Early diagnosis of a newborn with tuberous sclerosis caused by a genetic mutation

Objective Tuberous sclerosis (TSC) is an autosomal dominant disorder, often detected during childhood. We present the results of genetic testing in a newborn with suspected TSC. Methods A newborn with no specific clinical manifestations of TSC showed evidence of TSC on magnetic resonance imaging and echocardiography. Next-generation sequencing (NGS) and multiple ligation-dependent probe amplification (MLPA) of the TSC1 and TSC2 gene exons were carried out to confirm the diagnosis. Results The results of MLPA were negative, but NGS showed a heterozygous mutation in the TSC1 gene comprising insertion of a T residue at c.2165 (exon 17) to c.2166 (exon 17), indicating a loss of function mutation. These results were verified by Sanger sequencing. This genetic change was present in the newborn but the parental genotypes were wild-type, indicating a de novo mutation. Conclusions In this case, a case of TSC caused by a heterozygous mutation in the TSC1 gene was confirmed by NGS sequencing. This indicates the suitability of genetic testing for the early diagnosis of clinically rare and difficult-to-diagnose diseases, to guide clinical treatment.

Molecular-Genetic Characteristics and Genotype-Phenotype Correlations in Bulgarian Patients with Tuberous Sclerosis Complex

Objective The aim of the study was to determine the molecular-genetic characteristics of the autosomal dominant systematic disorder Tuberous Sclerosis Complex (TSC1 and TSC2) in Bulgarian patients and to derive some genotype-phenotype correlations. Material and Methods In total 42 patients/families with suspected clinical diagnosis of TSC were analyzed. We used direct sequencing and MLPA for the TSC1 and TSC2 gene analysis. Results In 38 families (90.5%) we confirmed the suspected clinical diagnosis – 15 with TSC1 (35.7%) and 23 (54.8%) with TSC2. In 4 families (9.5%) pathogenic variants were not found. In all 38 patients with proven diagnosis of TSC, we found 38 different mutations, 15 of which (39%) were detected for the first time by our research group. The mutation “hotspots“ in TSC1 gene are exons 9, 15, 17 and 18, where 73% of the TSC1 mutations are localized, while the TSC2 gene mutation “hotspots“ are exons 13 and 34, with 22% of the mutations situated there. In the TSC2 patients the common clinical findings include subcortical tubers, epilepsy with generalized tonic-clonic seizures, subependymal giant cell astrocytoma, facial angiofibromas, ungual fibromas, cardiac rhabdomyomas and renal angiomyolipomas, while in the TSC1 patients typically cortical tubers, cortical dysplasia and subependymal nodules were registered. In patients with aggressive frameshift and nonsense TSC1 and TSC2 mutations commonly hypomelanotic macules, cortical and subcortical tubers, cortical dysplasia, epilepsy with different types of seizures were found. Renal angiomyolipomas and cysts were detected mainly in patients with large deletions. Shagreen patches and intellectual disability were typically registered in equal degree in patients with frameshift, nonsense and missense mutations. Conclusion Although some genotype-phenotype correlations were derived, there is a great inter- and intrafamilial clinical variability in TSC, so it is impossible to predict the course of the disease on the basis of the detected molecular defect. The obtained results helped us to develop a diagnostic algorithm for proper molecular-genetic diagnostics which permits adequate genetic counseling, prophylaxis and treatment in the affected TSC families.

VEGFR Inhibitors for Uterine Metastatic Perivascular Epithelioid Tumors (PEComa) Resistant to mTOR Inhibitors. A Case Report and Review of Literature

Uterine perivascular epithelioid cell tumors (PEComas) are rare neoplasms. PI3K/AKT/mTOR pathway upregulation is critical for their pathogenesis and is often associated with TSC1/TSC2 inactivation. Although first line mTOR inhibitors are an effective treatment, metastatic PEComas eventually progress. A 53-year-old woman presented a 4-month history of post-menopausal vaginal bleeding. Clinical and radiological examination detected a uterine mass and a single S1 bone lesion. The patient underwent a radical hysterectomy and bone biopsy. The anatomopathological evaluation concluded to an oligo-metastatic uterine PEComa. The tumor harbored a heterozygous deletion of 9q34 that contains the TSC1 gene. Concerning the primary lesion, the resection was complete and the single bone metastasis was treated with radiotherapy. Three months later, the patient presented bone, lung and subcutaneous metastatic progression. An everolimus and denosumab treatment was initiated. After 2 years of treatment, a clinically significant bone, lung and subcutaneous progression was detected. Following a literature review of the possible therapeutic options, we initiated a second line treatment by pazopanib. This treatment resulted in regression of the subcutaneous lesions and stability of lung and bone metastases. In this challenging, rare setting, our report suggests single agent, anti-angiogenic, tyrosine kinase inhibitor to be effective as second line treatment of metastatic uterine PEComa progressing on mTOR inhibitors.

Neurobiologia do autismo infantil

Objetivo: discutir as características neurobiológicas do Transtorno do Espectro Autista (TEA) em crianças. Metodologia: foi realizada uma revisão narrativa com artigos dos últimos 10 anos (2010-2020), nas bases de dados PsycINFO, Medline, PubMed, SciELO, LILACS e Periódicos CAPES. Utilizaram-se como descritores “Autismo Infantil”, “Transtorno Autista”, “Infância”, “Ciclo Vital”, “Neurodesenvolvimento Infantil”, “Neurobiologia”, “Neurociências”, “Neuroanatomia”, “Neurofisiologia”, “Transtorno do Espectro Autista” e o Booleano “AND”. Todos estes materiais foram lidos na íntegra, categorizados e, posteriormente, analisados criticamente. Resultados e discussão: em indivíduos com TEA, há uma diminuição da conectividade entre o córtex pré-frontal medial e o córtex cingulado posterior; esta diminuição está relacionada a uma função social inferior quando comparada com crianças normais. Alteração semelhante foi encontrada em relação à rede frontoparietal relacionando-se aos comportamentos repetitivos observados no transtorno. Há, também, a hipótese relacionada a uma disfunção cerebelar e, mais especificamente, que a deleção da Proteína 1 do Complexo Esclerose Tuberosa (TSC1) – gene que codifica a proteína hamartina – nas células de Purkinje, reflita nos déficits sociais e comportamentais em indivíduos com TEA. Além disso, é sugerido que o fenótipo do transtorno esteja associado a uma perturbação nos circuitos que conectam o cerebelo ao córtex pré-frontal medial e, também, a uma maior ativação desta última região. Conclusão: ainda não se tem uma conclusão satisfatória e patognomônica da neurobiologia do TEA em crianças; as alterações neurofuncionais mais comuns no TEA estão relacionadas às redes cerebelares e às Células de Purkinje.

Methylation of Hypothalamic Tsc1-mTOR Signaling in Regulation of Obesity and Obesity Resistance

The Tsc1-mTOR signaling pathway is often related to obesity, and epigenetic modification may lead to expression changes of obesity-related gene. Therefore, we aim to investigate the methylation of the Tsc1-mTOR signaling pathway in regulation of obesity susceptibility. Wistar rats were fed a normal diet or a high-fat diet to develop animal models. Protein and mRNA expression levels of Tsc1-mTOR signaling in the hypothalamus were determined by Western blot and quantitative real-time PCR. Methylation of Tsc1 gene promoter was detected by bisulfite genomic sequence. Both mRNA and protein expression levels of Tsc1 in DIO group hypothalamus were lower; mTOR and its downstream targets S6K1, 4EBP1, and S6 protein expression levels were higher than those of the DIO-R group and the chow group. The Tsc1 gene promoter methylation rate in the hypothalamus was 92.05 ± 3.07 % in the DIO group, 87.27 ± 1.91 % in the DIO-R group, and 88.18 % ± 3.20 % in the chow group, respectively, with significantly higher levels in the DIO group. Both the expression levels of Tsc1 gene promoter methylation and Tsc1-mTOR signaling pathway in the hypothalamus of DIO rats and DIO-R rats are different. These findings may shed light on the potential mechanism for the differentiation of obesity susceptibility.

NFB-16. mTOROPATHIES AND SUBEPENDYMAL GIANT CELL ASTROCYTOMAS: PREDICTIVE VALUE OF GERMINAL TSC1/2 MUTATIONS SCREENING IN FAMILIAL CASES

mTOR controls several important aspects of cell function particularly in the nervous system. Its hyperactivation has been involved in tuberous sclerosis complex (TSC) and other mTORopathies as well as drug-resistant epilepsy. Mutations in TSC1 and TSC2 genes cause loss of normal inhibition of mTORC1 complex, leading to cell overgrowth and disruptions in synaptogenesis. Many children and adults with TSC harbour neurologic defects especially subependymal giant cell astrocytomas (SEGAs) in the brain. Here, we have performed mutational analysis followed by a genetic counselling for a Tunisian family from Sfax town harboring epileptic seizures associated to a neurocutaneous disorder. Index cases were referred for renal angiolipomas (RAL) associated to seizures crisis and were diagnosed as having TSC. The first 26-year-old patient complained of epilepsy since the age of 22 with left temporal crisis related to cortical tubers near the Heschl’s gyrus. His brother, a 36-year-old man presented more severe epileptic crisis (since 15 years-old), multiples RAL, subependymal nodules, and a rapid evolution of his mTORopathy with tumoral progression of his renal and central nerve lesions: renal cell carcinoma and SEGAs. TSC1 gene mutation screening showed heterozygous two bp deletion at codons 213 and 214 of exon 5. SEGAs are rare, low-grade glioneuronal brain tumors that occur almost exclusively in TSC patients but can lead to nervous complications. We showed through this report, the predictive value of germinal TSC mutations screening in familial cases, because early recognition of the molecular defect may lead to appropriate management of the tumoral progression.

Novel MicroRNA Binding Site SNPs and the Risk of Clear Cell Renal Cell Carcinoma (ccRCC): A Case-Control Study

Background: Renal cell carcinoma represents 3% of all adult malignancies. MicroRNAs exhibit specific functions in various biological processes through their interaction with cellular mRNA involved in apoptosis and cell cycle control. Recent studies have reported the potential association of single-nucleotide polymorphisms (SNPs) in miRNA-binding sites of VHL-HIF1α pathway genes with renal cancer development and progression. Objective: The objective of this study is to investigate SNPs invoking an alteration in the nature of interaction with miRNA binding sites of VHL-HIF1α pathway genes. Patients & Methods: Total 450 cases of histologically and clinically verified ccRCC and 490 controls were included in our study. Genotyping was performed using a TaqMan PCR allelic discrimination method. Kaplan-Meier method of statistical analysis was implemented to analyze the overall patient survival rate. Results : Polymorphism rs10491534 in TSC1 gene was significantly associated with risk of developing advanced ccRCC. Allele G of rs1642742 in VHL gene was significantly prevalent in ccRCC compared with control group aged 55 and older (OR = 1.5566; CI [1.1532-2.1019]). Results from the dominant model combining individuals with AG or AA genotype showed that the A allele bearers of CDCP1 rs6773576 exhibited higher risk of death compared to GG carriers (HR 3.93, 95% CI 1.76-17.21, log-rank P = 0.0033). Conclusion: The present study delineated the association of miRNA binding site variants in VHL-HIF1α pathway genes with the ccRCC risk, which may affect clinical outcome.

Mutation Spectrum of Tuberous Sclerosis Complex Patients in Indian Population

Tuberous sclerosis complex (TSC) is a multiorgan disorder characterized by formation of hamartomas and broad phenotypic spectrum including seizures, mental retardation, renal dysfunction, skin manifestations and brain tubers. It is inherited in an autosomal dominant pattern, caused due to mutation in either TSC1 or TSC2 genes. Seizures are one of the major presenting symptoms of TSC that helps in early diagnosis. The present study describes the mutation spectrum in TSC1 and TSC2 genes in TSC patients and their association with neurocognitive-behavioral phenotypes. Ninety-eight TSC patients were enrolled for TSC genetic testing after detailed clinical and neurobehavioral assessment. Large genomic rearrangement testing was performed by multiplex ligation-dependent probe amplification (MLPA) technique for all cases and Sanger sequencing was performed for MLPA negative cases. Large rearrangements were identified in approximately 1% in TSC1 and 14.3% in TSC2 genes. The present study observed the presence of duplications in two (2%) cases, both involving TSC2/PKD1 contiguous genes which to the best of our knowledge is reported for the first time. 8.1% of small variants were identified in the TSC1 gene and 85.7% in TSC2 gene, out of which 23 were novel variations and no variants were found in six (6.1%) cases. This study provides a representative picture of the distribution of variants in the TSC1 and TSC2 genes in Indian population along with the detailed assessment of neurological symptoms. This is the largest cohort study from India providing an overview of comprehensive clinical and molecular spectrum.

Mesenchymal Stem Cell-Specific and Preosteoblast-Specific Ablation of TSC1 in Mice Lead to Severe and Slight Spinal Dysplasia, Respectively

Background. TSC1-related signaling plays a pivotal role in intramembranous and endochondral ossification processes during skeletogenesis. This study was aimed at determining the significance of the TSC1 gene at different stages of spinal development. Materials and Methods. TSC1-floxed mice (TSC1flox/flox) were crossed with Prrx1-Cre or BGLAP-Cre transgenic mice or mesenchymal stem cell- and osteoblast-specific TSC1-deficient mice, respectively. Somatic and vertebral differences between WT and Prrx1-TSC1 null mice were examined at 4 weeks after birth. Results. No apparent body size abnormalities were apparent in newborn and 4-week- to 2-month-old mice with BGLAP-Cre driver-depleted TSC1. Vertebral and intervertebral discs displayed strong dysplasia in Prrx1-TSC1 null mice. In contrast, vertebrae were only slightly affected, and intervertebral discs from skeletal preparations displayed no apparent changes in BGLAP-TSC1 null mice. Conclusion. Our data suggest that the TSC1 gene is crucial for endochondral ossification during postnatal spine development but plays discriminative roles at different stages. Mesenchymal stem cell-specific ablation of TSC1 led to severe spinal dysplasia at early stages of endochondral ossification while osteoblast-specific deletion of TSC1 affected vertebrae slightly and had no detectable effects on intervertebral discs.