Genetic Epistasis Research Articles

Models from experiments: combinatorial drug perturbations of cancer cells

We present a novel method for deriving network models from molecular profiles of perturbed cellular systems. The network models aim to predict quantitative outcomes of combinatorial perturbations, such as drug pair treatments or multiple genetic alterations. Mathematically, we represent the system by a set of nodes, representing molecular concentrations or cellular processes, a perturbation vector and an interaction matrix. After perturbation, the system evolves in time according to differential equations with built-in nonlinearity, similar to Hopfield networks, capable of representing epistasis and saturation effects. For a particular set of experiments, we derive the interaction matrix by minimizing a composite error function, aiming at accuracy of prediction and simplicity of network structure. To evaluate the predictive potential of the method, we performed 21 drug pair treatment experiments in a human breast cancer cell line (MCF7) with observation of phospho-proteins and cell cycle markers. The best derived network model rediscovered known interactions and contained interesting predictions. Possible applications include the discovery of regulatory interactions, the design of targeted combination therapies and the engineering of molecular biological networks.

XylS–Pm Promoter Interactions through Two Helix–Turn–Helix Motifs: Identifying XylS Residues Important for DNA Binding and Activation

The XylS protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. XylS belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain, made up of seven α-helices conforming two helix–turn–helix DNA-binding domains. α-Helix 3 and α-helix 6 are the recognition helices. In consonance with XylS structural organization, Pm exhibits a bipartite DNA-binding motif consisting of two boxes, called A and B, whose sequences are TGCA and GGNTA, respectively. This bipartite motif is repeated at the Pm promoter so that one of the XylS monomers binds to each of the repeats. An extensive series of genetic epistasis assays combining mutant Pm promoters and XylS single substitution mutant proteins revealed that α-helix 3 contacts A box nucleotides, whereas α-helix 6 residues contact B box nucleotides. In α-helix 3, Asn246 and Arg242 are involved in specific contacts with the TG dinucleotide at box A, whereas Arg296 and Glu299 contact the second G and T nucleotides at box B. On the basis of our results and of the three-dimensional model of the XylS C-terminal domain, we propose that Ser243, Glu249 and Lys250 in α-helix 3, and Asn299 and Arg302 in α-helix 6 contact the phosphate backbones. Alanine substitutions at the predicted phosphate backbone-contacting residues yielded mutants with low levels of activity, suggesting that XylS–Pm binding specificity not only involves specific amino acid–base interactions, but also relies on secondary DNA structure, which, although at another level, also comes into play. We propose a model in which a XylS dimer binds to the direct repeats in Pm in a head-to-tail conformation that allows the direct interaction of the XylS proximal subunit with the RNA polymerase sigma factor.

An entropy-based approach for testing genetic epistasis underlying complex diseases

The genetic basis of complex diseases is expected to be highly heterogeneous, with complex interactions among multiple disease loci and environment factors. Due to the multi-dimensional property of interactions among large number of genetic loci, efficient statistical approach has not been well developed to handle the high-order epistatic complexity. In this article, we introduce a new approach for testing genetic epistasis in multiple loci using an entropy-based statistic for a case-only design. The entropy-based statistic asymptotically follows a χ 2 distribution. Computer simulations show that the entropy-based approach has better control of type I error and higher power compared to the standard χ 2 test. Motivated by a schizophrenia data set, we propose a method for measuring and testing the relative entropy of a clinical phenotype, through which one can test the contribution or interaction of multiple disease loci to a clinical phenotype. A sequential forward selection procedure is proposed to construct a genetic interaction network which is illustrated through a tree-based diagram. The network information clearly shows the relative importance of a set of genetic loci on a clinical phenotype. To show the utility of the new entropy-based approach, it is applied to analyze two real data sets, a schizophrenia data set and a published malaria data set. Our approach provides a fast and testable framework for genetic epistasis study in a case-only design.

Sex Differences in Genetic Variation in Weight: a Longitudinal Study of Body Mass Index in Adolescent Twins

Genes that influence a phenotype earlier in life may differ from those influencing the same phenotype later, particularly during significant development periods such as puberty, when it is known that new genetic and environmental influences may become important. In the present study, body mass index (BMI) data were collected from 470 monozygotic twin pairs and 673 dizygotic twin pairs longitudinally at ages 12, 14 and 16, roughly straddling puberty. In order to examine whether there are qualitative and quantitative differences in genetic and environmental influences affecting BMI in males and females, during development, a general sex-limitation simplex model (which represents the longitudinal time series of the data) was fitted to the repeated measurements of BMI. The ADE simplex model provided the best fit to the adolescent data, with disparity in the magnitude of additive genetic influences between sexes, but no differences in the non-additive genetic (epistasis or dominance) or environmental influences. Results found may reflect many genetic and environmental influences during puberty, including the possible complex interaction between genes involved in the biological mechanism of weight regulation and the development of likely peer pressured activities such as severe exercise and diet regimes. Although, over 1,000 pairs of twins were used, this study still lacked the power to properly discriminate between additive and non-additive genetic variance.

The molecular underpinnings of genetic phenomena

Epiphenomena are those processes that ostensibly have no precedent at lower levels of scientific organization. In this review, it is argued that many genetic processes, including ploidy, dominance, heritability, pleiotropy, epistasis, mutational load and recombination, all are at least analogous to biochemical events that were requisite features of the RNA world. Most, if not all, of these features of contemporary whole organisms and populations may have their ultimate evolutionary roots in the chemical repertoire of catalytic RNAs. Some of these phenomena will eventually prove to be not only analogous but homologous to ribozyme activities.

Six3 Represses Nodal Activity to Establish Early Brain Asymmetry in Zebrafish

The vertebrate brain is anatomically and functionally asymmetric; however, the molecular mechanisms that establish left-right brain patterning are largely unknown. In zebrafish, asymmetric left-sided Nodal signaling within the developing dorsal diencephalon is required for determining the direction of epithalamic asymmetries. Here, we show that Six3, a transcription factor essential for forebrain formation and associated with holoprosencephaly in humans, regulates diencephalic Nodal activity during initial establishment of brain asymmetry. Reduction of Six3 function causes brain-specific deregulation of Nodal pathway activity, resulting in epithalamic laterality defects. Based on misexpression and genetic epistasis experiments, we propose that Six3 acts in the neuroectoderm to establish a prepattern of bilateral repression of Nodal activity. Subsequently, Nodal signaling from the left lateral plate mesoderm alleviates this repression ipsilaterally. Our data reveal a Six3-dependent mechanism for establishment of correct brain laterality and provide an entry point to understanding the genetic regulation of Nodal signaling in the brain.

A linkage and family-based association analysis of a potential neurocognitive endophenotype of bipolar disorder

The identification of the genetic variants underpinning bipolar disorder (BPD) has been impeded by a complex pattern of inheritance characterized by genetic and phenotypic heterogeneity, genetic epistasis, and gene-environment interactions. In this paper two strategies were used to ameliorate these confounding factors. A unique South African sample including 190 individuals of the relatively, reproductively isolated Afrikaner population was assessed with a battery of neuropsychological tests in an attempt to identify a BPD-associated quantitative trait or endophenotype. BPD individuals performed significantly worse than their unaffected relatives on visual and verbal memory tasks, a finding congruent with the literature. Afocused linkage and family-based association study was carried out using this memory-related endophenotype. In the largest 77-strong Afrikaner pedigree significant evidence for linkage was detected on chromosome 22q11, a region previously implicated in BPD. The quantitative transmission disequilibrium tests-based association analysis suggested that functional variants of the DRD4 and MAO-A genes modulate memory-related cognition. We speculate that polymorphisms at these loci may predispose to a subtype of BPD characterized by memory-related deficits.

Longevity determined by developmental arrest genes in Caenorhabditis elegans

The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.

Modeling sequence–sequence interactions for drug response

Genetic interactions or epistasis may play an important role in the genetic etiology of drug response. With the availability of large-scale, high-density single nucleotide polymorphism markers, a great challenge is how to associate haplotype structures and complex drug response through its underlying pharmacodynamic mechanisms. We have derived a general statistical model for detecting an interactive network of DNA sequence variants that encode pharmacodynamic processes based on the haplotype map constructed by single nucleotide polymorphisms. The model was validated by a pharmacogenetic study for two predominant beta-adrenergic receptor (betaAR) subtypes expressed in the heart, beta1AR and beta2AR. Haplotypes from these two receptors trigger significant interaction effects on the response of heart rate to different dose levels of dobutamine. This model will have implications for pharmacogenetic and pharmacogenomic research and drug discovery. A computer program written in Matlab can be downloaded from the webpage of statistical genetics group at the University of Florida. Supplementary data are available at Bioinformatics online.

Genetic epistasis in the VLDL catabolic pathway is associated with deleterious variations on triglyceridemia in obese subjects

Abdominal obesity and hypertriglyceridemia (the hypertriglyceridemic-waist phenotype) increase cardiovascular risk. The very low-density lipoprotein (VLDL) is a triglyceride (TG)-rich particle. Frequent variations in the genes coding for enzymes and proteins involved in the VLDL catabolism have already been documented. The epistatic effect of such variants on the risk profile associated with abdominal obesity remains to be elucidated. This study aims to assess the effect of combinations of frequent single-nucleotide polymorphisms (SNPs) in the VLDL catabolic pathway on the relation between abdominal obesity and fasting TG. Only gene variants in the lipoprotein lipase, apolipoprotein (apo) CIII, hepatic lipase and apo E genes known to be frequent in the general population (allele frequency>5%) were included in this study. The presence of selected SNPs was detected by polymerase chain reaction-restriction fragment length polymorphism in a sample of 640 non-diabetic French Canadians at high cardiovascular risk (405 obese, 235 non-obese). Carrying more than two frequent gene variants involved in the VLDL catabolic pathway significantly increased the risk of hyperTG (odds ratio of TG>1.7 mmol/l=4.15; P=0.001). This effect was proportional to the number of SNPs and genes involved and was significantly amplified by the presence of abdominal obesity defined on the basis of waist circumference. When combined with abdominal obesity, epistasis in the VLDL pathway has a deleterious effect on fasting TG and coronary artery disease risk profile according to the TG threshold (1.7 mmol/l) used in medical guidelines for the assessment of the metabolic syndrome and associated risk.

C. elegansEVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

During C. elegans development, LIN-12 (Notch) signaling specifies the anchor cell (AC) and ventral uterine precursor cell (VU) fates from two equivalent pre-AC/pre-VU cells in the hermaphrodite gonad. Once specified, the AC induces patterned proliferation of vulva via expression of LIN-3 (EGF) and then invades into the vulval epithelium. Although these cellular processes are essential for the proper organogenesis of vulva and appear to be temporally regulated, the mechanisms that coordinate the processes are not well understood. We computationally identified egl-43 as a gene likely to be expressed in the pre-AC/pre-VU cells and the AC, based on the presence of an enhancer element similar to the one that transcribes lin-3 in the same cells. Genetic epistasis analyses reveal that egl-43 acts downstream of or parallel to lin-12 in AC/VU cell fate specification at an early developmental stage, and functions downstream of fos-1 as well as upstream of zmp-1 and him-4 to regulate AC invasion at a later developmental stage. Characterization of the egl-43 regulatory region suggests that EGL-43 is a direct target of LIN-12 and HLH-2 (E12/47), which is required for the specification of the VU fate during AC/VU specification. EGL-43 also regulates basement membrane breakdown during AC invasion through a FOS-1-responsive regulatory element that drives EGL-43 expression in the AC and VU cells at the later stage. Thus, egl-43 integrates temporally distinct upstream regulatory events and helps program cell fate specification and cell invasion.

The Ral/exocyst effector complex counters c-Jun N-terminal kinase-dependent apoptosis in Drosophila melanogaster.

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK.

Growth Regulation: A Beginning for the Hippo Pathway

A signaling pathway involving two protein kinases, Hippo and Warts, restricts the growth of imaginal discs in Drosophila. Four recent studies taken together show that the protocadherin Fat can regulate Warts in two different ways.

The Drosophila inhibitor of apoptosis protein DIAP2 functions in innate immunity and is essential to resist gram-negative bacterial infection.

The founding member of the inhibitor of apoptosis protein (IAP) family was originally identified as a cell death inhibitor. However, recent evidence suggests that IAPs are multifunctional signaling devices that influence diverse biological processes. To investigate the in vivo function of Drosophila melanogaster IAP2, we have generated diap2 null alleles. diap2 mutant animals develop normally and are fully viable, suggesting that diap2 is dispensable for proper development. However, these animals were acutely sensitive to infection by gram-negative bacteria. In Drosophila, infection by gram-negative bacteria triggers the innate immune response by activating the immune deficiency (imd) signaling cascade, a NF-kappaB-dependent pathway that shares striking similarities with the pathway of mammalian tumor necrosis factor receptor 1 (TNFR1). diap2 mutant flies failed to activate NF-kappaB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumbed to bacterial infection. Our genetic epistasis analysis places diap2 downstream of or in parallel to imd, Dredd, Tak1, and Relish. Therefore, DIAP2 functions in the host immune response to gram-negative bacteria. In contrast, we find that the Drosophila TNFR-associated factor (Traf) family member Traf2 is dispensable in resistance to gram-negative bacterial infection. Taken together, our genetic data identify DIAP2 as an essential component of the Imd signaling cascade, protecting the organism from infiltrating microbes.

Sli-3 negatively regulates the LET-23/epidermal growth factor receptor-mediated vulval induction pathway in Caenorhabditis elegans.

The LIN-3-LET-23-mediated inductive signaling pathway plays a major role during vulval development in C. elegans. Studies on the components of this pathway have revealed positive as well as negative regulators that function to modulate the strength and specificity of the signal transduction cascade. We have carried out genetic screens to identify new regulators of this pathway by screening for suppressors of lin-3 vulvaless phenotype. The screens recovered three loci including alleles of gap-1 and a new gene represented by sli-3. Our genetic epistasis experiments suggest that sli-3 functions either downstream or in parallel to nuclear factors lin-1 and sur-2. sli-3 synergistically interacts with the previously identified negative regulators of the let-23 signaling pathway and causes excessive cell proliferation. However, in the absence of any other mutation sli-3 mutant animals display wild-type vulval induction and morphology. We propose that sli-3 functions as a negative regulator of vulval induction and defines a branch of the inductive signaling pathway. We provide evidence that sli-3 interacts with the EGF signaling pathway components during vulval induction but not during viability and ovulation processes. Thus, sli-3 helps define specificity of the EGF signaling to induce the vulva.

Activated AKT/PKB signaling in C. elegansuncouples temporally distinct outputs of DAF-2/insulin-like signaling

BackgroundIn the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals.ResultsThis report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes.ConclusionA screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants.

Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo

In mammals, Pax3, Six4, Six1 and Six5 genes are co-expressed with Eya1, Eya2 and Eya4 genes during mouse somitogenesis. To unravel the functions of Eya genes during muscle development, we analyzed myogenesis in Eya2-/− and in Eya1−/− embryos. A delay in limb myogenesis was observed between E10 and E13 in Eya1−/− embryos only, that is later compensated. Compound E18 Eya1−/−Eya2−/+ fetuses present a muscle phenotype comparable with that of Six1−/− fetuses; lacking a diaphragm and with a specific absence of limb muscles, suggesting either genetic epistasis between Six and Eya genes, or biochemical interactions between Six and Eya proteins. We tested these two non-exclusive possibilities. First, we show that Six proteins recruit Eya proteins to drive transcription during embryogenesis in the dermomyotomal epaxial and hypaxial lips of the somites by binding MEF3 DNA sites. Second, we show that Pax3 expression is lost in the ventrolateral (hypaxial) dermomyotomes of the somite in both Eya1−/−Eya2−/− embryos and in Six1−/−Six4−/− embryos, precluding hypaxial lip formation. This structure, from which myogenic cells delaminate to invade the limb does not form in these double mutant embryos, leading to limb buds without myogenic progenitor cells. Eya1 and Eya2, however, are still expressed in the somites of Six1Six4 double mutant and in splotch embryos, and Six1 is expressed in the somites of Eya1Eya2 double mutant embryos and in splotch embryos. Altogether these results show that Six and Eya genes lie genetically upstream of Pax3 gene in the formation of ventrolateral dermomyotome hypaxial lips. No genetic links have been characterized between Six and Eya genes, but corresponding proteins activate key muscle determination genes (Myod, Myogenin and Mrf4). These results establish a new hierarchy of genes controlling early steps of hypaxial myogenic commitment in the mouse embryo.

The Predicted G-Protein-Coupled Receptor GPR-1 Is Required for Female Sexual Development inthe Multicellular Fungus Neurospora crassa

G-protein-coupled receptors (GPCRs) control important aspects of asexual and sexual development in eukaryotic organisms. We have identified a predicted GPCR in the filamentous fungus Neurospora crassa with similarity to cyclic AMP-receptor like GPCRs from Dictyostelium discoideum and GCR1 from Arabidopsis thaliana. Expression of gpr-1 is highest in female reproductive structures, and deletion of gpr-1 leads to defects during sexual development. Unfertilized female structures (protoperithecia) from Deltagpr-1 strains are weakly pigmented, small, and submerged in the agar. The perithecia produced after fertilization have deformed beaks that lack ostioles, the openings through which ascospores are discharged. Localization studies using a GPR-1-green fluorescent protein fusion protein showed that GPR-1 is targeted to female reproductive structures. Genetic epistasis experiments with the three Galpha genes were inconclusive due to the early block in mating exhibited by Deltagna-1 strains. Phenotypic analysis of mutants from a high-throughput N. crassa knockout project allowed identification of BEK-1, a homeodomain transcription factor that is a potential target of GPR-1. The perithecial defects of Deltabek-1 strains are similar to those of the Deltagpr-1 strain, and epistasis analysis indicates that bek-1 could function downstream of gpr-1 during postfertilization events. The effect must be posttranscriptional, as bek-1 transcript levels are not affected in Deltagpr-1 strains. The lack of ostioles in Deltagpr-1 and Deltabek-1 mutants has an undesirable effect on the ability to spread progeny (ascospores) by the normal ejection mechanism and would severely compromise the fitness of these strains in nature.

C. elegans GLA-3 is a novel component of the MAP kinase MPK-1 signaling pathway required for germ cell survival

During oocyte development in Caenorhabditis elegans, approximately half of all developing germ cells undergo apoptosis. While this process is evolutionarily conserved from worms to humans, the regulators of germ cell death are still largely unknown. In a genetic screen for novel genes involved in germline apoptosis in Caenorhabditis elegans, we identified and cloned gla-3. Loss of gla-3 function results in increased germline apoptosis and reduced brood size due to defective pachytene exit from meiosis I. gla-3 encodes a TIS11-like zinc-finger-containing protein that is expressed in the germline, from the L4 larval stage to adulthood. Biochemical evidence and genetic epistasis analysis revealed that GLA-3 participates in the MAPK signaling cascade and directly interacts with the C. elegans MAPK MPK-1, an essential meiotic regulator. Our results show that GLA-3 is a new component of the MAPK cascade that controls meiotic progression and apoptosis in the C. elegans germline and functions as a negative regulator of the MAPK signaling pathway during vulval development and in muscle cells.

Genetic context determines susceptibility to intraocular pressure elevation in a mouse pigmentary glaucoma.

BackgroundDBA/2J (D2) mice develop an age-related form of glaucoma. Their eyes progressively develop iris pigment dispersion and iris atrophy followed by increased intraocular pressure (IOP) and glaucomatous optic nerve damage. Mutant alleles of the Gpnmb and Tyrp1 genes are necessary for the iris disease, but it is unknown whether alleles of other D2 gene(s) are necessary for the distinct later stages of disease. We initiated a study of congenic strains to further define the genetic requirements and disease mechanisms of the D2 glaucoma.ResultsTo further understand D2 glaucoma, we created congenic strains of mice on the C57BL/6J (B6) genetic background. B6 double-congenic mice carrying D2-derived Gpnmb and Tyrp1 mutations develop a D2-like iris disease. B6 single-congenics with only the Gpnmb and Tyrp1 mutations develop milder forms of iris disease. Genetic epistasis experiments introducing a B6 tyrosinase mutation into the congenic strains demonstrated that both the single and double-congenic iris diseases are rescued by interruption of melanin synthesis. Importantly, our experiments analyzing mice at ages up to 27 months indicate that the B6 double-congenic mice are much less prone to IOP elevation and glaucoma than are D2 mice.ConclusionAs demonstrated here, the Gpnmb and Tyrp1 iris phenotypes are both individually dependent on tyrosinase function. These results support involvement of abnormal melanosomal events in the diseases caused by each gene. In the context of the inbred D2 mouse strain, the glaucoma phenotype is clearly influenced by more genes than just Gpnmb and Tyrp1. Despite the outward similarity of pigment-dispersing iris disease between D2 and the B6 double-congenic mice, the congenic mice are much less susceptible to developing high IOP and glaucoma. These new congenic strains provide a valuable new resource for further studying the genetic and mechanistic complexity of this form of glaucoma.