Characterization of deletions in a cohort with cleft lip and palate

Orofacial clefts are relatively common birth defects with an estimated incidence of 1 in 700 live births that carry a significant public health burden. The causal factors of the development of orofacial clefts are complex; monogenic, multifactorial, non-syndromic and syndromic forms are all described in the literature. In almost all cases, genetics are thought to play a role in the etiology. In this study, we used an international cohort of 2,141 orofacial cleft patients and their families to find individuals with microdeletions, utilizing genome wide SNP chips for genetic analyses. We identified 94 individuals with deletions greater than 750 kb and compared them to registries with detailed phenotypic features and medical and family histories. We divided the cohort into distinct groupings: (1) individuals with a highly–penetrant orofacial cleft-associated deletion syndrome, (2) individuals with a lower-penetrant orofacial cleft microdeletion, (3) individuals with a large deletion encompassing likely contributory genes, and (4) individuals with a deletion of unknown significance. This cohort helps to support previous literature describing patients with orofacial clefts and microdeletions, along with presenting rarer associations, including an individual with a 12q21.1 deletion and cardiofaciocutaneous-like phenotype, an individual with a 7q36.3 deletion within the SHH regulator region with an absent nasal bone and cartilage, and an individual with a 3p26.3 deletion with a family history of polydactyly and intellectual disability. With our cohort, we help to define the range of associated features in individuals with microdeletions and orofacial clefts

C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity

Tissue-specific splicing factor gene expression signatures

The alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific splicing decisions most likely result from differences in the concentration and/or activity of these proteins. However, large-scale data to systematically address this issue have just recently started to become available. Here we show that splicing factor gene expression signatures can be identified that reflect cell type and tissue-specific patterns of alternative splicing. We used a computational approach to analyze microarray-based gene expression profiles of splicing factors from mouse, chimpanzee and human tissues. Our results show that brain and testis, the two tissues with highest levels of alternative splicing events, have the largest number of splicing factor genes that are most highly differentially expressed. We further identified SR protein kinases and small nuclear ribonucleoprotein particle (snRNP) proteins among the splicing factor genes that are most highly differentially expressed in a particular tissue. These results indicate the power of generating signature-based predictions as an initial computational approach into a global view of tissue-specific alternative splicing regulation

Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1. CONCLUSIONS/SIGNIFICANCE: We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis

Gender Minority Stress and Psychological Inflexibility on Psychological Distress in Tgnc Adults

Transgender and gender nonconforming (TGNC) adults in the United States have disproportionately higher rates of mental health issues compared to the general population. Testa et al.’s (2015) Gender Minority Stress and Resilience (GMSR) model explains this discrepancy as the result of factors unique to gender identity; however, empirical tests of the model have yielded inconclusive results. The present study examined the mediation pathways of the GMSR model while including a newly suggested mediating factor, psychological inflexibility (see Lloyd et al., 2019). It was hypothesized that gender minority proximal (internal) stress as well as psychological inflexibility would mediate the relationship between distal (external) stress factors and the outcomes of depression, anxiety, and suicidal ideation. A sample of 91 American TGNC adults completed an online survey, and their data were analyzed using a series of individual mediation pathways. Results were partially consistent with past studies, finding full support for internalized transphobia and psychological inflexibility as mediators and partial support for negative expectations and nondisclosure as mediators. This pattern of results may indicate that internal stress factors relating to the self (as opposed to other people) may be more effective in mediating the effects of distal stress factors and therefore should receive more emphasis in therapeutic treatments. However, the study’s generalizability was limited by its cross-sectional design, inability to test the entire conceptual model at once, and sample demographics

Chronic nonpulsatile blood flow. III. Effects of pump flow rate on oxygen transport and utilization in chronic nonpulsatile biventricular bypass

AbstractThe relationship between blood flow and oxygen transport was studied in five calves with chronic nonpulsatile biventricular bypass. Seven days was allowed for recovery from the effects of anesthesia and operation; the natural heart was then fibrillated. Pump flows were maintained at nominal rates of 90, 100, or 120 ml ·kg-1 · min for 1 week each, with the sequence varied from experiment to experiment. Venous and arterial blood samples were taken at rest for blood gas analysis. Serum lactate analysis was done twice a week, on the third and seventh days after each pump flow change. Serum catecholamine levels were assayed on the seventh day of each flow rate. Progressive exercise tests were also conducted during each test segment. Basal oxygen consumption of a 4-month-old calf was 6.3 ± 0.3 ml · kg-1 · min-1. The mixed venous oxygen tension decreased when pump flow rate was reduced (29.6 ± 1.0, 28.3 ± 1.2, and 23.8 ± 0.9 mm Hg at 120, 100, and 90 ml · kg-1 · min-1 of pump flow, respectively), and oxygen extraction increased linearly when pump flow rate was reduced. Hemoglobin concentration significantly affected oxygen extraction rate. Serum lactate concentration increased significantly at a 90 ml · kg-1 · min-1 perfusion compared with concentrations at other pump flow rates (7.81 ± 2.42 mEq/L at 90 ml · kg-1 · min-1 vs 0.71 ± 0.19 and 0.73 ± 0.81 mEq/L at 100 and 120 ml · kg-1 · min-1, respectively; p < 0.01, analysis of variance, Scheffe F test). Maximum oxygen extraction during exercise was 78%. These results suggest that a critical flow level between 90 and 100 ml · kg-1 · min-1 maintains oxidative metabolism in the calf with chronic nonpulsatile flow. The resulting oxygen delivery was slightly higher than that indicated in the literature. Maximal oxygen extraction was normal. (J THORAC CARDIOVASC SURG 1996;111:863-72

The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x.

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells