Contrasting epistatic interactions between rat quantitative trait loci controlling mammary cancer development

We previously defined quantitative trait loci (QTLs) that control susceptibility to 7,12-dimethylbenz(α)anthracene-induced mammary carcinoma in SPRD-Cu3 (susceptible) and WKY (resistant) rats. Two of these QTLs, assigned to chromosomes (Chr) 10 and 18, control tumor growth rate and invasiveness. In this study we characterized a congenic strain in which a large segment of WKY Chr 10 was introduced in the SPRD-Cu3 genetic background and demonstrated that this chromosome segment controls this tumor trait. The WKY allele at this QTL (Mcsta1) reduces the growth rate of the fastest growing tumors by 26%. We also previously showed that two SPRD-Cu3-WKY congenic strains containing a WKY chromosome segment derived either from Chr 5 or from Chr 18 exhibit a reduction in tumor multiplicity (QTLs Msctm1 and Mcstm2, respectively) (with no reduction in tumor growth rate in the Chr 18 congenic). In this study we generated a double congenic strain, which contains the two WKY differential segments from Chr 5 and 18, to determine how these two segments interact with one another. Interestingly, two types of epistatic interactions were found: no additive effect was seen with respect to tumor multiplicity, while a reduction in tumor growth rate was observed. It thus appears that WKY alleles located on Chr 5 and Chr 8 interact epistatically in a contrasting manner to modulate tumor multiplicity (in a nonadditive manner) and growth rate (in a synergic manner). Tumor growth rate is thus influenced by two QTLs, on Chr 10 (Mcsta1) and on Chr 18 (Mcsta2), the action of the latter being dependent on the presence of the Chr5 QTL (Mcstm1). The expression level of positional and functional candidate genes was also analyzed. On Chr 5, Pla2g2a is subject to a syntenic control while expression of the Tp53 (Chr 10) and Pmai1/Noxa (Chr 18) genes appears to be controlled by several mammary cancer resistance QTLs.     

Quantitative trait locus analysis of unsaturated fatty acids in a recombinant inbred population of soybean

Soybean [Glycine max (L.) Merr.] is an important oilseed crop which produces about 30 % of the world’s edible vegetable oil. The quality of soybean oil is determined by its fatty acid composition. Soybean oil high in oleic and low in linolenic fatty acids is desirable for human consumption and other uses. The objectives of this study were to identify quantitative trait loci (QTLs) for unsaturated fatty acids and to evaluate the genetic effects of single QTL and QTL combinations in soybean. A population of recombinant inbred lines derived from the cross of SD02-4-59 × A02-381100 was evaluated for fatty acid content in seven environments. In total, 516 polymorphic single nucleotide polymorphism markers, 477 polymorphic simple sequence repeat markers and three GmFAD3 genes were used to genotype the mapping population. By using the composite interval mapping and/or the interval mapping method, a total of 15 QTLs for the three unsaturated fatty acids were detected in more than two environments. Two QTLs for oleic acid on linkage groups G [chromosome (Chr) 18] (qOLE-G) and J (Chr 16) (qOLE-J), three QTLs for linoleic acid on linkage groups A1 (Chr 5) (qLLE-A1) and G (Chr 18) (qLLE-G-1 and qLLE-G-2), and five QTLs for linolenic acid on linkage groups C2 (Chr 6), D1a (Chr 1), D1b (Chr 2), F (Chr 13) and G (Chr 18) were consistently detected in at least three individual environments and the average data over all environments. Significant QTL × QTL interactions were not detected. However, significant QTL × environment interactions were detected for all the QTLs which were repeatedly detected. Some QTLs reported previously were confirmed, and seven new QTLs (two for oleic acid, two for linoleic acid and three for linolenic acid) were identified in this study. Comparisons of two-locus and three-locus combinations indicated that cumulative effects of QTLs were significant for all the three unsaturated fatty acids. QTL pyramiding by molecular marker-assisted breeding would be an appropriate strategy for the improvement of unsaturated fatty acids in soybean.     

Multiple obesity QTLs identified in an intercross between the NZO (New Zealand obese) and the SM (small) mouse strains

The inheritance of adiposity levels has been investigated in an intercross of the obese, diabetes-prone NZO and the small, lean SM mouse strains. Adiposity index (AI) was defined as the sum of four fat pad weights divided by body weight. DNA pools from fat and lean mice were analyzed with microsatellite variants to screen the genome for quantitative trait loci (QTLs) affecting AI. Ten significant QTLs affecting AI were identified on Chromosome (Chr) 1 (three loci), Chr 2, Chr 5 (two loci), Chr 6 (two loci), Chr 7, and Chr 17. Most of the QTLs appear to be novel. Several QTLs differentially affect specific fat depots. Thus, Chr 2 and Chr 7 QTLs affect gonadal more than inguinal fat, while the converse is true for the Chr 17 QTL. Gender influences the expression of several of the QTLs. For example, effects of the proximal Chr 1 QTL (Obq7) on AI appears to be primarily in males. The proximal AI QTL on Chr 6 (Obq13) maps near the neuropeptide Y (Npy) locus. Sequence analysis of the Npy gene revealed a 1-nucleotide deletion within a highly conserved portion of the 3′ untranslated region in strain NZO. However, the deletion is polymorphic among mouse strains. Furthermore, lack of association between this same variant and AI in previously analyzed crosses raises doubt that it is the basis of Obq13. The present cross is the fourth in a series of intercrosses among 10 inbred strains arranged such that each strain is crossed with each adjacent strain within a circle. This design affords multiple opportunities to analyze each segregating QTL. Received: 17 July 2000 / Accepted: 9 October 2000     

The genetic basis of adrenal gland weight and structure in BXD recombinant inbred mice

Adrenal gland function is mediated through secreted hormones, which play a vital role in the autonomic and hypothalamic-pituitary-adrenal (HPA)-axis-mediated stress response. The genetic underpinnings of the stress response can be approached using a quantitative trait locus (QTL) analysis. This method has been used to investigate genomic regions associated with variation in complex phenotypes, but it has not been used to explore the structure of the adrenal. We used QTL analyses to identify candidate genes underlying adrenal weight and adrenal cortical zone and medulla widths. We used 64 BXD recombinant inbred (RI) strains of mice (n?=?528) and 2 parental strains (C57BL/6J and DBA/2J; n?=?20) to measure adrenal weights and adrenal zone widths. For adrenal weight, we found significant QTLs on chromosome 3 for females (Fawq1) and Chr 4 for males (Mawq1) and suggestive QTLs on Chrs 1, 3, 10, and 14 for females and Chrs 2, 4, 10, 17, and X for males. We identified a significant QTL on Chr 10 (Mawdq1) and a suggestive QTL on Chr 13 for male adrenal total width. For male adrenal medulla width, we found a significant QTL on Chr 5 (Mmwdq1) and a suggestive QTL on Chr 1. We also identified significant QTLs on Chrs 10 (Mxwdq1) and 14 (Mxwdq2) for male X-zone width. There are 113 genes that mapped within the significant QTL intervals, and we identified 4 candidate genes associated with adrenal structure and/or function. In summary, this study is an important first step for detecting genetic factors influencing the structure of the adrenal component of the HPA axis using QTL analyses, which may relate to adrenal function and provide further insights into elucidating genes critical for stress-related phenotypes.     

Genetic Modifiers ofLeprAssociated with Variability in Insulin Production and Susceptibility to NIDDM

In an attempt to identify the genetic basis for susceptibility to non-insulin-dependent diabetes mellitus within the context of obesity, we generated 401 genetically obeseLepr^fa/Lepr^faF2 WKY13M intercross rats that demonstrated wide variation in multiple phenotypic measures related to diabetes, including plasma glucose concentration, percentage of glycosylated hemoglobin, plasma insulin concentration, and pancreatic islet morphology. Using selective genotyping genome scanning approaches, we have identified three quantitative trait loci (QTLs) on Chr. 1 (LOD 7.1 for pancreatic morpholology), Chr. 12 (LOD 5.1 for body mass index and LOD 3.4 for plasma glucose concentration), and Chr. 16 (P< 0.001 for genotype effect on plasma glucose concentration). The obese F2 progeny demonstrated sexual dimorphism for these traits, with increased diabetes susceptibility in the males appearing at approximately 6 weeks of age, as sexual maturation occurred. For each of the QTLs, the linked phenotypes demonstrated sexual dimorphism (more severe affection in males). The QTL on Chr. 1 maps to a region vicinal to that previously linked to adiposity in studies of diabetes susceptibility in the nonobese Goto–Kakizaki rat, which is genetically closely related to the Wistar counterstrain we employed. Several candidate genes, including tubby (tub), multigenic obesity 1 (Mob1), adult obesity and diabetes (Ad), and insulin-like growth factor-2 (Igf2), map to murine regions homologous to the QTL region identified on rat Chr. 1.     

Genetic Analysis of Susceptibility to Dextran Sulfate Sodium-Induced Colitis in Mice

The genetic basis for differential sensitivity of inbred mice to inflammatory bowel disease induced by dextran sulfate sodium (DSS) is unknown. Susceptible C3H/HeJ were outcrossed to partially resistant C57BL/6J mice. F2 and N2 progeny were phenotyped by evaluating histopathologic lesions in large intestine detected 16 days after a 5-day period of feeding 3.5% DSS. Screening for DSS colitis (Dssc) loci revealed quantitative trait loci (QTL) on Chr 5 (Dssc1) and Chr 2 (Dssc2). These traits contributed additively, explaining 17.5% of the variation in total colonic lesions. Additional QTL on Chr 18 and 1 that collectively explained 11% of the variation in total colon lesions were indicated. In the cecum, only a putative QTL on Chr 11 was associated with pathology (lesion severity) in the cecum. Reduced DSS susceptibility was observed in congenic stocks in which the highly susceptible NOD/Lt strain carried putative resistance alleles from either B6 on Chr 2 or from the highly resistant NON/Lt strain on Chr 9. We conclude that multiple genes control susceptibility to DSS colitis in mice. PossibleDssccandidate genes are discussed in terms of current knowledge of inflammatory bowel disease susceptibility loci in humans.     

Physical confirmation and mapping of overlapping rat mammary carcinoma susceptibility QTLs, Mcs2 and Mcs6

Only a portion of the estimated heritability of breast cancer susceptibility has been explained by individual loci. Comparative genetic approaches that first use an experimental organism to map susceptibility QTLs are unbiased methods to identify human orthologs to target in human population-based genetic association studies. Here, overlapping rat mammary carcinoma susceptibility (Mcs) predicted QTLs, Mcs6 and Mcs2, were physically confirmed and mapped to identify the human orthologous region. To physically confirm Mcs6 and Mcs2, congenic lines were established using the Wistar-Furth (WF) rat strain, which is susceptible to developing mammary carcinomas, as the recipient (genetic background) and either Wistar-Kyoto (WKy, Mcs6) or Copenhagen (COP, Mcs2), which are resistant, as donor strains. By comparing Mcs phenotypes of WF.WKy congenic lines with distinct segments of WKy chromosome 7 we physically confirmed and mapped Mcs6 to ~33 Mb between markers D7Rat171 and gUwm64-3. The predicted Mcs2 QTL was also physically confirmed using segments of COP chromosome 7 introgressed into a susceptible WF background. The Mcs6 and Mcs2 overlapping genomic regions contain multiple annotated genes, but none have a clear or well established link to breast cancer susceptibility. Igf1 and Socs2 are two of multiple potential candidate genes in Mcs6. The human genomic region orthologous to rat Mcs6 is on chromosome 12 from base positions 71,270,266 to 105,502,699. This region has not shown a genome-wide significant association to breast cancer risk in pun studies of breast cancer susceptibility.     

Congenic strain confirms putative quantitative trait locus for body weight in the rat

Quantitative trait loci (QTLs) affecting body weight were investigated in the backcross population derived from nondiabetic BB/OK and spontaneously hypertensive rat (SHR) strains. The F₁ hybrids were backcrossed onto SHR rats, and QTL analysis was performed separately with the resulting backcross populations for each sex on Chromosomes (Chrs) 1, 3, 4, 10, 13, and 18. The body weight was determined at the age of 14 weeks, and the statistical analysis was performed with MAPMAKER/QTL 1.1b computer program. According to the stringent threshold for a lod score of 3.0, markers on Chr 1 were found to be linked with body weight. The QTL with a peak lod score (5.1) on Chr 1 for a male population was located within markers Igf2 and D1Mgh12. In contrast, in the female population the body weight affecting QTL (lod = 5.7) on Chr 1 was located between the D1Mit3 and Lsn markers. The existence of QTLs on Chr 1 affecting body weight in the male population was confirmed by congenic BB.Sa rats, carrying chromosomal region of SHR (Sa-Igf2) on the genetic background of BB rat. Received: 14 July 1997 / Accepted: 22 December 1997     

Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential β-Defensin Expression

Identification of genetic factors that modify complex traits is often complicated by gene-environment interactions that contribute to the observed phenotype. In model systems, the phenotypic outcomes quantified are typically traits that maximize observed variance, which in turn, should maximize the detection of quantitative trait loci (QTL) in subsequent mapping studies. However, when the observed trait is dependent on multiple interacting factors, it can complicate genetic analysis, reducing the likelihood that the modifying mutation will ultimately be found. Alternatively, by focusing on intermediate phenotypes of a larger condition, we can reduce a model’s complexity, which will, in turn, limit the number of QTL that contribute to variance. We used a novel method to follow angiogenesis in mice that reduces environmental variance by measuring endothelial cell growth from culture of isolated skin biopsies that varies depending on the genetic source of the tissue. This method, in combination with a backcross breeding strategy, is intended to reduce genetic complexity and limit the phenotypic effects to fewer modifier loci. We determined that our approach was an efficient means to generate recombinant progeny and used this cohort to map a novel s.c. angiogenesis QTL to proximal mouse chromosome (Chr.) 8 with suggestive QTL on Chr. 2 and 7. Global mRNA expression analysis of samples from parental reference strains revealed β-defensins as potential candidate genes for future study.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

Worse Renal Disease in Postmenopausal F2[Dahl S x R]-Intercross Rats: Detection of Novel QTLs Affecting Hypertensive Kidney Disease

The prevalence of hypertension increases after menopause with 75% of postmenopausal women developing hypertension in the United States, along with hypertensive end organ diseases. While human and animal model studies have indicated a protective role for estrogen against cardiovascular disease and glomerulosclerosis, clinical studies of hormone replacement therapy in postmenopausal women have shown polar results with some improvement in hypertension but worsening of hypertensive kidney disease, or no effect at all. These observations suggest that the pathogenesis of postmenopausal hypertension and its target organ complications is more complex than projected, and that loss of endogenous estrogens induces epigenetic changes that alter genetic susceptibility to end-organ complications per se resulting in pathogenetic mechanisms beyond correction by hormone replacement. We studied postmenopausal-induced changes in renal disease and performed a total genome scan for quantitative trait loci (QTLs) affecting kidney disease in postmenopausal 16m-old F2[Dahl S x R]-intercross female rats. We used glomerular injury score (GIS) as quantitative trait. We compared QTLs amongst premenopausal, ovariectomized and postmenopausal F2[Dahl S x R]-intercross rats using identical phenotype characterization. Postmenopausal F2[Dahl S x R]-intercross rats exhibited increased hypertensive glomerulosclerosis (P<0.01) and equivalent levels of kidney disease when compared to premenopausal and ovariectomized F2[Dahl S x R]-intercross rats respectively. We detected three significant to highly significant GIS-QTLs (GIS-pm1 on chromosome 4, LOD 3.54; GIS-pm2 on chromosome 3, LOD 2.72; GIS-pm3 on chromosome 5, LOD 2.37) and two suggestive GIS-QTLs (GIS-pm4 on chromosome 2, LOD 1.70; GIS-pm5 on chromosome 7, LOD 1.28), all of which were unique to this postmenopausal population. Detection of increased renal disease phenotype in postmenopausal and ovariectomized subjects suggests a protective role of ovarian hormones. Furthermore, the detection of distinct GIS-QTLs in postmenopausal intercross female rats suggests that distinct genetic mechanisms underlie hypertensive glomerulosclerosis in premenopausal and postmenopausal states.     

Mapping murine loci for seizure response to kainic acid

Mature DBA/2J (D2) mice are very sensitive to seizures induced by various chemical and physical stimuli, whereas C57BL/6J (B6) mice are relatively seizure resistant. We have conducted a genome-wide search for quantitative trait loci (QTLs) influencing the differential sensitivity of these strains to kainic acid (KA)-induced seizures by studying an F₂ intercross population. Parental, F₁, and F₂ mice (8–10 weeks of age) were injected subcutaneously with 25 mg/kg of KA and observed for 3 h. Latencies to focal and generalized seizures and status epilepticus were recorded and used to calculate an overall seizure score. Results of seizure testing indicated that the difference in susceptibility to KA-induced seizures between D2 and B6 mice is a polygenic phenomenon with at least 65% of the variance due to genetic factors. First-pass genome screening (10-cM marker intervals) in F₂ progeny (n = 257) documented a QTL of moderate effect on Chromosome (Chr) 1 with a peak LOD score of 5.5 (17% of genetic variance explained) localized between D1Mit30 and D1Mit16. Provisional QTLs of small effect were detected on Chr 11 (D11Mit224–D11Mit14), 15 (D15Mit6–D15Mit46) and 18 (D18Mit9–D18Mit144). Multiple locus models generally confirmed the Mapmaker/QTL results and also provided evidence for another QTL on Chr 4 (D4Mit9). Multilocus analysis of seizure severity suggested that additional loci on Chrs 5 (D5Mit11), 7 (D7Mit66), and 15 (D15Nds2) might also contribute to KA-induced seizure response. Overall, our results document a complex genetic determinism for KA-induced seizures in these mouse strains with contributions from as many as eight QTLs. Received: 16 April 1996 / Accepted: 21 October 1996     

A novel strategy for genetic dissection of complex traits: the population of specific chromosome substitution strains from laboratory and wild mice

The mouse is an irreplaceable model for understanding the precise genetic mechanisms of mammalian physiological pathways. Thousands of quantitative trait loci (QTLs) have been mapped onto the mouse genome during the last two decades. However, only a few genes’ underlying complex traits have been successfully identified, and rapid fine mapping of QTL genes still remains a challenge for mouse geneticists. Currently, the Collaborative Cross (CC) has proceeded to the goal of establishing more than 1,000 recombinant inbred strains for the sub-centimorgan mapping resolution of QTL loci. In this article, a novel complementary strategy, designated as population of specific chromosome substitution strains or PSCSS, is proposed for rapid fine mapping of QTLs on the substituted chromosome. One specific chromosome (Chr 1) of recipient mouse strain C57BL/6 J has been substituted by homologous counterparts from five different inbred strains (C3H/He, FVB/N, AKR, NOD/LtJ, NZW/LacJ), an outbred line Kunmin mouse in China, and 23 wild mice captured in different localities. The primary genetic studies on the structure of these wild donor chromosomes (Chr 1) show that these donor chromosomes harbor extensive genetic polymorphisms, with a high density of SNP distribution, abundant variations of STR alleles, and a high level of historical recombination accumulation. These specific chromosome substitution strains eventually form a special population that has the identical genetic background of the recipient strain and differs only in the donor chromosomes. With simple association studies, known QTLs on the donor chromosome can be rapidly mapped in high resolution without requirement of further crosses. This approach, taking advantage of the extensive genetic polymorphisms of wild resources and chromosome substitution strategy, brings a new outlook for genetic dissection of complex traits.     

Quantitative trait loci for growth traits in C57BL/6J × DBA/2J mice

Quantitative trait loci (QTLs) for body weight and tail length are mapped in an F₂ population of 927 C57BL/6J × DBA/2J mice. We test the concordance between the locations of the mapped QTLs with those detected by changes of marker frequency under artificial selection in a previous experiment with the same base population. The directions of effects of the QTLs are generally in agreement, and in many cases significant QTLs are found in similar map positions, but there are also discrepancies between the two experiments. There are indications of age-specific QTL effects on growth. For body weight traits, the genetic variation in the F₂ appears to result from many loci with relatively small effects. For tail length at 10 weeks, however, a single QTL on Chromosome (Chr) 1 with a peak LOD score of ∼33 contributes most of the genetic variation detected, changes the trait value by about 6%, and explains about 20% of the phenotypic variance of the trait. Received: 4 August 1998 / Accepted: 17 November 1998     

Identification of quantitative trait loci controlling resistance to gray leaf spot disease in maize

Breeding maize for gray leaf spot (GLS) resistance has been hindered by the quantitative nature of the inheritance of GLS resistance and by the limitations of selection under less than optimumal disease pressure. In order to identify the quantitative trait loci (QTLs) controlling GLS resistance, a cross was made between B73 (susceptible) and Va14 (resistant) to generate a large F₂ population. Six GLS disease assessments were made throughout the disease season for over 1000 F₂ plants in 1989, and for 600 F₂-derived F₃ lines replicated in two blocks in 1990. RFLP analysis for78 marker loci representing all ten maize chromosomes was conducted in 239 F₂ individuals including those with the extreme GLS disease phenotypes. The GLS disease scores of the three field evaluations, each averaged over six ratings, were separately used for the interval mapping in order to determine the consistency of the QTL effects. The heavy GLS disease pressure, meticulous disease ratings, and large population size of this study afforded us the sensitivity for detecting QTL effects. QTLs located on three chromosomes (1, 4, and 8) had large effects on GLS resistance, each explaining 35.0–56.0%, 8.8–14.3%, and 7.7–11.0% of the variance, respectively. These three QTL effects were remarkably consistent across three disease evaluations over 2 years and two generations. Smaller QTL effects were also found on chromosomes 2 and 5, but the chromosome-5 effect might be a false positive because it was not repeatable even in the same location. The chromosome-1 QTLs had the largest effect or highest R² reported for any quantitative trait to-date. Except for the chromosome-4 gene, which was from the susceptible parent B73, the resistance alleles at all QTL were derived from Va14. The resistance QTLs on chromosomes 1 and 2 appear to have additive effects, but those on chromosomes 4 and 8 are dominant and recessive, respectively. Significant interaction between the QTLs on chromosomes 1 and 4 was detected in all three evaluations. Cumulatively, the four QTLs identified in this study explained 44, 60, and 68% of the variance in F₂, and in F₃ replications 1 and 2, respectively.     

Genetic loci controlling breast cancer susceptibility in the Wistar-Kyoto rat

In this study, the Wistar-Kyoto (WKy) rat was genetically characterized for loci that modify susceptibility to mammary carcinogenesis. We used a genetic backcross between resistant WKy and susceptible Wistar-Furth (WF) rats as a panel for linkage mapping to genetically identify mammary carcinoma susceptibility (Mcs) loci underlying the resistance of the WKy rat. Rats were phenotyped for DMBA-induced mammary carcinomas and genotyped using microsatellite markers. To detect quantitative trait loci (QTL), we analyzed the genome scan data under both parametric and nonparametric distributional assumptions and used permutation tests to calculate significance thresholds. A generalized linear model analysis was also performed to test for interactions between significant QTL. This methodology was extended to identify interactions between the significant QTL and other genome locations. Chromosomes 5, 7, 10, and 14 were found to contain significant QTL, termed Mcs5, Mcs6, Mcs7, and Mcs8, respectively. The WKy alleles of Mcs5, -6, and -8 are associated with mammary carcinoma resistance; the WKy allele of Mcs7 is associated with an increased incidence of mammary cancer. In addition, we identified an interaction between Mcs8 and a region on chromosome 6 termed Mcsm1 (modifier of Mcs), which had no significant main effect on mammary cancer susceptibility in this genetic analysis.     

Association of a lithogenic Abcg5/Abcg8 allele on Chromosome 17 (Lith9) with cholesterol gallstone formation in PERA/EiJ mice

To examine further the genetic determinants of cholesterol gallstone susceptibility in inbred mice, we performed quantitative trait locus (QTL) analysis of an intercross of gallstone-susceptible PERA/EiJ and gallstone-resistant DBA/2J inbred mice. Three hundred twenty-four F₂ offspring were phenotyped for cholelithiasis during consumption of a lithogenic diet and genotyped using microsatellite markers. Linkage analysis was performed by interval mapping. In addition, we analyzed the combined datasets from this cross and from an independent cross of strain PERA and gallstone-resistant I/Ln mice. QTL mapping detected one significant new gallstone susceptibility (Lith) locus on Chromosome 13 (Lith15). A second significant QTL on Chr 6 (Lith16) confirmed a previous QTL. Furthermore, suggestive QTLs confirmed Lith loci from previous crosses on Chromosomes 1, 2, 5, 16 and X. QTL analysis of the dataset derived from the combined crosses increased the detection power and narrowed confidence intervals of Lith loci on Chromosomes 2, 6, 13, and 16. Moreover, the analysis of combined datasets revealed a shared QTL between both crosses on Chromosome 17 (Lith9). Significantly higher mRNA expression of Abcg5 and Abcg8 in strain PERA compared with strains I/Ln and DBA/2 further substantiated that the PERA allele of Abcg5/Abcg8 was responsible for lithogenicity underlying Lith9.     

Inferred expression regulator activities suggest genes mediating cardiometabolic genetic signals

Expression QTL (eQTL) analyses have suggested many genes mediating genome-wide association study (GWAS) signals but most GWAS signals still lack compelling explanatory genes. We have leveraged an adipose-specific gene regulatory network to infer expression regulator activities and phenotypic master regulators (MRs), which were used to detect activity QTLs (aQTLs) at cardiometabolic trait GWAS loci. Regulator activities were inferred with the VIPER algorithm that integrates enrichment of expected expression changes among a regulator’s target genes with confidence in their regulator-target network interactions and target overlap between different regulators (i.e., pleiotropy). Phenotypic MRs were identified as those regulators whose activities were most important in predicting their respective phenotypes using random forest modeling. While eQTLs were typically more significant than aQTLs in cis, the opposite was true among candidate MRs in trans. Several GWAS loci colocalized with MR trans-eQTLs/aQTLs in the absence of colocalized cis-QTLs. Intriguingly, at the 1p36.1 BMI GWAS locus the EPHB2 cis-aQTL was stronger than its cis-eQTL and colocalized with the GWAS signal and 35 BMI MR trans-aQTLs, suggesting the GWAS signal may be mediated by effects on EPHB2 activity and its downstream effects on a network of BMI MRs. These MR and aQTL analyses represent systems genetic methods that may be broadly applied to supplement standard eQTL analyses for suggesting molecular effects mediating GWAS signals.     

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.     

Quantitative trait locus analysis for obesity reveals multiple networks of interacting loci

Obesity is a highly heritable and genetically complex trait with hundreds of potential loci identified. An intercross of 513 F₂ progeny between the SM/J × NZB/BINJ inbred mouse strains was generated to identify quantitative trait loci (QTL) that are involved in the weight of four fat pads: mesenteric, inguinal, gonadal, and retroperitoneal. Sex and lean body weight were treated as covariates in the analysis of these fat pads. This analysis uncoupled genetic effects related to overall body size from those influencing the adiposity of a mouse. We identified multiple significant QTL. QTL alleles associated with increased lean body weight and individual fat pad weights are contributed by the NZB background. Adiposity loci are distinct from these body size QTLs and high-adiposity alleles are contributed by the SM background. An extended network of epistatic QTL is also observed. A QTL on Chr 19 is the center of a network of eight interacting QTL, Chr 4 is the center of six, and Chr 17 the center of four interacting QTL. We conclude that interacting networks of multiple genes characterize the regulation of fat pad depots and body weight. Haplotype patterns and a literature-driven approach were used to generate hypotheses regarding the identity of the genes and pathways underlying the QTL.