Whole-exome sequencing in multiplex preeclampsia families identifies novel candidate susceptibility genes.

OBJECTIVE: Preeclampsia is a common and serious heritable disorder of human pregnancy. Although there have been notable successes in identification of maternal susceptibility genes a large proportion of the heritability of preeclampsia remains unaccounted for. It is has been postulated that rare variation may account for some of this missing heritability. In this study, we performed whole-exome sequencing (WES) in multiplex families to identify rare exonic risk variants. METHODS: We conducted WES in 244 individuals from 34 Australian/New Zealand multiplex preeclampsia families. Variants were tested for association with preeclampsia using a threshold model and logistic regression. RESULTS: We found significant association for two moderately rare missense variants, rs145743393 (Padj = 0.0032, minor allele frequency = 0.016) in the chromosome 1 open reading frame 35 (C1orf35) gene, and rs34270076 (Padj = 0.0128, minor allele frequency = 0.024) in the pyroglutamylated RFamide peptide receptor (QRFPR) gene. To replicate these associations we performed imputation in our Australian genome wide association scan for preeclampsia and found no significant exonic variants in either C1orf35 or QRFPR. However, 11 variants demonstrating nominal significance (P < 0.05) in the genomic region between QRFPR and annexin A5 (ANXA5) were identified. We further leveraged publicly available genome-wide available summary data from the UK Biobank to investigate association of these two variants with the underlying clinical phenotypes of preeclampsia and detected nominal association of the QRFPR variant (rs34270076, P = 0.03) with protein levels in females. CONCLUSION: The study represents the first to use WES in multiplex families for preeclampsia and identifies two novel genes (QRFPR and C1orf35) not previously associated with preeclampsia and find nominal association of rs34270076 with protein levels, a key clinical feature of preeclampsia. We find further support for ANXA5 previously associated with pregnancy complications, including preeclampsia.

DNA methylation within the I.4 promoter region correlates with CYPl19A1 gene expression in human ex vivo mature omental and subcutaneous adipocytes.

BACKGROUND: DNA methylation at specific CpG sites within gene promoter regions is known to regulate transcriptional activity in vitro. In human adipose tissue, basal transcription of the aromatase (CYP19A1) gene is driven primarily by the I.4 promoter however the role of DNA methylation in regulating expression in ex vivo mature adipocytes is unknown. This observational study reports the correlation of DNA methylation within the I.4 promoter region of human mature subcutaneous and omental adipocytes with aromatase expression and body composition measures. METHODS: Omental and subcutaneous adipose tissue were collected from 25 obese subjects undergoing bariatric surgery and the mature adipocyte fraction purified. DNA methylation status of 5 CpG sites within a 550 base pair region encompassing the transcription start site (TSS) of promoter I.4 was determined using pyrosequencing. Relative aromatase and I.4 promoter specific mRNA expression was determined by qRT-PCR and whole body DXA performed in 25 participants. RESULTS: Site-specific DNA methylation varied from 21 ± 10% to 81 ± 11%. In omental adipocytes percentage methylation at the I.4.1 and I.4.2 CpG sites, but not other nearby sites, was negatively correlated with relative aromatase mRNA expression (R = - 0.52, P = 0.017 and R = - 0.52, P = 0.015). In contrast subcutaneous adipocytes percentage DNA methylation at the I.4.3 and I.4.5 sites were positively correlated with relative aromatase mRNA expression (R = 0.47, P = 0.022 and R = 0.55, P = 0.004). In a small subset of patients DNA methylation at the I.4.5 site was also positively correlated with whole body lean mass, bone mineral content and density. CONCLUSIONS: In conclusion in mature adipocytes, the primary source of estradiol after menopause, increasing DNA methylation was correlated with aromatase mRNA expression and thus estradiol biosynthesis. These findings support a tissue-specific epigenetic regulation of the basal promoter activity in mature adipocytes; the mechanisms influencing this regulation and its physiological role remain to be elucidated.

A non-synonymous coding change in the CYP19A1 gene Arg264Cys (rs700519) does not affect circulating estradiol, bone structure or fracture.

BACKGROUND: The biosynthesis of estrogens from androgens is catalyzed by aromatase P450 enzyme, coded by the CYP19A1 gene on chromosome 15q21.2. Genetic variation within the CYP19A1 gene sequence has been shown to alter the function of the enzyme. The aim of this study is to investigate whether a non-synonymous Arg264Cys (rs700519) single nucleotide polymorphism (SNP) is associated with altered levels of circulating estradiol, areal bone mineral density or fracture. METHODS: This population- based study of 1,022 elderly Caucasian women (mean age 74.95 ± 2.60 years) was genotyped for the rs700519 SNP were analyzed to detect any association with endocrine and bone phenotypes. RESULTS: The genotype frequencies were 997 wildtype (97.6%), 24 heterozygous (2.3%) and 1 homozygous (0.1%). When individuals were grouped by genotype, there was no association between the polymorphism and serum estradiol (wildtype 27.5 ± 16.0; variants 31.2 ± 18.4, P = 0.27). There was also no association seen on hip bone mineral density (wildtype 0.81 ± 0.12; 0.84 ± 0.14 for variants, P = 0.48) or femoral neck bone mineral density (0.69 ± 0.10 for wildtype; 0.70 ± 0.12 for variants, P = 0.54) before or after correction of the data with age, height, weight and calcium therapy. There were also no associations with quantitative ultrasound measures of bone structure (broadband ultrasound attenuation, speed of sound and average stiffness). CONCLUSIONS: In a cohort of 1,022 elderly Western Australian women, the presence of Arg264Cys (rs700519) polymorphism was not found to be associated with serum estradiol, bone structure or phenotypes.

Prior bovine immunodeficiency virus infection does not inhibit subsequent superinfection by the acutely pathogenic Jembrana disease virus.

In cattle the interaction between the two genetically and antigenically related bovine lentiviruses, the acutely pathogenic Jembrana disease virus (JDV) and the non-pathogenic Bovine immunodeficiency virus (BIV) has not been reported although both JDV and a BIV-like virus have been reported in the Bali cattle (Bos javanicus) population in Indonesia. The outcome of infection of Bali cattle with the R29 strain of BIV prior to superinfection 42 days later with JDV(TAB/87) was determined. All BIV-inoculated cattle were successfully infected and developed an antibody response to the TM and CA proteins. BIV infection did not prevent subsequent infection with JDV or ameliorate the clinical signs of Jembrana disease in the infected cattle. It did, however, modify the dynamics of the JDV infection with an earlier onset and end of the acute disease process, and a reduction in the duration of viremia that exceeded 10(6) genome copies/ml of plasma.

Bovine immunodeficiency virus produces a transient viraemic phase soon after infection in Bos javanicus.

Infection of Bali cattle (Bos javanicus) in Indonesia with a non-pathogenic bovine lentivirus similar to Bovine immunodeficiency virus (BIV) is suspected but efforts to detect the virus have been unsuccessful. To define the kinetics of BIV infection in Bali cattle, 13 were infected with the R-29 strain of BIV and monitored for 60 days. No clinical effects were detected. Proviral DNA was detected in peripheral blood mononuclear cells from 4 to 60 days with peak titres 20 days post-infection (dpi). There was a transient viraemia from 4 to 14 dpi with a maximum titre of 1x10(4)genome copies/ml plasma. An antibody response to the transmembrane (TM) glycoprotein commenced 12 dpi but an antibody response to the capsid (CA) protein was detected in one animal only and not until 34 dpi. The results indicated that detection of BIV in infected Bali cattle would have a greater chance of success soon after infection and prior to the onset of a CA antibody response.

In vivo infection of IgG-containing cells by Jembrana disease virus during acute infection.

Jembrana disease virus (JDV) is an unusual bovine lentivirus which causes a non-follicular proliferation of lymphocytes, a transient immunosuppression and a delayed humoral response in infected Bali cattle in Indonesia. A double-immunofluorescent labeling method was developed to identify the subset of mononuclear cells in which the viral capsid protein could be detected. Viral antigen was present in pleomorphic centroblast-like cells which were identified as IgG-containing cells, including plasma cells, in lymphoid tissues. There was no evidence of infection of CD3(+) T-cells or MAC387(+) monocytes in tissues but large vacuolated cells with a macrophage-like morphology in the lung were found to contain viral antigen although they could not be shown conclusively to be infected. The tropism of JDV for mature IgG-containing cells may be relevant to understanding the pathogenesis of Jembrana disease, the delayed antibody responses and the genetic composition of this atypical lentivirus.

Comparison of immunoassay and real-time PCR methods for the detection of Jembrana disease virus infection in Bali cattle.

A sensitive diagnostic assay for the detection of infections with the bovine lentivirus Jembrana disease virus (JDV) is required in Indonesia to control the spread of Jembrana disease. Immunoassays are used routinely but are compromised by cross-reactive epitopes in the capsid (CA) protein of JDV and the genetically related bovine immunodeficiency virus (BIV). JDV gag-specific primers were tested in a real-time PCR assay to detect proviral DNA in peripheral blood mononuclear cells from 165 cattle from the Tabanan district of Bali. JDV-specific amplicons were detected in 9% of the cattle and only 33% of the real-time PCR positive cattle were seropositive. The delayed seroconversion that occurs after infection with JDV could explain the low concordance between these assays but other factors may be responsible. BIV proviral DNA was not detected in any of the PBMC DNA samples. A high concordance value of 98.6% was found between the JDV plasma-derived antigen Western blot and the JDV p26-his recombinant protein ELISA. Only 21% of the seropositive cattle had detectable levels of proviral DNA suggesting that the proviral load in recovered cattle is low. A combination of real-time PCR and JDV p26-his ELISA is recommended for the detection of infection with JDV in Indonesia.

Analysis of Jembrana disease virus replication dynamics in vivo reveals strain variation and atypical responses to infection.

Jembrana disease virus (JDV) is an acute lentiviral infection of Bali cattle in Indonesia. Data generated during a series of cattle infection experiments was examined and significant differences were identified in the mean plasma viral load on the first and second days of the febrile response in cattle infected with JDV(TAB/87) compared to those infected with JDV(PUL/01). The peak and total viral loads >or=10(6) genome copies/ml during the acute stage of the disease were significantly higher in JDV(TAB/87) infected cattle. JDV(PUL/01) infected cattle developed peak rectal temperatures earlier than the JDV(TAB/87) cattle but there were no differences in the duration of the febrile responses observed for the 2 groups of animals. The plasma viremia was above 10(6) genome copies/ml for almost 3 days longer in JDV(TAB/87) compared to JDV(PUL/01) infected cattle. Atypical responses to infection occurred in approximately 15% of experimentally infected animals, characterized by reduced viral loads, lower or absent febrile responses and absence of p26-specific antibody responses. Most of these cattle developed normal Tm-specific antibody responses between 4-12 weeks post-infection.