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Background: Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. The phenomenon becomes very common in oldest-old individuals, in whom the implications of CHIP are not well defined. Here we investigate the relationships between CHIP and associated pathologies in people aged >80 years. Methods: We performed a mutational screening in 1794 oldest-old individuals enrolled in two population-based studies (“Health_&_Anemia” and “Monzino_80+”). Findings: Clonal mutations were observed in one third of oldest-old individuals and were associated with reduced survival and increased risk of cancers. Mutations in JAK2 and splicing genes (SF3B1, SRSF2, U2AF1, ZRSR2), multiple mutations (DNMT3A, TET2, ASXL1) combined with additional genetic lesions) and variant allele frequency ≥0.14 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1 or JAK2 (most occurring as single lesion) were associated with coronary heart disease and rheumatoid arthritis. Cytopenia (most including anemia) was a common finding in oldest-old population (>20%), the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly-specific mutation patterns was associated with a myeloid neoplasms-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of oldest-old subjects with cytopenia had presumptive evidence of myeloid neoplasm. Interpretation: Specific mutational patterns define different risk of developing cancers vs. inflammatory-associated diseases in oldest-old population. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms. Trial Registration: (ClinicalTrials.gov number:NCT03907553) Funding: Supported by European Union; Cariplo Foundation, Italy; Italian Association for Cancer Research; Italian Ministry of Health and Research. Declaration of Interests: The Authors declare no competing interests. Ethics Approval Statement: Study procedures were in accordance with the Declaration of Helsinki. Ethics Committees of Humanitas Research Hospital and Mario Negri Pharmacological Institute, Milan Italy approved the study. Written informed consent was obtained prior to blood sampling.

PHOX2B and its paralogue gene PHOX2A are two homeodomain proteins in the network regulating the development of autonomic ganglia that have been associated with the pathogenesis of neuroblastoma (NB), because of their over-expression in different NB cell lines and tumour samples. We used the SK-N-BE(2)C cell line to show that all-trans retinoic acid (ATRA), a drug that is widely used to inhibit growth and induce differentiation in NBs, regulates both PHOX2A and PHOX2B expression, albeit by means of different mechanisms: it up-regulates PHOX2A and down-regulates PHOX2B. Both mechanisms act at transcriptional level, but prolonged ATRA treatment selectively degrades the PHOX2A protein, whereas the corresponding mRNA remains up-regulated. Further, we show that PHOX2A is capable of modulating PHOX2B expression, but this mechanism is not involved in the PHOX2B down-regulation induced by retinoic acid. Our findings demonstrate that PHOX2A expression is finely controlled during retinoic acid differentiation and this, together with PHOX2B down-regulation, reinforces the idea that they may be useful biomarkers for NB staging, prognosis and treatment decision making., Highlights • RA regulates both PHOX2A and PHOX2B expression by means of different mechanisms. • PHOX2A and PHOX2B expression must be controlled during RA induced differentiation. • PHOX2A modulates PHOX2B, but does not mediate the RA-induced PHOX2B down-regulation. • RA induces PHOX2A protein degradation by means of the ubiquitin-proteasome system.

Mutations in the CACNA1A gene, which encodes the pore-forming α1A subunit of the CaV2.1 voltage-gated calcium channel, cause a number of human neurologic diseases including familial hemiplegic migraine. We have analyzed the functional impact of the E1015K amino acid substitution located in the "synprint" domain of the α1A subunit. This variant was identified in two families with hemiplegic migraine and in one patient with migraine with aura. The wild type (WT) and the E1015K forms of the GFP-tagged α1A subunit were expressed in cultured hippocampal neurons and HEK cells to understand the role of the variant in the transport activity and physiology of CaV2.1. The E1015K variant does not alter CaV2.1 protein expression, and its transport to the cell surface and synaptic terminals is similar to that observed for WT channels. Electrophysiological data demonstrated that E1015K channels have increased current density and significantly altered inactivation properties compared with WT. Furthermore, the SNARE proteins syntaxin 1A and SNAP-25 were unable to modulate voltage-dependent inactivation of E1015K channels. Overall, our findings describe a genetic variant in the synprint site of the CaV2.1 channel which is characterized by a gain-of-function and associated with both hemiplegic migraine and migraine with aura in patients.