SNP rs12794714 of CYP2R1 is associated with serum vitamin D levels and recurrent spontaneous abortion (RSA): a case-control study.

PURPOSE: Vitamin D (VD) deficiency seems to be associated with the risk of recurrent spontaneous abortion (RSA). Vitamin D receptor (VDR) and cytochrome P450 family 2 subfamily R member 1 (CYP2R1) are two genes which are vital for VD metabolism and actions. However, whether single-nucleotide polymorphisms (SNPs) in these genes are correlated with the risk of RSA are poorly understood. Therefore, we aimed to characterize the relationships among VDR SNPs, CYP2R1 SNPs and RSA. METHODS: This case-control study enrolled 75 RSA patients and 83 controls. Serum VD and some cytokines were detected with LC-MS/MS and flow cytometry, respectively. Genotyping for three SNPs of CYP2R1 (rs10741657, rs10766197 and rs12794714) and five SNPs of VDR (rs7975232, rs1544410, rs2189480, rs2228570 and rs2239179) was done with polymerase chain reaction (PCR) and high-throughput sequencing. All the data were analyzed with appropriate methods and in different models. RESULTS: The results revealed a significant correlation between the AG genotype of CYP2R1 rs12794714 and VD levels (OR 0.686; 95% CI 0.49-0.96; p = 0.028). Besides, the AG and GG genotypes of CYP2R1 rs12794714 were markedly related to the risk of RSA (OR 52.394, 59.497; 95% CI 2.683-1023.265, 3.110-1138.367; p = 0.009, 0.007, respectively). CONCLUSION: Our results indicate that CYP2R1 rs12794714 might be a risk factor for RSA. Hence, early screening of pregnant women for CYP2R1 rs12794714 is necessary to warrant proactive counseling and treatment against RSA.

In utero exposure to persistent and nonpersistent endocrine-disrupting chemicals and anogenital distance. A systematic review of epidemiological studies†.

Anti-androgenic endocrine-disrupting chemicals (EDCs) can cross the placenta to modify early offspring sexual dimorphic markers. These changes are linked to anogenital distance (AGD), which is an androgen-sensitive anthropometric parameter used as a biomarker of perineal growth and caudal migration of the genital tubercle. This review aimed to summarize strength of evidence for associations of in utero exposure to EDCs with AGD and to identify gaps and limitations in the literature so as to inform future research. We performed an electronic search of English literature in September 2019 in medical literature analysis and retrieval system online (MEDLINE), Web of Science and Toxline. We included epidemiological studies that examined in utero exposure to persistent and nonpersistent EDCs and considered AGD in offspring as an outcome. Our review contained 16 investigations examining exposure to persistent EDCs (nine studies) and nonpersistent EDCs (seven studies). Some individual studies reported an inverse association between exposure to bisphenol A (BPA), dioxins, perfluoroalkyl substances, and organochlorides and AGD in both male and female offspring. Meta-analysis of three studies found a small reduction of AGD in female offspring exposed to BPA. The number of studies per chemical is small, and number of subjects examined is limited; so, replication of these results is needed. To achieve more specificity and better replication of results, future studies should establish the association of nonpersistent EDCs using multiple urine samples, evaluate the cumulative impact of exposure to a mixture of anti-androgenic chemicals, and offer adequate consideration of more maternal- and children-related confounding factors.

Sensitive detection of low-abundance in-frame deletions in EGFR exon 19 using novel wild-type blockers in real-time PCR.

Epidermal growth factor receptor (EGFR) mutations are associated with response of tyrosine kinase inhibitors (TKIs) for patients with advanced non-small cell lung cancer (NSCLC). However, the existing methods for detection of samples having rare mutations(i.e. ~0.01%) have limits in terms of specificity, time consumption or cost. In the current study, novel wild-type blocking (WTB) oligonucleotides modified with phosphorothioate or inverted dT at the 5'-termini were designed to precisely detect 11 common deletion mutations in exon 19 of EGFR gene (E19del) using a WTB-PCR assay. And internal competitive leptin amplifications were further applied to enhance the specificity of the WTB-PCR system. Our results showed that WTB-PCR could completely block amplification of wild-type EGFR when 200 ng of DNA was used as template. Furthermore, the current WTB-PCR assay facilitated the detection of E19del mutations with a selectivity of 0.01% and sensitivity as low as a single copy. And, the results showed that the current WTB-PCR system exceeded detection limits afforded by the ARMS-PCR assay. In conclusion, the current WTB-PCR strategy represents a simple and cost-effective method to precisely detect various low-abundance deletion mutations.

Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions.