2. However, 10–15% TSC patients have no mutation identified with conventional molecular diagnostic studies. We used the ultra-deep pyrosequencing technique of 454 Sequencing to search for mosaicism in 38 TSC patients who had no TSC1 or TSC2 mutation identified by conventional methods. Two TSC2 mutations were identified, each at 5.3% read frequency in different patients, consistent with mosaicism. Both mosaic mutations were confirmed by several methods. Five of 38 samples were found to have heterozygous non-mosaic mutations, which had been missed in earlier analyses. Several other possible low-frequency mosaic mutations were identified by deep sequencing, but were discarded as artifacts by secondary studies. The low frequency of detection of mosaic mutations, two (6%) of 33, suggests that the majority of TSC patients who have no mutation identified are not due to mosaicism, but rather other causes, which remain to be determined. These findings indicate the ability of deep sequencing, coupled with secondary confirmatory analyses, to detect low-frequency mosaic mutations.     

Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by mutations in either of two genes, TSC1 and TSC2. Point mutations and small indels account for most TSC1 and TSC2 mutations. We examined 261 TSC DNA samples (209 small-mutation-negative and 52 unscreened) for large deletion/duplication mutations using multiplex ligation-dependent probe amplification (MLPA) probe sets designed to permit interrogation of all TSC1/2 exons, as well as 15–50 kb of flanking sequence. Large deletion/duplication mutations in TSC1 and TSC2 were identified in 54 patients, of which 50 were in TSC2, and 4 were in TSC1. All but two mutations were deletions. Only 13 deletions were intragenic in TSC2, and one in TSC1, so that 39 (73%) deletions extended beyond the 5′, 3′ or both ends of TSC1 or TSC2. Mutations were identified in 24% of small-mutation-negative and 8% of unscreened samples. Eight of 54 (15%) mutations were mosaic, affecting 34–62% of cells. All intragenic mutations were confirmed by LR-PCR. Genotype/phenotype analysis showed that all (21 of 21) patients with TSC2 deletions extending 3′ into the PKD1 gene had kidney cysts. Breakpoints of intragenic deletions were randomly distributed along the TSC2 sequence, and did not preferentially involve repeat sequence elements. Our own 20-plex probe sets gave more robust performance than the 40-plex probe sets from MRC-Holland. We conclude that large deletions in TSC1 and TSC2 account for about 0.5 and 6% of mutations seen in TSC patients, respectively, and MLPA is a highly sensitive and accurate detection method, including for mosaicism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Deletions in CCM2 are a common cause of cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities of the brain that can result in a variety of neurological disabilities, including hemorrhagic stroke and seizures. Mutations in the gene KRIT1 are responsible for CCM1, mutations in the gene MGC4607 are responsible for CCM2, and mutations in the gene PDCD10 are responsible for CCM3. DNA sequence analysis of the known CCM genes in a cohort of 63 CCM-affected families showed that a high proportion (40%) of these lacked any identifiable mutation. We used multiplex ligation-dependent probe analysis to screen 25 CCM1, -2, and -3 mutation-negative probands for potential deletions or duplications within all three CCM genes. We identified a total of 15 deletions: 1 in the CCM1 gene, 0 in the CCM3 gene, and 14 in the CCM2 gene. In our cohort, mutation screening that included sequence and deletion analyses gave disease-gene frequencies of 40% for CCM1, 38% for CCM2, 6% for CCM3, and 16% with no mutation detected. These data indicate that the prevalence of CCM2 is much higher than previously predicted, nearly equal to CCM1, and that large genomic deletions in the CCM2 gene represent a major component of this disease. A common 77.6-kb deletion spanning CCM2 exons 2-10 was identified, which is present in 13% of our entire CCM cohort. Eight probands exhibit an apparently identical recombination event in the CCM2 gene, involving an AluSx in intron 1 and an AluSg distal to exon 10. Haplotype analysis revealed that this CCM2 deletion occurred independently at least twice in our families. We hypothesize that these deletions occur in a hypermutable region because of surrounding repetitive sequence elements that may catalyze the formation of intragenic deletions.     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.     

Survivin expression in tuberous sclerosis complex cells

Tuberous Sclerosis Complex (TSC) is a tumor suppressor gene disorder with mutations of TSC1/TSC2 genes. This leads to the development of hamartomas that most frequently affect central nervous system, kidney, and skin. Angiomyolipomas are abdominal masses made up of muscle vessels and adipose tissues that grow mostly in proximity to kidneys and liver. Bleeding and kidney failure are the major justification for surgery. This study shows that angiomyolipoma-derived human smooth muscle TSC2-/- cells express the apoptosis inhibitor protein survivin when exposed to IGF-1. Survivin expression is also triggered whenever culture conditions perturb normal TSC2-/- cell function, such as the omission of EGF from the growth medium, the supplementation of anti-EGFR, blockade of PI3K and ERK, or inhibition of mTOR. Interestingly, single or simultaneous inhibition of PI3K by LY294002 and ERK by PD98059 does not prevent IGF-1-mediated survivin expression. Apoptogenic Smac/DIABLO, which is constitutively expressed by TSC2-/- A+ cells, is down-regulated by IGF-1 even in the presence of LY294002 and PD98059. These cells release IGF-1 by means of a negative feedback-regulated mechanism that is overrun when they are exposed to antibodies to IGF-1R, which increases the released amount by more than 400%. The autocrine release of IGF-1 may therefore be a powerful mechanism of survival of the tightly packed cells in the thick-walled vessels of TSC angiomyolipoma and in lymphangioleiomyomatosis (LAM) nodules. Future experimental therapies for TSC and LAM may result from the targeted inhibition of survivin, which may enhance sensitivity to TSC2 therapy.     

Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis

Tuberous sclerosis (TSC [MIM 191090 and MIM 191100]) is an autosomal dominant disorder characterized by hamartomas in many organs. Two thirds of cases are sporadic and are thought to represent new mutations. TSC is caused by mutations affecting either of the presumed tumor-suppressor genes, TSC1 and TSC2. Both appear to function as tumor suppressors, because somatic loss or intragenic mutation of the corresponding wild-type allele is seen in the associated hamartomas. Here we report the first comprehensive mutation analysis of TSC1 and TSC2 in a cohort of 150 unrelated TSC patients and their families, using heteroduplex and SSCP analysis of all coding exons and using pulsed-field gel electrophoresis and conventional Southern blot analysis and long PCR to screen for large rearrangements. Mutations were characterized in 120 (80%) of the 150 cases, affecting TSC1 in 22 cases and TSC2 in 98 cases. TSC1 mutations were significantly underrepresented in sporadic cases (P=. 000185). Twenty-two patients had TSC2 missense mutations that were found predominantly in the GAP-related domain (eight cases) and in a small region encoded in exons 16 and 17, between nucleotides 1849 and 1859 (eight cases), consistent with the presence of residues performing key functions at these sites. In contrast, all TSC1 mutations were predicted to be truncating, consistent with a structural or adapter role for the encoded protein. Intellectual disability was significantly more frequent in TSC2 sporadic cases than in TSC1 sporadic cases (P=.0145). These data provide the first representative picture of the distribution and spectrum of mutations across the TSC1 and TSC2 loci in clinically ascertained TSC and support a difference in severity of TSC1- and TSC2-associated disease.