Center for Medical Genomics: Abstracts for 2016 Retreat
Melissa Wilson Sayres (Arizona State Univ.)
Sex-biased genome evolution and medicine
Tremendous variation exists between the sexes for susceptibility to diseases, and for response to treatments. Sex-biased processes occur on a variety of levels, from the differentiation of our sex chromosomes, to population dynamics, to sex-specific mutation rates and expression. The inundation of genomic and transcriptomic sequences provide the opportunity to apply computational and statistical approaches to understand sex-biased processes. The human sex chromosomes, X and Y, were once an indistinguishable pair of autosomes, but over the past 160 million years have become quite different. The Y has lost 90% of the ancestral gene content, but still retains relics of its ancestral partnership with the X. The Y chromosome, inherited through the genetic paternal line, and being nearly devoid of homologous recombination, also experiences evolutionary processes differently that regions that recombine. As such, studying patterns of genome-wide diversity can provide a unique insight into the history of sex-biased demography, selection, and mutation. I will discuss medical implications of our research to: 1) understand the degradation of the Y, and how this process has affected the X chromosome; 2) illuminate the history of sex-biased demography and selection acting on the Y chromosome; and, 3) investigate the role of life history on genomic evolution.
Heather Lawson (Washington Univ. St. Louis)
Physiological and genetic evidence links hemopexin to triglycerides in metabolic disease
Elevated serum triglycerides predict insulin resistance and vascular disease, but how the inert triglyceride molecule is related to development of metabolic disease is unknown. To pursue novel potential mediators of metabolic disease, we used a forward genetics approach involving inbred mice. We identified the heme binding protein hemopexin as a differentially expressed gene within a quantitative trait locus associated with serum triglycerides in an advanced intercross between the LG/J and SM/J strains. Hemopexin expression in mouse adipose tissue but not liver was regulated by diet, and serum levels in mice were associated with triglyceride levels. Hemopexin increased in concert with adipogenesis in 3T3-L1 cells, and disruption of its expression impaired adipocyte differentiation. RNAseq data from the adipose tissue of obese humans showed differential expression of hemopexin based on metabolic disease status, and circulating hemopexin levels were correlated with serum triglycerides in these human subjects. Hemopexin was also found in an unbiased proteomic screen of human atherosclerotic plaques, and shown to display differential abundance based on extent of disease. These findings suggest that hemopexin is associated with triglyceride levels and provide a framework for understanding mechanisms underlying lipid metabolism and metabolic disease.
Andrew Patterson (Penn State UP)
Using metabolomics to decipher the metabolic chatter between the host and gut microbiota
The Patterson lab is focused on understanding the host-metabolite-microbiome axis — specifically how the manipulation of gut bacteria through diet and/or xenobiotic exposure impacts their metabolism and how the gut bacteria interact with host nuclear receptors. The lab employs a variety of cutting-edge tools, including 1 H NMR- and mass spectrometry-based metabolomics, high throughput sequencing, and conventional and gnotobiotic mice, to facilitate its study of these pathways and understand their impact on human health and disease. Examples from recent studies with the farnesoid X receptor and the aryl hydrocarbon receptor will be provided to highlight these approaches.
Piotr Janicki (Penn State Hershey)
100 genomes for a price of one — pooled next-generation sequencing strategy for discovery of rare functional variants in platelet activation pathway genes contributing to risk of large vessels ischemic stroke
Platelets play a significant role in the development of cerebral ischemia by through their participation in thromboemboli that may initiate stroke symptoms. In addition, several antiplatelet agents significantly reduce the incidence of ischemic stroke after initial transient ischemic attacks. The genetic architecture of the platelet response in ischemic stroke is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes in platelets we undertook a study to identify to identify rare and new functional genetic variants in the 82 candidate genetic loci previously associated with platelet activation and aggregation by the use of massive, parallel next generation sequencing of selected exons and their flanking sequences from pooled-DNA samples in Polish patients with a history of the ischemic stroke. The study population consisted of 500 patients with the diagnosis of the acute ischemic stroke based on clinical features according to the World Health Organization definition and always supported by brain imaging (CT or MRI). Stroke etiology has been determined according to TOAST (Trial of Org 10172 in Acute Stroke Treatment) classification and included patients with the stroke subsequent to the large artery atherosclerosis (according to TOAST classification); and patients with stroke of undetermined etiology providing a confirmed stenosis of internal carotid artery ipsilateral to the infarct site combined with a negative history and no evidence of atrial fibrillation. The controls consisted of 500 age- and gender-matched patients free of stroke. The target selected for genotyping consisted of 82 genes containing 1134 exons (213.734 kbp) selected using the human (H. sapiens, hg19, GRCh37, February 2009) database. The customized library was created by the SureDesign platform from Agilent Technologies and consisted of 4249 probes (total probes size 295.592 kbp) with the average coverage of 99.6% of the selected exons. Sequencing of 10 pools (5 for each stroke and group) was performed on the Illumina HiSeq2500 sequencer generated an average of 36.1 (22.7-45.9 range) million pair-end 101 bp reads and 5.3 (3-7 range) Gbp per pooled sample consisting of 100 subjects. After final reads assembly by Varscan software and passing quality filters, the subsequent screening of sequenced exons (in vcf format) against dbSNP138 using wAnnovar software demonstrated a total of 314 unique rare (single nucleotide variants and indels, minor allele frequency or MAF < 5%) non-synonymous variants in controls and 821 in stroke patients (p < 0.0001 by Chi-Square test) from which 109 and 617, respectively, were new (p < 0.0001 by Chi-Square test). Because vast majority of variants (>99%) represented single nucleotide polymorphisms (SNP), the subsequent analysis was focused on the SNP variants which potential damaging effects on the generated proteins (defined as damaging in 5 out of 9 used prediction algorithms). This analysis showed total of 287 rare and damaging SNP variants in both groups, from which 230 (including 25 known and 205 unknown) were in the stroke pools and 57 (including 31 known and 26 unknown) in the control pools (p < 0.0001, Chi-Square test). The results of the study demonstrate statistically significant differences in the genomic burden and previously unknown, rare variants distribution in exons of genes previously associated with the platelet function between patients with large vessels ischemic stroke and control patients. These results establish also the power and applicability of pooled re-sequencing for comparative rare SNP association analysis of target sub genomes in large populations and an association between multiple rare, non-synonymous SNPs and ischemic stroke.
Anna Di Rienzo (Univ. Chicago)
The genetics of inter-individual variation in transcriptional response traits: biomedical and evolutionary implications
A number of critical homeostatic processes, which maintain the proper balance between the organism and the changing environment, are mediated by transcriptional response mechanisms. Therefore, genetic variants affecting the transcriptional response to environmental factors are likely to have important biomedical and evolutionary significance. Our work has focused largely on glucocorticoids, which mediate the response to stress and are involved in the regulation of blood pressure and blood glucose, of the immune response. Glucocorticoids are of great medical interest also because they are widely used in the treatment of many immune-mediated diseases, such as asthma and inflammatory bowel disease, as well as lymphoid malignancies. The main mechanism through which glucocorticoids exert their effect is by changing the expression levels of their target genes, ultimately resulting in an array of changes at the cellular level. We developed experimental and statistical approaches to study the genetic bases of inter-individual variation in the transcriptional response to glucocorticoids in primary immune cells and to compare the transcriptional response across ethnic groups. We have applied these approaches to learn about the biology of glucocorticoid non-responsiveness in healthy donors and in patients with inflammatory bowel disease. We are currently studying also the inter-individual variation in the response to vitamin D, which also acts through a transcriptional mechanism. We have identified pathways that mediate the immunomodulatory effects of vitamin D and we are mapping the genetic bases of inter-individual variation in the response to vitamin D. This information is expected to improve the design and power of supplementation trials. In addition, given changes in UV exposure during human evolution, the vitamin D pathway is likely to have been remodeled in human populations. We are currently developing and applying statistical approaches to identify advantageous alleles affecting this pathway and to reconstruct the history of selective pressures acting on them.
Margherita Cantorna (Penn State UP)
Vitamin D and the microbiota cooperate to regulate gastrointestinal homeostasis and inflammatory bowel disease
Evidence from animal models of inflammatory bowel disease (IBD) clearly show that vitamin D deficiency results in more severe disease and that treatment with 1,25(OH)2D can suppress the symptoms. Epidemiological data shows that vitamin D deficiency is associated with a higher risk of having one of the IBDs. In addition, there are associations between 25(OH)D levels and disease activity in patients with Crohn's disease and the clinical trials that have been done in Crohn's disease have shown that vitamin D supplementation reduced relapse rate and disease activity. There have not been any interventions in ulcerative colitis patients. Vitamin D is an environmental factor that affects susceptibility and severity of experimental IBD in mice and likely Crohn's disease in humans. The mechanisms by which vitamin D would regulate IBD include regulation of innate and adaptive immunity and control of gastrointestinal permeability. Newer evidence shows that vitamin D, likely via regulation of immune cells, regulates the microbiota. The effectiveness of vitamin D to regulate IBD was inhibited by disruptions of the microbiota using broad spectrum antibiotics. More specifically the Helicobacteraceae family members within the Proteobacteria pyhlum were higher in vitamin D deficient mice and this was associated with more severe colitis. 1,25(OH)2D or antibiotics treatment reduced Helicobacteraceae numbers and was associated with less severe disease. The ability of the host to metabolize vitamin D depends on the microbiota. Colonization of germfree mice resulted in increased 25(OH)D and 24,25(OH)2D in the blood. The potential mechanisms by which the microbiota regulate systemic vitamin D metabolism will be discussed. The major finding of the present study is that there is a symbiotic relationship between vitamin D and the microbiota that influences the development of immune mediated diseases like IBD.
James Broach (Penn State Hershey)
The genetics of amyotrophic lateral sclerosis
Amyotrophic Lateral Sclerosis (ALS), or Motor Neuron Disease, is a devastating neurological disease for which no effective therapy exists. Previous observations have established that only 5-10% of ALS patients have a family history of the disease, which means that >90% of patients have no near relatives who have presented with the disease. However, twin studies have estimated ALS heritability to be 60-70%, suggesting that many patients who present with sporadic ALS (sALS) may have an underlying genetic cause. Accordingly, we have conducted genomic analysis of patients with sALS to determine whether unidentified genetic factors are contributing to the disease. We have sequenced the entire exomes of eleven sALS patients along with those of both parents of each patient (trios). Moreover, we performed CNV analysis for the trios. In addition, we have sequenced the exomes of another 75 sALS patients and 11 fALS patients. In all cases, we determined the repeat length of the hexanucleotide repeat in the c9orf72 gene, whose expansion is associated with increased incidence of ALS. Four of the eleven fALS patients carried variants previously identified by linkage analysis as causative of the disease and six of the remaining patients carried variants of unknown significance in genes in which other causative variants have been identified. Ten of the eleven patients for which we obtained sequence data for the parents had one or more genes in which both copies carried rare variants predicted to be deleterious. In addition, two of the patients carried de novo mutations, including single nucleotide polymorphisms as well as copy number variants, one of which spanned 10 Mb and rendered 10 genes hemizygous. Many of the genes carrying compound heterozygous variants encode proteins associated with vesicle transport in axons, disruption of which has been implicated in various neurodegenerative diseases. Our data suggest that the genetic basis of a significant fraction of sALS patients is the presence of rare inactivating variants in both copies of a gene critical for neuronal survival. Such a model would account for the apparent sporadic nature of what is a simple Mendelian pattern of inheritance. The identification of these genes and confirmation of their role in ALS will expand our understanding of the molecular basis of the disease and allow genetic stratification of the ALS patient population, which should facilitate therapeutic drug discovery.
Francisco Diaz (Penn State UP)
Effects of zinc deficiency on oocyte quality and developmental potential
Oocyte quality refers to the ability of the oocyte to support maturation, fertilization, and embryo development. The quality of the oocyte is established during oogenesis and is supported by multiple interactions with the somatic cells of the follicle and by endocrine and metabolic signals. Many environmental and physiologic factors can influence the course of oogenesis and hence can alter the quality of the oocyte. Nutritional factors are among the most important determinants of oocyte quality, but the mechanisms by which specific nutrients regulate oocyte development are just now coming into focus. Recent work by our lab and others using in vitro methods show unequivocally that the mineral zinc has profound effects on oocyte development and the production of high quality oocytes. Zinc is tightly regulated in the oocyte. Before ovulation, free intracellular zinc is low, but increases dramatically upons stimulation by ovulatory signals. Blocking this increase in zinc with a metal chelator, such as TPEN, causes severe spindle defects and meiotic arrest before metaphase II in a high proportion of oocytes. However, even oocytes that make it through meiosis are less competent to undergo fertilization and preimplantation embryo development. Importantly, our subsequent in vivo studies show that similar meiotic and preimplantation defects occur in female mice fed a zinc-deficient diet for 3-5 days before ovulation. Unexpectedly, developmental defects of acute preconception zinc deficiency continue after implantation as shown by defective placenta and fetal development as late as day 16 of pregnancy in the mouse. The mechanisms responsible for these in vivo defects of preconception zinc deficiency are likely complex because zinc binds to over 300 proteins in the cell. However, zinc deficiency impacts epigenetic processes through a decrease in synthesis of s-adenosylmethionine (SAM), a universal methyl donor required for DNA and histone methylation. SAM concentration is lower in liver of mothers fed a zinc deficient diet and this is associated with a global decrease in histone H3K4 trimethylation and DNA methylation in the oocyte. Supplementing zinc deficient oocytes with exogenous SAM during in vitro maturation increases DNA and histone methylation and improves fertilization rate indicating that SAM synthesis is one pathway altered by zinc deficiency. Thus, acute preconception zinc deficiency causes profound meiotic and developmental defects, but much more work is needed to gain a comprehensive understanding of the zinc-dependent pathways regulating oocyte development and function.
George Lucian Moldovan (Penn State Hershey)
Mechanisms regulating the response to replication stress and DNA damage
Accurate DNA replication is essential for maintaining genomic stability and suppressing mutagenesis and carcinogenesis. DNA lesions induced by DNA damaging agents, as well as difficult to replicate DNA regions such as common fragile sites, inhibit progression of replicative polymerases. Our laboratory is studying mechanisms that cells employ to stabilize and restart stalled replication forks, thus alleviating replication stress. We are focusing our studies on the replication co-factor PCNA, a central component of the replication machinery. In recent years, we have identified a number of novel PCNA-centered mechanisms promoting restart of stalled forks. These include: regulation of translesion synthesis through PARP14-mediated ADP-ribosylation, control of recombination-dependent fork restart by PARI and PARP14, and activation of checkpoint signaling at stalled forks through HUWE1-mediated ubiquitination of H2AX. Our work sheds light on the complex events that regulate mutagenesis during DNA replication.