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author:("portanje, Ron")
1.  Identification of Novel Genes Associated with Renal Tertiary Lymphoid Organ Formation in Aging Mice 
PLoS ONE  2014;9(3):e91850.
A hallmark of aging-related organ deterioration is a dysregulated immune response characterized by pathologic leukocyte infiltration of affected tissues. Mechanisms and genes involved are as yet unknown. To identify genes associated with aging-related renal infiltration, we analyzed kidneys from aged mice (≥20 strains) for infiltrating leukocytes followed by Haplotype Association Mapping (HAM) analysis. Immunohistochemistry revealed CD45+ cell clusters (predominantly T and B cells) in perivascular areas coinciding with PNAd+ high endothelial venules and podoplanin+ lymph vessels indicative of tertiary lymphoid organs. Cumulative cluster size increased with age (analyzed at 6, 12 and 20 months). Based on the presence or absence of clusters in male and female mice at 20 months, HAM analysis revealed significant associations with loci on Chr1, Chr2, Chr8 and Chr14 in male mice, and with loci on Chr4, Chr7, Chr13 and Chr14 in female mice. Wisp2 (Chr2) showed the strongest association (P = 5.00×10−137) in male mice; Ctnnbip1 (P = 6.42×10−267) and Tnfrsf8 (P = 5.42×10−245) (both on Chr4) showed the strongest association in female mice. Both Wisp2 and Ctnnbip1 are part of the Wnt-signaling pathway and the encoded proteins were expressed within the tertiary lymphoid organs. In conclusion, this study revealed differential lymphocytic infiltration and tertiary lymphoid organ formation in aged mouse kidneys across different inbred mouse strains. HAM analysis identified candidate genes involved in the Wnt-signaling pathway that may be causally linked to tertiary lymphoid organ formation.
doi:10.1371/journal.pone.0091850
PMCID: PMC3956762  PMID: 24637805
2.  Differences in Health Status Affect Susceptibility and Mapping of Genetic Loci for Atherosclerosis (Fatty Streak) in Inbred Mice 
Objective
We observed differences in atherosclerosis susceptibility in mouse inbred strains over the years as the health status of our animal rooms increased. Therefore, we investigated the effect of animal room health status on atherosclerosis susceptibility in different strains. As this data can also be used for genome-wide association mapping, we performed a mapping study and compared our results with previously found quantitative trait loci for atherosclerosis in mouse and human.
Methods and Results
Males and females from 48 inbred strains were housed in two animal rooms with different health status and given an atherogenic diet. We compared atherosclerosis susceptibility between animal rooms and between sexes and found that susceptibility is dependent on both health status and sex. Subsequently, the data were used for associations with loci on the mouse genome using 63,222 SNPs. Three loci in males and four loci in females were identified using the data from the low health status room. No significant associations were identified using the data from the high health status room.
Conclusion
Health status influences susceptibility to atherosclerosis and suggests that microbiological pressure plays an important role in the development of atherosclerosis in many strains. As we were only able to map susceptibility loci using the data from the lower health status room, we argue that susceptibility under these conditions is determined by a few key loci, while in the higher health status room different mechanisms might play a role in the differences in atherosclerosis susceptibility between strains and we did not have enough power to map the loci that are involved.
doi:10.1161/ATVBAHA.112.255703
PMCID: PMC3563286  PMID: 22837474
atherosclerosis; inbred strains; QTL
3.  The role of mouse strain differences in the susceptibility to fibrosis: a systematic review 
In humans, a number of genetic factors have been linked to the development of fibrosis in a variety of different organs. Seeking a wider understanding of this observation in man is ethically important. There is mounting evidence suggesting that inbred mouse strains with different genetic backgrounds demonstrate variable susceptibility to a fibrotic injury. We performed a systematic review of the literature describing strain and organ specific response to injury in order to determine whether genetic susceptibility plays a role in fibrogenesis. Data were collected from studies that were deemed eligible for analysis based on set inclusion criteria, and findings were assessed in relation to strain of mouse, type of injury and organ of investigation. A total of 44 studies were included covering 21 mouse strains and focusing on fibrosis in the lung, liver, kidney, intestine and heart. There is evidence that mouse strain differences influence susceptibility to fibrosis and this appears to be organ specific. For instance, C57BL/6J mice are resistant to hepatic, renal and cardiac fibrosis but susceptible to pulmonary and intestinal fibrosis. However, BALB/c mice are resistant to pulmonary fibrosis but susceptible to hepatic fibrosis. Few studies have assessed the effect of the same injury stimulus in different organ systems using the same strains of mouse. Such mouse strain studies may prove useful in elucidating the genetic as well as epigenetic factors in humans that could help determine why some people are more susceptible to the development of certain organ specific fibrosis than others.
doi:10.1186/1755-1536-6-18
PMCID: PMC3849643  PMID: 24294831
Mouse strain; Genetics; Fibrosis; Wound healing; Genetic susceptibility
4.  Decreased APOE-containing HDL subfractions and cholesterol efflux capacity of serum in mice lacking Pcsk9 
Background
Studies in animals showed that PCSK9 is involved in HDL metabolism. We investigated the molecular mechanism by which PCSK9 regulates HDL cholesterol concentration and also whether Pcsk9 inactivation might affect cholesterol efflux capacity of serum and atherosclerotic fatty streak volume.
Methods
Mass spectrometry and western blot were used to analyze the level of apolipoprotein E (APOE) and A1 (APOA1). A mouse model overexpressing human LDLR was used to test the effect of high levels of liver LDLR on the concentration of HDL cholesterol and APOE-containing HDL subfractions. Pcsk9 knockout males lacking LDLR and APOE were used to test whether LDLR and APOE are necessary for PCSK9-mediated HDL cholesterol regulation. We also investigated the effects of Pcsk9 inactivation on cholesterol efflux capacity of serum using THP-1 and J774.A1 macrophage foam cells and atherosclerotic fatty streak volume in the aortic sinus of Pcsk9 knockout males fed an atherogenic diet.
Results
APOE and APOA1 were reduced in the same HDL subfractions of Pcsk9 knockout and human LDLR transgenic male mice. In Pcsk9/Ldlr double-knockout mice, HDL cholesterol concentration was lower than in Ldlr knockout mice and higher than in wild-type controls. In Pcsk9/Apoe double-knockout mice, HDL cholesterol concentration was similar to that of Apoe knockout males. In Pcsk9 knockout males, THP-1 macrophage cholesterol efflux capacity of serum was reduced and the fatty streak lesion volume was similar to wild-type controls.
Conclusions
In mice, LDLR and APOE are important factors for PCSK9-mediated HDL regulation. Our data suggest that, although LDLR plays a major role in PCSK9-mediated regulation of HDL cholesterol concentration, it is not the only mechanism and that, regardless of mechanism, APOE is essential. Pcsk9 inactivation decreases the HDL cholesterol concentration and cholesterol efflux capacity in serum, but does not increase atherosclerotic fatty streak volume.
doi:10.1186/1476-511X-12-112
PMCID: PMC3751695  PMID: 23883163
Apolipoprotein E; Atherosclerotic fatty streak; Low-density lipoprotein receptor; Macrophage foam cell; Proprotein convertase subtilisin/kexin type 9
5.  Pla2g12b and Hpn Are Genes Identified by Mouse ENU Mutagenesis That Affect HDL Cholesterol 
PLoS ONE  2012;7(8):e43139.
Despite considerable progress understanding genes that affect the HDL particle, its function, and cholesterol content, genes identified to date explain only a small percentage of the genetic variation. We used N-ethyl-N-nitrosourea mutagenesis in mice to discover novel genes that affect HDL cholesterol levels. Two mutant lines (Hlb218 and Hlb320) with low HDL cholesterol levels were established. Causal mutations in these lines were mapped using linkage analysis: for line Hlb218 within a 12 Mbp region on Chr 10; and for line Hlb320 within a 21 Mbp region on Chr 7. High-throughput sequencing of Hlb218 liver RNA identified a mutation in Pla2g12b. The transition of G to A leads to a cysteine to tyrosine change and most likely causes a loss of a disulfide bridge. Microarray analysis of Hlb320 liver RNA showed a 7-fold downregulation of Hpn; sequencing identified a mutation in the 3′ splice site of exon 8. Northern blot confirmed lower mRNA expression level in Hlb320 and did not show a difference in splicing, suggesting that the mutation only affects the splicing rate. In addition to affecting HDL cholesterol, the mutated genes also lead to reduction in serum non-HDL cholesterol and triglyceride levels. Despite low HDL cholesterol levels, the mice from both mutant lines show similar atherosclerotic lesion sizes compared to control mice. These new mutant mouse models are valuable tools to further study the role of these genes, their affect on HDL cholesterol levels, and metabolism.
doi:10.1371/journal.pone.0043139
PMCID: PMC3422231  PMID: 22912808
6.  Identification of the Mhc Region as an Asthma Susceptibility Locus in Recombinant Congenic Mice 
Mouse models of allergic asthma are characterized by airway hyperreactivity (AHR), Th2-driven eosinophilic airway inflammation, high allergen-specific IgE (anti-OVA IgE) levels in serum, and airway remodeling. Because asthma susceptibility has a strong genetic component, we aimed to identify new asthma susceptibility genes in the mouse by analyzing the asthma phenotypes of the Leishmania major resistant (lmr) recombinant congenic (RC) strains. The lmr RC strains are derived from C57BL/6 and BALB/c intercrosses and carry congenic loci on chromosome 17 (lmr1) and 9 (lmr2) in both backgrounds. Whereas the lmr2 locus on chromosome 9 contributes to a small background-specific effect on anti-OVA IgE and AHR, the lmr1 locus on chromosome 17 mediates a strong effect on Th2-driven eosinophilic airway inflammation and background-specific effects on anti-OVA IgE and AHR. The lmr1 locus contains almost 600 polymorphic genes. To narrow down this number of candidate genes, we performed genome-wide transcriptional profiling on lung tissue from C.lmr1 RC mice and BALB/c control mice. We identified a small number of differentially expressed genes located within the congenic fragment, including a number of Mhc genes, polymorphic between BALB/c and C57Bl/6. The analysis of asthma phenotypes in the C.B10-H2b RC strain, carrying the C57Bl/6 haplotype of the Mhc locus in a BALB/c genetic background, reveals a strikingly similar asthma phenotype compared with C.lmr1, indicating that the differentially expressed genes located within the C.B10-H2b congenic fragment are the most likely candidate genes to contribute to the reduced asthma phenotypes associated with the C57Bl/6 allele of lmr1.
doi:10.1165/rcmb.2009-0369OC
PMCID: PMC3266060  PMID: 20971879
allergic asthma; quantitative trait locus; recombinant congenic mice; chromosome 17; mouse model
7.  Rat Ace allele variation determines susceptibility to AngII induced renal damage 
INTRODUCTION
Ace b/l polymorphism in rat is associated to differential tissue ACE expression and activity, and susceptibility to renal damage. Same polymorphism was recently found in outbred Wistar rat strain with b allele accounting for higher renal ACE, and provided a model for studying RAAS response behind the innate high or low ACE conditions.
METHODS
We investigated the reaction of these alleles on chronic Angiotensin II (AngII) infusion. Wistar rats were selected to breed male homozygotes for the b (WU-B) or l allele (WU-L) (n=12). For each allele, one group (n=6) received AngII infusion via osmotic minipump (435 ng/kg/min) for three weeks. Other group (n=6) served as control.
RESULTS
WU-B had higher ACE activity at baseline then WU-L. Interestingly, baseline renal ACE2 expression and activity were higher in WU-L. AngII infusion induced same increase in blood pressure in both genotypes, no proteinuria, but caused tubulo-interstitial renal damage with increased α-SMA and monocyte/macrophage influx only in WU-B (p<0.05). Low ACE WU-L rats did not develop renal damage.
CONCLUSION
AngII infusion causes proteinuria independent renal damage only in rats with genetically predetermined high ACE while rats with low ACE seemed to be protected against the detrimental effect of AngII. Differences in renal ACE2, mirroring those in ACE, might be involved.
doi:10.1177/1470320311415886
PMCID: PMC3356861  PMID: 21788250
ACE; ACE2; WU-L and WU-B; Angiotensin II induced renal injury
8.  Genome-Wide Association Mapping of Quantitative Traits in Outbred Mice 
G3: Genes|Genomes|Genetics  2012;2(2):167-174.
Recent developments in high-density genotyping and statistical analysis methods that have enabled genome-wide association studies in humans can also be applied to outbred mouse populations. Increased recombination in outbred populations is expected to provide greater mapping resolution than traditional inbred line crosses, improving prospects for identifying the causal genes. We carried out genome-wide association mapping by using 288 mice from a commercially available outbred stock; NMRI mice were genotyped with a high-density single-nucleotide polymorphism array to map loci influencing high-density lipoprotein cholesterol, systolic blood pressure, triglyceride levels, glucose, and urinary albumin-to-creatinine ratios. We found significant associations (P < 10−5) with high-density lipoprotein cholesterol and identified Apoa2 and Scarb1, both of which have been previously reported, as candidate genes for these associations. Additional suggestive associations (P < 10−3) identified in this study were also concordant with published quantitative trait loci, suggesting that we are sampling from a limited pool of genetic diversity that has already been well characterized. These findings dampen our enthusiasm for currently available commercial outbred stocks as genetic mapping resources and highlight the need for new outbred populations with greater genetic diversity. Despite the lack of novel associations in the NMRI population, our analysis strategy illustrates the utility of methods that could be applied to genome-wide association studies in humans.
doi:10.1534/g3.111.001792
PMCID: PMC3284324  PMID: 22384395
Mouse Genetic Resource
9.  Genetic analysis of albuminuria in the aging mouse and concordance with loci for diabetic nephropathy found in a genome-wide association scan 
Kidney international  2009;77(3):201-210.
Aging is a physiological process involving both genetic factors and environmental agents that can lead to function loss in organs. In the kidney, aging can cause leakage of proteins in urine, starting with albumin. Discovering molecular mechanisms responsible for albuminuria during aging could offer new perspectives on the etiology of this abnormality. Haplotype association mapping in the mouse is a novel approach which uses the haplotypes of the relatively closely related mouse inbred strains and the variation of the phenotypes among these strains to find associations between haplotypes and phenotype. Albumin-to-creatinine ratios, measures of urinary albumin excretion, were determined in 30 inbred mouse strains at 12, 18, and 24 months of age. To determine genetic loci that are involved in albuminuria, haplotype association mapping was performed for males and females separately at all 3 time points using a set of 63,222 SNPs. One significant and 8 suggestive loci were identified, some of which map to previously identified loci for traits associated with kidney damage in the mouse, but with a much higher resolution, which narrowed the mapped loci. These 9 loci were then investigated in the data of the genome-wide association scan for diabetic nephropathy in human type 1 diabetes. Two of the 9 mouse loci were found to be significantly associated with diabetic nephropathy, suggesting common underlying genes predisposing to kidney disease in mice and humans.
doi:10.1038/ki.2009.434
PMCID: PMC2807478  PMID: 19924099
10.  Candidate genes for obesity revealed from a C57BL/6J x129S1/SvImJ intercross 
Objective
To identify the genes controlling body fat, we carried out a quantitative trait locus (QTL) analysis using C57BL/6J (B6) and 129S1/SvImJ (129) mice, which differ in obesity susceptibility after consuming an atherogenic diet.
Methods
Mice were fed chow until 8 weeks and an atherogenic diet from 8 to 16 weeks; body fatness was measured by X-ray absorptiometry in 528 (B6x129) F2 at 8 and 16 weeks. A high-density genome scan was performed using 508 polymorphic markers. After identifying the genetic loci, we narrowed the QTL using comparative genomics and bioinformatics.
Results
The percentage of body fat was significantly linked to loci on chromosomes (Chr) 1 (22, 68 and 173 Mb), 4 (74 Mb), 5 (73 Mb), 7 (88 Mb), 8 (43 and 80 Mb), 9 (55 Mb), 11 (115 Mb) and 12 (32 Mb); three suggestive loci on Chrs 6 (76 Mb), 9 (30 Mb) and 16 (26 Mb) and two pairs of interacting loci (Chr 2 at 99.8 Mb with Chr 7; Chr 1 at 68 Mb with Chr 11). Comparative genomics narrowed the QTL intervals by 20–57% depending on the chromosome; in most cases, haplotype analysis further narrowed them by about 90%.
Conclusions
Our analysis identified 15 QTL for percentage of body fat. We narrowed the QTL using comparative genomics and haplotype analysis and suggest several candidate genes: Apcs on Chr 1, Ppargc1a on Chr 5, Ucp1 on Chr 8, Angptl6 on Chr 9 and Lpin1 on Chr 12.
doi:10.1038/ijo.2008.56
PMCID: PMC2782629  PMID: 18414419
quantitative trait loci; comparative genomics; haplotype; SNP; fat mass
11.  Quantitative Trait Loci for Bone Mineral Density in an SM/J by NZB/BlNJ Intercross Population and Identification of Trps1 as a Probable Candidate Gene 
Identification of genes that regulate bone mineral density (BMD) will enhance our understanding of osteoporosis and could provide novel molecular targets for treatment or prevention. We generated a mouse intercross population and carried out a quantitative trait locus (QTL) analysis of 143 female and 124 male F2 progeny from progenitor strains SM/J and NZB/BlNJ using the whole body and vertebral aBMD as measured by dual X-ray absorptiometry. We found that both whole body and vertebral aBMD were affected by two loci on chromosome 9, one with a significant epistatic interaction on distal chromosome 8 and the other with a sex specific effect. Two additional significant QTL were identified on chromosome 12, and several suggestive ones on chromosomes 5, 8, 15, and 19. The chromosome 9, 12, and 15 loci have been previously identified in other crosses. SNP based haplotype analysis of the progenitor strains identified blocks within the QTL region that distinguish the low allele strains from the high allele strains, significantly narrowing the QTL region and reducing the possible candidate genes to 98 for chromosome 9, 31 for chromosome 12, and only two for chromosome 15. Trps1 is the most probable candidate gene for the chromosome 15 QTL. The sex specific effects may help to elucidate the BMD differences between males and females. This study illustrates the power of statistical modeling to resolve linked QTL and the utility of haplotype analysis in narrowing the list of candidates.
doi:10.1359/JBMR.080414
PMCID: PMC2586053  PMID: 18442308
high-fat diet; peripheral dual-energy X-ray absorptiometry; quantitative trait gene; haplotype analysis
12.  Use of a Dense Single Nucleotide Polymorphism Map for In Silico Mapping in the Mouse 
PLoS Biology  2004;2(12):e393.
Rapid expansion of available data, both phenotypic and genotypic, for multiple strains of mice has enabled the development of new methods to interrogate the mouse genome for functional genetic perturbations. In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for adenylate cyclase 7.
A large resource of genetic markers, typed in 48 strains of mice, combined with statistical techniques, allow "in silico" mapping of genetic regions involved in interesting traits in mice
doi:10.1371/journal.pbio.0020393
PMCID: PMC526179  PMID: 15534693
13.  Albuminuria is associated with too few glomeruli and too much testosterone 
Kidney International  2013;83(6):1118-1129.
Normally, the glomerular filtration barrier almost completely excludes circulating albumin from entering the urine. Genetic variation and both pre- and postnatal environmental factors may affect albuminuria in humans. Here we determine whether glomerular gene expression in mouse strains with naturally occurring variations in albuminuria would allow identification of proteins deregulated in relatively ‘leaky' glomeruli. Albuminuria increased in female B6 to male B6 to female FVB/N to male FVB/N mice, whereas the number of glomeruli/kidney was the exact opposite. Testosterone administration led to increased albuminuria in female B6 but not female FVB/N mice. A common set of 39 genes, many expressed in podocytes, were significantly differentially expressed in each of the four comparisons: male versus female B6 mice, male versus female FVB/N mice, male FVB/N versus male B6 mice, and female FVB/N versus female B6 mice. The transcripts encoded proteins involved in oxidation/reduction reactions, ion transport, and enzymes involved in detoxification. These proteins may represent novel biomarkers and even therapeutic targets for early kidney and cardiovascular disease.
doi:10.1038/ki.2013.45
PMCID: PMC3674403  PMID: 23447063
albuminuria; gender difference; glomerulus; nephron number; podocyte

Results 1-13 (13)