Genome Sequencing Explores Complexity of Chromosomal Abnormalities in Recurrent Miscarriage.

Recurrent miscarriage (RM) affects millions of couples globally, and half of them have no demonstrated etiology. Genome sequencing (GS) is an enhanced and novel cytogenetic tool to define the contribution of chromosomal abnormalities in human diseases. In this study we evaluated its utility in RM-affected couples. We performed low-pass GS retrospectively for 1,090 RM-affected couples, all of whom had routine chromosome analysis. A customized sequencing and interpretation pipeline was developed to identify chromosomal rearrangements and deletions/duplications with confirmation by fluorescence in situ hybridization, chromosomal microarray analysis, and PCR studies. Low-pass GS yielded results in 1,077 of 1,090 couples (98.8%) and detected 127 chromosomal abnormalities in 11.7% (126/1,077) of couples; both members of one couple were identified with inversions. Of the 126 couples, 39.7% (50/126) had received former diagnostic results by karyotyping characteristic of normal human male or female karyotypes. Low-pass GS revealed additional chromosomal abnormalities in 50 (4.0%) couples, including eight with balanced translocations and 42 inversions. Follow-up studies of these couples showed a higher miscarriage/fetal-anomaly rate of 5/10 (50%) compared to 21/93 (22.6%) in couples with normal GS, resulting in a relative risk of 2.2 (95% confidence interval, 1.1 to 4.6). In these couples, this protocol significantly increased the diagnostic yield of chromosomal abnormalities per couple (11.7%) in comparison to chromosome analysis (8.0%, chi-square test p = 0.000751). In summary, low-pass GS identified underlying chromosomal aberrations in 1 in 9 RM-affected couples, enabling identification of a subgroup of couples with increased risk of subsequent miscarriage who would benefit from a personalized intervention.

Characterization of gut microbiota associated with clinical parameters in intrahepatic cholestasis of pregnancy.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder that specifically occurs in pregnancy. Elevated levels of liver transaminases aspartate aminotransferase, alanine aminotransferase and serum bilirubin levels are common biochemical characteristics in ICP. The disorder is associated with an increased risk of premature delivery and stillbirth. The characterization of the potential microbiota in ICP could go a long way in the prevention and treatment of this pregnancy disease. METHODS: A total of 58 patients were recruited for our study: 27 ICP patients and 31 healthy pregnant subjects with no ICP. The V3 and V4 regions of the 16S rDNA collected from fecal samples of both diseased and control groups were amplified. 16S rRNA gene amplicon sequencing was then performed on gut microbiota. Sequencing data were analyzed and the correlation between components of microbiota and patient ICP status was found. Related metabolic pathways, relative abundance and significantly different operational taxonomic units (OTUs) between ICP and controls were also identified. RESULTS: Elevated levels of total bile acid, ALT, AST, Dbil and Tbil were recorded or observed in ICP subjects as compared to the control. Gut microbiota in pregnant women was dominated by four major phyla and 27 core genera. PCoA analysis results indicated that there was no significant clustering in Bray-Curtis distance matrices. Our results showed that there was a correlation between specific OTUs and measured clinical parameters of pregnant women. Comparison at the different taxonomy levels revealed high levels of abundance of Blautia and Citrobacter in ICP patients. At the family level, Enterobacteriaceae and Leuconostocaceae were higher in ICP patients. 638 KEGG Orthologs and 138 pathways significantly differed in the two groups. PLS-DA model with VIP plots indicated a total of eight genera and seven species were key taxa in ICP and control groups. CONCLUSIONS: Our research indicated that although there was no significant clustering by PCoA analysis, patients with ICP have increased rare bacteria at different phylogenetic levels. Our results also illustrated that all 638 KEGG Orthologs and 136 in 138 KEGG pathways were less abundant in ICP patients compared to the controls.

Balanced Chromosomal Rearrangement Detection by Low-Pass Whole-Genome Sequencing.

Balanced chromosomal rearrangements (or balanced chromosome abnormalities, BCAs) are common chromosomal structural variants. Emerging studies have demonstrated the feasibility of using whole-genome sequencing (WGS) for detection of BCA-associated breakpoints, but the requirement for a priori knowledge of the rearranged regions from G-banded chromosome analysis limits its application. The protocols described here are based on low-pass WGS for detecting BCA events independent from chromosome analysis, and has been validated using genomic data from the 1000 Genomes Project. This approach adopts non-size-selected mate-pair library (3∼8 kb) with 2∼3 µg DNA as input, and requires only 30 million read-pairs (50 bp, equivalent to 1-fold base-coverage) for each sample. The complete procedure takes 13 days and the total cost is estimated to be less than $600 (USD) per sample. © 2018 by John Wiley & Sons, Inc.

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

Background: Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings: We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion: Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.