A signature of renal stress resistance induced by short-term dietary restriction, fasting, and protein restriction.

During kidney transplantation, ischemia-reperfusion injury (IRI) induces oxidative stress. Short-term preoperative 30% dietary restriction (DR) and 3-day fasting protect against renal IRI. We investigated the contribution of macronutrients to this protection on both phenotypical and transcriptional levels. Male C57BL/6 mice were fed control food ad libitum, underwent two weeks of 30%DR, 3-day fasting, or received a protein-, carbohydrate- or fat-free diet for various periods of time. After completion of each diet, renal gene expression was investigated using microarrays. After induction of renal IRI by clamping the renal pedicles, animals were monitored seven days postoperatively for signs of IRI. In addition to 3-day fasting and two weeks 30%DR, three days of a protein-free diet protected against renal IRI as well, whereas the other diets did not. Gene expression patterns significantly overlapped between all diets except the fat-free diet. Detailed meta-analysis showed involvement of nuclear receptor signaling via transcription factors, including FOXO3, HNF4A and HMGA1. In conclusion, three days of a protein-free diet is sufficient to induce protection against renal IRI similar to 3-day fasting and two weeks of 30%DR. The elucidated network of common protective pathways and transcription factors further improves our mechanistic insight into the increased stress resistance induced by short-term DR.

Restricted diet delays accelerated ageing and genomic stress in DNA-repair-deficient mice.

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.

Proteomics and Down syndrome screening: a validation study.

OBJECTIVE: In a previous discovery study, we identified seven potential screening markers for Down syndrome (DS). Here, we report on an extended study to validate the discriminative potential of these markers. METHODS: Concentrations of the seven analytes were measured using bead-based multiplexed immunoassays in maternal serum from 27 DS pregnancies and 27 matched controls. Control samples were matched to the cases by gestational age (exact day), maternal weight ( ± 5 kg), and maternal age ( ± 1 year) and by closest sample date. Prediction values were obtained for current screening markers [pregnancy-associated plasma protein A (PAPP-A), free beta human chorionic gonadotrophin (fß-hCG) and nuchal translucency (NT)] and seven markers identified before based on concentration fold ratios between DS and controls. Models were fitted based on data of the discovery study or this study and also tested on both datasets. RESULTS: A significantly higher fold ratio was only found for epidermal growth factor (EGF) (-1.96; p = 0.006). In the prediction model for the current dataset, EGF improved the detection rate (DR) of DS by 5.7% [at a fixed 5% false-positive rate (FPR)] when added to the currently used screening markers. CONCLUSIONS: Validation of previously identified biomarkers only confirmed EGF for further consideration as a DS screening marker. This underlines the importance of validating biomarkers; in this study, limiting the range of plausible biomarkers to only one suitable biomarker.

Glucose levels and genetic variants across transcriptional pathways: interaction effects with BMI.

OBJECTIVE: Much of the genetic variation in glucose levels remains to be discovered. Especially, research on gene-environment interactions is scarce. Overweight is one of the main risk factors for hyperglycemia. As transcriptional regulation is important for both weight maintenance and glucose control, we analyzed 353 single nucleotide polymorphisms (SNPs), occurring in transcriptional pathways of glucose and lipid metabolism in interaction with body mass index (BMI) on glucose levels. RESEARCH DESIGN AND METHODS: SNPs were measured in 3244 participants of the Doetichem cohort. Non-fasting glucose levels and BMI were measured twice in 6 years. SNP x BMI interactions were analyzed by mixed models and adjusted for age, sex, time since last meal, and follow-up time. False discovery rate (FDR) <0.2 was used to adjust for multiple testing. RESULTS: Two SNPs in the PPARGC1A gene (rs8192678, FDR=0.07; rs3755863, FDR=0.17) showed a significant interaction with BMI. The rare allele of both SNPs was associated with significantly lower glucose levels in subjects with a BMI28 kg m(-2). A small intervention study (n=120) showed similar, though non-significant, results. CONCLUSIONS: Using a pathway-based approach, we found that BMI significantly modified the association between two SNPs in the PPARGC1A gene and glucose levels. The association between glucose and PPARGC1A was only present in lean subjects. This suggests that the effect of the PPARGC1A gene, which is involved both in fatty acid oxidation and glucose metabolism, is modified by BMI.

Genetic variations in regulatory pathways of fatty acid and glucose metabolism are associated with obesity phenotypes: a population-based cohort study.

BACKGROUND: As nuclear receptors and transcription factors have an important regulatory function in adipocyte differentiation and fat storage, genetic variation in these key regulators and downstream pathways may be involved in the onset of obesity. OBJECTIVE: To explore associations between single nucleotide polymorphisms (SNPs) in candidate genes from regulatory pathways that control fatty acid and glucose metabolism, and repeated measurements of body mass index (BMI) and waist circumference in a large Dutch study population. METHODS: Data of 327 SNPs across 239 genes were analyzed for 3575 participants of the Doetinchem cohort, who were examined three times during 11 years, using the Illumina Golden Gate assay. Adjusted random coefficient models were used to analyze the relationship between SNPS and obesity phenotypes. False discovery rate q-values were calculated to account for multiple testing. Significance of the associations was defined as a q-value < or = 0.20. RESULTS: Two SNPs (in NR1H4 and SMARCA2 in women only) were significantly associated with both BMI and waist circumference. In addition, two SNPs (in SIRT1 and SCAP in women only) were associated with BMI alone. A functional SNP, in IL6, was strongly associated with waist. CONCLUSION: In this explorative study among participants of a large population-based cohort, five SNPs, mainly located in transcription mediator genes, were strongly associated with obesity phenotypes. The results from whole genome and candidate gene studies support the potential role of NR1H4, SIRT1, SMARCA2 and IL6 in obesity. Although replication of our findings and further research on the functionality of these SNPs and underlying mechanism is necessary, our data indirectly suggest a role of GATA transcription factors in weight control.

Bead-based multiplexed immunoassays to identify new biomarkers in maternal serum to improve first trimester Down syndrome screening.

OBJECTIVES: To identify new discriminative biomarkers for Down syndrome (DS) pregnancies using a bead-based multiplexed immunoassay, and to use the newly identified biomarkers to construct a prediction model for non-invasive DS screening. METHODS: Maternal serum samples of 14 DS pregnancies and 15 matched controls were analyzed with a bead-based multiplexed immunoassay containing immunoassays for 90 different analytes. Potential biomarkers were selected on the basis of concentration fold ratios between DS and control samples. For these markers and the current screening markers (pregnancy-associated plasma protein-A, PAPP-A; free beta subunit of human chorion gonadotrophin (fbeta-hCG) and nuchal translucency) prediction values were obtained and used to calculate detection rates (DR) at a 5% false positive rate. RESULTS: Seven potential biomarkers of which the fold ratio exceeded 1.3 or -1.3 were selected for further analysis. All 14 DS cases in this study were detected using the combination of all currently used and newly identified markers. The modelled DR for all markers extrapolated to the general pregnant population was 82.5%, compared to a modelled DR of 56.2% for the current screening markers. CONCLUSION: This study demonstrates the possibility of improving the performance of the current first-trimester DS screening by addition of new biomarkers, which were identified using bead-based multiplexed immunoassays.

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

Analysis of the inheritance of white spotting and the evaluation of KIT and EDNRB as spotting loci in Dutch boxer dogs.

The genetic basis of the white spotting pattern in Dutch boxer dogs is not known. We studied whether the segregation of white spotting in boxers follows a Mendelian inheritance pattern. Blood samples were collected, along with digital photographs in standard directions of (grand)parents (n=16) and offspring (n=52) from eight litters of Dutch boxers. In order to select heterozygous parents, we selected nonuniform litters, in which at least one puppy was extreme white. On the basis of criteria for the location, the extent of white spotting, and the mean percentage of pigmented area of the foot soles, we classified 10 dogs as solid colored, 27 as flashy, and 15 as extreme white. This was not a significant deviation from the expected 1:2:1 ratio. Because the flashy phenotype seems to be an intermediate between the two homozygotes, white spotting in the Dutch boxer can be considered to be due to a single gene effect, with incomplete dominance. We have evaluated candidate genes c-KIT (KIT) and EDNRB for segregation with white spotting phenotype in these litters. Using polymorphic markers, very near the KIT and EDNRB genes, we found that segregation of the white spotting pattern did not coincide with segregation of these polymorphic markers. Thus neither KIT nor EDNRB are likely to be responsible for white spotting in the Dutch population of boxers.

Exclusion of the lim homeodomain gene LHX4 as a candidate gene for pituitary dwarfism in German shepherd dogs.

Pituitary dwarfism in the German shepherd dog is an autosomal recessive inherited abnormality. We tested the hypothesis that a variant of the LIM homeodomain gene LHX4 is responsible for the dwarfism phenotype. To this end, we isolated Bacterial Artificial Chromosome clones for the canine LHX4 gene. Southern blotting experiments showed that the LHX4 gene is a single copy gene in the canine genome. A complex CA-repeat was isolated from the BAC clones and was found to be polymorphic in German shepherd dogs. Genotyping 5 litters in which the dwarfism was segregating showed disconcordance between the inheritance of the dwarfism phenotype and the DNA marker. It is concluded that the LHX4 gene does not play a primary role in the pituitary dwarfism in the German shepherd dogs.