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Preterm Birth Genome Project PREBIC 2009 Update

Genetic Basis for PTB?. Leading risk factor for spontaneous preterm labour and preterm birthPersonal or family history of PTBProband with PTB more closely related than random members of populationTwin studies: heritability for PTB 17 - 36%. Varner MW, Esplin MS. BJOG 2005;112(1):28-31Ward K, Arg

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Preterm Birth Genome Project PREBIC 2009 Update

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    1. Preterm Birth Genome Project PREBIC 2009 Update A/Prof Craig Pennell On behalf of the PGP Consortium

    2. Genetic Basis for PTB? Leading risk factor for spontaneous preterm labour and preterm birth Personal or family history of PTB Proband with PTB more closely related than random members of population Twin studies: heritability for PTB 17 - 36% Utah, large pedigrees, multiple wifes/children, longitudinal records Utah, large pedigrees, multiple wifes/children, longitudinal records

    3. Genetic Basis for PTB? Trans-generational studies <30 weeks – OR PTB 2.4 Single best predictor of PTB is previous PTB 1 prior PTB 15% recurrence 2 prior PTB 32% recurrence Racial differences persist after controlling for etiologic factors Risk of PTB tends to remain with mother If a woman herself is born preterm, she is at risk of PTB when she falls pregnant Risk highest, the earlier born preterm Single best predictor of PTB is previous PTB 1 prior PTB 15% recurrence 2 prior PTB 32% recurrence Tend to occur at the same gestation, risk highest if woman herself born preterm Predisposition does not apply to men who were born pretermIf a woman herself is born preterm, she is at risk of PTB when she falls pregnant Risk highest, the earlier born preterm Single best predictor of PTB is previous PTB 1 prior PTB 15% recurrence 2 prior PTB 32% recurrence Tend to occur at the same gestation, risk highest if woman herself born preterm Predisposition does not apply to men who were born preterm

    4. Candidate Gene Studies of PTB More than 80 genes > 1600 polymorphisms Maternal DNA / Fetal DNA Most of the associations not reproducible Limited success

    5. Meta-analysis of Candidate Gene Studies – PTB Gene Resource IL1RN 86bp VNTR maternal DNA ADRB2 maternal DNA F2 maternal DNA F2 newborn DNA IFNG maternal DNA IL1 receptor antagonist Adrenergic beta two surface receptor Coagulation factor 2 Interferon gammaIL1 receptor antagonist Adrenergic beta two surface receptor Coagulation factor 2 Interferon gamma

    6. GWAS vs. Candidate Gene Studies Single locus analyses have literally littered the field with errors and misunderstandings regarding the true causal variant Complexity of causation of preterm birth: Candidate gene selections are incomplete or uneven as many of the underlying pathways are either ignored or not yet understood Bias during the candidate gene selection stage may miss some causal variant

    7. 2008 – Year of the GWAS Criteria for successful GWAS: =100,000 SNPs in phase one Independent replication As of 23 April 2009: 304 publications > 200 specific conditions 1337 SNPs

    8. GWAS and Preterm Birth

    11. Preterm birth genome project PGP Consortium

    12. PGP Consortium Preliminary discussions within PREBIC members Established in Geneva on Sep 4th and 5th, 2007 Included investigators from 4 continents Established Memorandum of Agreement to collaborate on GWAS by pooling resources (DNA) Established database of phenotype definitions

    13. Goals of PGP Consortium Create a community of researchers to identify PTB susceptibility genes Pool resources from multiple investigators to conduct GWAS across multiple geographic populations Detailed phenotypic and environmental data Establish a large pool of replication samples Deep re-sequencing of genes with significant/ interesting findings in GWA

    14. PGP Consortium Structure

    15. Sample Resources Cases – ~ 5,000 Hispanic – 2,000 (Mexico, USA) Caucasian – 2,000 (UK, USA, Denmark, Australia, Norway, Canada) African Americans – 300 (USA) Asians – 400 (Korea) Controls - ~ 5,000 Hispanic – 2,000 (Mexico, USA) Caucasian – 2,000 (UK, USA, Australia, Norway, Canada) African Americans – 1000 (USA) Asians – 500 (Korea) Cases – ~ 5,000 Hispanic – 2,000 (Mexico, USA) Caucasian – 2,000 (UK, USA, Denmark, Australia, Norway, Canada) African Americans – 300 (USA) Asians – 400 (Korea) Controls - ~ 5,000 Hispanic – 2,000 (Mexico, USA) Caucasian – 2,000 (UK, USA, Australia, Norway, Canada) African Americans – 1000 (USA) Asians – 500 (Korea)

    17. PREBIC: Optimal Dataset

    18. Phase 1 Validation of existing resources for genetic analyses (60 mother:infant pair samples): Currently the PGP has samples that have been collected from twelve countries. This initial phase will evaluate sample quality for high throughput genotyping from Korea, Denmark, Mexico, Canada and Australia. Samples from these countries have been selected because they have been collected using different methods including whole blood, saliva and blood spots with whole genome amplification. The goal of this phase is to examine the quality of these samples for genome wide analyses on the specific genotyping platform that will be used for subsequent phases.   Phase 2. Proof of principle and preliminary analyses (1200 cases - 1200 controls): This phase will examine 1200 cases (600 Mexican, 600 Caucasian) and 1200 controls (600 Mexican, 600 Caucasian) utilising maternal samples using the platform validated in Phase 1. For this study cases will be defined as spontaneous preterm birth. Controls will be normal pregnancies delivered at term matched for recognized risk factors for preterm birth. Association analyses will be performed on the platform validated in Phase 1 and meta-analysis will be used to identify universal genetic risk factors across the two populations with adequate power. The positive results will be validated in PGP samples from other countries using at least 2000 cases and 2000 controls.   Phase 3 Global evaluation of PTB risk genes (5000 cases-5000 controls) This phase will evaluate 1000 cases of spontaneous preterm birth and 1000 matched controls in each of five populations to confirm the results from Phase 2 and to provide adequate power to identify other, potentially population specific genetic risk factors for PTB. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 4 Evaluation of Interactions between Maternal and Fetal Genomes in PTB (5000 cases- 5000 controls) Fetal DNA from cases and controls used in Phase 3 will be studied for association with PTB. In addition, interactions between maternal and fetal DNA will be evaluated for risk. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 5 Evaluation of Gene by Environment Interactions predisposing to PTB In recognition of the fact that genetic risk are dependent on environmental exposures the PGP will evaluate gene by environment interactions utilizing pre-existing epidemiological data in the cohorts such as smoking, drug and alcohol, infection status and stress measures. The sample size in this Phases 3 and 4 are adequate to evaluate the interactions between genetic variation and the environment. Phase 1 Validation of existing resources for genetic analyses (60 mother:infant pair samples): Currently the PGP has samples that have been collected from twelve countries. This initial phase will evaluate sample quality for high throughput genotyping from Korea, Denmark, Mexico, Canada and Australia. Samples from these countries have been selected because they have been collected using different methods including whole blood, saliva and blood spots with whole genome amplification. The goal of this phase is to examine the quality of these samples for genome wide analyses on the specific genotyping platform that will be used for subsequent phases.   Phase 2. Proof of principle and preliminary analyses (1200 cases - 1200 controls): This phase will examine 1200 cases (600 Mexican, 600 Caucasian) and 1200 controls (600 Mexican, 600 Caucasian) utilising maternal samples using the platform validated in Phase 1. For this study cases will be defined as spontaneous preterm birth. Controls will be normal pregnancies delivered at term matched for recognized risk factors for preterm birth. Association analyses will be performed on the platform validated in Phase 1 and meta-analysis will be used to identify universal genetic risk factors across the two populations with adequate power. The positive results will be validated in PGP samples from other countries using at least 2000 cases and 2000 controls.   Phase 3 Global evaluation of PTB risk genes (5000 cases-5000 controls) This phase will evaluate 1000 cases of spontaneous preterm birth and 1000 matched controls in each of five populations to confirm the results from Phase 2 and to provide adequate power to identify other, potentially population specific genetic risk factors for PTB. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 4 Evaluation of Interactions between Maternal and Fetal Genomes in PTB (5000 cases- 5000 controls) Fetal DNA from cases and controls used in Phase 3 will be studied for association with PTB. In addition, interactions between maternal and fetal DNA will be evaluated for risk. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 5 Evaluation of Gene by Environment Interactions predisposing to PTB In recognition of the fact that genetic risk are dependent on environmental exposures the PGP will evaluate gene by environment interactions utilizing pre-existing epidemiological data in the cohorts such as smoking, drug and alcohol, infection status and stress measures. The sample size in this Phases 3 and 4 are adequate to evaluate the interactions between genetic variation and the environment.

    19.   Phase 3 Global evaluation of PTB risk genes (5000 cases-5000 controls) This phase will evaluate 1000 cases of spontaneous preterm birth and 1000 matched controls in each of five populations to confirm the results from Phase 2 and to provide adequate power to identify other, potentially population specific genetic risk factors for PTB. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 4 Evaluation of Interactions between Maternal and Fetal Genomes in PTB (5000 cases- 5000 controls) Fetal DNA from cases and controls used in Phase 3 will be studied for association with PTB. In addition, interactions between maternal and fetal DNA will be evaluated for risk. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 5 Evaluation of Gene by Environment Interactions predisposing to PTB In recognition of the fact that genetic risk are dependent on environmental exposures the PGP will evaluate gene by environment interactions utilizing pre-existing epidemiological data in the cohorts such as smoking, drug and alcohol, infection status and stress measures. The sample size in this Phases 3 and 4 are adequate to evaluate the interactions between genetic variation and the environment.   Phase 3 Global evaluation of PTB risk genes (5000 cases-5000 controls) This phase will evaluate 1000 cases of spontaneous preterm birth and 1000 matched controls in each of five populations to confirm the results from Phase 2 and to provide adequate power to identify other, potentially population specific genetic risk factors for PTB. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 4 Evaluation of Interactions between Maternal and Fetal Genomes in PTB (5000 cases- 5000 controls) Fetal DNA from cases and controls used in Phase 3 will be studied for association with PTB. In addition, interactions between maternal and fetal DNA will be evaluated for risk. Positive findings will be explicitly tested in replication cohorts within the PGP consortium.   Phase 5 Evaluation of Gene by Environment Interactions predisposing to PTB In recognition of the fact that genetic risk are dependent on environmental exposures the PGP will evaluate gene by environment interactions utilizing pre-existing epidemiological data in the cohorts such as smoking, drug and alcohol, infection status and stress measures. The sample size in this Phases 3 and 4 are adequate to evaluate the interactions between genetic variation and the environment.

    20. Genotyping Platform Affymetrix Genome-Wide Human SNP Array 6.0 906,600 SNPs Unbiased selection of 482,000 SNPs (5.0 array) Additional 424,000 SNPs Tag SNPs SNPs from chromosomes X and Y Mitochondrial SNPs New SNPs added to the dbSNP database SNPs in recombination hotspots 946,000 probes for the detection of CNV 10 x more CNV than other SNP/CN platforms

    21. Phase 1- Validation of Existing Resources Four sites, 4 DNA extraction techniques Korea / Denmark / Mexico / Canada 15 maternal-fetal pairs 15 maternal samples (M1 to M15) split M1 to M15 sent at room temperature M1 to M15 sent on dry ice 15 fetal samples (F1-F15) sent on dry ice

    22. Phase 1: Total Arrays Performed 4 x M1-M15 shipped on dry ice (n=60) 4 x M1-M5 shipped on dry ice (n=20) 4 x M1-M5 shipped at room temp. (n=60) 4 x F1-F15 shipped on dry ice (n=60) Total of 200 arrays

    23. Results Phase 1 PGP: Extraction 98.86 0.49 98.86 0.49

    24. Results Phase 1 PGP: Shipping 98.86 0.49 98.86 0.49

    25. Results Phase 1 PGP: Replication 98.86 0.49 98.86 0.49

    26. Results Phase 1 PGP: Accuracy 98.86 0.49 98.86 0.49

    27. Conclusion – Phase 1 PGP Consortium can work successfully together DNA from blood, salivette and blood spot (WGA) suitable for genome wide analysis DNA from buccal swabs not suitable for GWAS Samples can be shipped at room temperature Platform utilised: Rapid, accurate, cost effective Samples from Korea, Mexico and Denmark suitable for Phases 2-5 98.86 0.49 98.86 0.49

    28. Ongoing PGP Work Phase 2: Samples from Denmark, Australia and Mexico Australian samples Call rates: 98.70% ± 0.39% Genotyping – Perth / Mexico Accuracy same samples two genotyping sites Sourcing additional funding Finalized WHO contracts Genotyping commences May 2009 Phenotype data (optimal dataset) entry 3 countries Analytic plan developed Replication samples

    29. Preterm birth genome project PGP Consortium

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