Variants In MTOR Research Articles

Compassionate use of everolimus for refractory epilepsy in a patient with MTOR mosaic mutation

The MTOR gene encodes the mechanistic target of rapamycin (mTOR), which is a core component of the PI3K-AKT-mTOR signaling pathway. Postzygotic MTOR variants result in various mosaic phenotypes, referred to in OMIM as Smith-Kinsgmore syndrome or focal cortical dysplasia. We report here the case of a patient, with an MTOR mosaic gain-of-function variant (p.Glu2419Lys) in the DNA of 41% skin cells, who received compassionate off-label treatment with everolimus for refractory epilepsy. This 12-year-old-girl presented with psychomotor regression, intractable seizures, hypopigmentation along Blaschko's lines (hypomelanosis of Ito), asymmetric regional body overgrowth, and ocular anomalies, as well as left cerebral hemispheric hypertrophy with some focal underlying migration disorders. In response to the patient's increasingly frequent epileptic seizures, everolimus was initiated (after approval from the hospital ethics committee) at 5 mg/day and progressively increased to 12.5 mg/day. After 5 months of close monitoring (including neuropsychological and electroencephalographic assessment), no decrease in seizure frequency was observed. Though the physiopathological rationale was good, no significant clinical response was noticed under everolimus treatment. A clinical trial would be needed to draw conclusions, but, because the phenotype is extremely rare, it would certainly need to be conducted on an international scale.

Molecular Dynamic Simulations for Mammalian Target of Rapamycin (mTOR) Variants to Characterize Potential Disruption of Porphyromonas gingivalis Induced Degradation

Gingipains are a protease secreted by Porphyromonas gingivalis, a major cause of adult periodontitis. P. gingivalis infections are associated with suppressed adaptive immunity and increased neuroinflammation among individuals with Alzheimer’s disease. Gingipains promote neuronal damage and are associated with immunoreactivity. The mammalian target of rapamycin (mTOR) is one protein potentially influenced by gingipain function. mTOR, a protein kinase, controls rates of protein synthesis and degradation, and may also play a significant role in the Alzheimer’s phenotype. Recent evidence suggests that P. gingivalis may inhibit mTOR signaling Alzheimer’s patients. We sought to investigate the role mTOR variants may play in the interaction with gingipains and Alzheimer’s disease. The objectives were to investigate the effect mTOR variants had on structure, function and interaction with gingipains. It was hypothesized that wild type mTOR may confer gingipain induced protection from Alzheimer’s disease, while variant phenotypes may contribute to disease progression. Molecular dynamic simulations were performed to measure hydrophobic collapse and the average movement in 10ns for each structure and compared to wild type. Results suggest structural alterations among the variants which may influence interactions with gingipains. These structural changes may lend evidence to suggest subsequent in vivo analyses investigating how P. gingivalis infection may influence mTOR signaling plays in the Alzheimer’s phenotype.

Dissecting the genetic basis of focal cortical dysplasia: a large cohort study

Genetic malformations of cortical development (MCDs), such as mild MCDs (mMCD), focal cortical dysplasia (FCD), and hemimegalencephaly (HME), are major causes of severe pediatric refractory epilepsies subjected to neurosurgery. FCD2 are characterized by neuropathological hallmarks that include enlarged dysmorphic neurons (DNs) and balloon cells (BCs). Here, we provide a comprehensive assessment of the contribution of germline and somatic variants in a large cohort of surgical MCD cases. We enrolled in a monocentric study 80 children with drug-resistant epilepsy and a postsurgical neuropathological diagnosis of mMCD, FCD1, FCD2, or HME. We performed targeted gene sequencing ( ≥ 2000X read depth) on matched blood–brain samples to search for low-allele frequency variants in mTOR pathway and FCD genes. We were able to elucidate 29% of mMCD/FCD1 patients and 63% of FCD2/HME patients. Somatic loss-of-function variants in the N-glycosylation pathway-associated SLC35A2 gene were found in mMCD/FCD1 cases. Somatic gain-of-function variants in MTOR and its activators (AKT3, PIK3CA, RHEB), as well as germline, somatic and two-hit loss-of-function variants in its repressors (DEPDC5, TSC1, TSC2) were found exclusively in FCD2/HME cases. We show that panel-negative FCD2 cases display strong pS6-immunostaining, stressing that all FCD2 are mTORopathies. Analysis of microdissected cells demonstrated that DNs and BCs carry the pathogenic variants. We further observed a correlation between the density of pathological cells and the variant-detection likelihood. Single-cell microdissection followed by sequencing of enriched pools of DNs unveiled a somatic second-hit loss-of-heterozygosity in a DEPDC5 germline case. In conclusion, this study indicates that mMCD/FCD1 and FCD2/HME are two distinct genetic entities: while all FCD2/HME are mosaic mTORopathies, mMCD/FCD1 are not caused by mTOR-pathway-hyperactivating variants, and ~ 30% of the cases are related to glycosylation defects. We provide a framework for efficient genetic testing in FCD/HME, linking neuropathology to genetic findings and emphasizing the usefulness of molecular evaluation in the pediatric epileptic neurosurgical population.

Association between single nucleotide polymorphisms in the PI3K/AKT/mTOR pathway and bladder cancer risk in a sample of Iranian population.

In the past few years several investigations have focused on the role of PI3K/AKT/mTOR pathway and its deregulations in different cancers. This study aimed to examine genetic polymorphisms of this pathway in bladder cancer (BC). In this case-control study, 235 patients with pathologically confirmed bladder cancer and 254 control subjects were examined. PIK3CA, AKT1 and mTOR variants were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The findings proposed that the PIK3CA rs6443624 SNP significantly decreased the risk of BC (OR=0.44, 95 % CI=0.30-0.65, p<0.0001 CA vs CC; OR=0.35, 95 % CI=0.16-0.78, p=0.0107, AA vs CC; OR=0.60, 95 % CI=0.46-0.79, p=0.0002, A vs T). The AKT1 rs2498801 variant is associated with a decreased risk of BC (OR=0.57, 95 % CI=0.39-0.82, p=0.003, AG vs AA; OR=0.74, 95 % CI=0.56-0.97, p=0.032, G vs A) while, AKT1 rs1130233 polymorphism considerably increased the risk of BC (OR=3.70, 95 % CI=2.52-5.43, p<0.0001, GA vs GG; OR=5.81, 95 % CI=1.53-21.97, p=0.010, AA vs GG; OR=2.71, 95 % CI=1.98-3.70, p<0.0001, A vs G). Additionally, mTOR rs2295080 variant notably increased the risk of BC (OR=2.25, 95 % CI=1.50-3.38, p<0.0001, GT vs GG; OR=4.75, 95 % CI=2.80-8.06, p<0.0001, TT vs GG; OR=3.10, 95 % CI=2.34-4.10, p<0.0001, T vs G). None of the other examined polymorphisms (AKT1 rs1130214, AKT1 rs3730358, mTOR rs1883965) revealed significant association with BC. In conclusion, our findings suggest that PIK3CA rs6443624, AKT1 rs2498801, AKT1 rs1130233, as well mTOR rs2295080 polymorphism may be related to bladder cancer development in a sample of Iranian population. Validation of our findings in larger sample sizes of different ethnicities would provide evidence on the role of variants of PI3K/AKT/mTOR pathway in developing BC.

Germline and somatic mutations in the MTOR gene in focal cortical dysplasia and epilepsy.

Objective:To assess the prevalence of somatic MTOR mutations in focal cortical dysplasia (FCD) and of germline MTOR mutations in a broad range of epilepsies.Methods:We collected 20 blood-brain paired samples from patients with FCD and searched for somatic variants using deep-targeted gene panel sequencing. Germline mutations in MTOR were assessed in a French research cohort of 93 probands with focal epilepsies and in a diagnostic Danish cohort of 245 patients with a broad range of epilepsies. Data sharing among collaborators allowed us to ascertain additional germline variants in MTOR.Results:We detected recurrent somatic variants (p.Ser2215Phe, p.Ser2215Tyr, and p.Leu1460Pro) in the MTOR gene in 37% of participants with FCD II and showed histologic evidence for activation of the mTORC1 signaling cascade in brain tissue. We further identified 5 novel de novo germline missense MTOR variants in 6 individuals with a variable phenotype from focal, and less frequently generalized, epilepsies without brain malformations, to macrocephaly, with or without moderate intellectual disability. In addition, an inherited variant was found in a mother–daughter pair with nonlesional autosomal dominant nocturnal frontal lobe epilepsy.Conclusions:Our data illustrate the increasingly important role of somatic mutations of the MTOR gene in FCD and germline mutations in the pathogenesis of focal epilepsy syndromes with and without brain malformation or macrocephaly.

A Second Generation, Multiple Myeloma-Specific, Targeted Sequencing Platform for Detecting Translocations, Copy Number Alterations, and Single Nucleotide Variants

Abstract Background: Multiple myeloma (MM) is a hematologic cancer characterized by a diversity of genetic lesions-translocations, copy number alterations (CNAs), and single nucleotide variants (SNVs). The prognostic value of translocations and of CNAs has been well established. Determining the clinical significance of SNVs, which are recurrently mutated at much lower frequencies, and how this significance is impacted by translocations and CNAs requires additional, large-scale correlative studies. Such studies can be facilitated by cost-effective targeted sequencing approaches. Hence, we designed a single-platform targeted sequencing approach capable of detecting all three variant types. Methods: We designed oligonucleotide probes complementary to the coding regions of 467 genes and to the IgH and MYC loci, allowing a probe to closely match at most 5 regions within the genome. Genes were selected if they were expressed in an independent RNA-seq MM data set and harbored germline SNP-filtered variants that: (1) occurred with frequency &gt;3%, (2) were clustered in hotspots, (3) occurred in recurrently mutated "cancer genes" (as annotated in COSMIC or MutSig), or (4) occurred in genes involved in DNA repair and/or B-cell biology. IgH and MYC tiling was unbiased (with respect to annotated features within the loci) and spanned from 50 kilobasepairs (kbps) upstream of both regions to 50 kbps downstream of IgH and 100 kbps downstream of MYC. Results: We performed targeted sequencing of 96 CD138-enriched samples derived from MM patients, as well as matched peripheral blood leukocyte normal controls. Sequencing depth (mean 107X) was commensurate with that of available exome sequencing data from these samples (mean 71X). Samples harbored a mean of 25 non-silent variants, including those in known MM-associated genes: NRAS (24%), KRAS (22%), FAM46C (17%), TP53 (10%), DIS3 (8%), and BRAF (3%). Variants detected by both platforms showed a strong correlation (r^2 = 0.8). The capture array detected activating, oncogenic variants in NRAS Q61K (n=3 patients) and KRAS G12C/D/R/V (n=5) that were not detected in exome data. Additionally, we found non-silent, capture-specific variants in MTOR (3%) and in two transcription-related genes that have been previously implicated in cancer: ZFHX4 (5%) and CHD3 (5%). To assess the potential role of deep subclonal variants and our ability to detect them, we performed additional sequencing (mean 565X) on six of the tumor/normal pairs. This revealed 14 manually-reviewed, non-silent variants that were not detected by the initial targeted sequencing. These had a mean variant allele frequency of 2.8% and included mutations in DNMT3A and FAM46C. At least one of these 14 variants occurred in five of the six re-sequenced samples. This highlights the importance of this additional depth, which will be used in future studies. Our approach successfully detected CNAs near expected frequencies, including hyperdiploidy (52%), del(13) (43%), and gain of 1q (35%). Similarly, it inferred IgH translocations at expected frequencies: t(4;14) (14%), t(6;14) (3%), t(11;14) (15%), and t(14;20) (1%). As expected, translocations occur predominantly within the IgH constant region, but also frequently 5' (i.e., telomeric) of the IGHM switch region, and occasionally within the V and D regions. We detected MYC -associated translocations, whose frequencies have been the subject of debate, at 10% (n=9 patients), with five involving IgH, three having both partners in or near MYC, and one having both types. Finally, our platform detected novel IgH translocations with partners near DERL3 (n=2), MYCN (n=1), and FLT3 (n=1). Additional evidence suggests that DERL3 and MYCN may be targets of IgH-induced overexpression: of 84 RNA-seq patient samples, six exhibited outlying expression of DERL3, including one sample in which we detected the translocation in corresponding DNA, and one exhibited outlying expression of MYCN. Conclusion: Our MM-specific targeted sequencing strategy is capable of detecting deeply subclonal SNVs, in addition to CNAs and IgH and MYC translocations. Though additional validation is required, particularly with respect to translocation detection, we anticipate that such technology will soon enable clinical testing on a single sequencing platform. Disclosures Vij: Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding.

Abstract 3280: Interaction between genetic variants in mTOR and body size on breast cancer risk in African-American and European-American women

Abstract Mammalian target of rapamycin (mTOR), a key protein in the phosphatidylinositol 3-kinase/Akt/mTOR (PI3K-AKT-mTOR) pathway promoting cell proliferation and angiogenesis, can be activated by overfeeding in animal models. However, epidemiological data on the mTOR pathway and breast cancer risk are very limited. We investigated the association of 13 single nucleotide polymorphisms (SNPs) in mTOR with breast cancer and potential effect modification of body size in the Women's Circle of Health Study (WCHS), a large case-control study in New York and New Jersey. Participants included 658 incident cases and 649 controls of European American background (EA) and 621 African-American (AA) cases and 744 matched controls, aged 20-75 y. Body size was approximated by body mass index (BMI) at recruitment measured by trained interviewers or obtained from self-report if the measurement was unavailable. Because allele frequencies varied between AAs and EAs, multivariable logistic regression was performed separately within groups, adjusting for age, family history, education, history of benign breast disease, cigarette smoking, proportion of European ancestry, and BMI (for main effects only). We observed higher risks of breast cancer associated with 3 SNPs in high linkage disequilibrium (r2 = 1) (rs2536 [3’-UTR], p=0.08; rs12116957 [intron], p=0.09; rs12125777 [intron], p=0.018) under a dominant genetic model among EA women. For rs12125777, variant allele carriers had a 71% higher breast cancer risk (odds ratio [OR]=1.71, 95% confidence interval (CI)=1.10-2.66) compared to non-carriers. The risk was more prominent among women who were overweight/obese (i.e., BMI ≥25 kg/m2; OR=3.34, 95% CI=1.67-6.69), compared to that among those with normal weight (OR=0.89, 95% CI=0.48-1.68; p-interaction=0.009). Compared to estrogen receptor (ER) positive cancers, the increased risk was more noticeable for ER negative cancers: the ORs comparing variant allele carriers to non-carriers were 2.68 (95% CI=1.24-5.79) overall, 8.49 (95% CI=2.85-25.3) among overweight/obese women, and 0.94 (95% CI=0.26-3.38) among normal-weight women; p-interaction=0.018. No association of genetic variants in mTOR with breast cancer was observed among AA women at a significant level of 0.1. Our findings suggest an etiologic role of overweight/obesity via promoting the mTOR pathway in breast carcinogenesis among EA women. Citation Format: Ting-Yuan D. Cheng, Jyoti Shankar, Gary Zirpoli, Song Yao, Michelle Roberts, Chi-Chen Hong, Elisa V. Bandera, Christine B. Ambrosone. Interaction between genetic variants in mTOR and body size on breast cancer risk in African-American and European-American women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3280. doi:10.1158/1538-7445.AM2014-3280

P0217 Potentially functional polymorphisms in the mTOR gene and gastric adenocarcinoma susceptibility in an eastern Chinese population

Background mTOR is an important intracellular mediator involved in the PI3K/AKT signalling pathway and a critical regulator of cell growth and proliferation in response to environmental and nutritional conditions. We evaluated potentially functional polymorphisms in the 5′ untranslated region (UTR) of the mTOR gene associated with the risk of gastric cancer. Methods In this case–control study with 1123 patients and 1113 healthy controls matched for age and gender, we genotyped four potentially functional single nucleotide polymorphisms (SNPs) located in 5′ UTR of the mTOR gene and evaluated their association with risk of gastric cancer. Findings In individual polymorphism analysis, the rs1034528 G > C was associated with 1.21-times increased risk of gastric cancer under a dominant model (adjusted odds ratio 1.21, 95% confidence interval [CI] 1.01–1.46). When the four polymorphisms were combined, the risk of gastric cancer increased in a dose-dependent manner as the number of risk genotypes increased (ptrend = 0.001). Patients with three or four risk genotypes had a significantly higher risk of gastric cancer than those with 0–2 risk genotypes (adjusted odds ratio 1.41, 95% CI 1.15–1.73). Stratification analysis indicated that the combined effect of risk genotypes was more evident in subgroups of older age (>59 years), men, smokers, non-drinkers, patients with gastric cardia adenocarcinoma, and those with clinical stage III and IV disease. Further analysis using mRNA expression data from the HapMap project suggested that both CG and CC carriers of rs1034528 G > C were associated with non-significantly decreased expression of mTOR mRNA in normal cell lines from 45 Chinese patients (p = 0.97 for CG carriers, p = 0.38 for CC carriers). Interpretation Functional mTOR variants may contribute to the risk of gastric cancer. Larger studies with different ethnic populations are warranted to validate these findings.