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INTRODUCTION: Next-generation sequencing (NGS) in family-based study designs will be pivotal in unlocking the missing heritability of common complex diseases. Whilst our prior linkage- and association-based positional cloning studies in family- and population-based Australian cohorts, respectively, have discovered novel preeclampsia candidate genes (INHBB,ACVR2A,LCT,LRP1B,RND3,GCA,ERAP2,TNFSF13B), the full complement of causal genetic variation remains largely unknown. We have now sequenced the exomes of two Australian preeclampsia families in another step forward to unlocking preeclampsia's complex allelic architecture. OBJECTIVES: Identify family-specific exon-centric loci segregating in preeclamptic women only. METHODS: The exomes of 18 women (7 preeclamptics,11 controls) from two Australian families contributing to our chromosome 5q (Family 1) and 13q (Family 2) susceptibility loci, respectively, were sequenced using Illumina's TruSeq Exome Enrichment assay and NGS technology. Sequence alignments, quality control assessment and variant calling were conducted on our 8000 parallel processor compute server, MEDUSA. As a first pass, we prioritized exome sequence data to non-synonymous variants within the 1-LOD drop intervals of our 5q and 13q loci. Prioritized exonic variants were also genotyped in the Western Australian Pregnancy (Raine) Cohort to assess their significance against a plethora of cardiovascular disease (CVD) related traits. RESULTS: In Family 1 we identified two missense SNPs and in Family 2 we identified one missense SNP to segregate in the preeclamptic women but not in the unaffected women. The first SNP in Family 1 (rs62375061) resides within the LYSMD3 gene, is predicted to "possibly" damage the focal protein and the only public record of this SNP is within the Watson genome. The second SNP in Family 1 (rs111033530) resides within the GPR98 gene, is predicted to "probably" damage the focal protein and is rare (1.7% population prevalence). The SNP in Family 2 (rs1805388) resides within the LIG4 gene, is predicted to be highly deleterious (F-SNP FSS=0.849) and is common (⩾17% population prevalence). In the Raine cohort the LIG4 SNP was also significantly associated with weight (p=0.0085), total cholesterol (p=0.0007), HDL cholesterol (p=0.0067) and LDL cholesterol (p=0.0324). CONCLUSION: Our preliminary exome data documents the substantial potential to rapidly identify likely functional variants that influence preeclampsia risk. The GPR98 finding is of major interest to us as a recent genome-wide association study reported a significant association with diastolic blood pressure for a SNP at this same gene locus. Furthermore, our findings implicate LIG4 as a novel candidate susceptibility gene for CVD and add weight to the hypothesis of shared genetic risk factors for preeclampsia and CVD.
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INTRODUCTION: There is compelling evidence to support the hypothesis that a maternal constitutional predisposition to cardiovascular disease (CVD) is a key component in development of preeclampsia. In particular, CVD and preeclampsia share pathological features such as endothelial dysfunction and inflammation, and have several metabolic abnormalities in common. In support of this hypothesis, our recent genetic dissection of the Australian preeclampsia susceptibility locus on chromosome 2q22 revealed shared novel genetic risk factors for preeclampsia and CVD-related traits. OBJECTIVES: To replicate association between our recently reported 2q22 preeclampsia risk variants and CVD-related traits in an independent Australian population based cohort. METHODS: Four independent SNPs from four genes, rs35821928 (LRP1B), rs17783344 (GCA), rs115015150 (RND3) and rs2322659 (LCT), were recently found to be significantly associated with preeclampsia susceptibility and CVD-related traits. These SNPs were genotyped in a large independent Australian cohort rich in quantitative CVD risk traits; The Western Australian Pregnancy Cohort (Raine) Study. This cohort comprises of blood samples from 1246 mothers and 1461 adolescents and clinical measures such as, but not limited to, anthropometric measures of adiposity and lipid-related measures. Genetic association analyses of these four potential preeclampsia susceptibility SNPs against the CVD-related risk traits were performed using the software package R. All statistical analyses assumed an additive model of gene action. RESULTS: Several significant associations (p<0.05) for all four SNPs with a variety of CVD-related risk traits were detected, both for the mothers and the adolescents. The LRP1B SNP was associated with HDL/cholesterol ratio, LDL cholesterol, triglycerides, skinfold measures and weight. The GCA SNP was associated with total cholesterol, HDL cholesterol, serum insulin, hemoglobin, blood glucose, BMI and skinfold measures. The RND3 SNP was associated with triglycerides and waist-hip ratio. The LCT SNP was associated with hemoglobin, blood glucose and abdominal skinfold. CONCLUSION: We have recently identified genetic variants within the LRP1B, GCA, RND3 and LCT genes to be significantly associated with preeclampsia susceptibility and CVD-related risk traits. We have now demonstrated thatthese specific genetic variants are associated with CVD-related risk traits in an independent population. Our collective findings provide substantial empirical data to support the hypothesis that genetic risk factors for preeclampsia and CVD are, at least in part, shared.
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OBJECTIVES: Endoplasmic reticulum (ER) stress has been implicated in both pre-eclampsia (PE) and fetal growth restriction (FGR), and is characterised by activation of three signalling branches: 1) PERK-pEIF2α, 2) ATF6 and 3) splicing of XBP1(U) into XBP1(S). To evaluate the contribution of ER stress in the pathogenesis of PE relative to FGR, we compared levels of ER stress markers in decidual tissue from pregnancies complicated by PE and/or FGR. STUDY DESIGN: Whole-genome transcriptional profiling was performed on decidual tissue from women with PE (n = 13), FGR (n = 9), PE+FGR (n = 24) and controls (n = 58), and used for pathway and targeted transcriptional analyses of ER stress markers. The expression and cellular localisation of ER stress markers was assesses by Western blot and immunofluorescence analyses. RESULTS: Increased ER stress was observed in FGR and PE+FGR, including both the PERK-pEIF2α and ATF6 signalling branches, whereas ER stress was less evident in isolated PE. However, these cases demonstrated elevated levels of XBP1(U) protein. ATF6 and XBP1 immunoreactivity was detected in most (>80%) extravillous trophoblasts, decidual cells and macrophages. No difference in the proportion of immunopositive cells or staining pattern was observed between study groups. CONCLUSIONS: Increased PERK-pEIF2α and ATF6 signalling have been associated with decreased cellular proliferation and may contribute to the impaired placental growth characterising pregnancies with FGR and PE+FGR. XBP1(U) has been proposed as a negative regulator of ER stress, and increased levels in PE may reflect a protective mechanism against the detrimental effects of ER stress.
Assuntos
Decídua/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Retardo do Crescimento Fetal/metabolismo , Pré-Eclâmpsia/metabolismo , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Estudos de Casos e Controles , Decídua/patologia , Estresse do Retículo Endoplasmático/genética , Feminino , Retardo do Crescimento Fetal/etiologia , Retardo do Crescimento Fetal/genética , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Pré-Eclâmpsia/etiologia , Pré-Eclâmpsia/genética , Gravidez , Complicações na Gravidez/etiologia , Complicações na Gravidez/genética , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia , Regulação para Cima/genética , Adulto JovemRESUMO
Variation in the Storkhead box-1 (STOX1) gene has previously been associated with pre-eclampsia. In this study, we assess candidate single nucleotide polymorphisms (SNPs) in STOX1 in an independent population cohort of pre-eclamptic (n = 1.139) and non-pre-eclamptic (n = 2.269) women (the HUNT2 study). We also compare gene expression levels of STOX1 and its paralogue, Storkhead box-2 (STOX2) in decidual tissue from pregnancies complicated by pre-eclampsia and/or fetal growth restriction (FGR) (n = 40) to expression levels in decidual tissue from uncomplicated pregnancies (n = 59). We cannot confirm association of the candidate SNPs to pre-eclampsia (P > 0.05). For STOX1, no differential gene expression was observed in any of the case groups, whereas STOX2 showed significantly lower expression in deciduas from pregnancies complicated by both pre-eclampsia and FGR as compared with controls (P = 0.01). We further report a strong correlation between transcriptional alterations reported previously in choriocarcinoma cells over expressing STOX1A and alterations observed in decidual tissue of pre-eclamptic women with FGR.