O1 The metabolomics approach to autism: identification of biomarkers for early detection of autism spectrum disorder

A. K. Srivastava1, Y. Wang2, R. Huang3, C. Skinner1, T. Thompson3, L. Pollard3, T. Wood3, F. Luo2, R. Stevenson1

1JCSRI, Greenwood Genetic Center, Greenwood, SC, USA; 2School of Computing, Clemson University, Clemson, SC, USA; 3Biochemical Genetics Laboratory, Greenwood Genetic Center, Greenwood, SC, USA

Correspondence: A. K. Srivastava – JCSRI, Greenwood Genetic Center, Greenwood, SC, USA

Objectives

From the first description by Leo Kanner [1], autism has been an enigmatic neurobehavioral phenomenon. The new genetic/genomic technologies of the past decade have not been as productive as originally anticipated in unveiling the mysteries of autism. The specific etiology of the majority of cases of autism spectrum disorder (ASD) is unknown, although numerous genetic/genomic variants and alterations of diverse cellular functions have been reported. Prompted by this failure, we have investigated whether the metabolomics approach might yield results which could simultaneously lead to a blood-based screening/diagnostic test and to treatment options.

Methods

Plasma samples from a clinically well-defined cohort of 100 male individuals, ages 2-16+ years, with ASD and 32 age-matched typically developing (TD) controls were subjected to global metabolomic analysis.

Results

We have identified more than 25 plasma metabolites among the approximately 650 metabolites analyzed, representing over 70 biochemical pathways, that can discriminate children with ASD as young as 2 years from children that are developing typically. The discriminating power was greatest in the 2–10 year age group and weaker in older age groups. The initial findings were validated in a second cohort of 83 children, males and females, ages 2–10 years, with ASD and 76 age and gender-matched TD children. The discriminant metabolites were associated with several key biochemical pathways suggestive of potential contributions of increased oxidative stress, mitochondrial dysfunction, inflammation and immune dysregulation in ASD. Further, targeted quantitative analysis of a subset of discriminating metabolites using tandem mass spectrometry provided a reliable laboratory method to detect children with ASD.

Conclusion

Metabolic profiling appears to be a robust technique to identify children with ASD ages 2–10 years and provides insights into the altered metabolic pathways in ASD, which could lead to treatment strategies.

References

1. Kanner, L. Autistic disturbances of affective contact. Nervous Child. 1943; 2: 217–250.

Disclosure of interest

None declared.

O2 Phenome-wide association study for smoking- and drinking-associated genes in 26,394 American women with African, Asian, European, and Hispanic descents

R. Polimanti1, J. Gelernter1,2,3

1Department Psychiatry, Yale Sch Med and VA CT Healthcare Center, West Haven, CT, USA; 2Department Genetics, Yale Sch Med and VA CT Healthcare Center, West Haven, CT, USA; 3Department Neurobiology, Yale Sch Med and VA CT Healthcare Center, West Haven, CT, USA

Correspondence: R. Polimanti – Dept Psychiatry, Yale Sch Med and VA CT Healthcare Center, West Haven, CT, USA

Objectives

To uncover novel traits associated with nicotine and alcohol use genetics, we performed a phenome-wide association study in a large multi-ethnic cohort.

Methods

We investigated 7,688 African-Americans (AFR), 1,133 Asian-Americans (ASN), 14,081 European-Americans (EUR), and 3,492 Hispanic-Americans (HISP) from the Women’s Health Initiative, analyzing risk alleles located in the CHRNA5CHRNA3 locus (rs8034191, rs1051730, rs12914385, rs2036527, and rs16969968) for nicotine-related traits and ADH1B (rs1229984 and rs2066702) and ALDH2 (rs671) for alcohol-related traits with respect to anthropometric characteristics, dietary habits, social status, psychological circumstances, reproductive history, health conditions, and nicotine- and alcohol-related traits.

Results

The investigated loci resulted associated with novel traits: rs1229984 were associated with family income (p=4.1*10−12), having a pet (p=6.5*10−11), partner education (p=1.8*10−10), “usually expect the best” (p=2.4*10−7), “felt calm and peaceful” (p=2.6*10−7), education (p=3.7*10−6), and number of term pregnancies (p=1.12*10−5) in EUR; rs1051730 and rs16969968 showed a suggestive association with “High cholesterol requiring pills ever” (p=3.8*10−4 and p=1.8*10−4) in trans-ethnic meta-analysis. We also replicated the known associations: rs80341911 was associated with cigarettes per day (CIGSDAY, p=3.4*10−8), smoking status (p=6.7*10−3), and “smoked at least 100 cigarettes” (p=7.3*10−3) in EUR; rs1051730 and rs16969968 were associated with CIGSDAY (p=9.1*10−8 and p=1.1*10−7) and lung cancer (p=7.3*10−3 and p=9.9*10−3) in EUR; rs2036527 was associated with CIGSDAY (p=3.5*10−3) in AFR; rs1229984 showed associations for alcohol servings (ALC, p=2.9*10−6), beer servings (p=3*10−6), wine servings (WINE, p=3.9*10−6), liquor servings (p=5.5*10−6), dietary alcohol (DIETALC, p=6.1*10−6), “Drinks alcohol (age 50)” (p=9.3*10−6) in EUR and for ALC (p=5.2*10−5) and DIETALC (p=9.6*10−5) in HISP; rs671 resulted associated with alcohol intake (p=3.8*10−8), DIETALC (p=1.9*10−7), ALC (p=1.3*10−6), WINE (p=1.1*10−5) and “Drank 12 alcoholic beverages ever” (p=1.2*10−5) in ASN.

Conclusion

We provided novel genetic data regarding the consequences of smoking and drinking behaviors and confirmed ethnic differences in their genetic predisposition.

Disclosure of interest

None declared.

O3 Effects of prenatal environment, genotype and DNA methylation on birth weight and subsequent postnatal outcomes: findings from GUSTO, an Asian birth cohort

X. Lin1, I. Y. Lim1, Y. Wu1, A. L. Teh1, L. Chen1, I. M. Aris1, S. E. Soh1, M. T. Tint2, J. L. MacIsaac3, F. Yap4, K. Kwek4, S. M. Saw2, M. S. Kobor3, M. J. Meaney1, K. M. Godfrey5, Y. S. Chong1, J. D. Holbrook1, Y. S. Lee1, P. D. Gluckman1,6, N. Karnani1, GUSTO study group

1Singapore Institute for Clinical Sciences, Singapore, Singapore; 2National University of Singapore, Singapore, Singapore; 3University of British Columbia, Vancouver, British Columbia, Canada; 4KK Women’s and Children’s Hospital, Singapore, Singapore; 5University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK; 6University of Auckland, Auckland, New Zealand

Correspondence: X. Lin – Singapore Institute for Clinical Sciences, Singapore, Singapore

Objectives

Prenatal environment and genetic polymorphism can have a lasting impact on offspring’s metabolic function by perturbing its epigenome. Birth weight is often used as a surrogate for the overall quality of the intrauterine environment. We present the first neonate epigenome-wide association study in an Asian mother-offspring cohort, that interrogates the effects of prenatal environment variables, umbilical cord DNA methylation and SNPs, on birth weight.

Methods

In GUSTO, a prospective mother-offspring cohort study (N=987), we examined the associations between DNA methylation, SNPs, birth weight and 11 prenatal environment variables. First, we investigated the association between perinatal methylome and birth weight to identify sites of variability in methylation. Second, we interrogated the contribution of genetic and prenatal environmental factors on this variability in the epigenome. Finally, we examined whether these methylation marks at birth were associated with offspring size and adiposity in early childhood.

Results

Methylation levels at 50 CpGs were significantly associated with birth weight, and a subset of these CpGs was located in genes and miRNA known to be involved in metabolic pathways/disorders. We further examined the influence of environmental and genetic factors on methylation at these 50 CpG sites. Sixteen CpGs were associated with both, an additional 24 CpGs were associated with only environmental factors, while only 3 CpGs were associated with genetic factors alone. Environmental factors associated with methylation were predominantly maternal-adiposity-related (pre-pregnancy body mass index, pregnancy weight gain and maternal glucose levels). Methylation levels at half of these CpGs were also associated with offspring size and adiposity in early childhood.

Conclusion

Developmental pathways to obesity begin before birth and involve genetic, epigenetic and environmental factors.

Disclosure of interest

X. Lin: None declared., I. Y. Lim: None declared., Y. Wu: None declared., A. L. Teh: None declared., L. Chen: None declared., I. M. Aris: None declared., S. E. Soh: None declared., M. T. Tint: None declared., J. L. MacIsaac: None declared., F. Yap: None declared., K. Kwek: None declared., S. M. Saw: None declared., M. S. Kobor: None declared., M. J. Meaney: None declared., K. M. Godfrey Conflict with: KMG has received reimbursement for speaking at conferences sponsored by companies selling nutritional products. He is part of an academic consortium that has received research funding from Abbott Nutrition, Nestec and Danone, Y. S. Chong Conflict with: YSC has received reimbursement for speaking at conferences sponsored by companies selling nutritional products. He is part of an academic consortium that has received research funding from Abbott Nutrition, Nestec and Danone, J. D. Holbrook: None declared., Y. S. Lee: None declared., P. D. Gluckman: None declared., N. Karnani: None declared.

O4 High-throughput identification of specific qt interval modulating enhancers at the SCN5A locus

A. Kapoor, D. Lee, A. Chakravarti

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Correspondence: A. Kapoor – McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Objectives

Genome-wide association studies (GWAS) have indicated that sequence variation in cis-regulatory elements (CRE) plays important roles in common disease risk/trait variation, but identification of these causal variants has remained a major challenge in complex trait genetics. Here, we performed reporter assays for all common variants at the QT interval associated SCN5A GWAS locus, with the goal of identifying the underlying causal variants.

Methods

A target region of ~500kb at SCN5A was defined based on recombination hotspots (rate>10cM/Mb; HapMap) flanking the 5 independent QT interval GWAS hits. Within the target region, all common variants (minor allele frequency >5%) from the 1000 Genomes European ancestry populations in moderate linkage disequilibrium (r2>0.3) with any of the 5 GWAS hits were selected. Both alleles of these variants were amplified with flanking sequences and cloned upstream of a minimal promoter driven firefly luciferase gene in pGL4.23. Human cardiomyocyte cells, AC16, were transfected with test constructs and Renilla luciferase vector (for transfection normalization) in triplicate and luciferase assays were performed 24h later. Reporter assays on a subset of variants were repeated for assessing allelic difference in regulatory activity. All cloning and reporter assays were performed in 96- and 24-well plates.

Results

Of a total 121 variants selected, 112 variants in 104 amplicons passed primer design (amplicon size 256-617bp; median 397bp), and we successfully cloned both alleles for 106 variants in 98 amplicons. In reporter assays, compared to empty vector, 24 and 40 amplicons showed enhancer (>2-fold) and suppressor (<0.5-fold) activities in AC16 cells, respectively. Of these only 4 were observed as open chromatin regions in heart tissue in NIH Epigenomics data. Overall, 12 variants showed nominally significant allelic difference (P<0.05) in reporter activity and were repeated with 18 replicates and 7 variants were identified to have repeated significant allelic difference in regulatory activity.

Conclusion

Independent of the available epigenomic data, which are of limited relevance, an unbiased in vitro reporter screen for CREs overlapping all common variants associated with QT interval at the SCN5A GWAS locus identified 7 common cis-regulatory variants. Our immediate next goals are to a) evaluate the effect of deleting these 7 CREs on SCN5A expression in AC16 cells and b) identify the trans-acting factors regulating their functions.

Disclosure of interest

None declared.

O5 Identification of extracellular matrix components inducing cancer cell migration in the supernatant of cultivated mesenchymal stem cells

C. Maercker1, F. Graf2, M. Boutros2

1Esslingen University of Applied Sciences, Esslingen, Germany; 2German Cancer Research Center, Heidelberg, Germany

Correspondence: C. Maercker – Esslingen University of Applied Sciences, Esslingen, Germany

Objectives

Some cancers show a strong tendency to metastasize to bone, a tissue of mesenchymal origin and a prominent site of mesenchymal stem cells (MSC) residing in the stem cell niche. With bone metastasis formation being one of the most detrimental steps in cancer progression, a better understanding of how bone metastases are initially formed is key to successfully targeting bone metastasis of, for example, prostate cancer. Recent reports have suggested that bone-metastasizing cancers may mimic the process of homing of hematopoietic stem cells to their bone niche.

Methods

In order to understand the role of MSC in metastasis formation, we investigated the interaction of primary human bone marrow MSC with established cancer cell lines able to metastasize to bone. With a trans-well migration assay we could show that MSC induced a rapid migration response of prostate and breast cancer cell lines already within two hours after start of the experiment. In order to identify factors stimulating cancer cell migration, MSC cell culture supernatant was separated by size exclusion and ion exchange chromatography. Migratory fractions then were further analyzed by mass spectrometry and antibody array analysis.

Results

With this approach we identified the extracellular matrix proteins type I and type III collagen, fibronectin and laminin 421 as potential drivers of cancer cell migration, which was confirmed by using recombinant proteins. RNAi experiments showed that the cancer cell extracellular matrix receptor beta 1 integrin obviously plays a pivotal role for cell migration.

Conclusion

From our results we conclude that the extracellular matrix as it is produced by MSC obviously plays a crucial role for cancer metastasis and therefore might be a promising anti-cancer drug target.

Disclosure of interest

None declared.

O6 Single cell allele specific expression (ASE) IN T21 and common trisomies: a novel approach to understand DOWN syndrome and other aneuploidies

G. Stamoulis1, F. Santoni2, P. Makrythanasis2, A. Letourneau1, M. Guipponi2, N. Panousis1, M. Garieri1, P. Ribaux1, E. Falconnet1, C. Borel1, S. E. Antonarakis1,2,3

1Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland; 2Geneva University Hospitals-HUG, Service of Genetic Medicine, Geneva, Switzerland; 3iGE3 Institute of Genetics and Genomics of Geneva, University of Geneva Medical School, Geneva, Switzerland

Correspondence: G. Stamoulis – Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland

Objectives

Trisomy 21 is a model disorder of altered gene expression. We have previously used a pair of monozygotic twins discordant for trisomy 21 to study the global dysregulation of gene expression, without the noise due to genetic variation among individuals (Nature:508; 345–350;2014). The majority of previous studies focused on aneuploidies were conducted οn cell populations or tissues. Our study focusing on gene and allelic expression behaviour of single cells (SC), aims to reveal biological insights regarding the cellular impact of aneuploidy and uncover the mechanisms of gene dosage.

Methods

We estimated the allele specific expression (ASE) from RNAseq of ~1000 single cells in different aneuploidies. We used 352 SC fibroblasts (173 Normal and 179 T21 cells) from the pair of monozygotic twins discordant for T21, 166 SC from a mosaic T21, 176 SC from a mosaic T18, 151 SC from a mosaic T8, and 146 SC from a mosaic T13.

Results

In the monozygotic twins, a considerable number of heterozygous sites at the non-chr21 genome showed monoallelic expression (MAE);(Normal: 73.5 % monoallelic in 564,668 observations, and T21: 78.7 % monoallelic in 549,799 observations). There was also considerable MAE for chr21 sites in Normal and, surprisingly, in T21 cells as well (Normal: 63,3 % monoallelic in 5,009 observations, and T21: 72.8 % monoallelic in 6,456 observations). We classified the genes on chr21 in 3 classes according to the level of the aggregate MAE of their corresponding sites (9 monoallelic, 29 intermediate, 2 biallelic). Similar results, i.e. extensive MAE on the supernumerary chromosome genes, were also observed in the other aneuploidies.

Conclusion

We hypothesize that each class of genes contributes in a specific way to the phenotypic variability of Down Syndrome. Our analysis showed that, for genes with monoallelic expression, the abnormal gene dosage induced by the aneuploid chromosome is maybe due to the number of cells expressing the gene. This difference in the fraction of expressing cells could contribute to the development and the variability of phenotypes in aneuploidies. This study provides a new fundamental understanding of the allele specific expression in T21 and other aneuploidies.

Disclosure of interest

None declared.

O7 Role of microRNA in LCL to IPSC reprogramming

S. Kumar1, J. Curran2, J. Blangero2

1South Texas Diabetes and Obesity Institute, School of Medicine, University of Texas Rio-Grande Valley, Edinburg, TX, USA; 2South Texas Diabetes and Obesity Institute, School of Medicine, University of Texas Rio-Grande Valley, Brownsville, TX, USA

Correspondence: S. Kumar – South Texas Diabetes and Obesity Institute, School of Medicine, University of Texas Rio-Grande Valley, Edinburg, TX, USA

Objectives

A large number of EBV immortalized lymphoblastoid cell lines (LCLs) have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in-vitro cell model. Recent successes in reprograming LCLs into induced pluripotent stem cells (iPSCs) have paved the way to generate more relevant in-vitro disease models using this existing bio-resource. However the effects of EBV encoded oncoproteins on cellular transcription and function make LCLs a unique biomaterial to reprogramme. Accumulating evidence now provides support that miRNAs play a critical role in transcription factor-induced reprogramming of iPSCs.

Methods

To investigate the role of miRNAs in regulating gene expression and ce