Consistent but indirect evidence has implicated genetic factors in smoking behavior1,2. We report meta-analyses of several smoking phenotypes within cohorts of the Tobacco and Genetics Consortium (n = 74,053). We also partnered with the European Network of Genetic and Genomic Epidemiology (ENGAGE) and Oxford-GlaxoSmithKline (Ox-GSK) consortia to follow up the 15 most significant regions (n > 140,000). We identified three loci associated with number of cigarettes smoked per day. The strongest association was a synonymous 15q25 SNP in the nicotinic receptor gene CHRNA3 (rs1051730[A], β = 1.03, standard error (s.e.) = 0.053, P = 2.8 × 10−73). Two 10q25 SNPs (rs1329650[G], β = 0.367, s.e. = 0.059, P = 5.7 × 10−10; and rs1028936[A], β = 0.446, s.e. = 0.074, P = 1.3 × 10−9) and one 9q13 SNP in EGLN2 (rs3733829[G], β = 0.333, s.e. = 0.058, P = 1.0 × 10−8) also exceeded genome-wide significance for cigarettes per day. For smoking initiation, eight SNPs exceeded genome-wide significance, with the strongest association at a nonsynonymous SNP in BDNF on chromosome 11 (rs6265[C], odds ratio (OR) = 1.06, 95% confidence interval (Cl) 1.04–1.08, P = 1.8 × 10−8). One SNP located near DBH on chromosome 9 (rs3025343[G], OR = 1.12, 95% Cl 1.08–1.18, P = 3.6 × 10−8) was significantly associated with smoking cessation.

Recent findings in tumor biopsies from lung adenocarcinoma patients suggest that somatic mutations in the genes encoding epidermal growth factor receptor (EGFR) and Kirsten ras (KRAS) confer sensitivity and resistance, respectively, to EGFR inhibition. Here, we provide evidence that these genetic mutations are not sufficient to modulate the biological response of lung adenocarcinoma cells to EGFR inhibition. We found high expression of ErbB family members, ErbB ligands, or both in three models that were sensitive to EGFR inhibition, including alveolar epithelial neoplastic lesions in mice that develop lung adenocarcinoma by oncogenic KRAS, human lung adenocarcinoma cell lines, and tumor biopsies from lung adenocarcinoma patients. Thus, lung adenocarcinoma cells that depend on EGFR for survival constitutively activate the receptor through a combination of genetic mutations and overexpression of EGFR dimeric partners and their ligands. (Cancer Res 2005; 65(24): 11478-85)

Smoking influences body weight such that smokers weigh less than non-smokers and smoking cessation often leads to weight increase. The relationship between body weight and smoking is partly explained by the effect of nicotine on appetite and metabolism. However, the brain reward system is involved in the control of the intake of both food and tobacco. We evaluated the effect of single-nucleotide polymorphisms (SNPs) affecting body mass index (BMI) on smoking behavior, and tested the 32 SNPs identified in a meta-analysis for association with two smoking phenotypes, smoking initiation (SI) and the number of cigarettes smoked per day (CPD) in an Icelandic sample (N=34 216 smokers). Combined according to their effect on BMI, the SNPs correlate with both SI (r=0.019, P=0.00054) and CPD (r=0.032, P=8.0 × 10−7). These findings replicate in a second large data set (N=127 274, thereof 76 242 smokers) for both SI (P=1.2 × 10−5) and CPD (P=9.3 × 10−5). Notably, the variant most strongly associated with BMI (rs1558902-A in FTO) did not associate with smoking behavior. The association with smoking behavior is not due to the effect of the SNPs on BMI. Our results strongly point to a common biological basis of the regulation of our appetite for tobacco and food, and thus the vulnerability to nicotine addiction and obesity.

The maintenance of normal body weight is disrupted in patients with anorexia nervosa (AN) for prolonged periods of time. Prior to the onset of AN, premorbid body mass index (BMI) spans the entire range from underweight to obese. After recovery, patients have reduced rates of overweight and obesity. As such, loci involved in body weight regulation may also be relevant for AN and vice versa. Our primary analysis comprised a cross-trait analysis of the 1000 single nucleotide polymorphisms (SNPs) with the lowest p-values in a genome-wide association meta-analysis (GWAMA) of AN (GCAN) for evidence of association in the largest published GWAMA for BMI (GIANT). Subsequently we performed sex-stratified analyses for these 1000 SNPs. Functional ex vivo studies on four genes ensued. Lastly, a look-up of GWAMA-derived BMI related loci was performed in the AN GWAMA. We detected significant associations (p-values < 5×10−5, Bonferroni corrected p < 0.05) for 9 SNP alleles at 3 independent loci. Interestingly, all AN susceptibility alleles were consistently associated with increased BMI. None of the genes (chr. 10: CTBP2, chr. 19: CCNE1, chr. 2: CARF and NBEAL1; the latter is a region with high linkage disequilibrium) nearest to these SNPs has previously been associated with AN or obesity. Sex-stratified analyses revealed that the strongest BMI signal originated predominantly from females (chr. 10 rs1561589; poverall: 2.47 × 10−06/pfemales: 3.45 × 10−07/pmales: 0.043). Functional ex vivo studies in mice revealed reduced hypothalamic expression of Ctbp2 and Nbeal1 after fasting. Hypothalamic expression of Ctbp2 was increased in diet induced obese (DIO) mice as compared to age-matched lean controls. We observed no evidence for associations for the look-up of BMI related loci in the AN GWAMA. A cross-trait analysis of AN and BMI loci revealed variants at three chromosomal loci with potential joint impact. The chromosome 10 locus is particularly promising given that the association with obesity was primarily driven by females. In addition, the detected altered hypothalamic expression patterns of Ctbp2 and Nbeal1 as a result of fasting and DIO implicate these genes in weight regulation.

Embryos lacking an epidermal growth factor receptor (EGFR) exhibit strain-specific defects in placental development that can result in mid-gestational embryonic lethality. To determine the level of EGFR signaling required for normal placental development, we characterized congenic strains homozygous for the hypomorphic Egfr wa2 allele or heterozygous for the antimorphic Egfr Wa5 allele. Egfr wa2 homozygous embryos and placentas exhibit strain-dependent growth restriction at 15.5 days post-coitus while Egfr Wa5 heterozygous placentas are only slightly reduced in size with no effect on embryonic growth. Egfr wa2 homozygous placentas have a reduced spongiotrophoblast layer in some strains, while spongiotrophoblasts and glycogen cells are almost completely absent in others. Our results demonstrate that more EGFR signaling occurs in Egfr Wa5 heterozygotes than in Egfr wa2 homozygotes and suggest that Egfr wa2 homozygous embryos model EGFR-mediated intrauterine growth restriction in humans. We also consistently observed differences between strains in wild-type placenta and embryo size as well as in the cellular composition and expression of trophoblast cell subtype markers and propose that differential expression in the placenta of Glut3, a glucose transporter essential for normal embryonic growth, may contribute to straindependent differences in intrauterine growth restriction caused by reduced EGFR activity.

Homozygosity for the Egfr(tm1Mag) null allele in mice leads to genetic background dependent placental abnormalities and embryonic lethality. Molecular mechanisms or genetic modifiers that differentiate strains with surviving versus non-surviving Egfr nullizygous embryos have yet to be identified. Egfr transcripts in wildtype placenta were quantified by ribonuclease protection assay (RPA) and the lowest level of Egfr mRNA expression was found to coincide with Egfr(tm1Mag) homozygous lethality. Immunohistochemical analysis of ERBB family receptors, ERBB2, ERBB3, and ERBB4, showed similar expression between Egfr wildtype and null placentas indicating that Egfr null trophoblast do not up-regulate these receptors to compensate for EGFR deficiency. Significantly fewer numbers of bromodeoxyuridine (BrdU) positive trophoblast were observed in Egfr nullizygous placentas and Cdc25a and Myc, genes associated with proliferation, were significantly down-regulated in null placentas. However, strains with both mild and severe placental phenotypes exhibit reduced proliferation suggesting that this defect alone does not account for strain-specific embryonic lethality. Consistent with this hypothesis, intercrosses generating mice null for cell cycle checkpoint genes (Trp53, Rb1, Cdkn1a, Cdkn1b or Cdkn2c) in combination with Egfr deficiency did not increase survival of Egfr nullizygous embryos. Since complete development of the spongiotrophoblast compartment is not required for survival of Egfr nullizygous embryos, reduction of this layer that is commonly observed in Egfr nullizygous placentas likely accounts for the decrease in proliferation.

Epidermal growth factor receptor (EGFR) is a member of the ERBB family of receptor tyrosine kinases that has been shown to play an important developmental and physiologic role in many aspects of pregnancy. We have previously shown in mice that Egfr(tm1Mag) nullizygous placentas have fewer proliferative trophoblasts than wild-type and exhibit strain-specific defects in the spongiotrophoblast and labyrinth layers. In this study we used mice with the hypermorphic Egfr(Dsk5) allele to study the effects of increased levels of EGFR signaling on placental development. On three genetic backgrounds, heterozygosity for Egfr(Dsk5) resulted in larger placental size with a more prominent spongiotrophoblast layer and increased expression of glycogen cell-specific genes. The C3HeB/FeJ strain showed additional placental enlargement of Egfr(Dsk5) homozygotes with a significant number of homozygous embryos dying prior to 15.5 days post-coitus (dpc). We also observed strain-specific subfertility in Egfr(Dsk5) heterozygous females and pregnancy loss was dependent on maternal factors rather than embryo genotype. Higher levels of phospho-EGFR were detected in the uterus of Egfr(Dsk5) heterozygotes but the structure of Egfr(Dsk5) heterozygous nonpregnant uteri appeared similar to wild-type. Collectively, our results demonstrate that mice with increased levels of EGFR signaling exhibit an extensive level of genetic background-dependent phenotypic variability. In addition, EGFR promotes growth of the placental spongiotrophoblast layer in mice, and EGFR expressed in the uterine stroma may play an underappreciated role in preparation of the uterus for embryo implantation.

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