Doctor of Philosophy, University of California San Francisco (2010)
Douglas Vollrath, Postdoctoral Faculty Sponsor
Retinal pigment epithelium (RPE) cell migration in response to disease has been reported for age-related macular degeneration, proliferative vitreoretinopathy, and proliferative diabetic retinopathy. The complex molecular process of RPE cell migration is regulated in part by growth factors and cytokines, and activation of the PI3/AKT/mTOR signaling pathway. Rapamycin, an allosteric mTOR inhibitor, has been shown to block only one of the primary downstream mTOR effectors, p70 S6 kinase 1, in many cell types. INK128, a selective mTOR ATP binding site competitor, blocks both p70 S6 kinase 1 and a second primary downstream effector, 4E-BP1. We performed scratch assays using differentiated ARPE-19 and primary porcine RPE cells to assess the effect of mTOR inhibition on cell migration. We found that INK128-mediated blocking of both p70 S6 kinase 1 and 4E-BP1 was much more effective at preventing RPE cell migration than rapamycin-mediated inhibition of p70 S6 kinase 1 alone.
View details for DOI 10.1007/978-3-319-17121-0_94
View details for Web of Science ID 000369715400094
View details for PubMedID 26427479
Common genetic variants 3' of MC4R within two large linkage disequilibrium (LD) blocks spanning 288 kb have been associated with common and rare forms of obesity. This large association region has not been refined and the relevant DNA segments within the association region have not been identified. In this study, we investigated whether common variants in the MC4R gene region were associated with adiposity-related traits in a biracial population-based study. Single nucleotide polymorphisms (SNPs) in the MC4R region were genotyped with a custom array and a genome-wide array and associations between SNPs and five adiposity-related traits were determined using race-stratified linear regression. Previously reported associations between lower BMI and the minor alleles of rs2229616/Val103Ile and rs52820871/Ile251Leu were replicated in white female participants. Among white participants, rs11152221 in a proximal 3' LD block (closer to MC4R) was significantly associated with multiple adiposity traits, but SNPs in a distal 3' LD block (farther from MC4R) were not. In a case-control study of severe obesity, rs11152221 was significantly associated. The association results directed our follow-up studies to the proximal LD block downstream of MC4R. By considering nucleotide conservation, the significance of association, and proximity to the MC4R gene, we identified a candidate MC4R regulatory region. This candidate region was sequenced in 20 individuals from a study of severe obesity in an attempt to identify additional variants, and the candidate region was tested for enhancer activity using in vivo enhancer assays in zebrafish and mice. Novel variants were not identified by sequencing and the candidate region did not drive reporter gene expression in zebrafish or mice. The identification of a putative insulator in this region could help to explain the challenges faced in this study and others to link SNPs associated with adiposity to altered MC4R expression.
View details for DOI 10.1371/journal.pone.0096805
View details for Web of Science ID 000336653300033
View details for PubMedID 24820477
Early cochlear development is marked by an exuberant outgrowth of neurites that innervate multiple targets. The establishment of mature cochlear neural circuits is, however, dependent on the pruning of inappropriate axons and synaptic connections. Such refinement also occurs in the central nervous system (CNS), and recently, genes ordinarily associated with immune and inflammatory processes have been shown to play roles in synaptic pruning in the brain. These molecules include the major histocompatibility complex class I (MHCI) genes, H2-Kb and H2-Db, and the complement cascade gene, C1qa. Since the mechanisms involved in synaptic refinement in the cochlea are not well understood, we investigated whether these immune system genes may be involved in this process and whether they are required for normal hearing function. Here we report that these genes are not necessary for normal synapse formation and refinement in the mouse cochlea. We further demonstrate that C1qa expression is not necessary for normal hearing in mice but the lack of expression of H2-Kb and H2-Db causes hearing impairment. These data underscore the importance of the highly polymorphic family of MHCI genes in hearing in mice and also suggest that factors and mechanisms regulating synaptic refinement in the cochlea may be distinct from those in the CNS.
View details for DOI 10.1371/journal.pone.0094549
View details for PubMedID 24804771
Heterozygous mutations in melanocortin-4 receptor (MC4R) are the most frequent genetic cause of obesity. Bariatric surgery is a successful treatment for severe obesity. The mechanisms of weight loss after bariatric surgery are not well understood.Ninety-two patients who had Roux-en-Y gastric bypass (RYGB) surgery were screened for MC4R mutations. We compared percent excess weight loss (%EWL) in the four MC4R mutation carriers with that of two control groups: 8 matched controls and with the remaining 80 patients who underwent RYGB.Four patients were heterozygous for functionally significant MC4R mutations. In patients with MC4R mutations, the %EWL after RYGB (66% EWL) was not significantly different compared to matched controls (70% EWL) and non-matched controls (60% EWL) after 1 year of follow-up.This study suggests that patients with heterozygous MC4R mutations also benefit from RYGB and that weight loss may be independent of the presence of such mutations.
View details for DOI 10.1007/s11695-010-0295-8
View details for Web of Science ID 000291981700019
View details for PubMedID 20957447
Functionally significant heterozygous mutations in the Melanocortin-4 receptor (MC4R) have been implicated in 2.5% of early onset obesity cases in European cohorts. The role of mutations in this gene in severely obese adults, particularly in smaller North American patient cohorts, has been less convincing. More recently, it has been proposed that mutations in a phylogenetically and physiologically related receptor, the Melanocortin-3 receptor (MC3R), could also be a cause of severe human obesity. The objectives of this study were to determine if mutations impairing the function of MC4R or MC3R were associated with severe obesity in North American adults. We studied MC4R and MC3R mutations detected in a total of 1821 adults (889 severely obese and 932 lean controls) from two cohorts. We systematically and comparatively evaluated the functional consequences of all mutations found in both MC4R and MC3R. The total prevalence of rare MC4R variants in severely obese North American adults was 2.25% (CI(95%): 1.44-3.47) compared with 0.64% (CI(95%): 0.26-1.43) in lean controls (P < 0.005). After classification of functional consequence, the prevalence of MC4R mutations with functional alterations was significantly greater when compared with controls (P < 0.005). In contrast, the prevalence of rare MC3R variants was not significantly increased in severely obese adults [0.67% (CI(95%): 0.27-1.50) versus 0.32% (CI(95%): 0.06-0.99)] (P = 0.332). Our results confirm that mutations in MC4R are a significant cause of severe obesity, extending this finding to North American adults. However, our data suggest that MC3R mutations are not associated with severe obesity in this population.
View details for DOI 10.1093/hmg/ddn431
View details for Web of Science ID 000263828100015
View details for PubMedID 19091795
The extent to which common variants contribute to common phenotypes and disease in humans has important consequences for the future of medical genomics. Two reports have recently clarified this issue for one of the most pressing public health concerns, obesity. These large and comprehensive genome-wide association studies find that common variants within at least 11 genes are associated with obesity. Interestingly, most of these genes are highly expressed in the central nervous system, further highlighting its role in the pathogenesis of obesity. However, the individual and combined effects of these variants explain only a small fraction of the inherited variability in obesity, suggesting that rare variants may contribute significantly to the genetic predisposition for this condition.
View details for DOI 10.1186/gm31
View details for PubMedID 19341502
Genetic inheritance plays a significant role in the interindividual variability of drug response. The field of pharmacogenomics seeks to identify genetic factors that influence drug response, including both those that are inherited and those that arise within tumors, and use this information to improve drug therapy. Candidate gene approaches have led to clinical tests for toxicity avoidance (eg, TPMT, UGT1A1) and efficacy prediction (eg, epidermal growth factor receptor-activating mutations). However, the "right" genes are not known for most anticancer drugs. Strategies for uncovering pharmacogenomic associations vary widely from monogenic candidate gene approaches to polygenic genome-wide approaches. This review will place in context clinically relevant pharmacogenomic discovery approaches, including the relative strengths and weaknesses and the challenges inherent with achieving the goal of individualized therapy.
View details for DOI 10.1200/JCO.2005.03.0825
View details for Web of Science ID 000232546200011
View details for PubMedID 16145062
Previous studies have highlighted the use of phenotype generation in immortalized lymphoblastoid cells from the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees as a powerful means of discovering genes involved in complex biological and pharmacological phenotypes. However, there is no data on how representative CEPH pedigrees are of the general population of European origin for genetic variants of pharmacogenetic significance. A vast amount of data in a population of restricted applicability would be of little value. Genotype and allele frequencies of 28 variants in 15 pharmacogenetically relevant genes were analyzed in germ-line DNA from European- and African-origin blood donors, and CEPH cell lines of European origin. The results demonstrate that allele frequencies for the 28 polymorphisms are highly similar between the CEPH and the European-origin populations. However, genotype frequencies in the CEPH population did not provide a high level of prediction for the African-origin population. These data support the usefulness of the CEPH panel in pharmacogenetic discovery efforts for European-derived populations.
View details for DOI 10.1517/14622422.214.171.124
View details for Web of Science ID 000227979900011
View details for PubMedID 15723606
Little is known about the heritability of chemotherapy activity or the identity of genes that may enable the individualization of cancer chemotherapy. Although numerous genes are likely to influence chemotherapy response, current candidate gene-based pharmacogenetics approaches require a priori knowledge and the selection of a small number of candidate genes for hypothesis testing. In this study, an ex vivo familial genetics strategy using lymphoblastoid cells derived from Centre d'Etude du Polymorphisme Humain reference pedigrees was used to discover genetic determinants of chemotherapy cytotoxicity. Cytotoxicity to the mechanistically distinct chemotherapy agents 5-fluorouracil and docetaxel were shown to be heritable traits, with heritability values ranging from 0.26 to 0.65 for 5-fluorouracil and 0.21 to 0.70 for docetaxel, varying with dose. Genome-wide linkage analysis was also used to map a quantitative trait locus influencing the cellular effects of 5-fluorouracil to chromosome 9q13-q22 [logarithm of odds (LOD) = 3.44], and two quantitative trait loci influencing the cellular effects of docetaxel to chromosomes 5q11-21 (LOD = 2.21) and 9q13-q22 (LOD = 2.73). Finally, 5-fluorouracil and docetaxel were shown to cause apoptotic cell death involving caspase-3 cleavage in Centre d'Etude du Polymorphisme Humain lymphoblastoid cells. This study identifies genomic regions likely to harbor genes important for chemotherapy cytotoxicity using genome-wide linkage analysis in human pedigrees and provides a widely applicable strategy for pharmacogenomic discovery without the requirement for a priori candidate gene selection.
View details for DOI 10.1073/pnas.0404580101
View details for Web of Science ID 000223276700051
View details for PubMedID 15282376
Although the mouse has great potential for pharmacogenomic discovery, little is known about variation in drug response or genetic variation in pharmacologically relevant genes between inbred mouse strains. We therefore assessed variation in gene sequence, mRNA expression and protein activity of thiopurine methyltransferase (TPMT) in multiple inbred mouse strains. TPMT activity was measured by high-performance liquid chromatography detection of 6-MMP produced by incubation of liver homogenates with 6-MP. Genetic variation was assessed by resequencing and single nucleotide polymorphism (SNP) genotyping using pyrosequencing technology. mRNA expression was measured by real-time polymerase chain reaction. We observed an almost five-fold variation in TPMT activity, with strains falling into distinct low and high activity groups. This pattern of TPMT activity was highly correlated with expression of TPMT mRNA among strains, and high TPMT expression is dominant in F1 hybrids. To correlate genotype with phenotype, 29 SNPs and one insertion/deletion were genotyped throughout the TPMT gene and upstream 10 kb. Only two haplotypes were observed across all 30 polymorphisms, corresponding to the low and high activity groups. These results suggest that differential mouse TPMT activity is due to variation in mRNA expression. In addition, the identified pattern of low haplotype diversity suggests that the mouse is likely to be useful for pharmacogenomic discovery by associating haplotype blocks with drug response phenotypes among inbred strains.
View details for DOI 10.1097/01.fpc.0000114722.42625.f7
View details for Web of Science ID 000221074700004
View details for PubMedID 15083069