Evaluation of low-pass genome sequencing in polygenic risk score calculation for Parkinson's disease.

BACKGROUND: Low-pass sequencing (LPS) has been extensively investigated for applicability to various genetic studies due to its advantages over genotype array data including cost-effectiveness. Predicting the risk of complex diseases such as Parkinson's disease (PD) using polygenic risk score (PRS) based on the genetic variations has shown decent prediction accuracy. Although ultra-LPS has been shown to be effective in PRS calculation, array data has been favored to the majority of PRS analysis, especially for PD. RESULTS: Using eight high-coverage WGS, we assessed imputation approaches for downsampled LPS data ranging from 0.5 × to 7.0 × . We demonstrated that uncertain genotype calls of LPS diminished imputation accuracy, and an imputation approach using genotype likelihoods was plausible for LPS. Additionally, comparing imputation accuracies between LPS and simulated array illustrated that LPS had higher accuracies particularly at rare frequencies. To evaluate ultra-low coverage data in PRS calculation for PD, we prepared low-coverage WGS and genotype array of 87 PD cases and 101 controls. Genotype imputation of array and downsampled LPS were conducted using a population-specific reference panel, and we calculated risk scores based on the PD-associated SNPs from an East Asian meta-GWAS. The PRS models discriminated cases and controls as previously reported when both LPS and genotype array were used. Also strong correlations in PRS models for PD between LPS and genotype array were discovered. CONCLUSIONS: Overall, this study highlights the potentials of LPS under 1.0 × followed by genotype imputation in PRS calculation and suggests LPS as attractive alternatives to genotype array in the area of precision medicine for PD.

Whole genome sequencing reveals a frameshift mutation and a large deletion in YY1AP1 in a girl with a panvascular artery disease.

BACKGROUND: Rare diseases are pathologies that affect less than 1 in 2000 people. They are difficult to diagnose due to their low frequency and their often highly heterogeneous symptoms. Rare diseases have in general a high impact on the quality of life and life expectancy of patients, which are in general children or young people. The advent of high-throughput sequencing techniques has improved diagnosis in several different areas, from pediatrics, achieving a diagnostic rate of 41% with whole genome sequencing (WGS) and 36% with whole exome sequencing, to neurology, achieving a diagnostic rate between 47 and 48.5% with WGS. This evidence has encouraged our group to pursue a molecular diagnosis using WGS for this and several other patients with rare diseases. RESULTS: We used whole genome sequencing to achieve a molecular diagnosis of a 7-year-old girl with a severe panvascular artery disease that remained for several years undiagnosed. We found a frameshift variant in one copy and a large deletion involving two exons in the other copy of a gene called YY1AP1. This gene is related to Grange syndrome, a recessive rare disease, whose symptoms include stenosis or occlusion of multiple arteries, congenital heart defects, brachydactyly, syndactyly, bone fragility, and learning disabilities. Bioinformatic analyses propose these mutations as the most likely cause of the disease, according to its frequency, in silico predictors, conservation analyses, and effect on the protein product. Additionally, we confirmed one mutation in each parent, supporting a compound heterozygous status in the child. CONCLUSIONS: In general, we think that this finding can contribute to the use of whole genome sequencing as a diagnosis tool of rare diseases, and in particular, it can enhance the set of known mutations associated with different diseases.

De novo assembly and phasing of a Korean human genome.

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

Divergent reprogramming routes lead to alternative stem-cell states.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Genome-wide characterization of the routes to pluripotency.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

Comprehensive Analysis of the Transcriptional and Mutational Landscape of Follicular and Papillary Thyroid Cancers.

Follicular thyroid carcinoma (FTC) and benign follicular adenoma (FA) are indistinguishable by preoperative diagnosis due to their similar histological features. Here we report the first RNA sequencing study of these tumors, with data for 30 minimally invasive FTCs (miFTCs) and 25 FAs. We also compared 77 classical papillary thyroid carcinomas (cPTCs) and 48 follicular variant of PTCs (FVPTCs) to observe the differences in their molecular properties. Mutations in H/K/NRAS, DICER1, EIF1AX, IDH1, PTEN, SOS1, and SPOP were identified in miFTC or FA. We identified a low frequency of fusion genes in miFTC (only one, PAX8-PPARG), but a high frequency of that in PTC (17.60%). The frequencies of BRAFV600E and H/K/NRAS mutations were substantially different in miFTC and cPTC, and those of FVPTC were intermediate between miFTC and cPTC. Gene expression analysis demonstrated three molecular subtypes regardless of their histological features, including Non-BRAF-Non-RAS (NBNR), as well as BRAF-like and RAS-like. The novel molecular subtype, NBNR, was associated with DICER1, EIF1AX, IDH1, PTEN, SOS1, SPOP, and PAX8-PPARG. The transcriptome of miFTC or encapsulated FVPTC was indistinguishable from that of FA, providing a molecular explanation for the similarly indolent behavior of these tumors. We identified upregulation of genes that are related to mitochondrial biogenesis including ESRRA and PPARGC1A in oncocytic follicular thyroid neoplasm. Arm-level copy number variations were correlated to histological and molecular characteristics. These results expanded the current molecular understanding of thyroid cancer and may lead to new diagnostic and therapeutic approaches to the disease.

Integrative analysis of oncogenic fusion genes and their functional impact in colorectal cancer.

BACKGROUND: Fusion genes are good candidates of molecular targets for cancer therapy. However, there is insufficient research on the clinical implications and functional characteristics of fusion genes in colorectal cancer (CRC). METHODS: In this study, we analysed RNA sequencing data of CRC patients (147 tumour and 47 matched normal tissues) to identify oncogenic fusion genes and evaluated their role in CRC. RESULTS: We validated 24 fusion genes, including novel fusions, by three algorithms and Sanger sequencing. Fusions from most patients were mutually exclusive CRC oncogenes and included tumour suppressor gene mutations. Eleven fusion genes from 13 patients (8.8%) were determined as oncogenic fusion genes by analysing their gene expression and function. To investigate their oncogenic impact, we performed proliferation and migration assays of CRC cell lines expressing fusion genes of GTF3A-CDK8, NAGLU- IKZF3, RNF121- FOLR2, and STRN-ALK. Overexpression of these fusion genes increased cell proliferation except GTF3A-CDK8. In addition, overexpression of NAGLU-IKZF3 enhanced migration of CRC cells. We demonstrated that NAGLU-IKZF3, RNF121-FOLR2, and STRN-ALK had tumourigenic effects in CRC. CONCLUSION: In summary, we identified and characterised oncogenic fusion genes and their function in CRC, and implicated NAGLU-IKZF3 and RNF121-FOLR2 as novel molecular targets for personalised medicine development.

Development of a common platform for the noninvasive prenatal diagnosis of X-linked diseases.

OBJECTIVE: The aim of this study was to develop a common targeted massively parallel sequencing platform for the noninvasive prenatal diagnosis (NIPD) of multiple X-linked diseases. METHOD: The custom capture probe was designed to target 33 genes and recombination hotspots. We tested the carrier mother and male proband pair of 6 families. Plasma DNA of the pregnant carrier mother was collected at different gestational weeks and sequenced. The fetal genotype of each family was determined by estimating the imbalance between the 2 maternal haplotypes constructed using a common custom-designed platform. RESULTS: The targeted sequencing of the maternal, proband, and fetal genomic DNAs and maternal plasma DNAs resulted in uniform coverage across the target region. Three to 5 recombination points were observed in each sample. However, these recombination points did not affect the haplotype dosage analysis for fetal genotype prediction. Consequently, all fetal genotypes in the 6 families obtained from haplotype dosage analysis of maternal plasma sequencing data were predicted correctly. CONCLUSIONS: Since a single platform that covers multiple diseases may prevent the need for disease-specific probes for the NIPD of individual disorders, this approach may provide a practical advantage for clinically implementing the NIPD of X-linked diseases.

Clinical application of genomic profiling to find druggable targets for adolescent and young adult (AYA) cancer patients with metastasis.

BACKGROUND: Although adolescent and young adult (AYA) cancers are characterized by biological features and clinical outcomes distinct from those of other age groups, the molecular profile of AYA cancers has not been well defined. In this study, we analyzed cancer genomes from rare types of metastatic AYA cancers to identify driving and/or druggable genetic alterations. METHODS: Prospectively collected AYA tumor samples from seven different patients were analyzed using three different genomics platforms (whole-exome sequencing, whole-transcriptome sequencing or OncoScan™). Using well-known bioinformatics tools (bwa, Picard, GATK, MuTect, and Somatic Indel Detector) and our annotation approach with open access databases (DAVID and DGIdb), we processed sequencing data and identified driving genetic alterations and their druggability. RESULTS: The mutation frequencies of AYA cancers were lower than those of other adult cancers (median = 0.56), except for a germ cell tumor with hypermutation. We identified patient-specific genetic alterations in candidate driving genes: RASA2 and NF1 (prostate cancer), TP53 and CDKN2C (olfactory neuroblastoma), FAT1, NOTCH1, and SMAD4 (head and neck cancer), KRAS (urachal carcinoma), EML4-ALK (lung cancer), and MDM2 and PTEN (liposarcoma). We then suggested potential drugs for each patient according to his or her altered genes and related pathways. By comparing candidate driving genes between AYA cancers and those from all age groups for the same type of cancer, we identified different driving genes in prostate cancer and a germ cell tumor in AYAs compared with all age groups, whereas three common alterations (TP53, FAT1, and NOTCH1) in head and neck cancer were identified in both groups. CONCLUSION: We identified the patient-specific genetic alterations and druggability of seven rare types of AYA cancers using three genomics platforms. Additionally, genetic alterations in cancers from AYA and those from all age groups varied by cancer type.

A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole-genome and transcriptome sequencing.

The identification of the molecular events that drive cancer transformation is essential to the development of targeted agents that improve the clinical outcome of lung cancer. Many studies have reported genomic driver mutations in non-small-cell lung cancers (NSCLCs) over the past decade; however, the molecular pathogenesis of >40% of NSCLCs is still unknown. To identify new molecular targets in NSCLCs, we performed the combined analysis of massively parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-yr-old lung adenocarcinoma patient, who is a never-smoker and has no familial cancer history. The cancer showed no known driver mutation in EGFR or KRAS and no EML4-ALK fusion. Here we report a novel fusion gene between KIF5B and the RET proto-oncogene caused by a pericentric inversion of 10p11.22-q11.21. This fusion gene overexpresses chimeric RET receptor tyrosine kinase, which could spontaneously induce cellular transformation. We identified the KIF5B-RET fusion in two more cases out of 20 primary lung adenocarcinomas in the replication study. Our data demonstrate that a subset of NSCLCs could be caused by a fusion of KIF5B and RET, and suggest the chimeric oncogene as a promising molecular target for the personalized diagnosis and treatment of lung cancer.

The transcriptional landscape and mutational profile of lung adenocarcinoma.

All cancers harbor molecular alterations in their genomes. The transcriptional consequences of these somatic mutations have not yet been comprehensively explored in lung cancer. Here we present the first large scale RNA sequencing study of lung adenocarcinoma, demonstrating its power to identify somatic point mutations as well as transcriptional variants such as gene fusions, alternative splicing events, and expression outliers. Our results reveal the genetic basis of 200 lung adenocarcinomas in Koreans including deep characterization of 87 surgical specimens by transcriptome sequencing. We identified driver somatic mutations in cancer genes including EGFR, KRAS, NRAS, BRAF, PIK3CA, MET, and CTNNB1. Candidates for novel driver mutations were also identified in genes newly implicated in lung adenocarcinoma such as LMTK2, ARID1A, NOTCH2, and SMARCA4. We found 45 fusion genes, eight of which were chimeric tyrosine kinases involving ALK, RET, ROS1, FGFR2, AXL, and PDGFRA. Among 17 recurrent alternative splicing events, we identified exon 14 skipping in the proto-oncogene MET as highly likely to be a cancer driver. The number of somatic mutations and expression outliers varied markedly between individual cancers and was strongly correlated with smoking history of patients. We identified genomic blocks within which gene expression levels were consistently increased or decreased that could be explained by copy number alterations in samples. We also found an association between lymph node metastasis and somatic mutations in TP53. These findings broaden our understanding of lung adenocarcinoma and may also lead to new diagnostic and therapeutic approaches.

Noninvasive prenatal diagnosis of duchenne muscular dystrophy: comprehensive genetic diagnosis in carrier, proband, and fetus.

BACKGROUND: Noninvasive prenatal diagnosis of monogenic disorders using maternal plasma and targeted massively parallel sequencing is being investigated actively. We previously demonstrated that comprehensive genetic diagnosis of a Duchenne muscular dystrophy (DMD) patient is feasible using a single targeted sequencing platform. Here we demonstrate the applicability of this approach to carrier detection and noninvasive prenatal diagnosis. METHODS: Custom solution-based target enrichment was designed to cover the entire dystrophin (DMD) gene region. Targeted massively parallel sequencing was performed using genomic DNA from 4 mother and proband pairs to test whether carrier status could be detected reliably. Maternal plasma DNA at varying gestational weeks was collected from the same families and sequenced using the same targeted platform to predict the inheritance of the DMD mutation by their fetus. Overrepresentation of an inherited allele was determined by comparing the allele fraction of 2 phased haplotypes after examining and correcting for the recombination event. RESULTS: The carrier status of deletion/duplication and point mutations was detected reliably through using a single targeted massively parallel sequencing platform. Whether the fetus had inherited the DMD mutation was predicted correctly in all 4 families as early as 6 weeks and 5 days of gestation. In one of these, detection of the recombination event and reconstruction of the phased haplotype produced a correct diagnosis. CONCLUSIONS: Noninvasive prenatal diagnosis of DMD is feasible using a single targeted massively parallel sequencing platform with tiling design.

Transcriptional activation of p21(WAF¹/CIP¹) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.

Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.

Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Epigenetic regulation of cholinergic receptor M1 (CHRM1) by histone H3K9me3 impairs Ca(2+) signaling in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expanded trinucleotide CAG repeat in the gene coding for huntingtin. Deregulation of chromatin remodeling is linked to the pathogenesis of HD but the mechanism remains elusive. To identify what genes are deregulated by trimethylated histone H3K9 (H3K9me3)-dependent heterochromatin, we performed H3K9me3-ChIP genome-wide sequencing combined with RNA sequencing followed by platform integration analysis in stable striatal HD cell lines (STHdhQ7/7 and STHdhQ111/111) cells. We found that genes involving neuronal synaptic transmission including cholinergic receptor M1 (CHRM1), cell motility, and neuronal differentiation pathways are downregulated while their promoter regions are highly occupied with H3K9me3 in HD. Moreover, we found that repression of CHRM1 gene expression by H3K9me3 impairs Ca(2+)-dependent neuronal signal transduction in stable cell lines expressing mutant HD protein. Thus, our data indicate that the epigenetic modifications, such as aberrant H3K9me3-dependent heterochromatin plasticity, directly contribute to the pathogenesis of HD.

Comprehensive genomic analyses associate UGT8 variants with musical ability in a Mongolian population.

BACKGROUND: Musical abilities such as recognising music and singing performance serve as means for communication and are instruments in sexual selection. Specific regions of the brain have been found to be activated by musical stimuli, but these have rarely been extended to the discovery of genes and molecules associated with musical ability. METHODS: A total of 1008 individuals from 73 families were enrolled and a pitch-production accuracy test was applied to determine musical ability. To identify genetic loci and variants that contribute to musical ability, we conducted family-based linkage and association analyses, and incorporated the results with data from exome sequencing and array comparative genomic hybridisation analyses. RESULTS: We found significant evidence of linkage at 4q23 with the nearest marker D4S2986 (LOD=3.1), whose supporting interval overlaps a previous study in Finnish families, and identified an intergenic single nucleotide polymorphism (SNP) (rs1251078, p = 8.4 × 10(-17)) near UGT8, a gene highly expressed in the central nervous system and known to act in brain organisation. In addition, a non-synonymous SNP in UGT8 was revealed to be highly associated with musical ability (rs4148254, p = 8.0 × 10(-17)), and a 6.2 kb copy number loss near UGT8 showed a plausible association with musical ability (p = 2.9 × 10(-6)). CONCLUSIONS: This study provides new insight into the genetics of musical ability, exemplifying a methodology to assign functional significance to synonymous and non-coding alleles by integrating multiple experimental methods.

TIARA: a database for accurate analysis of multiple personal genomes based on cross-technology.

High-throughput genomic technologies have been used to explore personal human genomes for the past few years. Although the integration of technologies is important for high-accuracy detection of personal genomic variations, no databases have been prepared to systematically archive genomes and to facilitate the comparison of personal genomic data sets prepared using a variety of experimental platforms. We describe here the Total Integrated Archive of Short-Read and Array (TIARA; http://tiara.gmi.ac.kr) database, which contains personal genomic information obtained from next generation sequencing (NGS) techniques and ultra-high-resolution comparative genomic hybridization (CGH) arrays. This database improves the accuracy of detecting personal genomic variations, such as SNPs, short indels and structural variants (SVs). At present, 36 individual genomes have been archived and may be displayed in the database. TIARA supports a user-friendly genome browser, which retrieves read-depths (RDs) and log2 ratios from NGS and CGH arrays, respectively. In addition, this database provides information on all genomic variants and the raw data, including short reads and feature-level CGH data, through anonymous file transfer protocol. More personal genomes will be archived as more individuals are analyzed by NGS or CGH array. TIARA provides a new approach to the accurate interpretation of personal genomes for genome research.

Comprehensive characterization of maternal, fetal, and neonatal microbiomes supports prenatal colonization of the gastrointestinal tract.

In this study, we aimed to comprehensively characterize the microbiomes of various samples from pregnant women and their neonates, and to explore the similarities and associations between mother-neonate pairs, sample collection sites, and obstetrical factors. We collected samples from vaginal discharge and amniotic fluid in pregnant women and umbilical cord blood, gastric liquid, and meconium from neonates. We identified 19,597,239 bacterial sequences from 641 samples of 141 pregnant women and 178 neonates. By applying rigorous filtering criteria to remove contaminants, we found evidence of microbial colonization in traditionally considered sterile intrauterine environments and the fetal gastrointestinal track. The microbiome distribution was strongly grouped by sample collection site, rather than the mother-neonate pairs. The distinct bacterial composition in meconium, the first stool passed by newborns, supports that microbial colonization occurs during normal pregnancy. The microbiome in neonatal gastric liquid was similar, but not identical, to that in maternal amnionic fluid, as expected since fetuses swallow amnionic fluid in utero and their urine returns to the fluid under normal physiological conditions. Establishing a microbiome library from various samples formed only during pregnancy is crucial for understanding human development and identifying microbiome modifications in obstetrical complications.