Background: Long QT syndrome (LQTS) is the first described and most common inherited arrhythmia. Over the last 25 years, multiple genes have been reported to cause this condition and are routinely tested in patients. Because of dramatic changes in our understanding of human genetic variation, reappraisal of reported genetic causes for LQTS is required. Methods: Utilizing an evidence-based framework, 3 gene curation teams blinded to each other’s work scored the level of evidence for 17 genes reported to cause LQTS. A Clinical Domain Channelopathy Working Group provided a final classification of these genes for causation of LQTS after assessment of the evidence scored by the independent curation teams. Results: Of 17 genes reported as being causative for LQTS, 9 ( AKAP9, ANK2, CAV3, KCNE1, KCNE2, KCNJ2, KCNJ5, SCN4B, SNTA1 ) were classified as having limited or disputed evidence as LQTS-causative genes. Only 3 genes ( KCNQ1, KCNH2, SCN5A ) were curated as definitive genes for typical LQTS. Another 4 genes ( CALM1, CALM2, CALM3, TRDN ) were found to have strong or definitive evidence for causality in LQTS with atypical features, including neonatal atrioventricular block. The remaining gene ( CACNA1C ) had moderate level evidence for causing LQTS. Conclusions: More than half of the genes reported as causing LQTS have limited or disputed evidence to support their disease causation. Genetic variants in these genes should not be used for clinical decision-making, unless accompanied by new and sufficient genetic evidence. The findings of insufficient evidence to support gene-disease associations may extend to other disciplines of medicine and warrants a contemporary evidence-based evaluation for previously reported disease-causing genes to ensure their appropriate use in precision medicine.

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Hedgehog pathway regulates tissue patterning and cell proliferation. Gli1 transcription factor is the major effector of Hedgehog signaling and its deregulation is often associated to medulloblastoma formation. Proteolytic processes represent a critical mechanism by which this pathway is turned off. Here, we characterize the regulation of an ubiquitin-mediated mechanism of Gli1 degradation, promoted by the coordinated action of the E3 ligase Itch and the adaptor protein Numb. We show that Numb activates the catalytic activity of Itch, releasing it from an inhibitory intramolecular interaction between its homologous to E6-AP C-terminus and WW domains. The consequent activation of Itch, together with the recruitment of Gli1 through direct binding with Numb, allows Gli1 to enter into the complex, resulting in Gli1 ubiquitination and degradation. This process is mediated by a novel Itch-dependent degron, composed of a combination of two PPXYs and a phospho-serine/proline motifs, localized in Gli1 C-terminal region, indicating the role of two different WW docking sites in Gli1 ubiquitination. Remarkably, Gli1 protein mutated in these modules is no longer regulated by Itch and Numb, and determines enhanced Gli1-dependent medulloblastoma growth, migration and invasion abilities, as well as in vitro transforming activity. Our data reveal a novel mechanism of regulation of Gli1 stability and function, which influences Hedgehog/Gli1 oncogenic potential. Oncogene (2011) 30, 65-76; doi: 10.1038/onc.2010.394; published online 6 September 201

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