CXM: a new tool for mapping breast cancer risk in the tumor microenvironment.

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3(IL2Rγ) consomic model compared with the SS(IL2Rγ) parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3(IL2Rγ) background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk.

Refined mapping of a hypertension susceptibility locus on rat chromosome 12.

Previously, we found that transferring 6.1 Mb of salt-sensitive (SS) chromosome 12 (13.4-19.5 Mb) onto the consomic SS-12(BN) background significantly elevated mean arterial pressure in response to an 8% NaCl diet (178±7 versus 144±2 mm Hg; P<0.001). Using congenic mapping, we have now narrowed the blood pressure locus by 86% from a 6.1-Mb region containing 133 genes to an 830-kb region (chr12:14.36-15.19 Mb) with 14 genes. Compared with the SS-12(BN) consomic, the 830-kb blood pressure locus was associated with a ∆+15 mm Hg (P<0.01) increase in blood pressure, which coincided with elevated albuminuria (∆+32 mg/d; P<0.001), proteinuria (∆+48 mg/d; P<0.01), protein casting (∆+154%; P<0.05), and renal fibrosis (∆+79%; P<0.05). Of the 14 genes residing in the 830-kb locus, 8 were differentially expressed, and among these, Chst12 (carbohydrate chondroitin 4 sulfotransferase 12) was most consistently downregulated by 2.6- to 4.5-fold (P<0.05) in both the renal medulla and cortex under normotensive and hypertensive conditions. Moreover, whole genome sequence analysis of overlapping blood pressure loci revealed an ≈86-kb region (chr12:14 541 567-14 627 442 bp) containing single-nucleotide variants near Chst12 that are unique to the hypertensive SS strain when compared with the normotensive Brown Norway, Dahl salt-resistant, and Wistar-Kyoto strains. Finally, the 830-kb interval is syntenic to a region on human chromosome 7 that has been genetically linked to blood pressure, suggesting that insight gained from our SS-12(BN) congenic strain may be translated to a better understanding of human hypertension.

Genome sequencing reveals loci under artificial selection that underlie disease phenotypes in the laboratory rat.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.

SORCS1 contributes to the development of renal disease in rats and humans.

Many lines of evidence demonstrate that genetic variability contributes to chronic kidney disease susceptibility in humans as well as rodent models. Little progress has been made in discovering causal kidney disease genes in humans mainly due to genetic complexity. Here, we use a minimal congenic mapping strategy in the FHH (fawn hooded hypertensive) rat to identify Sorcs1 as a novel renal disease candidate gene. We investigated the hypothesis that genetic variation in Sorcs1 influences renal disease susceptibility in both rat and human. Sorcs1 is expressed in the kidney, and knocking out this gene in a rat strain with a sensitized genome background produced increased proteinuria. In vitro knockdown of Sorcs1 in proximal tubule cells impaired protein trafficking, suggesting a mechanism for the observed proteinuria in the FHH rat. Since Sorcs1 influences renal function in the rat, we went on to test this gene in humans. We identified associations between single nucleotide polymorphisms in SORCS1 and renal function in large cohorts of European and African ancestry. The experimental data from the rat combined with association results from different ethnic groups indicates a role for SORCS1 in maintaining proper renal function.

Genetic basis of the impaired renal myogenic response in FHH rats.

This study examined the effect of substitution of a 2.4-megabase pair (Mbp) region of Brown Norway (BN) rat chromosome 1 (RNO1) between 258.8 and 261.2 Mbp onto the genetic background of fawn-hooded hypertensive (FHH) rats on autoregulation of renal blood flow (RBF), myogenic response of renal afferent arterioles (AF-art), K(+) channel activity in renal vascular smooth muscle cells (VSMCs), and development of proteinuria and renal injury. FHH rats exhibited poor autoregulation of RBF, while FHH.1BN congenic strains with the 2.4-Mbp BN region exhibited nearly perfect autoregulation of RBF. The diameter of AF-art from FHH rats increased in response to pressure but decreased in congenic strains containing the 2.4-Mbp BN region. Protein excretion and glomerular and interstitial damage were significantly higher in FHH rats than in congenic strains containing the 2.4-Mbp BN region. K(+) channel current was fivefold greater in VSMCs from renal arterioles of FHH rats than cells obtained from congenic strains containing the 2.4-Mbp region. Sequence analysis of the known and predicted genes in the 2.4-Mbp region of FHH rats revealed amino acid-altering variants in the exons of three genes: Add3, Rbm20, and Soc-2. Quantitative PCR studies indicated that Mxi1 and Rbm20 were differentially expressed in the renal vasculature of FHH and FHH.1BN congenic strain F. These data indicate that transfer of this 2.4-Mbp region from BN to FHH rats restores the myogenic response of AF-art and autoregulation of RBF, decreases K(+) current, and slows the progression of proteinuria and renal injury.

Identification of hypertension susceptibility loci on rat chromosome 12.

Previous studies have identified multiple blood pressure and renal disease quantitative trait loci located on rat chromosome 12. In the present study, we narrowed blood pressure loci using a series of overlapping Dahl salt-sensitive/Mcwi (SS)-12 Brown Norway (BN) congenic lines. We found that transferring 6.1 Mb of SS chromosome 12 (13.4-19.5 Mb) onto the consomic SS-12BN background significantly elevated blood pressure on 1% NaCl (146±6 versus 127±1 mm Hg; P<0.001) and 8% NaCl diets (178±7 versus 144±2 mm Hg; P<0.001). Compared with the SS-12BN consomic, these animals also had significantly elevated albumin (218±31 versus 104±8 mg/d; P<0.001) and protein excretion (347±41 versus 195±12 mg/d; P<0.001) on a 1% NaCl diet. Elevated blood pressure, albuminuria, and proteinuria coincided with greater renal and cardiac damage, demonstrating that SS allele(s) within the 6.1 Mb congenic interval are associated with strong cardiovascular disease phenotypes. Sequence analysis of the 6.1 Mb congenic region revealed 12 673 single nucleotide polymorphisms between SS and BN rats. Of these polymorphisms, 293 lie within coding regions, and 18 resulted in nonsynonymous changes in conserved genes, of which 5 were predicted to be potentially damaging to protein function. Syntenic regions in human chromosome 7 have also been identified in multiple linkage and association studies of cardiovascular disease, suggesting that genetic variants underlying cardiovascular phenotypes in this congenic strain can likely be translated to a better understanding of human hypertension.

A 4.1-Mb congenic region of Rf-4 contributes to glomerular permeability.

The combined transfer of two renal function quantitative trait loci (QTLs), Rf-1 (rat chromosome 1) and Rf-4 (rat chromosome 14), from the Fawn-hooded hypertensive rat onto the August Copenhagen Irish genetic background significantly increases proteinuria and demonstrates an interaction between these QTLs. Because the original Rf-4 congenic region is 61.9 Mbp, it is necessary to reduce this interval to feasibly search for variants responsible for renal susceptibility in this region. Here, we generated a minimal congenic line (Rf-1a+4_a) to identify a 4.1-Mb region of the Rf-4 QTL that significantly contributes to the severity of proteinuria in the Fawn-hooded hypertensive rat. Rf-1a+4_a animals have an increased glomerular permeability to albumin without significant changes in BP, indicating that at least one genetic element in this refined region directly affects renal function. Sequence analysis revealed no variants predicted to damage protein function, implying that regulatory elements are responsible for the Rf-4 phenotype. Multiple human studies, including recent genome-wide association studies, link the homologous human region with susceptibility to renal disease, suggesting that this congenic line is an important model for studying pathways that contribute to the progression of kidney disease.

Characterization of the genomic structure and function of regions influencing renin and angiogenesis in the SS rat.

Impaired regulation of renin in Dahl salt-sensitive rats (SS/JRHsdMcwi, SS) contributes to attenuated angiogenesis in this strain. This study examined angiogenic function and genomic structure of regions surrounding the renin gene using subcongenic strains of the SS and BN/NHsdMcwi (BN) rat to identify important genomic variations between SS and BN involved in angiogenesis. Three candidate regions on Chr 13 were studied: two congenic strains containing 0.89 and 2.62 Mb portions of BN Chr 13 that excluded the BN renin allele and a third strain that contained a 2.02 Mb overlapping region that included the BN renin allele. Angiogenesis induced by electrical stimulation of the tibialis anterior muscle was attenuated in the SS compared with the BN. Congenics carrying the SS renin allele had impaired angiogenesis, while strains carrying the BN renin allele had angiogenesis restored. The exception was a congenic including a region of BN genome 0.4 Mb distal to renin that restored both renin regulation and angiogenesis. This suggests that there is a distant regulatory element in the BN capable of restoring normal regulation of the SS renin allele. The importance of ANG II in the restored angiogenic response was demonstrated by blocking with losartan. Sequencing of the 4.05 Mb candidate region in SS and BN revealed a total of 8,850 SNPs and other sequence variants. An analysis of the genes and their variants in the region suggested a number of pathways that may explain the impaired regulation of renin and angiogenesis in the SS rat.