Tumour evolution inferred by single-cell sequencing

Genomic analysis provides insights into the role of copy number variation in disease, but most methods are not designed to resolve mixed populations of cells. In tumours, where genetic heterogeneity is common, very important information may be lost that would be useful for reconstructing evolutionary history. Here we show that with flow-sorted nuclei, whole genome amplification and next generation sequencing we can accurately quantify genomic copy number within an individual nucleus. We apply single-nucleus sequencing to investigate tumour population structure and evolution in two human breast cancer cases. Analysis of 100 single cells from a polygenomic tumour revealed three distinct clonal subpopulations that probably represent sequential clonal expansions. Additional analysis of 100 single cells from a monogenomic primary tumour and its liver metastasis indicated that a single clonal expansion formed the primary tumour and seeded the metastasis. In both primary tumours, we also identified an unexpectedly abundant subpopulation of genetically diverse 'pseudodiploid' cells that do not travel to the metastatic site. In contrast to gradual models of tumour progression, our data indicate that tumours grow by punctuated clonal expansions with few persistent intermediates.     

Influence of the enteric surface coat on the unidirectional flux of acetamide across the wall of rat small intestine

In vivo treatment of the jejunal mucosa with glycosidic enzymes seems to remove the enteric surface coat of the enterocyte. As a consequence, the mucosa-to-serosa unidirectional flux of acetamide increases remarkably. The glycocalyx probably represents a barrier to the diffusion of small hydrosoluble solutes.     

Relationship between passive permeability to nonelectrolytes and active transport activity in the isolated intestinal wall

Riassunto La permeabilità all'acetamide dell'epitelio intestinale di digiuno di ratto non sembra influenzata dal grado di rigonfiamento delle cellule epiteliali.

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This work has been supported by a research grant of the Consiglio Nazionale delle Ricerche, Rome, Italy.     

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.     

In vivo intestinal absorption of sugars and electrolytes

Riassunto Nell'intestino tenue di ratto in vivo è stato osservato che la concentrazione intracellulare di glucosio o di 3-O-metilglucosio, durante l'assorbimento di questi zuccheri, è sempre minore che nel siero. Ciò lascia presumere che esista una pompa per l'estrusione degli zuccheri a livello della membrana serosale delle cellule assorbenti intestinali.

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This work was supported by a research grant of the Consiglio Nazionale delle Ricerche (CNR), Rome.