Increased serum iron levels in pregnant women with preeclampsia: a meta-analysis of observational studies.

Our study aimed to investigate whether the serum iron levels in patients with preeclampsia were higher than in healthy pregnant women and to evaluate potential heterogeneities. We searched Pubmed, Embase, Web of Science and Medline databases for studies before September 2016. The standardised mean difference (SMD) and 95% confidence interval (CI) were used to combine results across the studies, in addition to the random-effect model. A total of 10 studies involving 363 patients with preeclampsia and 370 healthy controls were eligible through the inclusion criteria. In comparison with healthy pregnant women, the serum iron levels are higher in the patients with preeclampsia [summary SMD = 0.28, 95% CI = 0.11-0.44], and this association was also significant in the case-control studies. The serum iron levels were higher in the pregnant women with preeclampsia than in the healthy controls in both the Asian and European populations. Our study provides significant evidence of higher serum iron levels in the patients with preeclampsia than in healthy pregnant women. Impact Statement What is already known on this subject? Serum iron levels have inconsistent associations with a risk of preeclampsia in pregnant women. What the results of this study add? Compared with healthy pregnant women, serum iron levels are higher in patients with preeclampsia. What the implications are of these findings for clinical practice and/or further research? Further studies across large numbers of cases and increased patient populations are necessary to confirm our findings.

Expression, SNP identification, linkage disequilibrium, and haplotype association analysis of the growth suppressor gene ZBED6 in Qinchuan beef cattle.

Zinc finger, BED-type containing 6 (ZBED6) is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle myogenesis and development. The aims of this study were to determine ZBED6 expression level and examine the association of the ZBED6 polymorphism with growth traits in Qinchuan beef cattle. The bovine ZBED6 mRNA was detected in eight tissues by quantitative real-time PCR (qPCR), being highly expressed in skeletal muscle. Three single nucleotide polymorphisms (SNPs) were identified the bovine ZBED6 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (forced PCR-RFLP) methods. In this study, we reported one mutation in the promoter and two missense mutations in the coding regions within the bovine ZBED6 gene, and the haplotype variability and extent of linkage disequilibrium (LD) in 817 individuals from the Qinchuan (QC) and Chinese Holstein (CH). We also investigated haplotype structure and linkage disequilibrium coefficients for three SNPs of ZBED6 in the study populations. The result of haplotype analysis of three SNPs showed that eight different haplotypes were identified in two breeds. The wild-type haplotype (Hap 1: GCA) and mutant-type haplotype (Hap 8: AGG) shared by two populations accounted for 29.8%, 57.5%, and 8.6%, 0% of all haplotypes observed in QC and CH, respectively. The statistical analyses indicated that three SNPs, 23 combined genotypes, and 8 haplotypes were significantly associated with different growth traits in the QC cattle population (P < 0.05 or P < 0.01). The mutant-type variants and mutant haplotype were superior for growth traits; the heterozygote diplotype was associated with higher growth traits compared to the wild-type homozygote. The results of this study suggest that the ZBED6 gene possibly is a strong candidate gene that affects growth traits in QC beef cattle breeding program.

Comparative analysis of the IGF2 and ZBED6 gene variants and haplotypes reveals significant effect of growth traits in cattle.

Muscle growth is a complex phenomenon regulated by many factors, whereby net growth results from the combined action of synthesis and turnover. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation; Zinc finger, BED-type containing 6 (ZBED6) is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle. In this study, a total of seven single nucleotide polymorphisms (SNPs) were identified, four SNPs in intron 8 of IGF2 and one promoter SNP and two missense mutations in the coding region of ZBED6, two of which were in complete linkage disequilibrium (LD) in the bovine IGF2. The 58 haplotypes were inferred in 1522 individuals representing four purebred cattle breeds from China. The seven SNPs, 79 and 66 combined diplotypes were revealed for association with body mass in Nanyang and Jiaxian cattle populations at five different ages (P < 0.05 or 0.01). The mutant-type variants and haplotype 58 (likely in LD with the beneficial quantitative trait nucleotide allele) was superior for body mass; the heterozygote diplotype of the most common haplotypes 58 was associated with higher body mass compared to either heterozygote or homozygote. The statistical analyses indicated that the mutant-type variants and haplotypes are significantly associated with body mass in study cattle populations at different ages. These data demonstrate that variants and haplotypes are associated with growth traits, and these results may provide important biological insights into the phenotypic differentiation that is associated with adaptation and specialization of cattle breeds.

A novel c.-274C>G polymorphism in bovine SIRT1 gene contributes to diminished promoter activity and is associated with increased body size.

SIRT1, a mammalian homologue for yeast silent information regulator 2 (SIR2), is a NAD(+) -dependent deacetylase that belongs to the class III histone deacetylases. It plays an important role in diverse cellular processes, including stress resistance, mitochondrial function, suppression of inflammation and DNA repair. In this study, we screened and identified a novel polymorphism (c.-274C>G) in the SIRT1 promoter region. In silico prediction reveals that this SNP is in the core of cell cycle-dependent element (CDE)-binding motif. Interestingly, the G allele abolished a CDE-binding site, which suggested its functional significance. In the luciferase assay system, we found that the G allele-containing construct displayed a strikingly lower promoter activity compared with the C allele, which may downregulate SIRT1 expression levels. Additionally, we observed a significant association between the c.-274C>G polymorphism and growth traits in Nanyang cattle, suggesting that anomalous transcription factor-based repression of SIRT1 may increase bovine fat mass and body size.

Tetra-primer ARMS-PCR identified four pivotal genetic variations in bovine PNPLA3 gene and its expression patterns.

Patatin-like phospholipase domain-containing protein 3 (PNPLA3), a member of the patatin like phospholipase domain-containing (PNPLA) family, plays an important role in energy balance, fat metabolism regulation, glucose metabolism and fatty liver disease. Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a new method offering fast detection and extreme simplicity at a negligible cost for SNP genotyping. In this paper, we investigated the genetic variations at different ages of 660 Chinese indigenous cattle belonging to three breeds (QC, NY, JX) and applied T-ARMS-PCR and PCR-RFLP methods to genotype four SNPs, SNP1: g.A2980G, SNP2: g.A2996T, SNP3: g.A36718G, SNP4: g.G36850A. The statistical analyses indicated that these 4 SNPs affected growth traits markedly (P<0.05) in QC population, whereas combined haplotypes were not (P>0.05). The qPCR (quantitative PCR) indicated that bovine PNPLA3 gene was exclusively expressed in fat tissues. Besides, the analysis between SNP and mRNA expression revealed that, in SNP1, the expression of AG was much higher than AA and GG (P<0.05), which was in accordance with the results of growth traits association analysis, while the results of SNP4 was not. These results supported high potential that SNPs of bovine PNPLA3 gene might be utilized as genetic markers in marker-assisted selection (MAS) for Chinese cattle breeding programs.

Haplotypes in the promoter region of the CIDEC gene associated with growth traits in Nanyang cattle.

Cell death-inducing DFFA-like effector c (CIDEC, also known as Fsp27) has emerged as an important regulator of metabolism associated with lipodystrophy, diabetes, and hepatic steatosis. It is required for unilocular lipid droplet formation and optimal energy storage. The mechanism between this gene and livestock growth traits, however, has yet to be reported. In this study, we found ten novel single nucleotide polymorphisms (SNPs) in the 5' transcriptional region of CIDEC in Nanyang (NY) cattle, which are located in the recognition sequences (potential cis-acting elements) of 22 transcription factors, and the nine haplotypes represent nine different combinations of polymorphic potential cis-acting elements. The results indicated that individuals with the H8-H8 diplotype had heavier body weights and faster growth rates (P < 0.01) at 18th months than those with H1-H8. We evaluated the transcriptional activities of different haplotypes in vitro, the results were consistent with the association analysis. The H8 haplotype had 1.88-fold (P < 0.001) higher transcriptional activity than the H1 haplotype. We speculate that the haplotypes of the potential cis-acting elements may affect the transcriptional activity of CIDEC, thus affecting the growth traits of cattle. This information may be used in molecular marker-assisted selection of cattle breeding in the future.

Tetra-primer ARMS-PCR identifies the novel genetic variations of bovine HNF-4α gene associating with growth traits.

Hepatocyte nuclear factor-4α (HNF-4α), a member of the hepatocyte nuclear factor family, plays an important role in regulating the expression of genes involved in the development, differentiation and normal function of liver and pancreatic ß cells, as well as the maintenance of glucose homeostasis. Tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a new method offering fast detection and extreme simplicity at a negligible cost for SNP genotyping. In this paper, we characterize the polymorphisms of the bovine HNF-4α gene in three Chinese indigenous cattle breeds (n=660). Six novel SNPs were identified including 1 mutation in the coding region and others in introns. The statistical analyses indicated that 4 SNPs (g.T53729C, g.A53861G, g.A65188C and g.T65444C) affected growth traits markedly (P<0.05) in Qinchuan cattle (2 years after birth). Besides, haplotypes involving these 4 SNP sites in the bovine HNF-4α gene were identified and their effects on growth traits were also analyzed. The results showed that haplotypes 2, 7, 9 and 11 were predominant and accounted for 73.2%, 59.6%, and 67.1% in Qinchuan, Nanyang and Jiaxian cattle breeds, respectively. Hap9 (TAAT) was extremely predominant in all test populations, which suggested that individuals with Hap9 were more adapted to the environment. Furthermore, 4 combined haplotypes were constructed to guarantee the reliability of analysis results in Qinchuan cattle. There were also significant differences in body length (P<0.05). These findings will benefit for the application of DNA marker related to the growth traits on marker-assisted selection (MAS), and improve the performance of beef cattle.

Intragenic DNA methylation status down-regulates bovine IGF2 gene expression in different developmental stages.

DNA methylation is a key epigenetic modification in mammals and has an essential and important role in muscle development. Insulin-like growth factor 2 (IGF2) is a fetal growth and differentiation factor that plays an important role in muscle growth and in myoblast proliferation and differentiation. The aim of this study was to evaluate the expression of IGF2 and the methylation pattern on the differentially methylated region (DMR) of the last exon of IGF2 in six tissues with two different developmental stages. The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The quantitative real-time PCR (qPCR) analysis indicated that IGF2 has a broad tissue distribution and the adult bovine group showed significant lower mRNA expression levels than that in the fetal bovine group (P<0.05 or P<0.01). Moreover, the DNA methylation level analysis showed that the adult bovine group exhibited a significantly higher DNA methylation levels than that in the fetal bovine group (P<0.05 or P<0.01). These results indicate that IGF2 expression levels were negatively associated with the methylation status of the IGF2 DMR during the two developmental stages. Our results suggest that the methylation pattern in this DMR may be a useful parameter to investigate as a marker-assisted selection for muscle developmental in beef cattle breeding program and as a model for studies in other species.

Transcription factor ZBED6 mediates IGF2 gene expression by regulating promoter activity and DNA methylation in myoblasts.

Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.

Relationship of polymorphisms within ZBED6 gene and growth traits in beef cattle.

ZBED6 is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle. The aim of this study was to examine the association of the ZBED6 polymorphism with growth traits in beef cattle breed. Three single nucleotide polymorphisms (SNPs) were identified in the bovine ZBED6 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (Forced PCR-RFLP) methods. Overall, we reported one mutation (SNP1) in the promoter region and two missense mutations (SNP2 and 3) in the coding region (single exon) within the bovine ZBED6 gene, and the haplotype variability and extent of linkage disequilibrium (LD) in 1522 individuals representing four main cattle breeds from China (Nanyang, NY; Qinchuan, QC; Jiaxian, JX; and Chinese Holstein, CH). We also investigated haplotype frequencies and linkage disequilibrium coefficients for three SNPs in all study populations. LD and haplotype structure of ZBED6 were different between breeds. The result of haplotype analysis of three SNPs showed that eight different haplotypes were identified in all breeds. The wild-type haplotype (Hap 1: GCA) and mutant-type haplotype (Hap 8: AGG) shared by all four populations accounted for 15.1, 29.8, 21.7, 57.5% and 9.5, 8.6, 16.7, 0% of all haplotypes were observed in NY, QC, JX and CH, respectively. The statistical analyses indicated that three SNPs were significantly associated with growth traits in NY cattle population (P<0.05 or P<0.01) at five different ages. The results of this study suggest that the ZBED6 gene possibly is a strong candidate gene that affects growth traits in beef cattle breeding program.

A 5'-regulatory region and two coding region polymorphisms modulate promoter activity and gene expression of the growth suppressor gene ZBED6 in cattle.

Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. Polymorphisms in its promoter and coding regions are likely to impact ZBED6 transcription and growth traits. In this study, rapid amplification of 5' cDNA ends (5'-RACE) analysis revealed two transcription start sites (TSS) for the bovine ZBED6 starting within exon 1 of the ZC3H11A gene (TSS-1) and upstream of the translation start codon of the ZBED6 gene (TSS-2). There was one SNP in the promoter and two missense mutations in the coding region of the bovine ZBED6 by sequencing of the pooled DNA samples (Pool-Seq, n = 100). The promoter and coding region are the key regions for gene function; polymorphisms in these regions can alter gene expression. Quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution in cattle and is highly expressed in skeletal muscle. Eleven promoter-detection vectors were constructed, which enabled the cloning of putative promoter sequences and analysis of ZBED6 transcriptional activity by luciferase reporter gene assays. The core region of the basal promoter of bovine ZBED6 is located within region -866 to -556. The activity of WT-826G-pGL3 in driving reporter gene transcription is significantly higher than that of the M-826A-pGL3 construct (P < 0.01). Analysis of gene expression patterns in homozygous full-sibling Chinese Qinchuan cattle showed that the mutant-type Hap-AGG exhibited a lower mRNA level than the wild-type Hap-GCA (P < 0.05) in longissimus dorsi muscle (LDM). Moreover, ZBED6 mRNA expression was low in C2C12 cells overexpressing the mutant-type ZBED6 (pcDNA3.1(+)-Hap-GG) (P < 0.01). Our results suggest that the polymorphisms in the promoter and coding regions may modulate the promoter activity and gene expression of bovine ZBED6 in the skeletal muscles of these cattle breeds.