A multi-omic analysis of MCF10A cells provides a resource for integrative assessment of ligand-mediated molecular and phenotypic responses.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Bayesian Network Inference Modeling Identifies TRIB1 as a Novel Regulator of Cell-Cycle Progression and Survival in Cancer Cells.

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G1-S transition. The model identified known components of the cell-cycle machinery, such as CCND1, CCNE2, and CDC25A, as well as revealed novel regulators of G1-S transition, IER2, TRIB1, TRIM27. Experimental validation of model predictions confirmed 10 of 12 predicted genes to have a role in G1-S progression. Further analysis showed that TRIB1 regulated the cyclin D1 promoter via NFκB and AP-1 sites and sensitized cells to TRAIL-induced apoptosis. In clinical specimens of breast cancer, TRIB1 levels correlated with expression of NFκB and its target genes (IL8, CSF2), and TRIB1 copy number and expression were predictive of clinical outcome. Together, our results establish a critical role of TRIB1 in cell cycle and survival that is mediated via the modulation of NFκB signaling. Cancer Res; 77(7); 1575-85. ©2017 AACR.

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer.

BACKGROUND: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors. METHODS: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA). RESULTS: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup. CONCLUSIONS: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.

Genome-wide linkage scan of antisocial behavior, depression, and impulsive substance use in the UCSF family alcoholism study.

OBJECTIVE: Epidemiological and clinical studies suggest that the rates of antisocial behavior, depression, and impulsive substance use are increased among individuals diagnosed with alcohol dependence relative to those who are not. Thus, the present study conducted genome-wide linkage scans of antisocial behavior, depression, and impulsive substance use in the University of California at San Francisco Family Alcoholism Study. METHODS: Antisocial behavior, depressive symptoms, and impulsive substance use were assessed using three scales from the Minnesota Multiphasic Personality Inventory - 2nd ed.: the Antisocial Practices content scale, the Depression content scale, and the revised MacAndrew Alcoholism scale. Linkage analyses were carried out using a variance components approach. RESULTS: Suggestive evidence of linkage to three genomic regions independent of alcohol and cannabis dependence diagnostic status was observed: the Antisocial Practices content scale showed evidence of linkage to chromosome 13 at 11 cM, the MacAndrew Alcoholism scale showed evidence of linkage to chromosome 15 at 47 cM, and all three scales showed evidence of linkage to chromosome 17 at 57-58 cM. CONCLUSION: Each of these regions has shown previous evidence of linkage and association to substance dependence as well as other psychiatric disorders such as mood and anxiety disorders, attention-deficit hyperactivity disorder, and schizophrenia, thus suggesting potentially broad relations between these regions and psychopathology.

Subtype and pathway specific responses to anticancer compounds in breast cancer.

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy.

Pancreatic ductal adenocarcinoma (PDA) is a lethal disease. Overall survival is typically 6 months from diagnosis. Numerous phase 3 trials of agents effective in other malignancies have failed to benefit unselected PDA populations, although patients do occasionally respond. Studies in other solid tumors have shown that heterogeneity in response is determined, in part, by molecular differences between tumors. Furthermore, treatment outcomes are improved by targeting drugs to tumor subtypes in which they are selectively effective, with breast and lung cancers providing recent examples. Identification of PDA molecular subtypes has been frustrated by a paucity of tumor specimens available for study. We have overcome this problem by combined analysis of transcriptional profiles of primary PDA samples from several studies, along with human and mouse PDA cell lines. We define three PDA subtypes: classical, quasimesenchymal and exocrine-like, and we present evidence for clinical outcome and therapeutic response differences between them. We further define gene signatures for these subtypes that may have utility in stratifying patients for treatment and present preclinical model systems that may be used to identify new subtype specific therapies.

Linkage scan of alcohol dependence in the UCSF Family Alcoholism Study.

Ample data suggest that alcohol dependence represents a heritable condition, and several research groups have performed linkage analysis to identify genomic regions influencing this disorder. In the present study, a genome-wide linkage scan for alcohol dependence was conducted in a community sample of 565 probands and 1080 first-degree relatives recruited through the UCSF Family Alcoholism Study. The Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) was used to derive DSM-IV alcohol dependence diagnoses. Although no loci achieved genome-wide significance (i.e., LOD score > 3.0), several linkage peaks of interest (i.e., LOD score > 1.0) were identified. When the strict DSM-IV alcohol dependence diagnosis requiring the temporal clustering of symptoms served as the phenotype, linkage peaks were identified on chromosomes 1p36.31-p36.22, 2q37.3, 8q24.3, and 18p11.21-p11.2. When the temporal clustering of symptoms was not required, linkage peaks were again identified on chromosomes 1p36.31-p36.22 and 8q24.3 as well as novel loci on chromosomes 1p22.3, 2p24.3-p24.1, 9p24.1-p23, and 22q12.3-q13.1. Follow-up analyses were conducted by performing linkage analysis for the 12 alcohol dependence symptoms assessed by the SSAGA across the support intervals for the observed linkage peaks. These analyses demonstrated that different collections of symptoms often assessing distinct aspects of alcohol dependence (e.g., uncontrollable drinking and withdrawal vs. tolerance and drinking despite health problems) contributed to each linkage peak and often yielded LOD scores exceeding that reported for the alcohol dependence diagnosis. Such findings provide insight into how specific genomic regions may influence distinct aspects of alcohol dependence.

The investigation into CYP2E1 in relation to the level of response to alcohol through a combination of linkage and association analysis.

BACKGROUND: A low level of response to alcohol during an individual's early experience with alcohol is associated with an increase risk of alcoholism. A family-based genome-wide linkage analysis using sibling pairs that underwent an alcohol challenge where the level of response to alcohol was measured with the Subjective High Assessment Scale (SHAS) implicated the 10q terminal (10qter) region. CYP2E1, a gene known for its involvement with ethanol metabolism, maps to this region. METHODS: Variance component multipoint linkage analysis was performed on a combined map of single-nucleotide polymorphism (SNP) and microsatellite data. To account for the heterogeneity evident in the dataset, a calculation assuming locus heterogeneity was made using the Heterogeneity Log of Odds (HLOD) score. Association between SNP marker allele counts and copy number and SHAS scores were evaluated using a logistic regression model. RESULTS: Linkage analysis detected significant linkage to CYP2E1, which was diminished because of apparent locus heterogeneity traced to a single family with extreme phenotypes. In retrospect, circumstances recorded during testing for this family suggest that their phenotype data are likely to be unreliable. Significant allelic associations were detected for several CYP2E1 polymorphisms and the SHAS score. DNA sequencing from families that contributed the greatest evidence for linkage did not detect any changes directly affecting the primary amino acid sequence. With the removal of a single family, combined evidence from microsatellites and SNPs offers significant linkage between the level of response to alcohol and the region on the end of chromosome 10. CONCLUSION: Combined linkage and association indicate that sequence changes in or near CYP2E1 affect the level of response to alcohol providing a predictor of risk of alcoholism. The absence of coding sequence changes indicates that regulatory sequences are responsible. Implicating CYP2E1 in the level of response to alcohol allows inferences to be made about how the brain perceives alcohol.

Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.

The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM). We describe a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural subtypes, respectively. Gene signatures of normal brain cell types show a strong relationship between subtypes and different neural lineages. Additionally, response to aggressive therapy differs by subtype, with the greatest benefit in the Classical subtype and no benefit in the Proneural subtype. We provide a framework that unifies transcriptomic and genomic dimensions for GBM molecular stratification with important implications for future studies.

Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer.

PURPOSE: Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer. EXPERIMENTAL DESIGN: We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies. RESULTS: In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone. CONCLUSIONS: These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer.

A systems analysis of the chemosensitivity of breast cancer cells to the polyamine analogue PG-11047.

BACKGROUND: Polyamines regulate important cellular functions and polyamine dysregulation frequently occurs in cancer. The objective of this study was to use a systems approach to study the relative effects of PG-11047, a polyamine analogue, across breast cancer cells derived from different patients and to identify genetic markers associated with differential cytotoxicity. METHODS: A panel of 48 breast cell lines that mirror many transcriptional and genomic features present in primary human breast tumours were used to study the antiproliferative activity of PG-11047. Sensitive cell lines were further examined for cell cycle distribution and apoptotic response. Cell line responses, quantified by the GI50 (dose required for 50% relative growth inhibition) were correlated with the omic profiles of the cell lines to identify markers that predict response and cellular functions associated with drug sensitivity. RESULTS: The concentrations of PG-11047 needed to inhibit growth of members of the panel of breast cell lines varied over a wide range, with basal-like cell lines being inhibited at lower concentrations than the luminal cell lines. Sensitive cell lines showed a significant decrease in S phase fraction at doses that produced little apoptosis. Correlation of the GI50 values with the omic profiles of the cell lines identified genomic, transcriptional and proteomic variables associated with response. CONCLUSIONS: A 13-gene transcriptional marker set was developed as a predictor of response to PG-11047 that warrants clinical evaluation. Analyses of the pathways, networks and genes associated with response to PG-11047 suggest that response may be influenced by interferon signalling and differential inhibition of aspects of motility and epithelial to mesenchymal transition.

Immutable functional attributes of histologic grade revealed by context-independent gene expression in primary breast cancer cells.

Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a "paracrine-independent expression of grade-associated" (PEGA) gene signature. PEGA members extended beyond "proliferation-driven" signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed.

Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells.

Normal human epithelial cells in culture have generally shown a limited proliferative potential of approximately 10 to 40 population doublings before encountering a stress-associated senescence barrier (stasis) associated with elevated levels of cyclin-dependent kinase inhibitors p16 and/or p21. We now show that simple changes in medium composition can expand the proliferative potential of human mammary epithelial cells (HMEC) initiated as primary cultures to 50 to 60 population doublings followed by p16-positive, senescence-associated beta-galactosidase-positive stasis. We compared the properties of growing and senescent pre-stasis HMEC with growing and senescent post-selection HMEC, that is, cells grown in a serum-free medium that overcame stasis via silencing of p16 expression and that display senescence associated with telomere dysfunction. Cultured pre-stasis populations contained cells expressing markers associated with luminal and myoepithelial HMEC lineages in vivo in contrast to the basal-like phenotype of the post-selection HMEC. Gene transcript and protein expression, DNA damage-associated markers, mean telomere restriction fragment length, and genomic stability differed significantly between HMEC populations at the stasis versus telomere dysfunction senescence barriers. Senescent isogenic fibroblasts showed greater similarity to HMEC at stasis than at telomere dysfunction, although their gene transcript profile was distinct from HMEC at both senescence barriers. These studies support our model of the senescence barriers encountered by cultured HMEC in which the first barrier, stasis, is retinoblastoma-mediated and independent of telomere length, whereas a second barrier (agonescence or crisis) results from telomere attrition leading to telomere dysfunction. Additionally, the ability to maintain long-term growth of genomically stable multilineage pre-stasis HMEC populations can greatly enhance experimentation with normal HMEC.

Basal subtype and MAPK/ERK kinase (MEK)-phosphoinositide 3-kinase feedback signaling determine susceptibility of breast cancer cells to MEK inhibition.

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies.

Comparative analyses of gene copy number and mRNA expression in glioblastoma multiforme tumors and xenografts.

Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors, genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.

A genome-wide screen for nicotine dependence susceptibility loci.

Genome-wide model free linkage analysis was conducted for nicotine dependence and tobacco use phenotypes in 607 members of 158 nuclear families consisting of at least two ever smokers (100 or more cigarettes smoked in lifetime). DNA from whole blood was genotyped for 739 autosomal microsatellite polymorphisms with an average inter-marker distance of 4.6 cM. A peak LOD score of 2.7 was observed on chromosome 6 for scores for the Fagerström Test for Nicotine Dependence. Exploratory analyses were conducted to determine whether sequence variation at other loci affected other measures of dependence or tobacco use. Four additional loci with LOD scores of 2.7 or more were associated with alternative measures of nicotine dependence, one with current frequency of use, and one with smoking cessation. Several of the corresponding support intervals were near putative loci reported previously (on chromosomes 6, 7, and 8) while others appear to be novel (on chromosomes 5, 16, and 19).

Autosomal linkage analysis for the level of response to alcohol.

BACKGROUND: The level of response (LR) to alcohol is a genetically-influenced phenotype related to the alcoholism risk. Usually measured by evaluating psychological and physiological changes that follow the administration of alcohol, the heritability of LR is estimated to be between 0.4 and 0.6, and efforts are being made to find genes related to this phenotype. This paper presents data from a family-based genome with linkage analysis focusing on alcohol challenge determinants of LR. METHODS: The subjects were 18-to-29-year-old sibling pairs with at least one parent who was alcohol-dependent and who had experience with alcohol but were not yet alcohol-dependent themselves. Both members of the sibling pairs were given oral alcohol challenges (0.75-0.90 ml/kg of ethanol for females and males, respectively), with LR established using the Subjective High Assessment Scale (SHAS) and changes in body sway (BS) repeatedly over a 3.5-hr. period. Blood samples from siblings and at least one parent were genotyped using 811 microsatellite markers, with results evaluated using several related variance component approaches as implemented in SOLAR for continuous traits. In addition, association was tested using single nucleotide polymorphisms (SNPs) within the KCNMA1, HTR7 and SLC18A2 genes that may relate to a finding on chromosome 10. RESULTS: Data were generated from 238 sib-pairs representing 365 individuals (41.6% were males) from 165 families. The most consistent results across methods and samples were observed for SHAS on chromosome 10 between 120 and 140 cM (with a maximum LOD score of 2.6 at 122 cM), and a second region of possible interest at 173 cM (LOD = 1.2). Statistical analysis with the KCNMA1, HTR7 and SLC18A2 genes, which lie in the support region of interest revealed no evidence for association after correction for multiple comparisons. CONCLUSIONS: These evaluations from the largest known alcohol challenge-based genetic study to date highlight the potential importance of genes on chromosome 10 as possible contributors to the low LR to alcohol as a risk factor for alcoholism.

Support for previously identified alcoholism susceptibility Loci in a cohort selected for smoking behavior.

BACKGROUND: Alcohol consumption and alcoholism are heritable traits. Previous linkage analyses for alcoholism and related traits have identified several putative susceptibility loci. In this paper we use, for the first time, linkage analysis to search for alcoholism-related phenotypes in a family sample selected for smoking behavior. METHODS: Genome-wide model free linkage analysis was conducted for a variety of phenotypes related to alcohol consumption in 158 nuclear families ascertained for having at least two first-degree relatives who smoked 100 or more cigarettes in their lifetime. The phenotypes included dichotomous, ordinal, and continuous traits. Because the traits were typically not normally distributed the QTL score statistic as implemented in Merlin was employed to deal with deviations from normality. Simulation analysis determined that the QTL score statistic is robust to deviations from normality. RESULTS: Linkage analysis detected three loci, one on chromosome 2 and two on chromosome 4, with nominal significance (LOD score > 2.7). These loci appear to be in close proximity to loci reported in other studies. CONCLUSIONS: While these findings did not reach genome-wide significance (LOD >4.0 given multiple comparisons) we have confidence that genes in these regions affect alcohol consumption. Two of the three significant findings in this analysis have been reported previously as alcoholism susceptibility loci. Simulation analysis shows that the most widely replicated finding on chromosome 4 is strongly supported (p=0.01) even with correction for multiple comparisons. These findings suggest that previously reported linkage results are robust to the effects of different approaches to sample ascertainment and definition.