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Autism spectrum disorder (ASD) has a complex genetic architecture involving contributions from both de novo and inherited variation. Few studies have been designed to address the role of rare inherited variation or its interaction with common polygenic risk in ASD. Here, we performed whole-genome sequencing of the largest cohort of multiplex families to date, consisting of 4,551 individuals in 1,004 families having two or more autistic children. Using this study design, we identify seven previously unrecognized ASD risk genes supported by a majority of rare inherited variants, finding support for a total of 74 genes in our cohort and a total of 152 genes after combined analysis with other studies. Autistic children from multiplex families demonstrate an increased burden of rare inherited protein-truncating variants in known ASD risk genes. We also find that ASD polygenic score (PGS) is overtransmitted from nonautistic parents to autistic children who also harbor rare inherited variants, consistent with combinatorial effects in the offspring, which may explain the reduced penetrance of these rare variants in parents. We also observe that in addition to social dysfunction, language delay is associated with ASD PGS overtransmission. These results are consistent with an additive complex genetic risk architecture of ASD involving rare and common variation and further suggest that language delay is a core biological feature of ASD.

Abstract KRAS is involved in the stability and expression of PD-L1. We investigated the expression of circulating mRNA (cmRNA) of KRAS4A and KRAS4B and the possible impact on progression-free survival (PFS) of patients with metastatic lung adenocarcinoma treated with immunotherapy. Patients without driver mutations undergoing Pembrolizumab (P) or P plus chemotherapy (PC) were prospectively accrued for liquid biopsy analysis of KRAS4A, KRAS4B, and PD-L1 cmRNA. Both KRAS isoforms were also studied for association with PD-L1 cmRNA. Of 56 patients, 28 received P and 28 PC. Patients with high levels of both KRAS isoforms showed significantly better PFS. The median PFS for KRAS4A was 29 months (95% CI 22–29 months) and KRAS4B 24 months (95% CI 13–29 months), respectively. The median PFS of patients with low levels of both isoforms was 12 months (95% CI 6–15 months for KRAS4A and 95% CI 5–20 months for KRAS4B). High KRAS4A retained a significant positive association with PFS in the multivariate model. An exploratory analysis in treatment subgroups found a positive association between high KRAS4A and KRAS4B with PFS in patients treated with P. PD-L1 cmRNA was significantly higher in patients with high KRAS isoforms levels and this effect was pronounced for high KRAS4A carriers. KRAS4A deserves further investigation as a potential marker for defining patients who may benefit the most from immune checkpoint inhibitors therapy and improving personalized cancer immunotherapeutic strategies.

Abstract Background Molecular predictors of bevacizumab efficacy in colorectal cancer have not been identified yet. Specific VEGF polymorphisms may affect gene transcription and therefore indirectly influence the efficacy of bevacizumab. Methods Genomic DNA of 111 consecutive metastatic colorectal cancer patients treated with first-line FOLFIRI plus bevacizumab was obtained from blood samples. VEGF -2578 C/A, -1498 C/T, + 405 C/G, + 936 C/T polymorphisms were analyzed by means of PCR-RFLP. DNA samples from 107 patients treated with FOLFIRI alone served as historical control group. The relation of VEGF polymorphisms with PFS, evaluated through Kaplan-Meier method and log-rank test, was the primary end-point. An interaction test with a Cox model has been performed in order to demonstrate the heterogeneity of the effect of VEGF -1498 C/T polymorphism between bevacizumab-and control group. Results In the bevacizumab-group median PFS and OS of patients carrying VEGF -1498 C/C, C/T and T/T allelic variants were, respectively, 12.8, 10.5, 7.5 months (p = 0.0046, log-rank test) and 27.3, 20.5, 18.6 months (p = 0.038, log-rank test). VEGF -1498 T/T genotype was associated with shorter PFS (HR = 2.13, [1.41-5.10], p = 0.0027). In the control group no significant association of VEGF -1498 C/T allelic variants and PFS or OS was found. Interaction between VEGF -1498 C/T variants and treatment effect suggested that the relation of VEGF -1498 T/T genotype with shorter PFS was caused by the effect of bevacizumab (p = 0.011). Other investigated polymorphisms did not affect the outcome. Conclusions These data suggest a possible role for VEGF -1498 C/T variants in predicting the efficacy of bevacizumab in the up-front treatment of metastatic colorectal cancer patients. A molecular tool for selecting subjects candidate to benefit from the anti-VEGF could be important for clinical practice. The retrospective and exploratory design of the present study, coupled with the non-randomized nature of the comparison between treated and untreated patients, imply that these results should be considered as hypothesis generators. A prospective validating trial is currently ongoing.

Abstract Background Inherited genetic factors such as E-cadherin (CDH1) promoter variants are believed to influence the risk towards sporadic diffuse gastric cancer (DGC). Recently, a new regulatory region essential for CDH1 transcription has been identified in CDH1 intron 2. Methods We genotyped all known polymorphisms located within conserved sequences of CDH1 intron 2 (rs10673765, rs9932686, rs1125557, rs9282650, rs9931853) in an Italian population consisting of 134 DGC cases and 100 healthy controls (55 patient relatives and 45 unrelated, matched individuals). The influence of individual variants on DGC risk was assessed using χ2-tests and logistic regression. The relative contribution of alleles was estimated by haplotype analysis. Results We observed a significant (p < 0.0004) association of the CDH1 163+37235G>A variant (rs1125557) with DGC risk. Odds ratios were 4.55 (95%CI = 2.09–9.93) and 1.38 (95%CI = 0.75–2.55) for AA and GA carriers, respectively. When adjusted for age, sex, smoking status, alcohol intake and H. pylori infection, the risk estimates remained largely significant for AA carriers. Haplotype analysis suggested the 163+37235A-allele contributes to disease risk independently of the other variants studied. Conclusion The CDH1 163+37235G>A polymorphism may represent a novel susceptibility variant for sporadic DGC if confirmed in other populations. Considering the broad expression of E-cadherin in epithelia, this exploratory study encourages further evaluation of the 163+37235A-allele as a susceptibility variant in other carcinomas.

Regulation of embryonic diapause, dormancy that interrupts the tight connection between developmental stage and time, is still poorly understood. Here, we characterize the transcriptional and metabolite profiles of mouse diapause embryos and identify unique gene expression and metabolic signatures with activated lipolysis, glycolysis, and metabolic pathways regulated by AMPK. Lipolysis is increased due to mTORC2 repression, increasing fatty acids to support cell survival. We further show that starvation in pre-implantation ICM-derived mouse ESCs induces a reversible dormant state, transcriptionally mimicking the in vivo diapause stage. During starvation, Lkb1, an upstream kinase of AMPK, represses mTOR, which induces a reversible glycolytic and epigenetically H4K16Ac-negative, diapause-like state. Diapause furthermore activates expression of glutamine transporters SLC38A1/2. We show by genetic and small molecule inhibitors that glutamine transporters are essential for the H4K16Ac-negative, diapause state. These data suggest that mTORC1/2 inhibition, regulated by amino acid levels, is causal for diapause metabolism and epigenetic state.

We performed RNA sequencing on 40,000 cells to create a high-resolution single-cell gene expression atlas of developing human cortex, providing the first single-cell characterization of previously uncharacterized cell types, including human subplate neurons, comparisons with bulk tissue, and systematic analyses of technical factors. These data permit deconvolution of regulatory networks connecting regulatory elements and transcriptional drivers to single-cell gene expression programs, significantly extending our understanding of human neurogenesis, cortical evolution, and the cellular basis of neuropsychiatric disease. We tie cell-cycle progression with early cell fate decisions during neurogenesis, demonstrating that differentiation occurs on a transcriptomic continuum; rather than only expressing a few transcription factors that drive cell fates, differentiating cells express broad, mixed cell-type transcriptomes before telophase. By mapping neuropsychiatric disease genes to cell types, we implicate dysregulation of specific cell types in ASD, ID, and epilepsy. We developed CoDEx, an online portal to facilitate data access and browsing.

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

Refinement of genetic variants within the major histocompatibility complex reveals three distinct genetic influences on schizophrenia risk and sheds light on the disease’s neurobiology.

Infantile spasms (IS) and Lennox-Gastaut syndrome (LGS) are epileptic encephalopathies characterized by early onset, intractable seizures, and poor developmental outcomes. De novo sequence mutations and copy number variants (CNVs) are causative in a subset of cases. We used exome sequence data in 349 trios with IS or LGS to identify putative de novo CNVs. We confirm 18 de novo CNVs in 17 patients (4.8%), 10 of which are likely pathogenic, giving a firm genetic diagnosis for 2.9% of patients. Confirmation of exome-predicted CNVs by array-based methods is still required due to false-positive rates of prediction algorithms. Our exome-based results are consistent with recent array-based studies in similar cohorts and highlight novel candidate genes for IS and LGS.

Altered cellular energetic metabolism has recently emerged as important feature of neoplastic cells. Indeed, interfering with cancer cell metabolism might represent a suitable therapeutic strategy. In this study, we aimed to assess glucose metabolism activation in human lymphomas and evaluate how metformin can exert its action on lymphoma cells. We studied a large series of human lymphomas (N = 252) and an in vitro model of Burkitt lymphoma (BL) cells. We combined molecular biology techniques, including global gene expression profiling (GEP) analysis, quantitative PCR (qPCR) and Western blotting, and biochemical assays, aimed to assess pentose phosphate pathway, tricarboxylic acid (TCA) cycle, and aerobic glycolysis rates. We found that glucose metabolism is overall enhanced in most lymphoma subtypes, based on gene expression profiling (GEP), with general shift to aerobic glycolysis. By contrast, normal B cells only showed an overall increase in glucose usage during germinal center transition. Interestingly, not only highly proliferating aggressive lymphomas but also indolent ones, like marginal zone lymphomas, showed the phenomenon. Consistently, genes involved in glycolysis were confirmed to be overexpressed in BL cells by qPCR. Biochemical assays showed that while aerobic glycolysis is increased, TCA cycle is reduced. Finally, we showed that metformin can induce cell death in BL cells by stressing cellular metabolism through the induction of GLUT1, PKM2, and LDHA. In conclusion, we unveiled glucose metabolism abnormalities in human lymphomas and characterized the mechanism of action of metformin in Burkitt lymphoma model.

Tyrosine kinase inhibitors (TKIs) have radically changed the outcome of chronic myeloid leukemia (CML) patients in the last 20 years. Moreover, the advent of second generation TKIs, namely nilotinib and dasatinib, have largely increased the number of CML patients achieving deep and sustained molecular responses. However, the possible mechanisms capable of influencing the maintenance of the long-term molecular response are not yet fully known and understood. In this light, polymorphisms in MDR-ABC transporters may influence the efficacy and safety of TKIs. In this study, we examined seven single nucleotide polymorphisms (SNPs) in four ABC transporter genes: ABCC1 rs212090 (5463T>A), ABCC2 rs3740066 (3972C>T), ABCC2 rs4148386 G>A, ABCC2 rs1885301 (1549G>A), ABCG2 rs2231137 (34G>A), ABCG2 rs2231142 G>C, ABCB1 rs1045642 (3435C>T), to determine their effect on the achievement and/or loss of molecular response in 90 CML patients treated with nilotinib. We found that ABCC2 rs3740066 CC and CT as well as the ABCB1 rs1045642 TT genotypes correlated with a higher probability to achieve MR3 in a shorter time (p=0.02, p=0.004, and p=0.01), whereas ABCG2 rs2231137 GG was associated with lower probability of MR3 achievement (p=0.005). Moreover, ABCC2 rs3740066 CC genotype, the ABCB1 rs1045642 CC and TT genotypes were positively correlated with MR4 achievement (p=0.02, p=0.007, and p=0.003). We then generated a predictive model incorporating the information of four genotypes, to evaluate the combined effect of the SNPs. The combination of SNPs present in the model affected the probability and the time to molecular response. This model had a high prognostic significance for both MR3 and MR4 (p=0.005 and p=0.008, respectively). Finally, we found ABCG2 rs2231142 GG genotype to be associated with a decrease risk of MR3 loss. In conclusion, MDR-transporters SNPs may significantly affect the achievement and loss of molecular response in CML patients treated with nilotinib.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Output-only methods are widely used to characterize the dynamic behavior of very diverse structures. However, their application to floating structures may be limited due to their strong nonlinear behavior. Therefore, since there is very little experience on the application of these experimental tools to these very peculiar structures, it is very important to develop studies, either based on numerical simulations or on real experimental data, to better understand their potential and limitations. In an initial phase, the use of numerical simulations permits a better control of all the involved variables. In this work, the Covariance-driven Stochastic Subspace Identification (SSI-COV) algorithm is applied to numerically simulated data of two different solutions to Floating Offshore Wind Turbines (FOWT) and for its capability of tracking the rigid body motion modal properties and susceptibility to different modeling restrictions and environmental conditions tested. The feasibility of applying the methods in an automated fashion in the processing of a large number of datasets is also evaluated. While the structure natural frequencies were consistently obtained from all the simulations, some difficulties were observed in the estimation of the mode shape components in the most changeling scenarios. The estimated modal damping coefficients were in good agreement with the expected results. From all the results, it can be concluded that output-only methods are capable of characterizing the dynamic behavior of a floating structure, even in the context of continuous dynamic monitoring using automated tracking of the modal properties, and should now be tested under uncontrolled environmental loads.