Victoria L. Stevens

Multiple Independent Loci at Chromosome 15q25.1 Affect Smoking Quantity: a Meta-Analysis and Comparison with Lung Cancer and COPD

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10−35 and <10−8 respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10−6). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10−20) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

Modulation of protein kinase C and diverse cell functions by sphingosine — a pharmacologically interesting compound linking sphingolipids and signal transduction

Sphingosine, the backbone moiety of sphingomyelin, gangliosides and other complex sphingolipids, is a potent inhibitor of protein kinase C in vitro and of cellular events dependent on this enzyme. The systems that have been found, thus far, to be affected by sphingosine encompass various components of host defense system, including the activation of platelets, neutrophils and natural killer cells; the cytolytic activity of pathogens and expression of viral genes; cell growth and differentiation in several cell types, including leukemic and neuronal cells; insulin stimulated hexose transport and metabolism in adipocytes; ion-transport systems in various models; the response of neuronal cells to excitatory compounds; and receptor desensitization. While sphingosine has appeared to be a relatively potent and specific inhibitor of protein kinase C in the systems studied, recent findings with the epidermal growth factor receptor indicate that it may serve as a pleotrophic modulator of cell functions. New strategies for the design of pharmacologically active agents should arise from further studies of the action of long-chain (sphingoid) bases. Furthermore, since free sphingosine is a natural constituent of cells and the levels can be modulated by phorbol esters and other factors, a cycle of complex sphingolipid hydrolysis and resynthesis to regulate the amount of free sphingosine may constitute one mechanism of action of these compounds.

Fumonisin B1-induced Sphingolipid Depletion Inhibits Vitamin Uptake via the Glycosylphosphatidylinositol-anchored Folate Receptor

The folate receptor, like many glycosylphosphatidylinositol-anchored proteins, is found associated with membrane domains that are insoluble in Triton X-100 at low temperature and that are enriched in cholesterol and sphingolipids. Depletion of cellular cholesterol has been shown to inhibit vitamin uptake by this receptor (Chang, W.-J., Rothberg, K. G., Kamen, B. A., and Anderson, R. G. W. (1993) J. Cell Biol.118, 63–69), suggesting that these domains regulate this process. In this study, the importance of sphingolipids for folate receptor function was investigated in Caco-2 cells using fumonisin B1, a mycotoxin that inhibits the biosynthesis of these lipids. The folate receptor-mediated transport of 5-methyltetrahydrofolate was almost completely blocked in cells in which sphingolipids had been reduced by ∼40%. This inhibition was dependent on the concentration and duration of the treatment with the mycotoxin and was mediated by the sphingolipid decrease. Neither receptor-mediated nor facilitative transport was inhibited by fumonisin B1 treatment, indicating that the effect of sphingolipid depletion was specific for folate receptor-mediated vitamin uptake. A concurrent loss in the total amount of folate binding capacity in the cells was seen as sphingolipids were depleted, suggesting a causal relationship between folate receptor number and vitamin uptake. These findings suggest that dietary exposure to fumonisin B1could adversely affect folate uptake and potentially compromise cellular processes dependent on this vitamin. Furthermore, because folate deficiency causes neural tube defects, some birth defects unexplained by other known risk factors may be caused by exposure to fumonisin B1.

Muscle mass: reliable indicator of protein-energy malnutrition severity and outcome

Summary In summary, muscle harbors a unique functional protein pool that can be clinically measured as an index of overall PEM sever-ity, regardless of the underlying cause of neg- ative energy balance. The anthropometricdata must be rigorously collected in selected patients by trained personnel and interpreta- tion must include recognition of how massand composition of muscle are interrelated.Muscle indices for use on patients in whomanthropometry is very inaccurate are una-vailable or inadequately validated (Table 2).A simple method of measuring muscle massthat is more accurate and more widely appli-cable than anthropometry is needed. Musclemass can predict clinical outcome when the target is death secondary to fuel depletion, but provides only a background index in theinfection or dehiscence prone trauma or sur-gical patient. U The authors gratefully recognize the clinical assistanceof Drs. Leonard Brubaker and Daniel M. Nixon incollecting the data. References 1. Lehninger AL. Biochemistry. 2nd ed. The molecularbasis of cell structure and function. New York:

Dairy, Calcium, and Vitamin D Intake and Postmenopausal Breast Cancer Risk in the Cancer Prevention Study II Nutrition Cohort

Background: Calcium, vitamin D, and dairy products are highly correlated factors, each with potential roles in breast carcinogenesis. Few prospective studies have examined these relationships in postmenopausal women. Methods: Participants in the Cancer Prevention Study II Nutrition Cohort completed a detailed questionnaire on diet, vitamin and mineral supplement use, medical history, and lifestyle in 1992 to 1993. After exclusion of women with a history of cancer and incomplete dietary data, 68,567 postmenopausal women remained for analysis. During follow-up through August 31, 2001, we identified 2,855 incident cases of breast cancer. Multivariate-adjusted rate ratios (RR) were calculated using Cox proportional hazards models. Results: Women with the highest intake of dietary calcium (>1,250 mg/d) were at a lower risk of breast cancer than those reporting ≤500 mg/d [RR, 0.80; 95% confidence interval (95% CI), 0.67-0.95; Ptrend = 0.02]; however, neither use of supplemental calcium nor vitamin D intake was associated with risk. Consumption starting at two or more servings of dairy products per day was likewise inversely associated with risk (RR, 0.81; 95% CI, 0.69-0.95; Ptrend = 0.002, compared with <0.5 servings/d). The associations were slightly stronger in women with estrogen receptor–positive tumors comparing highest to lowest intake: dietary calcium (RR, 0.67; 95% CI, 0.51-0.88; Ptrend = 0.004); dairy products (RR, 0.73; 95% CI, 0.57-0.93; Ptrend = 0.0003), and dietary vitamin D (RR, 0.74; 95% CI, 0.59-0.93; Ptrend = 0.006). Conclusions: Our results support the hypothesis that dietary calcium and/or some other components in dairy products may modestly reduce risk of postmenopausal breast cancer. The stronger inverse associations among estrogen receptor–positive tumors deserve further study. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2898–904)

Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5

Nicotine dependence risk and lung cancer risk are associated with variants in a region of chromosome 15 encompassing genes encoding the nicotinic receptor subunits CHRNA5, CHRNA3 and CHRNB4. To identify potential biological mechanisms that underlie this risk, we tested for cis-acting eQTLs for CHRNA5, CHRNA3 and CHRNB4 in human brain. Using gene expression and disease association studies, we provide evidence that both nicotine-dependence risk and lung cancer risk are influenced by functional variation in CHRNA5. We demonstrated that the risk allele of rs16969968 primarily occurs on the low mRNA expression allele of CHRNA5. The non-risk allele at rs16969968 occurs on both high and low expression alleles tagged by rs588765 within CHRNA5. When the non-risk allele occurs on the background of low mRNA expression of CHRNA5, the risk for nicotine dependence and lung cancer is significantly lower compared to those with the higher mRNA expression. Together, these variants identify three levels of risk associated with CHRNA5. We conclude that there are at least two distinct mechanisms conferring risk for nicotine dependence and lung cancer: altered receptor function caused by a D398N amino acid variant in CHRNA5 (rs16969968) and variability in CHRNA5 mRNA expression.

Nicotinic receptor gene variants influence susceptibility to heavy smoking.

Heavy smoking is a strong predictor of nicotine dependence, which is a major impediment to smoking cessation. Although both heavy smoking and nicotine dependence are highly heritable, previous attempts to identify genes influencing these phenotypes have been largely unsuccessful until very recently. We studied 1,452 heavy smokers (defined as smoking at least 30 cigarettes per day for at least 5 years) and 1,395 light smokers (defined as smoking <5 cigarettes per day for at least 1 year) to investigate the association of common variants in nicotinic receptor subunit genes with smoking behavior. Compared with the most common allele, two separate groups of single nucleotide polymorphisms (SNP) in the CHRNA5-CHRNA3-CHRNB4 gene cluster were associated with heavy smoking with a very high statistical significance. One group of eight SNPs, which included a nonsynonymous SNP in the CHRNA5 gene, was in strong linkage disequilibrium and associated with increased risk of heavy smoking. A second group of SNPs not strongly correlated with the first was associated with decreased risk of heavy smoking. Analyses that combined both groups of SNPs found associations with heavy smoking that varied by >2-fold. Our findings identify two loci in the CHRNA5-CHRNA3-CHRNB4 gene cluster that predict smoking behavior and provide strong evidence for the involvement of the α5 nicotinic receptor in heavy smoking. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3517–25)

Genome-wide association study identifies new prostate cancer susceptibility loci

Prostate cancer (PrCa) is the most common non-skin cancer diagnosed among males in developed countries and the second leading cause of cancer mortality, yet little is known regarding its etiology and factors that influence clinical outcome. Genome-wide association studies (GWAS) of PrCa have identified at least 30 distinct loci associated with small differences in risk. We conducted a GWAS in 2782 advanced PrCa cases (Gleason grade ≥ 8 or tumor stage C/D) and 4458 controls with 571 243 single nucleotide polymorphisms (SNPs). Based on in silico replication of 4679 SNPs (Stage 1, P < 0.02) in two published GWAS with 7358 PrCa cases and 6732 controls, we identified a new susceptibility locus associated with overall PrCa risk at 2q37.3 (rs2292884, P= 4.3 × 10(-8)). We also confirmed a locus suggested by an earlier GWAS at 12q13 (rs902774, P= 8.6 × 10(-9)). The estimated per-allele odds ratios for these loci (1.14 for rs2292884 and 1.17 for rs902774) did not differ between advanced and non-advanced PrCa (case-only test for heterogeneity P= 0.72 and P= 0.61, respectively). Further studies will be needed to assess whether these or other loci are differentially associated with PrCa subtypes.

GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment

Glycosylphosphatidylinositol (GPI) anchoring is important for the function of several proteins in the context of their membrane trafficking pathways. We have shown previously that endocytosed GPI‐anchored proteins (GPI‐APs) are recycled to the plasma membrane three times more slowly than other membrane components. Recently, we found that GPI‐APs are delivered to endocytic organelles, devoid of markers of the clathrin‐mediated pathway, prior to their delivery to a common recycling endosomal compartment (REC). Here we show that the rate‐limiting step in the recycling of GPI‐APs is their slow exit from the REC; replacement of the GPI anchor with a transmembrane protein sequence abolishes retention in this compartment. Depletion of endogenous sphingolipid levels using sphingolipid synthesis inhibitors or in a sphingolipid‐synthesis mutant cell line specifically enhances the rate of endocytic recycling of GPI‐APs to that of other membrane components. We have shown previously that endocytic retention of GPI‐APs is also relieved by cholesterol depletion. These findings strongly suggest that functional retention of GPI‐APs in the REC occurs via their association with sphingolipid and cholesterol‐enriched sorting platforms or ‘rafts’.

Rare variants of large effect in BRCA2 and CHEK2 affect risk of lung cancer

We conducted imputation to the 1000 Genomes Project of four genome-wide association studies of lung cancer in populations of European ancestry (11,348 cases and 15,861 controls) and genotyped an additional 10,246 cases and 38,295 controls for follow-up. We identified large-effect genome-wide associations for squamous lung cancer with the rare variants BRCA2 p.Lys3326X (rs11571833, odds ratio (OR) = 2.47, P = 4.74 × 10−20) and CHEK2 p.Ile157Thr (rs17879961, OR = 0.38, P = 1.27 × 10−13). We also showed an association between common variation at 3q28 (TP63, rs13314271, OR = 1.13, P = 7.22 × 10−10) and lung adenocarcinoma that had been previously reported only in Asians. These findings provide further evidence for inherited genetic susceptibility to lung cancer and its biological basis. Additionally, our analysis demonstrates that imputation can identify rare disease-causing variants with substantive effects on cancer risk from preexisting genome-wide association study data.