Contribution of Water from Food and Fluids to Total Water Intake: Analysis of a French and UK Population Surveys

Little has been published on the contribution of food moisture (FM) to total water intake (TWI); therefore, the European Food Safety Authority assumed FM to contribute 20%–30% to TWI. The aim of the present analysis was to estimate and compare TWI, the percentage of water from FM and from fluids in population samples of France and UK. Data from 2 national nutrition surveys (Enquête Comportements et Consommations Alimentaires en France (CCAF) 2013 and the National Diet and Nutrition Survey (NDNS) 2008/2009–2011/2012) were analyzed for TWI and the contribution of water from FM and fluids. Children and adults TWI were significantly lower in France than in the UK. The contribution of water from foods was lower in the UK than in France (27% vs. 36%). As TWI increased, the proportion of water from fluids increased, suggesting that low drinkers did not compensate by increasing intake of water-rich foods. In addition, 80%–90% of the variance in TWI was explained by differences in water intake from fluids. More data on the contribution of FM to TWI is needed to develop more robust dietary recommendations on TWI and guidance on fluid intake for the general public

Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer

Abstract Background Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. Methods Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. Results Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. Conclusions Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies

Additional file 3: Figure S3. of Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer

a Western blots were loaded with whole cell lysates of cultured human cells (RPE-1) that had been transfected either with scrambled control siRNA or with two different siRNAs targeting FLCN. Staining with the anti-FLCN antibody shows one specific band at the expected molecular weight the intensity of which is strongly reduced by FLCN knockdown (left panel). Anti-Actin staining of the same membrane was used to control for equal loading (right panel). b To check whether the antibody was suitable for IHC as well we stained cell pellets of the established FLCN knockout cell line UOK257 lentivirally transduced to express emGFP (left image) showing little to no signal. UOK257cells that had been lentivirally transduced to express FLCN show a strong signal (magnification 40×). c IHC in human tissue shows that FLCN can still be detected in the chromophobe renal cell carcinoma of the BHD patient (middle panel). Normal human kidney tissue from a control (left side) was stained for comparison and shows a stronger signal. A control staining without the FLCN first antibody is shown on the right side of the panel (magnification 40×). (PDF 9710 kb

Additional file 2: Figure S2. of Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer

Exon 10, intron 10 and exon 11 of FLCN were cloned into an expression vector. After transfection into mIMCD cells (of mouse origin) RNA and afterwards cDNA was prepared and subjected to sequencing and PCR. As expected in the wildtype (WT) situation, intron 10 (= 565 bp) is spliced out, resulting in a PCR product of 197 bp when using primers that are located in exon10 and exon11. The patient mutation leads to a not functional splice acceptor site in front of exon 11. As a consequence, intron 10 is not spliced out leading to larger PCR product of 762 bp. Human cDNA and human genomic DNA served as controls for the PCR products. Sanger sequencings confirmed the results, electropherograms of the crucial junctions are shown. (PDF 929 kb