Panel Of Ancestry Informative Markers Research Articles

Challenges in selecting admixture models and marker sets to infer genetic ancestry in a Brazilian admixed population

The inference of genetic ancestry plays an increasingly prominent role in clinical, population, and forensic genetics studies. Several genotyping strategies and analytical methodologies have been developed over the last few decades to assign individuals to specific biogeographic regions. However, despite these efforts, ancestry inference in populations with a recent history of admixture, such as those in Brazil, remains a challenge. In admixed populations, proportion and components of genetic ancestry vary on different levels: (i) between populations; (ii) between individuals of the same population, and (iii) throughout the individual's genome. The present study evaluated 1171 admixed Brazilian samples to compare the genetic ancestry inferred by tri-/tetra-hybrid admixture models and evaluated different marker sets from those with small numbers of ancestry informative markers panels (AIMs), to high-density SNPs (HDSNP) and whole-genome-sequence (WGS) data. Analyses revealed greater variation in the correlation coefficient of ancestry components within and between admixed populations, especially for minority ancestral components. We also observed positive correlation between the number of markers in the AIMs panel and HDSNP/WGS. Furthermore, the greater the number of markers, the more accurate the tri-/tetra-hybrid admixture models.

Association of microsatellite instability (MSI) status with the 5-year outcome and genetic ancestry in a large Brazilian cohort of colorectal cancer.

Colorectal cancer (CRC) has a high incidence and mortality worldwide. Microsatellite instability (MSI) is crucial in CRC, with distinct molecular and clinicopathological features in patients. Nowadays, it is a predictive marker for immunotherapy. We proposed to evaluate the 5-year outcome of MSI status in 1002 Brazilian CRC, and associate it with genetic ancestry, molecular and clinicopathological features. MSI evaluation was performed using molecular markers. MSI+ tumors were analyzed for alterations in 23 MSI-targeted genes. Genetic ancestry was evaluated using an Ancestry-Informative markers panel. MSI status was analyzed in relation to CRC specific survival and other clinical and genetic variables. MSI+ status was observed in 10.5% of cases. MSI+ status was significantly associated with the anatomic site right colon, mucinous histological type, clinical stage II, histological grade III/undifferentiated, no recurrence of disease, and live cases without cancer. No association of MSI status with genetic ancestry components was observed. MSI-targeted genes analyses showed the most frequently altered genes: ATM, EGFR, MRE11, ROCK1, and TGFBRII. There was a statistically significant difference in cancer-specific survival between cases according to MSI status. This study constitutes the most comprehensive analyses of the MSI impact on the Brazilian CRC. MSI+ frequency in Brazilian CRC agreed with the literature and was associated with several clinicopathological features related with less aggressive tumors, independently of their genetic ancestry.

Recapitulating whole genome based population genetic structure for Indian wild tigers through an ancestry informative marker panel.

Identification of genetic structure within wildlife populations have implications in their conservation and management. Accurately inferring population genetic structure requires whole-genome data across the geographical range of the species, which can be resource-intensive. A cheaper strategy is to employ a subset of markers that can efficiently recapitulate the population genetic structure inferred by the whole genome data. Such ancestry informative markers (AIMs), have rarely been developed for endangered species such as tigers utilizing single nucleotide polymorphisms (SNPs). Here, we first identify the population structure of the Indian tiger using whole-genome sequences and then develop an AIMs panel with a minimum number of SNPs that can recapitulate this structure. We identified four population clusters of Indian tigers with North-East, North-West, and South Indian tigers forming three separate groups, and Terai and Central Indian tigers forming a single cluster. To evaluate the robustness of our AIMs, we applied it to a separate dataset of tigers from across India. Out of 92 SNPs present in our AIMs panel, 49 were present in the new dataset. These 49 SNPs were sufficient to recapitulate the population genetic structure obtained from the whole genome data. To the best of our knowledge, this is the first-ever SNP-based AIMs panel for big cats, which can be used as a cost-effective alternative to whole-genome sequencing for detecting the biogeographical origin of Indian tigers. Our study can be used as a guideline for developing an AIMs panel for the management of other endangered species where obtaining whole genome sequences are difficult.

Development and Evaluation of the Ancestry Informative Marker Panel of the VISAGE Basic Tool.

We detail the development of the ancestry informative single nucleotide polymorphisms (SNPs) panel forming part of the VISAGE Basic Tool (BT), which combines 41 appearance predictive SNPs and 112 ancestry predictive SNPs (three SNPs shared between sets) in one massively parallel sequencing (MPS) multiplex, whereas blood-based age analysis using methylation markers is run in a parallel MPS analysis pipeline. The selection of SNPs for the BT ancestry panel focused on established forensic markers that already have a proven track record of good sequencing performance in MPS, and the overall SNP multiplex scale closely matched that of existing forensic MPS assays. SNPs were chosen to differentiate individuals from the five main continental population groups of Africa, Europe, East Asia, America, and Oceania, extended to include differentiation of individuals from South Asia. From analysis of 1000 Genomes and HGDP-CEPH samples from these six population groups, the BT ancestry panel was shown to have no classification error using the Bayes likelihood calculators of the Snipper online analysis portal. The differentiation power of the component ancestry SNPs of BT was balanced as far as possible to avoid bias in the estimation of co-ancestry proportions in individuals with admixed backgrounds. The balancing process led to very similar cumulative population-specific divergence values for Africa, Europe, America, and Oceania, with East Asia being slightly below average, and South Asia an outlier from the other groups. Comparisons were made of the African, European, and Native American estimated co-ancestry proportions in the six admixed 1000 Genomes populations, using the BT ancestry panel SNPs and 572,000 Affymetrix Human Origins array SNPs. Very similar co-ancestry proportions were observed down to a minimum value of 10%, below which, low-level co-ancestry was not always reliably detected by BT SNPs. The Snipper analysis portal provides a comprehensive population dataset for the BT ancestry panel SNPs, comprising a 520-sample standardised reference dataset; 3445 additional samples from 1000 Genomes, HGDP-CEPH, Simons Foundation and Estonian Biocentre genome diversity projects; and 167 samples of six populations from in-house genotyping of individuals from Middle East, North and East African regions complementing those of the sampling regimes of the other diversity projects.

The Theoretical Framework for the Panels of DNA Markers Formation in the Forensic Determination of an Individual Ancestral Origin

The knowledge of the ancestry of an unknown individual from forensic DNA may provide important information for law enforcement investigation. This information can be acquired from biological traces by means of massive parallel sequencing (MPS) technologies and developed arrays of ancestry informative DNA markers (AIM panels). Development of forensic AIM panels takes two steps: (1) obtainment of information on DNA polymorphism of individuals from different populations; (2) selection of polymorphic DNA markers (autosomal, Y-, X-chromosomal) and their validation. The necessity of keeping medical secrecy for the protection of human rights may limit the usage of ancestry informative markers suitable for forensic needs if they are also associated with genetic diseases and physical traits. The present article deals with the analysis of published strategies for selecting markers capable of differentiating populations to design a panel of ancestry informative markers and applying it in forensic analysis in the Republic of Belarus.

Developing ancestry informative marker panel for Nigeria-Cameroonian chimpanzees

Chimpanzees (Pan troglodytes), with a dwindling population size, are distributed across sub-Saharan Africa. They are classified into two biogeographical clusters comprising of four subspecies: a western African cluster that includes P. t. verus and P. t. ellioti and a central/eastern African cluster that includes P. t. troglodytes and P. t. schweinfurthii. While the genetic distinctness of Nigeria-Cameroonian chimpanzees (P. t. ellioti) from western chimpanzees has been known for a while, the fine structures within P. t. ellioti population has remained under-studied. In this study, we developed the first ever ancestry informative marker (AIMs) panel that can detect the fine population structure within Nigeria-Cameroonian chimpanzees with high resolution. We compared four commonly used AIMs determining strategies, namely Infocalc algorithm, Wright's FST, smart principal component analysis (SmartPCA) and ADMIXTURE to first identify the best approach and then developed an AIMs panel of 435 SNPs employing the consensus of the four approaches (n = 129),with additional supplements from the best two approaches (Infocalc and ADMIXTURE). To the best of our knowledge, we have developed the first-ever AIMs panel for chimpanzees, which can greatly aid in their planned reintroduction to the natural habitat, maintaining their genetic integrity through planned captive breeding, and in tracking illegal trading across the globe.

The association of Native American genetic ancestry and high-density lipoprotein cholesterol: A representative study of a highly admixed population.

Hispanic populations typically show a high prevalence of dyslipidemias, especially of low high-density lipoproteins (HDLs) or HDL cholesterol. Highly admixed populations are ideal groups to clarify the role of genetic ancestry on HDL concentrations, isolating it from that of other factors. The objective of this study was to estimate the association between Native American genetic ancestry and HDL-cholesterol levels independent of socioeconomic factors in a representative sample of the Mexican population. We used data from the Mexican National Health Survey 2000, analyzing 1647 subjects from whom stored DNA samples and HDL measurements were available. To estimate proportional genetic ancestry (Native American, African, and European), we used a 107 genetic ancestry informative marker panel with the software STRUCTURE. To estimate the association between genetic ancestry and low HDL levels, we fitted logistic regression models with the percentage of Native American genetic ancestry, in quartiles, as the main predictor. Mean HDL levels were 38.9 mg/dL, with 62% of subjects having levels below 40 mg/dL. Participants had on average 53.6% Native American, 39% European, and 7.3% African genetic ancestry. Those in the fourth quartile of Native American genetic ancestry had 35% higher odds of having low HDL-cholesterol relative to those in the first quartile (odds ratio, 1.35; 95% confidence interval, 0.99-1.81) after adjustment for socioeconomic level and other covariates, although the association is clearly nonlinear. Native American genetic ancestry seems to play a small but distinct role in the development of low HDL cholesterol levels.

An ancestry informative marker panel design for individual ancestry estimation of Hispanic population using whole exome sequencing data

BackgroundEuropeans and American Indians were major genetic ancestry of Hispanics in the U.S. These ancestral groups have markedly different incidence rates and outcomes in many types of cancers. Therefore, the genetic admixture may cause biased genetic association study with cancer susceptibility variants specifically in Hispanics. For example, the incidence rate of liver cancer has been shown with substantial disparity between Hispanic, Asian and non-Hispanic white populations. Currently, ancestry informative marker (AIM) panels have been widely utilized with up to a few hundred ancestry-informative single nucleotide polymorphisms (SNPs) to infer ancestry admixture. Notably, current available AIMs are predominantly located in intron and intergenic regions, while the whole exome sequencing (WES) protocols commonly used in translational research and clinical practice do not cover these markers. Thus, it remains challenging to accurately determine a patient’s admixture proportion without additional DNA testing.ResultsIn this study we designed an unique AIM panel that infers 3-way genetic admixture from three distinct and selective continental populations (African (AFR), European (EUR), and East Asian (EAS)) within evolutionarily conserved exonic regions. Initially, about 1 million exonic SNPs from selective three populations in the 1000 Genomes Project were trimmed by their linkage disequilibrium (LD), restricted to biallelic variants, and finally we optimized to an AIM panel with 250 SNP markers, or the UT-AIM250 panel, using their ancestral informativeness statistics. Comparing to published AIM panels, UT-AIM250 performed better accuracy when we tested with three ancestral populations (accuracy: 0.995 ± 0.012 for AFR, 0.997 ± 0.007 for EUR, and 0.994 ± 0.012 for EAS). We further demonstrated the performance of the UT-AIM250 panel to admixed American (AMR) samples of the 1000 Genomes Project and obtained similar results (AFR, 0.085 ± 0.098; EUR, 0.665 ± 0.182; and EAS, 0.250 ± 0.205) to previously published AIM panels (Phillips-AIM34: AFR, 0.096 ± 0.127, EUR, 0.575 ± 0.290, and EAS, 0.330 ± 0.315; Wei-AIM278: AFR, 0.070 ± 0.096, EUR, 0.537 ± 0.267, and EAS, 0.393 ± 0.300). Subsequently, we applied the UT-AIM250 panel to a clinical dataset of 26 self-reported Hispanic patients in South Texas with hepatocellular carcinoma (HCC). We estimated the admixture proportions using WES data of adjacent non-cancer liver tissues (AFR, 0.065 ± 0.043; EUR, 0.594 ± 0.150; and EAS, 0.341 ± 0.160). Similar admixture proportions were identified from corresponding tumor tissues. In addition, we estimated admixture proportions of The Cancer Genome Atlas (TCGA) collection of hepatocellular carcinoma (TCGA-LIHC) samples (376 patients) using the UT-AIM250 panel. The panel obtained consistent admixture proportions from tumor and matched normal tissues, identified 3 possible incorrectly reported race/ethnicity, and/or provided race/ethnicity determination if necessary.ConclusionsHere we demonstrated the feasibility of using evolutionarily conserved exonic regions to infer admixture proportions and provided a robust and reliable control for sample collection or patient stratification for genetic analysis. R implementation of UT-AIM250 is available at https://github.com/chenlabgccri/UT-AIM250.

Ancestry informative markers derived from discriminant analysis of principal components provide important insights into the composition of crossbred cattle

The cost of SNP genotyping to screen different breeds and to estimate the exact proportion of ancestry level is quite high, which can be compensated through deriving a small panel of ancestry informative markers (AIMs). Hence, we carried out the present study to provide an insight into ancestry level inferred from a panel of informative markers in the crossbred Vrindavani population developed at ICAR-IVRI, India. We have performed a new method i.e., discriminant analysis of principal components (DAPC) for the first time on the dataset of Vrindavani cattle. To confirm our method, we had performed DAPC on two other well-known crossbred cattle, i.e., Frieswal and Beefmaster. Three sets of panels (500, 1000 and 2000 markers) were tested for clustering of individuals. Among all the panels, we found the panel (1000 markers) with DAPC based contribution method was of the smallest size and comparatively of the highest accuracy.

A panel of 32 AIMs suitable for population stratification correction and global ancestry estimation in Mexican mestizos

BackgroundAssociation studies are useful to unravel the genetic basis of common human diseases. However, the presence of undetected population structure can lead to both false positive results and failures to detect genuine associations. Even when most of the approaches to deal with population stratification require genome-wide data, the use of a well-selected panel of ancestry informative markers (AIMs) may appropriately correct for population stratification. Few panels of AIMs have been developed for Latino populations and most contain a high number of markers (> 100 AIMs). For some association studies such as candidate gene approaches, it may be unfeasible to genotype a numerous set of markers to avoid false positive results. In such cases, methods that use fewer AIMs may be appropriate.ResultsWe validated an accurate and cost-effective panel of AIMs, for use in population stratification correction of association studies and global ancestry estimation in Mexicans, as well as in populations having large proportions of both European and Native American ancestries.Based on genome-wide data from 1953 Mexican individuals, we performed a PCA and SNP weights were calculated to select subsets of unlinked AIMs within percentiles 0.10 and 0.90, ensuring that all chromosomes were represented. Correlations between PC1 calculated using genome-wide data versus each subset of AIMs (16, 32, 48 and 64) were r2 = 0.923, 0.959, 0.972 and 0.978, respectively. When evaluating PCs performance as population stratification adjustment covariates, no correlation was found between P values obtained from uncorrected and genome-wide corrected association analyses (r2 = 0.141), highlighting that population stratification correction is compulsory for association analyses in admixed populations. In contrast, high correlations were found when adjusting for both PC1 and PC2 for either subset of AIMs (r2 > 0.900). After multiple validations, including an independent sample, we selected a minimal panel of 32 AIMs, which are highly informative of the major ancestral components of Mexican mestizos, namely European and Native American ancestries. Finally, the correlation between the global ancestry proportions calculated using genome-wide data and our panel of 32 AIMs was r2 = 0.972.ConclusionsOur panel of 32 AIMs accurately estimated global ancestry and corrected for population stratification in association studies in Mexican individuals.

OR23 HLA class II genes correlate with protective neutralizing antibody titers in a dengue vaccine efficacy trial

Aim A tetravalent, live attenuated dengue vaccine demonstrated efficacy, safety and immunogenicity in several clinical trials in Asia and Latin America. Efficacy differed based on infecting serotypes, presence of pre-existing dengue neutralizing antibody (NAb) titers and age. HLA class II molecules expressed on antigen presenting cells mediate CD4+ T cell stimulation of antibody production by B cells involved in vaccine-induced responses. We hypothesized that the differences in observed vaccine efficacy could be due to variation in NAb immune responses in conjunction with host HLA class II genes. Methods Samples were available from a subset of subjects that took part in the first tetravalent dengue vaccine efficacy trial conducted in Thailand. DNA was extracted from 335 saliva samples and HLA genotyping was performed using next-generation sequencing (NGS) of full-length genes. A panel of ancestry informative markers (AIMs) was genotyped to assess population stratification. Serotype-specific NAb titers were measured by plaque-reduction neutralization test 28 days after last injection. The association of NAb titers and HLA class II on dengue infection was tested by logistic regression. Linear regression was used to test association of HLA class II alleles with NAb levels after accounting for sex, age, and serotype as covariates. A minimal false discovery rate to account for multiple comparisons, with a two-sided p-value Results NGS identified 197 HLA class I and II alleles in the Thai Dengue vaccine trial. AIMs analysis did not identify population stratification comparing cases and controls. Magnitude of NAb levels post vaccination was significantly higher in the presence of HLA-DRB1*11 (p = 0.002, q = 0.08). HLA-DPB1*03:01 and *05:01 presence correlated with pre-existing NAb titers in the placebos (p = 0.005, q = 0.09; p = 0.001, q = 0.04). We did not observe a direct effect of HLA on dengue infection in either the placebo or treatment arm. Conclusions These findings suggest that specific HLA class II alleles modulate protective NAb titers in dengue infection. This is an exploratory study to identify signals to replicate in other dengue vaccine clinical studies. Understanding this HLA class II mechanism will enable improved vaccine design.

Abstract 5698: Infiltrating immune cells in breast cancer subtypes

Abstract In 2017, breast cancer (BC) affected around 29% of Hispanic women in the US according to the American Cancer Society. Among Hispanics, Puerto Rican women are one of the most diverse groups in terms of ancestry and culture. This diversity adds complexity to the BC etiology in this population. Several population studies have suggested ancestry as a BC risk factor which needs to be considered in clinical trial design aiming at selecting better therapies. In contrast to chemotherapeutic treatments known to have significant toxicities, some immunotherapies are recognized by having modest toxicity grades with a high capacity for individualized medicine. Monoclonal antibodies such as trastuzumab are used to treat BC by stratifying patients according to their molecular subtypes. Depending on whether BC tumors are (+) or (-) for the estrogen (ER), progesterone (PR), and HER2, four molecular subtypes have been identified: luminal A (ER+, PR+/−, HER2-), luminal B (ER+, PR+/−, HER2+), HER2+ (ER−, PR−, HER2+), and basal-like (ER−, PR−, HER2−). The objective of this study is to elucidate the distribution of molecular BC subtypes and ancestry markers in Puerto Rican women and to establish the relationship between these distributions with their tumor infiltrating lymphocytes (TIL). Our overall hypothesis is that Hispanic women with BC will have distinct molecular subtypes and ancestry distributions that will be associated with their TIL signature. By combining samples collected through the Puerto Rico Bio-Bank and from our BC cohort, 129 blood and tumor tissue samples were selected. Molecular BC subtypes were classified using PAM50. A panel of ancestry informative markers was genotyped on iPLEX technologies and global ancestry proportions were estimated using ADMIXTURE. PanCancer Immune Panel was used to characterize the TIL based on RNA signatures. The initial results obtained through PAM50, showed that 45% of the tumors were luminal A, 27% luminal B, 18% basal-like, and 7% HER2+. Global ancestry proportions for each patient grouped by PAM50 subtype were performed. Overall, the ancestry proportions were: 67% European (SD ± 12%), 19% African (SD ± 11%), and 14% Native American (SD ± 7%). To evaluate the TIL RNA signature among subtypes, a principal component analysis (PCA) was performed followed by an immune score using the PCA2. The PCA2 score was able to distinguish among the basal-like, luminal A and luminal B subtypes; however, the HER2+ samples could not be clearly distinguished from the luminal A or B subtypes. These results show a qualitative tendency intrinsic of each molecular BC subtype and the TIL signature. Long-term, this approach of studying BC subtypes, ancestry, and immune infiltrates while increasing sample size is expected to provide data needed to improve prognostic tools to support the design of immunotherapies for Hispanic women with BC. This study was supported by: #S06GM008239-20, 9SC1CA182846-04, #U54CA163068, 2U54CA163071-06, and R003050/MD007579. Citation Format: Jaime L. Matta, Jarline Encarnacion, Carmen Ortiz, Julie Dutil, Amy Weber, Shari Pilon-Thomas. Infiltrating immune cells in breast cancer subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5698.

Genetic structure, divergence and admixture of Han Chinese, Japanese and Korean populations

BackgroundHan Chinese, Japanese and Korean, the three major ethnic groups of East Asia, share many similarities in appearance, language and culture etc., but their genetic relationships, divergence times and subsequent genetic exchanges have not been well studied.ResultsWe conducted a genome-wide study and evaluated the population structure of 182 Han Chinese, 90 Japanese and 100 Korean individuals, together with the data of 630 individuals representing 8 populations wordwide. Our analyses revealed that Han Chinese, Japanese and Korean populations have distinct genetic makeup and can be well distinguished based on either the genome wide data or a panel of ancestry informative markers (AIMs). Their genetic structure corresponds well to their geographical distributions, indicating geographical isolation played a critical role in driving population differentiation in East Asia. The most recent common ancestor of the three populations was dated back to 3000 ~ 3600 years ago. Our analyses also revealed substantial admixture within the three populations which occurred subsequent to initial splits, and distinct gene introgression from surrounding populations, of which northern ancestral component is dominant.ConclusionsThese estimations and findings facilitate to understanding population history and mechanism of human genetic diversity in East Asia, and have implications for both evolutionary and medical studies.

Influence of pharmacogenetic polymorphisms and demographic variables on metformin pharmacokinetics in an admixed Brazilian cohort.

To identify pharmacogenetic and demographic variables that influence the systemic exposure to metformin in an admixed Brazilian cohort. The extreme discordant phenotype was used to select 106 data sets from nine metformin bioequivalence trials, comprising 256 healthy adults. Eleven single-nucleotide polymorphisms in SLC22A1, SLC22A2, SLC47A1 SLC47A2 and in transcription factor SP1 were genotyped and a validated panel of ancestry informative markers was used to estimate the individual proportions of biogeographical ancestry. Two-step (univariate followed by multivariate) regression modelling was developed to identify covariates associated with systemic exposure to metformin, accessed by the area under the plasma concentration-time curve, between 0 and 48h (AUC0-48h ), after single oral doses of metformin (500 or 1000mg). The individual proportions of African, Amerindian and European ancestry varied widely, as anticipated from the structure of the Brazilian population The dose-adjusted, log-transformed AUC0-48h 's (nghml-1 mg-1 ) differed largely in the two groups at the opposite ends of the distribution histogram, namely 0.82, 0.79-0.85 and 1.08, 1.06-1.11 (mean, 95% confidence interval; P=6.10-26 , t test). Multivariate modelling revealed that metformin AUC0-48h increased with age, food and carriage of rs12208357 in SLC22A1 but was inversely associated with body surface area and individual proportions of African ancestry. A pharmacogenetic marker in OCT1 (SLC22A1 rs12208357), combined with demographic covariates (age, body surface area and individual proportion of African ancestry) and a food effect explained 29.7% of the variability in metformin AUC0-48h .

Autosomal and Y-STR: A combinatory approach to ethnicity inference in Singapore

The advent of massively parallel sequencing and the development of ancestry informative marker panels have advanced forensic DNA analyses beyond being a mere tool for matching individuals to crime casework profiles. Now, there is the potential to make ethnicity, age, and even phenotype predictions for intelligence to assist law enforcement in cases with no suspects. This technology is, however, relatively time consuming and not readily available in most crime laboratories unlike the standard STR typing platform. The present study evaluates the concomitant use of autosomal and Y-STR allele frequencies for ethnicity inference to facilitate quick investigation and apprehension before the perpetrator can re-offend or escape the jurisdiction.

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OBJECTIVES/SPECIFIC AIMS: The preliminary analysis sought to retrospectively characterize the role of hypocretin receptor 2 (HCRTR2) in the development and prognosis of AD along with associated behavioral measures including smoking, self-reported drinking history, and neuroticism. Given the results in this study along with the paucity of information regarding the functional significance of rs2653349, we intend to comprehensively characterize HCRTR2 using haplotype analyses. We will then identify relationships between our haplotype analysis and IV alcohol self-administration using the Computer-Assisted Infusion System, and phenotypes identified in a sleep study. Furthermore, we aim at identifying functional loci in the hypocretin/orexin system by investigating differential allele expression in the orexin receptors in hippocampus tissue obtained from postmortem human brains. METHODS/STUDY POPULATION: This study examined 1569 European American and African American individuals between 18 and 65 years old, 922 of whom with a current diagnosis of AD. Participants were genotyped for HCRTR2 rs2653349 and ancestry was determined via a genome-wide panel of ancestry informative markers. AD was diagnosed using the Structured Clinical Interviews for DSM-IV (SCID-IV) for psychiatric disorders and recent alcohol use was assessed by 90-day Timeline Follow-back (TLFB) interviews. Smoking was assessed using the Fagerström Test for Nicotine Dependence and neuroticism was measured using the NEO Personality Inventory. RESULTS/ANTICIPATED RESULTS: In European Americans, a significant difference was found in current AD diagnosis between AX carriers and GG carriers (z=−2.390, p=0.017). This relationship remained significant in a logistic regression model controlled for age and gender (R2=0.269, p=0.015). TLFB drinking measures were compared based on the median values to correct for the ceiling effect resulting from the assessment covering the past 90 days. Total drinks (U=8.280, p=0.004), number of drinking days (U=6.983, p=0.008), and average drinks per days (U=7.221, p=0.007) were all noted to significantly differ between the two allele groups among Caucasians. The associations between rs2653349 and total drinks (R2=0.115, p=0.023) and heavy drinking days (R2=0.190, p=0.015) remained significant in linear regressions controlled for age and gender. Furthermore, Caucasian AX carriers had a higher median number of drinking days relative to GG homozygotes among current AD positive subjects (U=6.937, p=0.012) and a lower median number of drinking days among current AD negative subjects (U=4.430, p=0.035). Among Caucasian AD negative subjects, there was a significantly greater frequency of smokers (χ2=3.550, p=0.046). In African American participants, there were no significant differences in AD diagnosis and in measures of AD severity by genotype. African American males diagnosed with current AD had higher rates of smoking in the AX group (χ2=4.969, p=0.017). No significant associations were found between rs2653349 and neuroticism in any of the cohorts analyzed in this sample. DISCUSSION/SIGNIFICANCE OF IMPACT: The results suggest that, among Caucasians, AX carriers have an increased risk to develop AD independently of their age and gender. In addition, among individuals with a diagnosis of AD, AX carriers reported a greater number of drinking days, as measured by the TLFB, suggesting that this polymorphism also exerts an effect on the severity of the disease. This effect on increased alcohol consumption was absent in Caucasian AX carriers without current AD diagnosis. In future analysis, we will explore how different genetic profiles in HCRTR2, and also HCRTR1, may alter the orexin signaling pathway and how such alterations may predispose patients to develop AD and exacerbate AD once it develops.

Genetic ancestry and prostate cancer susceptibility SNPs in Puerto Rican and African American men.

The Puerto Rican (PR) population is a racially admixed population that has a high prostate cancer (PCa) mortality rate. We hypothesized in this pilot study that West African Ancestry (WAA) was associated with PCa in this heterogeneous (PR) population. A case/case and case/control study was performed. Controls, 207 African American (AA) and 133 PR were defined as men with no PCa, a serum PSA < 2.5 ng/mL and a negative rectal examination. Cases were patients with pathological specimens from radical prostatectomies (RP) (291 PR and 200 AA). DNA was extracted from whole blood of controls and from paraffin embedded normal seminal vesicle from the RPs. We assessed the association of PCa and aggressiveness with genetic ancestry using an ancestry informative marker panel (AIMs) and Wilcoxon rank-sum test and the association of PCa and aggressiveness with 15 previously PCa associated SNPs using Chi square test. Gleason Score (GS) and tumor stage (TS) were used to define low risk (GS ≤ 7[3 + 4]), TS ≤ pT2) and high risk (GS≥ 7[4 + 3], TS > pT2) PCa. Statistical analyses were done using SAS. No difference in overall percent WAA was found between PR cases and controls. Among PR or AA cases WAA was not associated with disease severity based upon risk group, Gleason score or stage. Among AA controls WAA was significantly higher than in cases. The SNP rs7824364 (chromosome 8q24) PCa risk allele was significantly increased among cases versus controls for both AA (P < 0.0001) and PR (P = 0.0001) men. PR men with ≥1 risk allele exhibited a higher percent of WAA (39% vs 29%, P = 0.034). The SNP rs7824364, a local marker of WAA in the 8q24 region was associated with PCa among both AA and PR men and with increased WAA among PR men. This novel relationship of PCA risk loci, WAA with PCa and its phenotype among PR men deserves further study.

Rare mutations and potentially damaging missense variants in genes encoding fibrillar collagens and proteins involved in their production are candidates for risk for preterm premature rupture of membranes.

Preterm premature rupture of membranes (PPROM) is the leading identifiable cause of preterm birth with ~ 40% of preterm births being associated with PPROM and occurs in 1% - 2% of all pregnancies. We hypothesized that multiple rare variants in fetal genes involved in extracellular matrix synthesis would associate with PPROM, based on the assumption that impaired elaboration of matrix proteins would reduce fetal membrane tensile strength, predisposing to unscheduled rupture. We performed whole exome sequencing (WES) on neonatal DNA derived from pregnancies complicated by PPROM (49 cases) and healthy term deliveries (20 controls) to identify candidate mutations/variants. Genotyping for selected variants from the WES study was carried out on an additional 188 PPROM cases and 175 controls. All mothers were self-reported African Americans, and a panel of ancestry informative markers was used to control for genetic ancestry in all genetic association tests. In support of the primary hypothesis, a statistically significant genetic burden (all samples combined, SKAT-O p-value = 0.0225) of damaging/potentially damaging rare variants was identified in the genes of interest—fibrillar collagen genes, which contribute to fetal membrane strength and integrity. These findings suggest that the fetal contribution to PPROM is polygenic, and driven by an increased burden of rare variants that may also contribute to the disparities in rates of preterm birth among African Americans.

Severity of alcohol dependence is associated with the fatty acid amide hydrolase Pro129Thr missense variant.

The endocannabinoid system plays an important role in reward and addiction. One of the two main endocannabinoid neurotransmitters, anandamide, is metabolized by fatty acid amide hydrolase, an enzyme with a functional genetic polymorphism (FAAH Pro129Thr, rs324420). The Thr129 allele has been linked to problem drug and alcohol use, but the association has not been widely replicated and may be stronger for clinical measures of severity rather than categorical diagnosis. In the present study, we sought to determine whether the Thr129 allele was associated with both alcohol dependence (AD) diagnosis and severity in a sample of 1434 European American and African American individuals, 952 of whom were diagnosed with lifetime AD. Participants were genotyped for FAAH rs324420, and ancestry was determined via a genome-wide panel of ancestry informative markers. Subjects participated in Structured Clinical Interviews for psychiatric disorders and 90-day Timeline Followback interviews to assess recent alcohol use. European American participants with current AD had a higher Thr129 allele frequency than non-dependent controls. In European Americans with lifetime AD, there were significantly different distributions of drinking days and binge drinking days between the two genotype groups, with Thr129 carriers reporting a median of 10 fewer abstinent days and 13 more binge drinking days than Pro129/Pro129 homozygotes. In African American participants, there were no significant differences between Thr129 allele frequency in cases and controls and no significant differences in measures of AD severity by genotype. These findings provide evidence that the Pro129Thr missense variant is associated with AD severity in European Americans.

Minimal SNP overlap among multiple panels of ancestry informative markers argues for more international collaboration

The century-old use of genetic markers to determine population relationships has morphed in modern forensics into use of markers to determine the ancestry of an individual from a DNA sample. Researchers have identified sets of SNPs that have frequency differences among populations and many sets of SNPs have been published for the purpose of inferring ancestry. Such inference also requires reference datasets for the particular set of SNPs selected. We have identified 21 largely independent published panels of ancestry informative SNPs (AISNPs) and examined their union of 1397 SNPs. No SNP occurs in more than 6 panels. The 1397 SNPs in 21 panels yield a largely empty matrix that is inhibiting progress on more refined ability to infer ancestry for a forensic sample. The most common set of reference populations is the HGDP set of 52 small population samples totaling a thousand individuals. Only 46 (3%) of the 1397 SNPs occur in three or more panels. We assembled a new dataset for 44 of those SNPs involving 4,559 individuals from 73 populations. Analyses of this dataset provided clear differentiation of only five biogeographic regions: sub-Saharan Africa, Europe and SW Asia, South Asia, East Asia, and the Americas. This is an inadequate level of biogeographic resolution already exceeded by other panels. We conclude that more such AISNP panels are not needed and that the forensic community must collaborate to develop a common set of highly differentiating AISNPs typed on a very large number of population samples. How that can be accomplished will be the subject of future discussion.