Robert Makowsky

Beyond Missing Heritability: Prediction of Complex Traits

Despite rapid advances in genomic technology, our ability to account for phenotypic variation using genetic information remains limited for many traits. This has unfortunately resulted in limited application of genetic data towards preventive and personalized medicine, one of the primary impetuses of genome-wide association studies. Recently, a large proportion of the “missing heritability” for human height was statistically explained by modeling thousands of single nucleotide polymorphisms concurrently. However, it is currently unclear how gains in explained genetic variance will translate to the prediction of yet-to-be observed phenotypes. Using data from the Framingham Heart Study, we explore the genomic prediction of human height in training and validation samples while varying the statistical approach used, the number of SNPs included in the model, the validation scheme, and the number of subjects used to train the model. In our training datasets, we are able to explain a large proportion of the variation in height (h2 up to 0.83, R2 up to 0.96). However, the proportion of variance accounted for in validation samples is much smaller (ranging from 0.15 to 0.36 depending on the degree of familial information used in the training dataset). While such R2 values vastly exceed what has been previously reported using a reduced number of pre-selected markers (<0.10), given the heritability of the trait (∼0.80), substantial room for improvement remains.

Macaques Vaccinated with Simian Immunodeficiency Virus SIVmac239Δnef Delay Acquisition and Control Replication after Repeated Low-Dose Heterologous SIV Challenge

ABSTRACT An effective human immunodeficiency virus (HIV) vaccine will likely need to reduce mucosal transmission and, if infection occurs, control virus replication. To determine whether our best simian immunodeficiency virus (SIV) vaccine can achieve these lofty goals, we vaccinated eight Indian rhesus macaques with SIVmac239Δnef and challenged them intrarectally (i.r.) with repeated low doses of the pathogenic heterologous swarm isolate SIVsmE660. We detected a significant reduction in acquisition of SIVsmE660 in comparison to that for naïve controls (log rank test; P = 0.023). After 10 mucosal challenges, we detected replication of the challenge strain in only five of the eight vaccinated animals. In contrast, seven of the eight control animals became infected with SIVsmE660 after these 10 challenges. Additionally, the SIVsmE660-infected vaccinated animals controlled peak acute virus replication significantly better than did the naïve controls (Mann-Whitney U test; P = 0.038). Four of the five SIVsmE660 vaccinees rapidly brought virus replication under control by week 4 postinfection. Unfortunately, two of these four vaccinated animals lost control of virus replication during the chronic phase of infection. Bulk sequence analysis of the circulating viruses in these animals indicated that recombination had occurred between the vaccine and challenge strains and likely contributed to the increased virus replication in these animals. Overall, our results suggest that a well-designed HIV vaccine might both reduce the rate of acquisition and control viral replication.

A striking lack of genetic diversity across the wide-ranging amphibian Gastrophryne carolinensis (Anura: Microhylidae)

We examine phylogeographic structure across a wide-ranging microhylid frog (Gastrophrynecarolinensis) using both mitochondrial (mtDNA) and nuclear (AFLP) data. Species with similar ecological characteristics such as large range size, low vagility, or existence across known biogeographic barriers, often are comprised of multiple, cryptic lineages. Surprisingly, our analyses of both portions of the genome show very little phylogeographic or population genetic structure. The family Microhylidae is one of the largest families of anurans with over 60 genera and around 400 species distributed across much of the world (Americas, Asia, Africa, and Madagascar), but very few phylogeographic studies have assessed intraspecific genetic diversity across the mitochondrial and nuclear genomes. Our results suggest that G. carolinensis, one of only three species of microhylid native to the US, has experienced a severe population bottleneck with subsequent range expansion. Comparable molecular data from closely related microhylids, in addition to demographic and ecological analyses, will provide valuable insight into patterns of genetic diversity and the processes driving phylogeographic diversity in these wide-ranging frogs.

Intramyocellular Lipid and Insulin Resistance: Differential Relationships in European and African Americans

Insulin resistance has been associated with the accumulation of fat within skeletal muscle fibers as intramyocellular lipid (IMCL). Here, we have examined in a cross‐sectional study the interrelationships among IMCL, insulin sensitivity, and adiposity in European Americans (EAs) and African Americans (AAs). In 43 EA and 43 AA subjects, we measured soleus IMCL content with proton‐magnetic resonance spectroscopy, insulin sensitivity with hyperinsulinemic–euglycemic clamp, and body composition with dual‐energy X‐ray absorptiometry. The AA and EA subgroups had similar IMCL content, insulin sensitivity, and percent fat, but only in EA was IMCL correlated with insulin sensitivity (r = −0.47, P < 0.01), BMI (r = 0.56, P < 0.01), percent fat (r = 0.35, P < 0.05), trunk fat (r = 0.47, P < 0.01), leg fat (r = 0.40, P < 0.05), and waist and hip circumferences (r = 0.54 and 0.55, respectively, P < 0.01). In a multiple regression model including IMCL, race, and a race by IMCL interaction, the interaction was found to be a significant predictor (t = 1.69, DF = 1, P = 0.0422). IMCL is related to insulin sensitivity and adiposity in EA but not in AA, suggesting that IMCL may not function as a pathophysiological factor in individuals of African descent. These results highlight ethnic differences in the determinants of insulin sensitivity and in the pathogenesis of the metabolic syndrome trait cluster.

Nuclear-mitochondrial discordance and gene flow in a recent radiation of toads.

Natural hybridization among recently diverged species has traditionally been viewed as a homogenizing force, but recent research has revealed a possible role for interspecific gene flow in facilitating species radiations. Natural hybridization can actually contribute to radiations by introducing novel genes or reshuffling existing genetic variation among diverging species. Species that have been affected by natural hybridization often demonstrate patterns of discordance between phylogenies generated using nuclear and mitochondrial markers. We used Amplified Fragment Length Polymorphism (AFLP) data in conjunction with mitochondrial DNA in order to examine patterns of gene flow and nuclear-mitochondrial discordance in the Anaxyrus americanus group, a recent radiation of North American toads. We found high levels of gene flow between putative species, particularly in species pairs sharing similar male advertisement calls that occur in close geographic proximity, suggesting that prezygotic reproductive isolating mechanisms and isolation by distance are the primary determinants of gene flow and genetic differentiation among these species. Additionally, phylogenies generated using AFLP and mitochondrial data were markedly discordant, likely due to recent and/or ongoing natural hybridization events between sympatric populations. Our results indicate that the putative species in the A. americanus group have experienced high levels of gene flow, and suggest that their North American radiation could have been facilitated by the introduction of beneficial genetic variation from admixture between divergent populations coming into secondary contact after glacial retreats.

Phylogeographic analysis and environmental niche modeling of the plain-bellied watersnake (Nerodia erythrogaster) reveals low levels of genetic and ecological differentiation

Species that exhibit geographically defined phenotypic variation traditionally have been divided into subspecies. Subspecies based on phenotypic features may not comprise monophyletic groups due to selection, gene flow, and/or convergent evolution. In many taxonomic groups the number of species once designated as widespread is dwindling rapidly, and many workers reject the concept of subspecies altogether. We tested whether currently recognized subspecies in the plain-bellied watersnake Nerodia erythrogaster are concordant with relationships based on mitochondrial markers, and whether it represents a single widespread species. The range of this taxon spans multiple potential biogeographic barriers (especially the Mississippi and Apalachicola Rivers) that correspond with lineage breaks in many species, including other snakes. We sequenced three mitochondrial genes (NADH-II, Cyt-b, Cox-I) from 156 geo-referenced specimens and developed ecological niche models using Maxent and spatially explicit climate data to examine historical and ecological factors affecting variation in N. erythrogaster across its range. Overall, we found little support for the recognized subspecies as either independent evolutionary lineages or geographically circumscribed units and conclude that although some genetic and niche differentiation has occurred, most populations assigned to N. erythrogaster appear to represent a single, widespread species. However, additional sampling and application of nuclear markers are necessary to clarify the status of the easternmost populations.

Analyzing the relationship between sequence divergence and nodal support using Bayesian phylogenetic analyses.

Determining the appropriate gene for phylogeny reconstruction can be a difficult process. Rapidly evolving genes tend to resolve recent relationships, but suffer from alignment issues and increased homoplasy among distantly related species. Conversely, slowly evolving genes generally perform best for deeper relationships, but lack sufficient variation to resolve recent relationships. We determine the relationship between sequence divergence and Bayesian phylogenetic reconstruction ability using both natural and simulated datasets. The natural data are based on 28 well-supported relationships within the subphylum Vertebrata. Sequences of 12 genes were acquired and Bayesian analyses were used to determine phylogenetic support for correct relationships. Simulated datasets were designed to determine whether an optimal range of sequence divergence exists across extreme phylogenetic conditions. Across all genes we found that an optimal range of divergence for resolving the correct relationships does exist, although this level of divergence expectedly depends on the distance metric. Simulated datasets show that an optimal range of sequence divergence exists across diverse topologies and models of evolution. We determine that a simple to measure property of genetic sequences (genetic distance) is related to phylogenic reconstruction ability in Bayesian analyses. This information should be useful for selecting the most informative gene to resolve any relationships, especially those that are difficult to resolve, as well as minimizing both cost and confounding information during project design.

Phylogenetic systematics of the genus Gonatodes (Squamata: Sphaerodactylidae) in the Guayana region, with description of a new species from Venezuela

The phylogenetic relationships and species boundaries of the diurnal geckos of the genus Gonatodes in the Guayana region are examined. Seven species of Gonatodes are recognized for the region, six of which are endemic to this region and form a monophyletic group together with G. hasemani from the southern part of the Amazon Basin. Two of the six species in this group are new to science, one of which is endemic to Venezuela and it is described here. Taxonomic accounts are provided for all the Venezuelan species of Gonatodes that are endemic to the Guayana region. The phylogenetic relationships of Gonatodes suggest that the diversity of this genus in the Guayana region has resulted mostly from in situ diversification rather than multiple colonization events by different lineages. The phylogenetic analyses also support that G. antillensis, the only nocturnal species in the genus, is indeed nested within Gonatodes, indicating re-evolution of some traits associated with nocturnal life. Finally, we comment on a recently proposed framework of temporal diversification in Gonatodes and suggest that we are still a long way from attaining a complete understanding about the systematics of this genus.

FcγR gene copy number in Kawasaki disease and intravenous immunoglobulin treatment response.

Objective Kawasaki disease (KD), response to intravenous immunoglobulin (IVIG) therapy, and associated coronary artery disease progression have been associated with genetic polymorphisms in Fc gamma receptor (Fc&ggr;R) genes. However, it is not known whether the existing gene copy number (GCN) variability relates to KD treatment response, susceptibility, or associated sequelae. Methods The copy number of individuals with KD (n=510) and their family members (n=808) for three variable Fc&ggr;Rs was assessed using pyrosequencing. We performed the transmission disequilibrium test to examine the association of GCN for Fc&ggr;Rs (Fc&ggr;R2C, Fc&ggr;R3A, and Fc&ggr;R3B) with susceptibility and used logistic regression models to determine its association with IVIG treatment outcomes. Results Fc&ggr;R2C and Fc&ggr;R3B GCN were significantly associated with KD susceptibility. IVIG response was associated with GCN variations of Fc&ggr;R3B in Whites and Fc&ggr;R2C in Hispanics, and gene risk score based on single nucleotide polymorphism and GCN in Fc&ggr;Rs were significantly different between IVIG responders and nonresponders among Whites. We found no significant associations between coronary artery disease and any of the Fc&ggr;R copy numbers. Conclusion GCN of Fc&ggr;R2C and Fc&ggr;R3B influences IVIG treatment response and predisposes individuals to KD, providing potential insights into understanding the mechanism of the Fc&ggr;R gene family in the IVIG pathway.

Effect of Diet Quality on Nutrient Allocation to the Test and Aristotle's Lantern in the Sea Urchin Lytechinus variegatus (LAMARCK, 1816)

ABSTRACT Small, adult (19.50 ± 2.01g wet weight) Lytechinus variegatus (Lamarck, 1816) were fed 8 formulated diets with different protein (12–36% dry weight as fed) and carbohydrate (21–39% dry weight) levels. Each sea urchin (n = 8 per treatment) was fed a daily ration of 1.5% of the average body weight of all individuals for 9 wk. Akaike information criterion (AIC) scores were used to compare 6 different dietary composition hypotheses for 8 growth measurements. For each physical growth response, different mathematical models representing a priori hypotheses were compared using the AIC score. The AIC is one of many information—theoretical approaches that allows for direct comparison of nonnested models with varying numbers of parameters. Dietary protein level and protein:energy ratio were the best models for prediction of test diameter increase. Dietary protein level was the best model of test with spines wet weight gain and test with spines dry matter production. When the Aristotles lantern was corrected for size of the test, there was an inverse relationship with dietary protein level. Log-transformed lantern to test with spines index was also best associated with the dietary protein model. Dietary carbohydrate level was a poor predictor for growth parameters. However, the protein × carbohydrate interaction model was the best model of organic content (percent dry weight) of the test without spines. These data suggest that there is a differential allocation of resources when dietary protein is limiting and the test, but not the Aristotles lantern, is affected by availability of dietary nutrients.