Muscle growth in young horses: Effects of age, cytokines, and growth factors.

Success as equine athletes requires proper muscle growth in young horses. Muscle hypertrophy occurs through protein synthesis and the contribution of muscle satellite cells, which can be stimulated or inhibited by cytokines and growth factors present during exercise and growth. The hypotheses of this study were that 1) the LM area in young horses would increase over 1 yr, and 2) specific cytokines and growth factors (IL-1ß, IL-6, tumor necrosis factor [TNF]-α, IGF-I, and fibroblast growth factor [FGF]-2) would alter proliferation and differentiation of satellite cells isolated from young horses. Fourteen horses were divided into 3 age groups: weanlings ( = 5), yearlings to 2 yr olds ( = 4), and 3 to 4 yr olds ( = 5). The area, height, and subcutaneous fat depth of the LM were measured using ultrasonography, and BW and BCS were taken in October (Fall1), April (Spring), and October of the following year (Fall2). Satellite cells obtained from 10-d-old foals ( = 4) were cultured in the presence of IL-6, IL-1ß, TNF-α, IGF-I, or FGF-2 before evaluation of proliferation and differentiation. Data were analyzed using PROC MIXED in SAS. Body weight increased from Fall1 to Spring in weanlings ( < 0.001) and increased in all horses from Spring to Fall2 ( ≤ 0.02). Area and height of the LM increased over time ( < 0.001) and with increasing age group of horse ( ≤ 0.03), although there was no interaction of time and age ( > 0.61). There was a significant increase in LM area in all animals from Spring to Fall2 ( < 0.001) but not from Fall1 to Spring. Interleukin-6 and TNF-α decreased satellite cell proliferation by 14.9 and 11.5%, respectively ( ≤ 0.01). Interleukin-6 increased fusion 6.2%, whereas TNF-α decreased fusion 8.7% compared with control cells ( ≤ 0.001). Interleukin-1ß had no effect on proliferation ( = 0.32) but tended to decrease fusion ( = 0.06). Satellite cell proliferation was increased 28.8 and 73.0% by IGF-I and FGF-2, respectively ( < 0.0001). Differentiation was decreased 13.1% in the presence of FGF-2 but increased 3.5% in the presence of IGF-I ( ≤ 0.01). In summary, the LM area increases over the course of a year in young horses with the most growth occurring in summer. By stimulating or inhibiting proliferation and differentiation of satellite cells, IL-6, TNF-α, IL-1ß, IGF-I, and FGF-2 may alter muscle growth in young horses, thereby impacting athletic potential.

Age effects on blood gas, spirometry, airway reactivity, and bronchoalveolar lavage fluid cytology in clinically healthy horses.

BACKGROUND: Despite the increasing number of geriatric horses attended by veterinarians, there is a lack of understanding of aging-related changes on the respiratory system of horses. OBJECTIVE: To identify aging-related changes on the respiratory function and bronchoalveolar lavage fluid (BALF) cytology of horses. ANIMALS: Fifteen healthy young adult (2-11 years) and 16 healthy aged (≥20 years) horses. METHODS: The respiratory system was examined by measurement of arterial blood gases (ABG), use of respiratory inductive plethysmography (RIP) for assessment of breathing pattern and ventilatory parameters, histamine bronchoprovocation, and BALF cytology. RESULTS: No significant differences were detected with regard to values obtained by ABG or bronchoprovocation of young adult and aged healthy horses. In aged horses, there were significant differences in mean ± SD of the following parameters when compared to young horses: prolonged expiratory time (Te) measured by RIP (3.9 ± 1.5 s versus 3.0 ± 0.6 s), decreased percentage of alveolar macrophages (40.6 ± 11.3% versus 53.5 ± 9.6%), and increased percentage of lymphocytes (53.4 ± 9.5% versus 43.9 ± 11.0%). No correlations between airway reactivity and ventilatory parameters, ABG, or BALF cytology were found in this asymptomatic population. CONCLUSIONS: These results suggest that aging does not cause changes in the results obtained by ABG, most RIP-derived variables, and bronchoprovocation in the horse. A decreased percentage of macrophage and an increased percentage of lymphocytes in the BALF cytology may be expected in the asymptomatic geriatric horse and may be a result of aging.

Spontaneous metastasis in mouse models of testicular germ-cell tumours.

Testicular germ-cell tumours (TGCTs) are the most common cancer in young men; the incidence is increasing worldwide and they have an unusually high rate of metastasis. Despite significant work on TGCTs and their metastases in humans, absence of a mouse model of spontaneous metastasis has greatly limited our understanding of the mechanisms by which metastatic potential is acquired and on their modes of dissemination. We report a new model of spontaneous TGCT metastasis in the 129 family of mice and provide evidence that these are true metastases derived directly from primary testicular cancers rather than independently from ectopic stem cells. These putative metastases (pMETs) occur at similar frequencies among TGCT-affected males in six genetically distinct TGCT-susceptible strains and were largely found in anatomical sites that are consistent with patterns of TGCT metastasis in humans. Various lines of evidence support their pluripotency and germ-cell origin, including presence of multiple endodermal, mesodermal and ectodermal derivatives as well as cells showing OCT4 and SSEA-1 pluripotency markers. In addition, pMETs were never found in males that did not have a TGCT, suggesting that metastases are derived from primary tumours. Finally, pMETS and primary TGCTs shared several DNA copy number variants suggesting a common cellular and developmental origin. Together, these results provide the first evidence for spontaneous TGCT metastasis in mice and show that these metastases originate from primary TGCTs rather than independently from ectopic stem cells.

Uptake of CdSe and CdSe/ZnS quantum dots into bacteria via purine-dependent mechanisms.

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.

Comparison of endoscopic, necropsy and histology scoring of equine gastric ulcers.

Equine gastric ulcer syndrome (EGUS) represents a major health problem in performance horses. Much debate exists regarding endoscopic gastric ulcer scoring systems and their ability accurately to predict severity or depth of gastric ulcers. The purpose of this study was to evaluate the ability of an endoscopist to count gastric ulcers and predict gastric ulcer severity or depth using 2 endoscopic scoring systems and compare them to the same gastric ulcers see on necropsy and histopathology. Endoscopic examination of the stomach was performed under general anaesthesia on 23 mixed breed yearling horses, after feed was withheld for 24 h. Gastric ulcers were scored using 2 systems, number/severity-scoring (N/S) and practitioner simplified (PS) systems. After endoscopy, the horses were subjected to euthanasia and the stomach mucosa examined blindly and scored again at necropsy using above scoring systems. Representative gastric ulcers were then placed in 10% formalin and processed routinely for histopathology. The gastric ulcers were scored using a histopathology system (HSS) based on ulcer depth. Number scores in the N/S scoring system and PS on endoscopic and necropsy examinations were compared using Friedman 2 way analysis of variance. Where significant differences between variables were found a post hoc analysis was conducted using a Tukey's Studentised range (HSD) test. Severity scores using the N/S (ENGS) and PS scores recorded for the stomach via endoscopy and scores from HSS were evaluated for significant association using a Mantel-Haenszel Chi-square and Pearson moment correlation coefficient analysis. Significance was P < 0.05. All horses had gastric ulcers in the nonglandular mucosa via endoscopic examination and at necropsy examination. Mean nonglandular ulcer number (ENGN) score was significantly (P = 0.0024) lower on endoscopic examination compared to the score at necropsy (NNGN); whereas PS scores were not significantly different on endoscopy when compared to necropsy examination. A significant but weak association was found between ENGS and HSS (3.89, P = 0.048; r = 0.453, P = 0.045) and no correlation was found between PS and HSS (1.2, P = 0.272; r = 0.117; P = 0.622). Only 1/23 horses had glandular ulcers observed via endoscopic examination whereas, 6/23 horses had glandular ulcers at necropsy and on histopathology. The prevalence of EGUS is high in stalled yearling horses. The endoscopist may underestimate the number of gastric ulcers and may not be able accurately to predict the severity or depth of those ulcers present in the nonglandular equine stomach. Furthermore, the endoscopist may miss glandular gastric ulcers.

Sensitized polygenic trait analysis.

Genetic variation in many biological processes and evolutionary adaptations is caused by polygenes--genes that act in combination to affect a particular trait. Despite the recent identification of several polygenes, many remain to be found, suggesting that new experimental and analytical methods are needed to facilitate their discovery. Here we discuss sensitized polygenetic trait analysis, a method that has emerged recently for simplifying the genetic analysis of polygenic traits. The method uses a known single gene mutation in linkage testing crosses to 'sensitize' the analysis. By increasing the frequency of affected individuals in segregating populations, linkages are more readily detected. This method has considerable potential, especially given the increasing variety of mutations that can be used to sensitize the genetic analysis of polygenic traits.

Disorganization in mice and humans.

Disorganization (Ds) is an autosomal dominant mouse mutant that produces a remarkable array of birth defects. So variable is the phenotype that no two mice appear identical. Ds also has markedly reduced penetrance, with 85-99% of Ds mice having no apparent anomalies. Paired structures are often affected, but always asymmetrically. Although the Ds gene has yet to be identified, it is thought that Ds is a gain-of-function mutation, and that Ds malformations are thought to arise through a two-hit mechanism. Unlike the two-hit model that has been used to describe the development of retinoblastoma, the "second hit" for Ds is thought not to arise in the other Ds allele. Although there is a long list of anomalies seen in Ds mice, two stand out as most characteristic: hamartomatous skin papillae, and mirror-image limb duplications. Through the observation of these unusual anomalies in human cases, the possibility of a human homologue of Ds was suggested. However, in reviewing types of anomalies seen in Ds mice, it is apparent that cases with these unusual defects represent only one end of the spectrum of the Ds phenotype. Ds may be the genetic basis for more usual and seemingly sporadic human birth defects as well.

Modifier genes in mice and humans.

An emerging theme of studies with spontaneous, engineered and induced mutant mice is that phenotypes often depend on genetic background, implying that genetic modifiers have a role in guiding the functional consequences of genetic variation. Understanding the molecular and cellular basis by which modifier genes exert their influence will provide insights into developmental and physiological pathways that are critical to fundamental biological processes, as well as into novel targets for therapeutic interventions in human diseases.

129/Sv mice--a model system for studying germ cell biology and testicular cancer.

Some forms of testicular germ cell tumors (TGCTs) arise from primordial germ cells (PGCs) during fetal development. In both humans and mice, genetic control of susceptibility is complex, involving both Mendelian and polygenic factors. Identification and characterization of TGCT genes will provide insight not only into the basis for inherited susceptibility, but also into the genetic control of the development of the PGC lineage. Recent work has revealed the identity of several susceptibility genes that are inherited as Mendelian traits, the chromosomal location of yet-to-be identified TGCT susceptibility genes, as well as clues to the nature of developmental pathways involved in tumorigenesis. In this review we summarize current understanding of the biology and genetics of TGCTs in mice and discuss the relevance of this work to testicular cancer in humans.

Betaine-homocysteine methyltransferase-2: cDNA cloning, gene sequence, physical mapping, and expression of the human and mouse genes.

Anomalies in folate and homocysteine metabolism can result in homocysteinemia and are implicated in disorders ranging from vascular disease to neural tube defects. Two enzymes are known to methylate homocysteine, vitamin B(12)-dependent methionine synthase (MTR) and betaine-homocysteine methyltransferase (BHMT). BHMT uses betaine, an intermediate of choline oxidation, as a methyl donor and is expressed primarily in the liver and kidney. We report the discovery of a novel betaine-homocysteine methyltransferase gene in humans and mice. The human BHMT2 gene is predicted to encode a 363-amino-acid protein (40.3 kDa) that shows 73% amino acid identity to BHMT. The BHMT2 transcript in humans is most abundant in adult liver and kidney and is found at reduced levels in the brain, heart, and skeletal muscle. The mouse Bhmt2 gene shows 69% amino acid identity and 79% similarity to the mouse Bhmt gene and 82% amino acid identity and 87% similarity to the human BHMT2 gene. Bhmt2 is expressed in fetal heart, lung, liver, kidney and eye. The discovery of a third gene with putative homocysteine methyltransferase activity is important for understanding the biochemical balance in using methyltetrahydrofolate and betaine as methyl donors as well as the metabolic flux between folate and choline metabolism in health and disease.

Evaluation of diet as a cause of gastric ulcers in horses.

OBJECTIVE: To measure pH, volatile fatty acid (VFA) concentrations, and lactate concentrations in stomach contents and determine number and severity of gastric lesions in horses fed bromegrass hay and alfalfa hay-grain diets. ANIMALS: Six 7-year-old horses. PROCEDURE: A gastric cannula was inserted in each horse. Horses were fed each diet, using a randomized crossover design. Stomach contents were collected immediately after feeding and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, and 24 hours after feeding on day 14. The pH and VFA and lactate concentrations were measured in gastric juice Number and severity of gastric lesions were scored during endoscopic examinations. RESULTS: The alfalfa hay-grain diet caused significantly higher pH in gastric juice during the first 5 hours after feeding, compared with that for bromegrass hay. Concentrations of acetic, propionic, and isovaleric acid were significantly higher in gastric juice, and number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. Valeric acid, butyric acid, and propionic acid concentrations and pH were useful in predicting severity of nonglandular squamous gastric lesions in horses fed alfalfa hay-grain, whereas valeric acid concentrations and butyric acid were useful in predicting severity of those lesions in horses fed bromegrass hay. CONCLUSIONS AND CLINICAL RELEVANCE: An alfalfa hay-grain diet induced significantly higher pH and VFA concentrations in gastric juice than did bromegrass hay. However, number and severity of nonglandular squamous gastric lesions were significantly lower in horses fed alfalfa hay-grain. An alfalfa hay-grain diet may buffer stomach acid in horses.

Luteinizing hormone induction of ovarian tumors: oligogenic differences between mouse strains dictates tumor disposition.

The use of fertility drugs has continued to grow since their introduction in the 1960s. Accompanying this increase has been the speculation that repetitive use of these drugs can cause ovarian tumors or cancer. We recently reported that transgenic mice with chronically elevated luteinizing hormone (LH), an analog of which is commonly used in fertility regimens, develop granulosa cell (GC) tumors. In this report we show that LH induction of these tumors is highly dependent on genetic background. In CF-1 mice, chronically elevated LH invariably causes GC tumors by 5 months of age. However, in hybrid mice generated by crossing CF-1 males with C57BL/6, SJL, or CD-1 females, elevated levels of this same hormone cause a completely different phenotype resembling a luteoma of pregnancy. We also show that three genes likely control these alternative hormonal responses. This clinical correlate of elevated LH reveals remarkably distinct, strain-dependent, ovarian phenotypes. In addition, these results support the rare incidence of GC tumors in the human population, and suggest that the ability of certain fertility drugs to cause ovarian tumors may depend on an individual's genetic predisposition.

A mouse model for valproate teratogenicity: parental effects, homeotic transformations, and altered HOX expression.

Valproate (VPA) is one of several effective anti-epileptic and mood-stabilizing drugs, many of which are also potent teratogens in humans and several other mammalian species. Variable teratogenicity among inbred strains of laboratory mice suggests that genetic factors influence susceptibility. While studying the genetic basis for VPA teratogenicity in mice, we discovered that parental factors influence fetal susceptibility to induced malformations. Detailed examination of these malformations revealed that many were homeotic transformations. To test whether VPA, like retinoic acid (RA), alters HOX expression, pluripotent human embryonal carcinoma cells were treated with VPA or RA and Hox expression assessed. Altered expression of specific Hox genes may thus account for the homeotic transformations and other malformations found in VPA-treated fetuses.

Real-time, sequence-specific detection of nucleic acids during strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on opposite sides by a fluorophore and a quencher. The probes also contain target-binding sequences located 3' to the BsoBI site. Fluorophore and quencher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during SDA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent for SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real time during the SDA reaction. Probe performance may be enhanced by embedding the fluorogenic BsoBI site within the loop of a folded hairpin structure. The new probe designs permit detection of as few as 10 target copies within 30 min in a closed-tube, real-time format, eliminating the possibility of carry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color competitive SDA format permits accurate quantification of target levels from the real-time fluorescence data.

Modulation by drugs of human hepatic sodium-dependent bile acid transporter (sodium taurocholate cotransporting polypeptide) activity.

Adequate bile flow, maintained in part by the efficient enterohepatic recirculation of bile acids, is critical for normal liver function. One important component of this process is the uptake of bile acids from the portal circulation into hepatocytes by the bile acid uptake transporter sodium taurocholate cotransporting polypeptide (NTCP). Thus, the expression and functional activity of this transporter may affect the rate of bile acid removal from the portal circulation. Accordingly, we assessed NTCP mRNA expression from human livers using a sensitive RNase protection assay. In addition, the ability of various bile acids and drugs to inhibit NTCP activity was determined using a recombinant vaccinia expression system. A 40-fold interindividual variability was found in NTCP mRNA levels determined in eight liver samples of disease-free donors. Expressed NTCP exhibited high-affinity, sodium-dependent uptake of taurocholate, and as expected, this was markedly inhibited by bile acids and organic anions. A number of drugs, including peptidomimetic renin inhibitors, propranolol, cyclosporin, and progesterone, were found to be potent inhibitors, whereas antiarrhythmic agents, including bupivicaine, lidocaine, and quinidine, were found to enhance NTCP activity. Accordingly, these results indicate that large interindividual variability exists in NTCP mRNA level and that a number of drugs currently in clinical use have the potential to interact with and alter NTCP activity, thereby affecting hepatic bile acid uptake.

Susceptibility to testicular germ-cell tumours in a 129.MOLF-Chr 19 chromosome substitution strain.

The identification of genes that control susceptibility to testicular germ-cell tumours (TGCTs), the most common cancer affecting young men, has been difficult. In laboratory mice, TGCTs arise from primordial germ cells in only the 129 inbred strains, and susceptibility is under multigenic control. The spontaneously arising mutation Ter (ref. 5) on mouse chromosome 18 (Refs 6,7) increases TGCT frequency on a 129/Sv background. We originally used Ter in genetic crosses to identify loci that control tumorigenesis. A genome scan of tumour-bearing progeny from backcrosses between the 129/Sv-Ter/+ and MOLF/Ei strains provided modest evidence that MOLF-derived alleles on chromosome 19 enhance development of bilateral TGCTs (ref. 9). To obtain independent evidence for linkage to the MOLF chromosome, we made an autosomal chromosome substitution strain (CSS; or 'consomic strain') in which chromosome 19 of 129/Sv+/+ was replaced by its MOLF-derived homologue. The unusually high frequency of TGCTs in this CSS (even in the absence of the Ter mutation) provides evidence confirming the genome survey results, identifies linkage for a naturally occurring strain variant allele that confers susceptibility to TGCTs and illustrates the power of CSSs in complex trait analysis.

Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene.

Fibroblast growth factor 9 (FGF9), originally cloned as glial-activating factor from human glioma cells, is expressed in adult rat brain and kidney. Here we report the chromosomal localization, genomic organization, and embryonic expression pattern of the mouse Fgf9 gene. Fgf9 maps to chromosome 14 near the Ctla6 locus. The gene spans more than 34 kb and contains three exons and two introns. Translation initiation occurs in exon 1, and translation termination occurs in exon 3. Fgf9 RNA was detected during mouse embryogenesis in several tissues in which Fgf gene expression has not been previously described, including intermediate mesoderm of late-stage gastrulation, ventricular myocardium, lung pleura, skeletal myoblasts in the early limb bud, spinal cord motor neurons, olfactory bulb, and gut lumenal epithelium. Fgf9 is coexpressed with other Fgf genes in some skeletal myoblasts, in limb apical ectoderm, in craniofacial ectoderm, and in the retina, inner ear, and tooth bud. Dev Dyn 1999;216:72-88.

Clinical experience over 48 years with pheochromocytoma.

OBJECTIVE: To analyze the presentation, localization, surgical management, pathology, and long-term outcome of a large series of patients with pheochromocytomas. SUMMARY BACKGROUND DATA: There are several areas of controversy pertaining to pheochromocytomas. Although many studies report a higher rate of malignancy for extraadrenal pheochromocytomas than for adrenal pheochromocytomas, the number of patients with the former tumor are small and statistical analysis is lacking. There has also been recent debate as to whether microscopic features of the tumor may be predictive of future behavior. METHODS: From 1950 to 1998, the authors observed 108 pheochromocytomas in 104 patients. The outcome of these patients has been followed prospectively. The medical records of these patients were reviewed for data on the presentation, localization, surgical management, pathology, and outcome. Patient survival was analyzed using Kaplan-Meier survival distributions. RESULTS: This study included 66 female patients and 38 male patients. The average age at surgery was 42.3 years. Sporadic cases accounted for 84% of the patients; the other 16% had multiple endocrine neoplasia type 2, von Recklinghausen's disease, von Hippel-Lindau disease, or Carney's syndrome. Of 64 adrenal tumors, 55 were initially considered benign, 6 had microscopic malignant features, and 3 had malignant disease. Mean patient follow-up was 12.6 years. To date, in five additional patients (none with microscopic disease) malignant disease developed (13% overall rate of malignancy). Recurrence occurred as late as 15 years after resection. Of 26 extraadrenal pheochromocytomas, 14 were initially considered benign, 8 had microscopic malignant features, and 4 had malignant disease. Thus, 46% of patients had either malignant disease or tumors with malignant features. Mean patient follow-up was 11.5 years. In one patient with benign disease and in one patient with malignant features, malignant disease developed (23% overall rate of malignancy). The difference in the rate of malignancy was not statistically significant between adrenal and extraadrenal pheochromocytomas. Patients with adrenal and extraadrenal pheochromocytomas also had similar rates of survival (p = NS). CONCLUSIONS: The data suggest that patients with extraadrenal pheochromocytomas have the same risk of malignancy and the same overall survival as patients with adrenal pheochromocytomas. Lifelong follow-up of these patients is mandatory.

Multigenic and imprinting control of ovarian granulosa cell tumorigenesis in mice.

Spontaneous juvenile ovarian granulosa cell (GC) tumors that occur in young girls are similar to GC carcinomas that develop in SWR-derived inbred mice. We analyzed female offspring from a series of matings among SWR and SJL inbred mice for chromosomal loci underlying tumor susceptibility. Intercross F2 female mice were produced by reciprocal matings of (SWR x SJL)F1 and (SJL x SWR)F1 parents. Tumorigenesis in these F2 mice as well as in SWXJ recombinant inbred and congenic strains of mice derived from SWR and SJL showed significant (P < 0.001) association with Gct1, a dominant susceptibility locus on chromosome (CHR) 4 and with Gct2 on CHR 12. Suggestive (P < 0.01) association was found with Gct3 on CHR 15. A fourth susceptibility locus, Gct4 on CHR X, was demonstrated with a strong parent-of-origin effect associated with the paternal genotype. Imprinting and complex interactions among these four loci combine to establish the probability for GC tumorigenesis in this mouse model.