S-CAP extends pathogenicity prediction to genetic variants that affect RNA splicing.

Exome analysis of patients with a likely monogenic disease does not identify a causal variant in over half of cases. Splice-disrupting mutations make up the second largest class of known disease-causing mutations. Each individual (singleton) exome harbors over 500 rare variants of unknown significance (VUS) in the splicing region. The existing relevant pathogenicity prediction tools tackle all non-coding variants as one amorphic class and/or are not calibrated for the high sensitivity required for clinical use. Here we calibrate seven such tools and devise a novel tool called Splicing Clinically Applicable Pathogenicity prediction (S-CAP) that is over twice as powerful as all previous tools, removing 41% of patient VUS at 95% sensitivity. We show that S-CAP does this by using its own features and not via meta-prediction over previous tools, and that splicing pathogenicity prediction is distinct from predicting molecular splicing changes. S-CAP is an important step on the path to deriving non-coding causal diagnoses.

Inverting the Topology of a Transmembrane Protein by Regulating the Translocation of the First Transmembrane Helix.

TM4SF20 (transmembrane 4 L6 family 20) is a polytopic membrane protein that inhibits proteolytic processing of CREB3L1 (cAMP response element-binding protein 3-like 1), a membrane-bound transcription factor that blocks cell division and activates collagen synthesis. Here we report that ceramide stimulates CREB3L1 cleavage by inverting the orientation of TM4SF20 in membranes. In the absence of ceramide, the N terminus of the first transmembrane helix of TM4SF20 is inserted into the endoplasmic reticulum (ER) lumen. This translocation requires TRAM2 (translocating chain-associated membrane protein 2), a membrane protein containing a putative ceramide-interacting domain. In the presence of ceramide, the N terminus of the first transmembrane domain of TM4SF20 is exposed to cytosol. Consequently, the membrane topology of TM4SF20 is inverted, and this form of TM4SF20 stimulates CREB3L1 cleavage. In the presence of ceramide, translocation of TM4SF20 is TRAM2-independent. We designate this mechanism-causing regulated inversion of the membrane topology as "regulated alternative translocation."

Sufficient production of geranylgeraniol is required to maintain endotoxin tolerance in macrophages.

Endotoxin tolerance allows macrophages to produce large quantities of proinflammatory cytokines immediately after their contact with lipopolysaccharides (LPSs), but prevents their further production after repeated exposure to LPSs. While this response is known to prevent overproduction of proinflammatory cytokines, the mechanism through which endotoxin tolerance is established has not been identified. In the current study, we demonstrate that sufficient production of geranylgeraniol (GGOH) in macrophages is required to maintain endotoxin tolerance. We show that increased synthesis of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) protein following LPS treatment is required to produce enough GGOH to inhibit expression of Malt1, a protein known to stimulate expression of proinflammatory cytokines, in macrophages repeatedly exposed to LPSs. Depletion of GGOH caused by inhibition of HMGCR led to increased Malt1 expression in macrophages subjected to repeated exposure to LPSs. Consequently, endotoxin tolerance was impaired, and production of interleukin 1-ß and other proinflammatory cytokines was markedly elevated in these cells. These results suggest that insufficient production of GGOH in macrophages may cause autoinflammatory diseases.