Functional characterisation of the TSC1–TSC2 complex to assess multiple TSC2 variants identified in single families affected by tuberous sclerosis complex

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). METHODS: We have used a combination of different assays to characterise the effects of a number of pathogenic TSC2 amino acid substitutions on TSC1-TSC2 complex formation and mTOR signalling. RESULTS: We used these assays to compare the effects of 9 different TSC2 variants (S132C, F143L, A196T, C244R, Y598H, I820del, T993M, L1511H and R1772C) identified in individuals with symptoms of TSC from 4 different families. In each case we were able to identify the pathogenic mutation. CONCLUSION: Functional characterisation of TSC2 variants can help identify pathogenic changes in individuals with TSC, and assist in the diagnosis and genetic counselling of the index cases and/or other family members

Biallelic variants in FLII cause pediatric cardiomyopathy by disrupting cardiomyocyte cell adhesion and myofibril organization

Pediatric cardiomyopathy (CM) represents a group of rare, severe disorders that affect the myocardium. To date, the etiology and mechanisms underlying pediatric CM are incompletely understood, hampering accurate diagnosis and individualized therapy development. Here, we identified biallelic variants in the highly conserved flightless-I (FLII) gene in 3 families with idiopathic, early-onset dilated CM. We demonstrated that patient-specific FLII variants, when brought into the zebrafish genome using CRISPR/Cas9 genome editing, resulted in the manifestation of key aspects of morphological and functional abnormalities of the heart, as observed in our patients. Importantly, using these genetic animal models, complemented with in-depth loss-of-function studies, we provided insights into the function of Flii during ventricular chamber morphogenesis in vivo, including myofibril organization and cardiomyocyte cell adhesion, as well as trabeculation. In addition, we identified Flii function to be important for the regulation of Notch and Hippo signaling, crucial pathways associated with cardiac morphogenesis and function. Taken together, our data provide experimental evidence for a role for FLII in the pathogenesis of pediatric CM and report biallelic variants as a genetic cause of pediatric CM.</p

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases.

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies

Anti-EGFR Antibody Efficiently and Specifically Inhibits Human TSC2−/− Smooth Muscle Cell Proliferation. Possible Treatment Options for TSC and LAM

BACKGROUND: Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2(-/-) ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2(-/-) ASM cell proliferation is EGF-dependent. METHODS AND FINDINGS: Effects of EGF on proliferation of TSC2(-/-) ASM cells and TSC2(-/-) ASM cells transfected with TSC2 gene were determined. In contrast to TSC2(-/-) ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2(-/-) ASM cells. Exposure of TSC2(-/-) ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2(-/-) cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2(-/-) ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2(-/-) ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed. CONCLUSION: Our results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC(-/-) ASM cells, and such EGF-dependency is the result of the lack of tuberin

Pathogenic <i>SPTBN1</i> variants cause an autosomal dominant neurodevelopmental syndrome

SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system

Folliculin mutations are not associated with severe COPD

<p>Abstract</p> <p>Background</p> <p>Rare loss-of-function folliculin (<it>FLCN</it>) mutations are the genetic cause of Birt-Hogg-Dubé syndrome, a monogenic disorder characterized by spontaneous pneumothorax, fibrofolliculomas, and kidney tumors. Loss-of-function folliculin mutations have also been described in pedigrees with familial spontaneous pneumothorax. Because the majority of patients with folliculin mutations have radiographic evidence of pulmonary cysts, folliculin has been hypothesized to contribute to the development of emphysema.</p> <p>To determine whether folliculin sequence variants are risk factors for severe COPD, we genotyped seven previously reported Birt-Hogg-Dubé or familial spontaneous pneumothorax associated folliculin mutations in 152 severe COPD probands participating in the Boston Early-Onset COPD Study. We performed bidirectional resequencing of all 14 folliculin exons in a subset of 41 probands and subsequently genotyped four identified variants in an independent sample of345 COPD subjects from the National Emphysema Treatment Trial (cases) and 420 male smokers with normal lung function from the Normative Aging Study (controls).</p> <p>Results</p> <p>None of the seven previously reported Birt-Hogg-Dubé or familial spontaneous pneumothorax mutations were observed in the 152 severe, early-onset COPD probands. Exon resequencing identified 31 variants, including two non-synonymous polymorphisms and two common non-coding polymorphisms. No significant association was observed for any of these four variants with presence of COPD or emphysema-related phenotypes.</p> <p>Conclusion</p> <p>Genetic variation in folliculin does not appear to be a major risk factor for severe COPD. These data suggest that familial spontaneous pneumothorax and COPD have distinct genetic causes, despite some overlap in radiographic characteristics.</p

Deficiency of FLCN in Mouse Kidney Led to Development of Polycystic Kidneys and Renal Neoplasia

The Birt–Hogg–Dubé (BHD) disease is a genetic cancer syndrome. The responsible gene, BHD, has been identified by positional cloning and thought to be a novel tumor suppressor gene. BHD mutations cause many types of diseases including renal cell carcinomas, fibrofolliculomas, spontaneous pneumothorax, lung cysts, and colonic polyps/cancers. By combining Gateway Technology with the Ksp-Cre gene knockout system, we have developed a kidney-specific BHD knockout mouse model. BHDflox/flox/Ksp-Cre mice developed enlarged kidneys characterized by polycystic kidneys, hyperplasia, and cystic renal cell carcinoma. The affected BHDflox/flox/Ksp-Cre mice died of renal failure at approximate three weeks of age, having blood urea nitrogen levels over tenfold higher than those of BHD flox/+/Ksp-Cre and wild-type littermate controls. We further demonstrated that these phenotypes were caused by inactivation of BHD and subsequent activation of the mTOR pathway. Application of rapamycin, which inhibits mTOR activity, to the affected mice led to extended survival and inhibited further progression of cystogenesis. These results provide a correlation of kidney-targeted gene inactivation with renal carcinoma, and they suggest that the BHD product FLCN, functioning as a cyst and tumor suppressor, like other hamartoma syndrome–related proteins such as PTEN, LKB1, and TSC1/2, is a component of the mTOR pathway, constituting a novel FLCN-mTOR signaling branch that regulates cell growth/proliferation

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway

Genomewide Analysis of Inherited Variation Associated with Phosphorylation of PI3K/AKT/mTOR Signaling Proteins

While there exists a wealth of information about genetic influences on gene expression, less is known about how inherited variation influences the expression and post-translational modifications of proteins, especially those involved in intracellular signaling. The PI3K/AKT/mTOR signaling pathway contains several such proteins that have been implicated in a number of diseases, including a variety of cancers and some psychiatric disorders. To assess whether the activation of this pathway is influenced by genetic factors, we measured phosphorylated and total levels of three key proteins in the pathway (AKT1, p70S6K, 4E-BP1) by ELISA in 122 lymphoblastoid cell lines from 14 families. Interestingly, the phenotypes with the highest proportion of genetic influence were the ratios of phosphorylated to total protein for two of the pathway members: AKT1 and p70S6K. Genomewide linkage analysis suggested several loci of interest for these phenotypes, including a linkage peak for the AKT1 phenotype that contained the AKT1 gene on chromosome 14. Linkage peaks for the phosphorylated:total protein ratios of AKT1 and p70S6K also overlapped on chromosome 3. We selected and genotyped candidate genes from under the linkage peaks, and several statistically significant associations were found. One polymorphism in HSP90AA1 was associated with the ratio of phosphorylated to total AKT1, and polymorphisms in RAF1 and GRM7 were associated with the ratio of phosphorylated to total p70S6K. These findings, representing the first genomewide search for variants influencing human protein phosphorylation, provide useful information about the PI3K/AKT/mTOR pathway and serve as a valuable proof of concept for studies integrating human genomics and proteomics

Existence of a lens-shaped cluster of surfaces self-shrinking by mean curvature

We rigorously show the existence of a rotationally and centrally symmetric "lens-shaped" cluster of three surfaces, meeting at a smooth common circle, forming equal angles of 120 degrees, self-shrinking under the motion by mean curvature.Comment: 22 pages, 2 figure