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Background Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. Methods and findings We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo, in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). Interpretation Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. Fund This work was financially supported by grants from "Ministero della Salute Italiano"(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from "Associazione Italiana Cancro" (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.

Multigenerational diabetes of adulthood is a mostly overlooked entity, simplistically lumped into the large pool of type 2 diabetes. The general aim of our research in the past few years is to unravel the genetic causes of this form of diabetes. Identifying among families with multigenerational diabetes those who carry mutations in known monogenic diabetes genes is the first step to then allow us to concentrate on remaining pedigrees in which to unravel new diabetes genes. Targeted next-generation sequencing of 27 monogenic diabetes genes was carried out in 55 family probands and identified mutations verified among their relatives by Sanger sequencing. Nine variants (in eight probands) survived our filtering/prioritization strategy. After likelihood of causality assessment by established guidelines, six variants were classified as “pathogenetic/likely pathogenetic” and two as “of uncertain significance.” Combining present results with our previous data on the six genes causing the most common forms of maturity-onset diabetes of the young allows us to infer that 23.6% of families with multigenerational diabetes of adulthood carry mutations in known monogenic diabetes genes. Our findings indicate that the genetic background of hyperglycemia is unrecognized in the vast majority of families with multigenerational diabetes of adulthood. These families now become the object of further research aimed at unraveling new diabetes genes.

Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. Here we show how tumor-initiating, human CRC stem-like cells (CCSCs) can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, circulating and metastatic CCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. Funding Statement: This work was supported by grants from AIRC (IG-14368) and MIUR (GR-2011-02351534, RC1703IC36). Declaration of Interests: The authors declare no competing interests. A.L. Vescovi has ownership interest in Hyperstem SA. Ethics Approval Statement: Fresh human colorectal cancer and blood specimens were collected from consenting patients at IRCCS “Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori” (IRST) and IRCCS “Casa Sollievo della Sofferenza”. All samples were obtained under protocols approved by the Research Ethics Boards (B063 and 94/CE). All animal procedures were carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committees (IACUCs) at the University of Milan-Bicocca.

Oral-facial-digital type VI syndrome (OFDVI) is a rare phenotype of Joubert syndrome (JS). Recently, C5orf42 was suggested as the major OFDVI gene, being mutated in 9 of 11 families (82 %). We sequenced C5orf42 in 313 JS probands and identified mutations in 28 (8.9 %), most with a phenotype of pure JS. Only 2 out of 17 OFDVI patients (11.7 %) were mutated. A comparison of mutated vs. non-mutated OFDVI patients showed that preaxial and mesoaxial polydactyly, hypothalamic hamartoma and other congenital defects may predict C5orf42 mutations, while tongue hamartomas are more common in negative patients. Electronic supplementary material The online version of this article (doi:10.1007/s00439-014-1508-3) contains supplementary material, which is available to authorized users.

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. DOI: http://dx.doi.org/10.7554/eLife.06602.001, eLife digest Joubert syndrome is a rare disorder that affects the brain and causes physical, mental, and sometimes visual impairments. In individuals with this condition, two parts of the brain called the cerebellar vermis and the brainstem do not develop properly. This is thought to be due to defects in the development and maintenance of tiny hair-like structures called cilia, which are found on the surface of cells. Currently, mutations in 25 different genes are known to be able to cause Joubert syndrome. However, these mutations only account for around 50% of the cases that have been studied, and the ‘unexplained’ cases suggest that mutations in other genes may also cause the disease. Here, Roosing et al. used a technique called a ‘genome-wide siRNA screen’ to identify other genes regulating the formation of cilia that might also be connected with Joubert syndrome. This approach identified almost 600 candidate genes. The data from the screen were combined with gene sequence data from 145 individuals with unexplained Joubert syndrome. Roosing et al. found that individuals with Joubert syndrome from 15 different families had mutations in a gene called KIAA0586. In chickens and mice, this gene—known as Talpid3—is required for the formation of cilia. Roosing et al.'s findings reveal a new gene that is involved in Joubert syndrome and also provides a list of candidate genes for future studies of other conditions caused by defects in the formation of cilia. The next challenges are to find out what causes the remaining unexplained cases of the disease and to understand what roles the genes identified in this study play in cilia. DOI: http://dx.doi.org/10.7554/eLife.06602.002

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655TA [p.Leu552(∗)] and c.280GA [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552(∗) alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3β phosphorylation that is observed after wild-type APPL1 transfection. These findings-linking APPL1 mutations to familial forms of diabetes-reaffirm the critical role of APPL1 in glucose homeostasis.