Publications by authors named "Mary Carbone"

35 Publications

Heat shock proteins and small nucleolar RNAs are dysregulated in a Drosophila model for feline hypertrophic cardiomyopathy.

G3 (Bethesda) 2021 Jan;11(1)

The Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27695, USA.

In cats, mutations in myosin binding protein C (encoded by the MYBPC3 gene) have been associated with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms linking these mutations to HCM remain unknown. Here, we establish Drosophila melanogaster as a model to understand this connection by generating flies harboring MYBPC3 missense mutations (A31P and R820W) associated with feline HCM. The A31P and R820W flies displayed cardiovascular defects in their heart rates and exercise endurance. We used RNA-seq to determine which processes are misregulated in the presence of mutant MYBPC3 alleles. Transcriptome analysis revealed significant downregulation of genes encoding small nucleolar RNA (snoRNAs) in exercised female flies harboring the mutant alleles compared to flies that harbor the wild-type allele. Other processes that were affected included the unfolded protein response and immune/defense responses. These data show that mutant MYBPC3 proteins have widespread effects on the transcriptome of co-regulated genes. Transcriptionally differentially expressed genes are also candidate genes for future evaluation as genetic modifiers of HCM as well as candidate genes for genotype by exercise environment interaction effects on the manifestation of HCM; in cats as well as humans.
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http://dx.doi.org/10.1093/g3journal/jkaa014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849908PMC
January 2021

Genotype by environment interaction for gene expression in Drosophila melanogaster.

Nat Commun 2020 10 28;11(1):5451. Epub 2020 Oct 28.

Program in Genetics, Department of Biological Sciences, W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC, 27695-7614, USA.

The genetics of phenotypic responses to changing environments remains elusive. Using whole-genome quantitative gene expression as a model, here we study how the genetic architecture of regulatory variation in gene expression changed in a population of fully sequenced inbred Drosophila melanogaster strains when flies developed in different environments (25 °C and 18 °C). We find a substantial fraction of the transcriptome exhibited genotype by environment interaction, implicating environmentally plastic genetic architecture of gene expression. Genetic variance in expression increases at 18 °C relative to 25 °C for most genes that have a change in genetic variance. Although the majority of expression quantitative trait loci (eQTLs) for the gene expression traits in the two environments are shared and have similar effects, analysis of the environment-specific eQTLs reveals enrichment of binding sites for two transcription factors. Finally, although genotype by environment interaction in gene expression could potentially disrupt genetic networks, the co-expression networks are highly conserved across environments. Genes with higher network connectivity are under stronger stabilizing selection, suggesting that stabilizing selection on expression plays an important role in promoting network robustness.
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http://dx.doi.org/10.1038/s41467-020-19131-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595129PMC
October 2020

Gene expression networks in the Genetic Reference Panel.

Genome Res 2020 03 6;30(3):485-496. Epub 2020 Mar 6.

Program in Genetics, W.M. Keck Center for Behavioral Biology and Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina 27695-7614, USA.

A major challenge in modern biology is to understand how naturally occurring variation in DNA sequences affects complex organismal traits through networks of intermediate molecular phenotypes. This question is best addressed in a genetic mapping population in which all molecular polymorphisms are known and for which molecular endophenotypes and complex traits are assessed on the same genotypes. Here, we performed deep RNA sequencing of 200 Genetic Reference Panel inbred lines with complete genome sequences and for which phenotypes of many quantitative traits have been evaluated. We mapped expression quantitative trait loci for annotated genes, novel transcribed regions, transposable elements, and microbial species. We identified host variants that affect expression of transposable elements, independent of their copy number, as well as microbiome composition. We constructed sex-specific expression quantitative trait locus regulatory networks. These networks are enriched for novel transcribed regions and target genes in heterochromatin and euchromatic regions of reduced recombination, as well as genes regulating transposable element expression. This study provides new insights regarding the role of natural genetic variation in regulating gene expression and generates testable hypotheses for future functional analyses.
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http://dx.doi.org/10.1101/gr.257592.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111517PMC
March 2020

Context-dependent genetic architecture of Drosophila life span.

PLoS Biol 2020 03 5;18(3):e3000645. Epub 2020 Mar 5.

Program in Genetics, W. M. Keck Center for Behavioral Biology and Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America.

Understanding the genetic basis of variation in life span is a major challenge that is difficult to address in human populations. Evolutionary theory predicts that alleles affecting natural variation in life span will have properties that enable them to persist in populations at intermediate frequencies, such as late-life-specific deleterious effects, antagonistic pleiotropic effects on early and late-age fitness components, and/or sex- and environment-specific or antagonistic effects. Here, we quantified variation in life span in males and females reared in 3 thermal environments for the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and an advanced intercross outbred population derived from a subset of DGRP lines. Quantitative genetic analyses of life span and the micro-environmental variance of life span in the DGRP revealed significant genetic variance for both traits within each sex and environment, as well as significant genotype-by-sex interaction (GSI) and genotype-by-environment interaction (GEI). Genome-wide association (GWA) mapping in both populations implicates over 2,000 candidate genes with sex- and environment-specific or antagonistic pleiotropic allelic effects. Over 1,000 of these genes are associated with variation in life span in other D. melanogaster populations. We functionally assessed the effects of 15 candidate genes using RNA interference (RNAi): all affected life span and/or micro-environmental variance of life span in at least one sex and environment and exhibited sex-and environment-specific effects. Our results implicate novel candidate genes affecting life span and suggest that variation for life span may be maintained by variable allelic effects in heterogeneous environments.
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http://dx.doi.org/10.1371/journal.pbio.3000645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077879PMC
March 2020

Lisinopril Preserves Physical Resilience and Extends Life Span in a Genotype-Specific Manner in Drosophila melanogaster.

J Gerontol A Biol Sci Med Sci 2019 11;74(12):1844-1852

Biology of Healthy Aging Program, Division of Geriatric Medicine and Gerontology, School of Medicine, Johns Hopkins University, Baltimore.

Physical resiliency declines with age and comorbid conditions. In humans, angiotensin-converting enzyme (ACE) has been associated with attenuation of the decline in physical performance with age. ACE-inhibitor compounds, commonly prescribed for hypertension, often have beneficial effects on physical performance however the generality of these effects are unclear. Here, we tested the effects of the ACE-inhibitor Lisinopril on life span, and age-specific speed, endurance, and strength using three genotypes of the Drosophila melanogaster Genetic Reference Panel. We show that age-related decline in physical performance and survivorship varies with genetic background. Lisinopril treatment increased mean life span in all Drosophila Genetic Reference Panel lines, but its effects on life span, speed, endurance, and strength depended on genotype. We show that genotypes with increased physical performance on Lisinopril treatment experienced reduced age-related protein aggregation in muscle. Knockdown of skeletal muscle-specific Ance, the Drosophila ortholog of ACE, abolished the effects of Lisinopril on life span, implying a role for skeletal muscle Ance in survivorship. Using transcriptome profiling, we identified genes involved in stress response that showed expression changes associated with genotype and age-dependent responsiveness to Lisinopril. Our results demonstrate that Ance is involved in physical decline and demonstrate genetic variation in phenotypic responses to an ACE inhibitor.
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http://dx.doi.org/10.1093/gerona/glz152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329186PMC
November 2019

Regulation of Drosophila Lifespan by bellwether Promoter Alleles.

Sci Rep 2017 06 23;7(1):4109. Epub 2017 Jun 23.

Program in Genetics, W. M. Keck Center for Behavioral Biology, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, USA.

Longevity varies among individuals, but how natural genetic variation contributes to variation in lifespan is poorly understood. Drosophila melanogaster presents an advantageous model system to explore the genetic underpinnings of longevity, since its generation time is brief and both the genetic background and rearing environment can be precisely controlled. The bellwether (blw) gene encodes the α subunit of mitochondrial ATP synthase. Since metabolic rate may influence lifespan, we investigated whether alternative haplotypes in the blw promoter affect lifespan when expressed in a co-isogenic background. We amplified 521 bp upstream promoter sequences containing alternative haplotypes and assessed promoter activity both in vitro and in vivo using a luciferase reporter system. The AG haplotype showed significantly greater expression of luciferase than the GT haplotype. We then overexpressed a blw cDNA construct driven by either the AG or GT haplotype promoter in transgenic flies and showed that the AG haplotype also results in greater blw cDNA expression and a significant decrease in lifespan relative to the GT promoter haplotype, in male flies only. Thus, our results show that naturally occurring regulatory variants of blw affect lifespan in a sex-specific manner.
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http://dx.doi.org/10.1038/s41598-017-04530-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482829PMC
June 2017

Genetic architecture of natural variation in visual senescence in Drosophila.

Proc Natl Acad Sci U S A 2016 10 10;113(43):E6620-E6629. Epub 2016 Oct 10.

Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695; W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695; Program in Genetics, North Carolina State University, Raleigh, NC 27695; Initiative in Biological Complexity, North Carolina State University, Raleigh, NC 27695

Senescence, i.e., functional decline with age, is a major determinant of health span in a rapidly aging population, but the genetic basis of interindividual variation in senescence remains largely unknown. Visual decline and age-related eye disorders are common manifestations of senescence, but disentangling age-dependent visual decline in human populations is challenging due to inability to control genetic background and variation in histories of environmental exposures. We assessed the genetic basis of natural variation in visual senescence by measuring age-dependent decline in phototaxis using Drosophila melanogaster as a genetic model system. We quantified phototaxis at 1, 2, and 4 wk of age in the sequenced, inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and found an average decline in phototaxis with age. We observed significant genetic variation for phototaxis at each age and significant genetic variation in senescence of phototaxis that is only partly correlated with phototaxis. Genome-wide association analyses in the DGRP and a DGRP-derived outbred, advanced intercross population identified candidate genes and genetic networks associated with eye and nervous system development and function, including seven genes with human orthologs previously associated with eye diseases. Ninety percent of candidate genes were functionally validated with targeted RNAi-mediated suppression of gene expression. Absence of candidate genes previously implicated with longevity indicates physiological systems may undergo senescence independent of organismal life span. Furthermore, we show that genes that shape early developmental processes also contribute to senescence, demonstrating that senescence is part of a genetic continuum that acts throughout the life span.
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http://dx.doi.org/10.1073/pnas.1613833113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087026PMC
October 2016

Spontaneous mutations and the origin and maintenance of quantitative genetic variation.

Elife 2016 05 23;5. Epub 2016 May 23.

Program in Genetics, North Carolina State University, Raleigh, United States.

Mutation and natural selection shape the genetic variation in natural populations. Here, we directly estimated the spontaneous mutation rate by sequencing new Drosophila mutation accumulation lines maintained with minimal natural selection. We inferred strong stabilizing natural selection on quantitative traits because genetic variation among wild-derived inbred lines was much lower than predicted from a neutral model and the mutational effects were much larger than allelic effects of standing polymorphisms. Stabilizing selection could act directly on the traits, or indirectly from pleiotropic effects on fitness. However, our data are not consistent with simple models of mutation-stabilizing selection balance; therefore, further empirical work is needed to assess the balance of evolutionary forces responsible for quantitative genetic variation.
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http://dx.doi.org/10.7554/eLife.14625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929002PMC
May 2016

Genetic basis of natural variation in body pigmentation in Drosophila melanogaster.

Fly (Austin) 2015 ;9(2):75-81

a Department of Biological Sciences ; North Carolina State University ; Raleigh , NC USA.

Body pigmentation in insects and other organisms is typically variable within and between species and is often associated with fitness. Regulatory variants with large effects at bab1, t and e affect variation in abdominal pigmentation in several populations of Drosophila melanogaster. Recently, we performed a genome wide association (GWA) analysis of variation in abdominal pigmentation using the inbred, sequenced lines of the Drosophila Genetic Reference Panel (DGRP). We confirmed the large effects of regulatory variants in bab1, t and e; identified 81 additional candidate genes; and validated 17 candidate genes (out of 28 tested) using RNAi knockdown of gene expression and mutant alleles. However, these analyses are imperfect proxies for the effects of segregating variants. Here, we describe the results of an extreme quantitative trait locus (xQTL) GWA analysis of female body pigmentation in an outbred population derived from light and dark DGRP lines. We replicated the effects on pigmentation of 28 genes implicated by the DGRP GWA study, including bab1, t and e and 7 genes previously validated by RNAi and/or mutant analyses. We also identified many additional loci. The genetic architecture of Drosophila pigmentation is complex, with a few major genes and many other loci with smaller effects.
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http://dx.doi.org/10.1080/19336934.2015.1102807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826111PMC
October 2016

Genetic basis of transcriptome diversity in Drosophila melanogaster.

Proc Natl Acad Sci U S A 2015 Nov 19;112(44):E6010-9. Epub 2015 Oct 19.

Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695; Program in Genetics, North Carolina State University, Raleigh, NC 27695; W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695

Understanding how DNA sequence variation is translated into variation for complex phenotypes has remained elusive but is essential for predicting adaptive evolution, for selecting agriculturally important animals and crops, and for personalized medicine. Gene expression may provide a link between variation in DNA sequence and organismal phenotypes, and its abundance can be measured efficiently and accurately. Here we quantified genome-wide variation in gene expression in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP), increasing the annotated Drosophila transcriptome by 11%, including thousands of novel transcribed regions (NTRs). We found that 42% of the Drosophila transcriptome is genetically variable in males and females, including the NTRs, and is organized into modules of genetically correlated transcripts. We found that NTRs often were negatively correlated with the expression of protein-coding genes, which we exploited to annotate NTRs functionally. We identified regulatory variants for the mean and variance of gene expression, which have largely independent genetic control. Expression quantitative trait loci (eQTLs) for the mean, but not for the variance, of gene expression were concentrated near genes. Notably, the variance eQTLs often interacted epistatically with local variants in these genes to regulate gene expression. This comprehensive characterization of population-scale diversity of transcriptomes and its genetic basis in the DGRP is critically important for a systems understanding of quantitative trait variation.
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http://dx.doi.org/10.1073/pnas.1519159112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640795PMC
November 2015

The Genomic Basis of Postponed Senescence in Drosophila melanogaster.

PLoS One 2015 17;10(9):e0138569. Epub 2015 Sep 17.

Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, 27695, United States of America; Program in Genetics, North Carolina State University, Raleigh, North Carolina, 27695, United States of America; W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, 27695, United States of America.

Natural populations harbor considerable genetic variation for lifespan. While evolutionary theory provides general explanations for the existence of this variation, our knowledge of the genes harboring naturally occurring polymorphisms affecting lifespan is limited. Here, we assessed the genetic divergence between five Drosophila melanogaster lines selected for postponed senescence for over 170 generations (O lines) and five lines from the same base population maintained at a two week generation interval for over 850 generations (B lines). On average, O lines live 70% longer than B lines, are more productive at all ages, and have delayed senescence for other traits than reproduction. We performed population sequencing of pools of individuals from all B and O lines and identified 6,394 genetically divergent variants in or near 1,928 genes at a false discovery rate of 0.068. A 2.6 Mb region at the tip of the X chromosome contained many variants fixed for alternative alleles in the two populations, suggestive of a hard selective sweep. We also assessed genome wide gene expression of O and B lines at one and five weeks of age using RNA sequencing and identified genes with significant (false discovery rate < 0.05) effects on gene expression with age, population and the age by population interaction, separately for each sex. We identified transcripts that exhibited the transcriptional signature of postponed senescence and integrated the gene expression and genetic divergence data to identify 98 (175) top candidate genes in females (males) affecting postponed senescence and increased lifespan. While several of these genes have been previously associated with Drosophila lifespan, most are novel and constitute a rich resource for future functional validation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138569PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574564PMC
May 2016

The Effects of Royal Jelly on Fitness Traits and Gene Expression in Drosophila melanogaster.

PLoS One 2015 30;10(7):e0134612. Epub 2015 Jul 30.

Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, United States of America; W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, United States of America; Program in Genetics, North Carolina State University, Raleigh, North Carolina, United States of America.

Royal Jelly (RJ) is a product made by honey bee workers and is required for queen differentiation and accompanying changes in queen body size, development time, lifespan and reproductive output relative to workers. Previous studies have reported similar changes in Drosophila melanogaster in response to RJ. Here, we quantified viability, development time, body size, productivity, lifespan and genome wide transcript abundance of D. melanogaster reared on standard culture medium supplemented with increasing concentrations of RJ. We found that lower concentrations of RJ do induce significant differences in body size in both sexes; higher concentrations reduce size, increase mortality, shorten lifespan and reduce productivity. Increased concentrations of RJ also consistently lengthened development time in both sexes. RJ is associated with changes in expression of 1,581 probe sets assessed using Affymetrix Drosophila 2.0 microarrays, which were enriched for genes associated with metabolism and amino acid degradation. The transcriptional changes are consistent with alterations in cellular processes to cope with excess nutrients provided by RJ, including biosynthesis and detoxification, which might contribute to accelerated senescence and reduced lifespan.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134612PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520568PMC
May 2016

Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior.

Proc Natl Acad Sci U S A 2015 Jul 22;112(27):E3555-63. Epub 2015 Jun 22.

Department of Biological Sciences, Program in Genetics and W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695-7614

Aggression is an evolutionarily conserved complex behavior essential for survival and the organization of social hierarchies. With the exception of genetic variants associated with bioamine signaling, which have been implicated in aggression in many species, the genetic basis of natural variation in aggression is largely unknown. Drosophila melanogaster is a favorable model system for exploring the genetic basis of natural variation in aggression. Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci, including gustatory receptor 63a (Gr63a), which encodes a subunit of the receptor for CO2, and genes associated with development and function of the nervous system. Although genes from the two association analyses were largely nonoverlapping, they mapped onto a genetic interaction network inferred from an analysis of pairwise epistasis in the DGRP. We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics.
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http://dx.doi.org/10.1073/pnas.1510104112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500262PMC
July 2015

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 1--small molecules by LCMS).

Bioanalysis 2014 ;6(22):3039-49

Pfizer, Pearl River, NY, USA.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for small molecule bioanalysis using LCMS. Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' input) and Part 3 (Large molecules bioanalysis using LBA and Immunogenicity) will be published in the upcoming issues of Bioanalysis.
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http://dx.doi.org/10.4155/bio.14.265DOI Listing
July 2015

8th GCC: consolidated feedback to US FDA on the 2013 draft FDA guidance on bioanalytical method validation.

Bioanalysis 2014 ;6(22):2957-63

Covance Laboratories, Chantilly, VA, USA.

The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.
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http://dx.doi.org/10.4155/bio.14.287DOI Listing
July 2015

Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines.

Genome Res 2014 Jul 8;24(7):1193-208. Epub 2014 Apr 8.

Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina 27595, USA;

The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia, and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.
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http://dx.doi.org/10.1101/gr.171546.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4079974PMC
July 2014

A molecular mechanism for glaucoma: endoplasmic reticulum stress and the unfolded protein response.

Trends Mol Med 2013 Oct 19;19(10):586-93. Epub 2013 Jul 19.

Department of Biological Sciences and W.M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695-7617, USA. Electronic address:

Primary open angle glaucoma (POAG) is a common late-onset neurodegenerative disease. Ocular hypertension represents a major risk factor, but POAG etiology remains poorly understood. Some cases of early-onset congenital glaucoma and adult POAG are linked to mutations in myocilin, a secreted protein of poorly defined function. Transgenic overexpression of myocilin in Drosophila and experiments in mice and human populations implicate the unfolded protein response (UPR) in the pathogenesis of glaucoma. We postulate that compromised ability of the UPR to eliminate misfolded mutant or damaged proteins, including myocilin, causes endoplasmic reticulum stress, resulting in functional impairment of trabecular meshwork cells that regulate intraocular pressure. This mechanism of POAG is reminiscent of other age-dependent neurodegenerative diseases that involve accumulation of protein aggregates.
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http://dx.doi.org/10.1016/j.molmed.2013.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795998PMC
October 2013

Epistasis dominates the genetic architecture of Drosophila quantitative traits.

Proc Natl Acad Sci U S A 2012 Sep 4;109(39):15553-9. Epub 2012 Sep 4.

Department of Genetics, North Carolina State University, Raleigh, NC 27695, USA.

Epistasis-nonlinear genetic interactions between polymorphic loci-is the genetic basis of canalization and speciation, and epistatic interactions can be used to infer genetic networks affecting quantitative traits. However, the role that epistasis plays in the genetic architecture of quantitative traits is controversial. Here, we compared the genetic architecture of three Drosophila life history traits in the sequenced inbred lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and a large outbred, advanced intercross population derived from 40 DGRP lines (Flyland). We assessed allele frequency changes between pools of individuals at the extremes of the distribution for each trait in the Flyland population by deep DNA sequencing. The genetic architecture of all traits was highly polygenic in both analyses. Surprisingly, none of the SNPs associated with the traits in Flyland replicated in the DGRP and vice versa. However, the majority of these SNPs participated in at least one epistatic interaction in the DGRP. Despite apparent additive effects at largely distinct loci in the two populations, the epistatic interactions perturbed common, biologically plausible, and highly connected genetic networks. Our analysis underscores the importance of epistasis as a principal factor that determines variation for quantitative traits and provides a means to uncover genetic networks affecting these traits. Knowledge of epistatic networks will contribute to our understanding of the genetic basis of evolutionarily and clinically important traits and enhance predictive ability at an individualized level in medicine and agriculture.
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http://dx.doi.org/10.1073/pnas.1213423109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465439PMC
September 2012

The Drosophila melanogaster Genetic Reference Panel.

Nature 2012 Feb 8;482(7384):173-8. Epub 2012 Feb 8.

Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA.

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype-phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype-phenotype mapping using the power of Drosophila genetics.
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http://dx.doi.org/10.1038/nature10811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683990PMC
February 2012

Complex genetic architecture of Drosophila aggressive behavior.

Proc Natl Acad Sci U S A 2011 Oct 26;108(41):17070-5. Epub 2011 Sep 26.

Laboratory of Developmental Genetics, Center for Human Genetics, Catholic University Leuven and Flemish Institute for Biotechnology, B-3000 Leuven, Belgium.

Epistasis and pleiotropy feature prominently in the genetic architecture of quantitative traits but are difficult to assess in outbred populations. We performed a diallel cross among coisogenic Drosophila P-element mutations associated with hyperaggressive behavior and showed extensive epistatic and pleiotropic effects on aggression, brain morphology, and genome-wide transcript abundance in head tissues. Epistatic interactions were often of greater magnitude than homozygous effects, and the topology of epistatic networks varied among these phenotypes. The transcriptional signatures of homozygous and double heterozygous genotypes derived from the six mutations imply a large mutational target for aggressive behavior and point to evolutionarily conserved genetic mechanisms and neural signaling pathways affecting this universal fitness trait.
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http://dx.doi.org/10.1073/pnas.1113877108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3193212PMC
October 2011

Genes of the unfolded protein response pathway harbor risk alleles for primary open angle glaucoma.

PLoS One 2011 31;6(5):e20649. Epub 2011 May 31.

Department of Genetics, North Carolina State University, Raleigh, North Carolina, United States of America.

The statistical power of genome-wide association (GWA) studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG). Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG) remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR) upon overexpression of transgenic human glaucoma-associated myocilin (MYOC). We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105107PMC
October 2011

Quantitative and molecular genetic analyses of mutations increasing Drosophila life span.

PLoS Genet 2010 Jul 29;6(7):e1001037. Epub 2010 Jul 29.

Department of Genetics and W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina, United States of America.

Understanding the genetic and environmental factors that affect variation in life span and senescence is of major interest for human health and evolutionary biology. Multiple mechanisms affect longevity, many of which are conserved across species, but the genetic networks underlying each mechanism and cross-talk between networks are unknown. We report the results of a screen for mutations affecting Drosophila life span. One third of the 1,332 homozygous P-element insertion lines assessed had quantitative effects on life span; mutations reducing life span were twice as common as mutations increasing life span. We confirmed 58 mutations with increased longevity, only one of which is in a gene previously associated with life span. The effects of the mutations increasing life span were highly sex-specific, with a trend towards opposite effects in males and females. Mutations in the same gene were associated with both increased and decreased life span, depending on the location and orientation of the P-element insertion, and genetic background. We observed substantial--and sex-specific--epistasis among a sample of ten mutations with increased life span. All mutations increasing life span had at least one deleterious pleiotropic effect on stress resistance or general health, with different patterns of pleiotropy for males and females. Whole-genome transcript profiles of seven of the mutant lines and the wild type revealed 4,488 differentially expressed transcripts, 553 of which were common to four or more of the mutant lines, which include genes previously associated with life span and novel genes implicated by this study. Therefore longevity has a large mutational target size; genes affecting life span have variable allelic effects; alleles affecting life span exhibit antagonistic pleiotropy and form epistatic networks; and sex-specific mutational effects are ubiquitous. Comparison of transcript profiles of long-lived mutations and the control line reveals a transcriptional signature of increased life span.
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http://dx.doi.org/10.1371/journal.pgen.1001037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912381PMC
July 2010

Alcohol sensitivity in Drosophila: translational potential of systems genetics.

Genetics 2009 Oct 3;183(2):733-45, 1SI-12SI. Epub 2009 Aug 3.

Department of Biology, W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, NC 27695, USA.

Identification of risk alleles for human behavioral disorders through genomewide association studies (GWAS) has been hampered by a daunting multiple testing problem. This problem can be circumvented for some phenotypes by combining genomewide studies in model organisms with subsequent candidate gene association analyses in human populations. Here, we characterized genetic networks that underlie the response to ethanol exposure in Drosophila melanogaster by measuring ethanol knockdown time in 40 wild-derived inbred Drosophila lines. We associated phenotypic variation in ethanol responses with genomewide variation in gene expression and identified modules of correlated transcripts associated with a first and second exposure to ethanol vapors as well as the induction of tolerance. We validated the computational networks and assessed their robustness by transposon-mediated disruption of focal genes within modules in a laboratory inbred strain, followed by measurements of transcript abundance of connected genes within the module. Many genes within the modules have human orthologs, which provides a stepping stone for the identification of candidate genes associated with alcohol drinking behavior in human populations. We demonstrated the potential of this translational approach by identifying seven intronic single nucleotide polymorphisms of the Malic Enzyme 1 (ME1) gene that are associated with cocktail drinking in 1687 individuals of the Framingham Offspring cohort, implicating that variation in levels of cytoplasmic malic enzyme may contribute to variation in alcohol consumption.
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http://dx.doi.org/10.1534/genetics.109.107490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2766331PMC
October 2009

A transcriptional network associated with natural variation in Drosophila aggressive behavior.

Genome Biol 2009 16;10(7):R76. Epub 2009 Jul 16.

Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA.

Background: Aggressive behavior is an important component of fitness in most animals. Aggressive behavior is genetically complex, with natural variation attributable to multiple segregating loci with allelic effects that are sensitive to the physical and social environment. However, we know little about the genes and genetic networks affecting natural variation in aggressive behavior. Populations of Drosophila melanogaster harbor quantitative genetic variation in aggressive behavior, providing an excellent model system for dissecting the genetic basis of naturally occurring variation in aggression.

Results: Correlating variation in transcript abundance with variation in complex trait phenotypes is a rapid method for identifying candidate genes. We quantified aggressive behavior in 40 wild-derived inbred lines of D. melanogaster and performed a genome-wide association screen for quantitative trait transcripts and single feature polymorphisms affecting aggression. We identified 266 novel candidate genes associated with aggressive behavior, many of which have pleiotropic effects on metabolism, development, and/or other behavioral traits. We performed behavioral tests of mutations in 12 of these candidate genes, and show that nine indeed affected aggressive behavior. We used the genetic correlations among the quantitative trait transcripts to derive a transcriptional genetic network associated with natural variation in aggressive behavior. The network consists of nine modules of correlated transcripts that are enriched for genes affecting common functions, tissue-specific expression patterns, and/or DNA sequence motifs.

Conclusions: Correlations among genetically variable transcripts that are associated with genetic variation in organismal behavior establish a foundation for understanding natural variation for complex behaviors in terms of networks of interacting genes.
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http://dx.doi.org/10.1186/gb-2009-10-7-r76DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728530PMC
January 2010

Co-regulated transcriptional networks contribute to natural genetic variation in Drosophila sleep.

Nat Genet 2009 Mar 22;41(3):371-5. Epub 2009 Feb 22.

Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA.

Sleep disorders are common in humans, and sleep loss increases the risk of obesity and diabetes. Studies in Drosophila have revealed molecular pathways and neural tissues regulating sleep; however, genes that maintain genetic variation for sleep in natural populations are unknown. Here, we characterized sleep in 40 wild-derived Drosophila lines and observed abundant genetic variation in sleep architecture. We associated sleep with genome-wide variation in gene expression to identify candidate genes. We independently confirmed that molecular polymorphisms in Catsup (Catecholamines up) are associated with variation in sleep and that P-element mutations in four candidate genes affect sleep and gene expression. Transcripts associated with sleep grouped into biologically plausible genetically correlated transcriptional modules. We confirmed co-regulated gene expression using P-element mutants. Quantitative genetic analysis of natural phenotypic variation is an efficient method for revealing candidate genes and pathways.
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http://dx.doi.org/10.1038/ng.330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2683981PMC
March 2009

Systems genetics of complex traits in Drosophila melanogaster.

Nat Genet 2009 Mar 22;41(3):299-307. Epub 2009 Feb 22.

Department of Genetics, North Carolina State University, Raleigh, North Carolina 27695, USA.

Determining the genetic architecture of complex traits is challenging because phenotypic variation arises from interactions between multiple, environmentally sensitive alleles. We quantified genome-wide transcript abundance and phenotypes for six ecologically relevant traits in D. melanogaster wild-derived inbred lines. We observed 10,096 genetically variable transcripts and high heritabilities for all organismal phenotypes. The transcriptome is highly genetically intercorrelated, forming 241 transcriptional modules. Modules are enriched for transcripts in common pathways, gene ontology categories, tissue-specific expression and transcription factor binding sites. The high degree of transcriptional connectivity allows us to infer genetic networks and the function of predicted genes from annotations of other genes in the network. Regressions of organismal phenotypes on transcript abundance implicate several hundred candidate genes that form modules of biologically meaningful correlated transcripts affecting each phenotype. Overlapping transcripts in modules associated with different traits provide insight into the molecular basis of pleiotropy between complex traits.
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http://dx.doi.org/10.1038/ng.332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752214PMC
March 2009

Overexpression of myocilin in the Drosophila eye activates the unfolded protein response: implications for glaucoma.

PLoS One 2009 16;4(1):e4216. Epub 2009 Jan 16.

Department of Genetics, North Carolina State University, Raleigh, North Carolina, USA.

Background: Glaucoma is the world's second leading cause of bilateral blindness with progressive loss of vision due to retinal ganglion cell death. Myocilin has been associated with congenital glaucoma and 2-4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. Among several hypotheses, activation of the unfolded protein response (UPR) has emerged as a possible disease mechanism.

Methodology / Principal Findings: We used a transgenic Drosophila model to analyze whole-genome transcriptional profiles in flies that express human wild-type or mutant MYOC in their eyes. The transgenic flies display ocular fluid discharge, reflecting ocular hypertension, and a progressive decline in their behavioral responses to light. Transcriptional analysis shows that genes associated with the UPR, ubiquitination, and proteolysis, as well as metabolism of reactive oxygen species and photoreceptor activity undergo altered transcriptional regulation. Following up on the results from these transcriptional analyses, we used immunoblots to demonstrate the formation of MYOC aggregates and showed that the formation of such aggregates leads to induction of the UPR, as evident from activation of the fluorescent UPR marker, xbp1-EGFP. CONCLUSIONS / SIGNIFICANCE: Our results show that aggregation of MYOC in the endoplasmic reticulum activates the UPR, an evolutionarily conserved stress pathway that culminates in apoptosis. We infer from the Drosophila model that MYOC-associated ocular hypertension in the human eye may result from aggregation of MYOC and induction of the UPR in trabecular meshwork cells. This process could occur at a late age with wild-type MYOC, but might be accelerated by MYOC mutants to account for juvenile onset glaucoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004216PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615221PMC
March 2009

Gene-environment contributions to the development of infant vagal reactivity: the interaction of dopamine and maternal sensitivity.

Child Dev 2008 Sep-Oct;79(5):1377-94

Center for Developmental Science, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599-8115, USA.

This study investigated dopamine receptor genes (DRD2 and DRD4) and maternal sensitivity as predictors of infant respiratory sinus arrhythmia (RSA) and RSA reactivity, purported indices of vagal tone and vagal regulation, in a challenge task at 3, 6, and 12 months in 173 infant-mother dyads. Hierarchical linear modeling (HLM) revealed that at 3 and 6 months, RSA withdrawal in response to maternal separation was greater (suggesting expected physiological regulation) in infants without the DRD2 risk allele than those with the risk allele. At 12 months, infants with the risk allele who were also exposed to maternal sensitivity showed levels of RSA withdrawal comparable to infants who were not at genetic risk. Findings demonstrate the importance of developmental analysis of gene-environment interaction.
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http://dx.doi.org/10.1111/j.1467-8624.2008.01194.xDOI Listing
February 2009

Phenotypic plasticity and genotype by environment interaction for olfactory behavior in Drosophila melanogaster.

Genetics 2008 Jun 27;179(2):1079-88. Epub 2008 May 27.

W. M. Keck Center for Behavioral Biology, North Carolina State University, Raleigh, North Carolina 27695, USA.

Genotype by environment interactions (GEI) play a major part in shaping the genetic architecture of quantitative traits and are confounding factors in genetic studies, for example, in attempts to associate genetic variation with disease susceptibility. It is generally not known what proportion of phenotypic variation is due to GEI and how many and which genes contribute to GEI. Behaviors are complex traits that mediate interactions with the environment and, thus, are ideally suited for studies of GEI. Olfactory behavior in Drosophila melanogaster presents an opportunity to systematically dissect GEI, since large numbers of genetically identical individuals can be reared under defined environmental conditions and the olfactory system of Drosophila and its behavioral response to odorants have been well characterized. We assessed variation in olfactory behavior in a population of 41 wild-derived inbred lines and asked to what extent different larval-rearing environments would influence adult olfactory behavior and whether GEI is a minor or major contributing source of phenotypic variation. We found that approximately 50% of phenotypic variation in adult olfactory behavior is attributable to GEI. In contrast, transcriptional analysis revealed that only 20 genes show GEI at the level of gene expression [false discovery rate (FDR) < 0.05], some of which are associated with physiological responses to environmental chemicals. Quantitative complementation tests with piggyBac-tagged mutants for 2 of these genes (CG9664 and Transferrin 1) demonstrate that genes that show transcriptional GEI are candidate genes for olfactory behavior and that GEI at the level of gene expression is correlated with GEI at the level of phenotype.
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http://dx.doi.org/10.1534/genetics.108.086769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2429861PMC
June 2008