FGR-associated placental insufficiency and capillary angiogenesis involves disruptions in human placental miRNAs and mRNAs.

Fetal growth restriction (FGR) is one of the most common pregnancy complications culminating in adverse fetal outcome, including preterm birth, neonatal mortality and stillbirth. Compromised placental development and function, especially disruption in angiogenesis and inadequate nutrient supply are contributing factors. Fetal sex also influences placental function. Knowledge of gene expression changes and epigenetic factors contributing to placental dysfunction in FGR pregnancies will help identify biomarkers and help target interventions. This study tested the hypothesis that FGR pregnancies are associated with disruptions in miRNA - an epigenetic factor and mRNAs involving key mediators of angiogenesis and microvessel development. Changes in expression of key genes/proteins involved in placental dysfunction by RT-PCR and immunohistochemistry and miRNA changes by RNA sequencing were undertaken with term placenta from 12 control and 20 FGR pregnancies. Findings showed changes in expression of genes involved in steroidogenesis, steroid action, IGF family members, inflammatory cytokines and angiogenic factors in FGR pregnancies. In addition, upregulation of MIR451A and downregulation of MIR543 in placentas from FGR group with female newborns and upregulation of MIR520G in placentas from FGR group with male newborns were also noted. MIR451A and MIR543 have been implicated in angiogenesis. Consistent with gene changes, CD34, the microvessel angiogenesis marker, also showed reduced staining only in female FGR group. These findings provide evidence that epigentically regulated gene expression changes in angiogenesis and capillary development influence placental impairment in FGR pregnancies. Our preliminary observations also support for these changes to be driven in a sex-specific manner.

The association between DNA methylation and human height and a prospective model of DNA methylation-based height prediction.

As a vital anthropometric characteristic, human height information not only helps to understand overall developmental status and genetic risk factors, but is also important for forensic DNA phenotyping. We utilized linear regression analysis to test the association between each CpG probe and the height phenotype. Next, we designed a methylation sequencing panel targeting 959 CpGs and subsequent height inference models were constructed for the Chinese population. A total of 11,730 height-associated sites were identified. By employing KPCA and deep neural networks, a prediction model was developed, of which the cross-validation RMSE, MAE and R2 were 5.62 cm, 4.45 cm and 0.64, respectively. Genetic factors could explain 39.4% of the methylation level variance of sites used in the height inference models. Collectively, we demonstrated an association between height and DNA methylation status through an EWAS analysis. Targeted methylation sequencing of only 959 CpGs combined with deep learning techniques could provide a model to estimate human height with higher accuracy than SNP-based prediction models.

Differences of circular RNA expression profiles between monozygotic twins' blood, with the forensic application in bloodstain and saliva.

Monozygotic twins (MZTs) possess identical genomic DNA sequences and are usually indistinguishable through routine forensic DNA typing methods, which can be relevant in criminal and paternity cases. Recently, novel epigenetic methods involving DNA methylation and microRNA analysis have been introduced to differentiate MZTs. In this study, we explore the potential of using epigenetic markers, specifically circular RNAs (circRNAs), a type of non-coding RNA (ncRNA), to identify MZTs, and investigate the unique expression patterns of circRNAs within pairs of MZTs, enabling effective differentiation. Epigenetics regulates gene expression at the post-transcriptional level and plays a crucial role in cell growth and aging. CircRNAs, a recently characterized subclass of ncRNA, have a distinct covalent loop structure without the typical 5' cap or 3' tail. They have been reported to modulate various cellular processes and play roles in embryogenesis and eukaryotic development. To achieve this, we conducted a comprehensive circRNA sequencing analysis (circRNA-seq) using total RNA extracted from the blood samples of five pairs of MZTs. We identified a total of 15,257 circRNAs in all MZTs using circRNA-seq. Among them, 3, 21, 338, and 2967 differentially expressed circRNAs (DEcircRNAs) were shared among five, four, three, and two pairs of MZTs, respectively. Subsequently, we validated twelve selected DEcircRNAs using real-time quantitative polymerase chain reaction (RT-qPCR) assays, which included hsa_circ_0004724, hsa_circ_0054196, hsa_circ_004964, hsa_circ_0000591, hsa_circ_0005077, hsa_circ_0054853, hsa_circ_0054716, hsa_circ_0002302, hsa_circ_0004482, hsa_circ_0001103, novel_circ_0030288 and novel_circ_0056831. Among them, hsa_circ_0005077 and hsa_circ_0004482 exhibited the best performance, showing differences in 7 out of 10 pairs of MZTs. These twelve differentially expressed circRNAs also demonstrated strong discriminative power when tested on saliva samples from 10 pairs of MZTs. Notably, hsa_circ_0004724 displayed differential expression in 8 out of 10 pairs of MZTs in their saliva. Additionally, we evaluated the detection sensitivity, longitudinal temporal stability, and suitability for aged bloodstains of these twelve DEcircRNAs in forensic scenarios. Our findings highlight the potential of circRNAs as molecular markers for distinguishing MZTs, emphasizing their suitability for forensic application.

Prediction of BMI traits in the Chinese population based on the gut metagenome.

BACKGROUND: Identifying individual characteristics based on trace evidence left at a crime scene is crucial in forensic identification. Microbial communities found in fecal traces have high individual specificity and could serve as potential markers for forensic characterization. Previous research has established that predicting body type based on the relative abundance of the gut microbiome is relatively accurate. However, the long-term stability and high individual specificity of the gut microbiome are closely linked to changes at the genome level of the microbiome. No studies have been conducted to deduce body shape from genetic traits. Therefore, in this study, the vital role of gut bacterial community characteristics and genetic traits in predicting body mass index (BMI) was investigated using gut metagenomic data from a healthy Chinese population. RESULTS: Regarding the gut microbial community, the underweight group displayed increased α-diversity in comparison to the other BMI groups. There were significant differences in the relative abundances of 19 species among these three BMI groups. The BMI prediction model, based on the 31 most significant species, showed a goodness of fit (R2) of 0.56 and a mean absolute error (MAE) of 2.09 kg/m2. The overweight group exhibited significantly higher α-diversity than the other BMI groups at the level of gut microbial genes. Furthermore, there were significant variations observed in the single-nucleotide polymorphism (SNP) density of 732 contigs between these three BMI groups. The BMI prediction model, reliant on the 62 most contributing contigs, exhibited a model R2 of 0.72 and an MAE of 1.56 kg/m2. The model predicting body type from 44 contigs correctly identified the body type of 93.55% of the study participants. CONCLUSION: Based on metagenomic data from a healthy Chinese population, we demonstrated the potential of genetic traits of gut bacteria to predict an individual's BMI. The findings of this study suggest the effectiveness of a novel method for determining the body type of suspects in forensic applications using the genetic traits of the gut microbiome and holds great promise for forensic individual identification.

Feature selection with a genetic algorithm can help improve the distinguishing power of microbiota information in monozygotic twins' identification.

Introduction: Personal identification of monozygotic twins (MZT) has been challenging in forensic genetics. Previous research has demonstrated that microbial markers have potential value due to their specificity and long-term stability. However, those studies would use the complete information of detected microbial communities, and low-value species would limit the performance of previous models. Methods: To address this issue, we collected 80 saliva samples from 10 pairs of MZTs at four different time points and used 16s rRNA V3-V4 region sequencing to obtain microbiota information. The data formed 280 inner-individual (Self) or MZT sample pairs, divided into four groups based on the individual relationship and time interval, and then randomly divided into training and testing sets with an 8:2 ratio. We built 12 identification models based on the time interval ( ≤ 1 year or ≥ 2 months), data basis (Amplicon sequence variants, ASVs or Operational taxonomic unit, OTUs), and distance parameter selection (Jaccard distance, Bray-Curist distance, or Hellinger distance) and then improved their identification power through genetic algorithm processes. The best combination of databases with distance parameters was selected as the final model for the two types of time intervals. Bayes theory was introduced to provide a numerical indicator of the evidence's effectiveness in practical cases. Results: From the 80 saliva samples, 369 OTUs and 1130 ASVs were detected. After the feature selection process, ASV-Jaccard distance models were selected as the final models for the two types of time intervals. For short interval samples, the final model can completely distinguish MZT pairs from Self ones in both training and test sets. Discussion: Our findings support the microbiota solution to the challenging MZT identification problem and highlight the importance of feature selection in improving model performance.

Identification Strategy of Biological Half Sibling Relationship.

OBJECTIVES: To compare the application value of the likelihood ratio (LR) method and identity by state (IBS) method in the identification involving half sibling relationships, and to provide a reference for the setting of relevant standards for identification of half sibling relationship. METHODS: (1) Based on the same genetic marker combinations, the reliability of computer simulation method was verified by comparing the distributions of cumulated identity by state score (CIBS) and combined full sibling index in actual cases with the distributions in simulated cases. (2) In different numbers of three genetic marker combinations, the simulation of full sibling, half sibling and unrelated individual pairs, each 1 million pairs, was obtained; the CIBS, as well as the corresponding types of cumulative LR parameters, were calculated. (3) The application value of LR method was compared with that of IBS method, by comparing the best system efficiency provided by LR method and IBS method when genetic markers in different amounts and of different types and accuracy were applied to distinguish the above three relational individual pairs. (4) According to the existing simulation data, the minimum number of genetic markers required to distinguish half siblings from the other two relationships using different types of genetic markers was estimated by curve fitting. RESULTS: (1) After the rank sum test, under the premise that the real relationship and the genetic marker combination tested were the same, there was no significant difference between the simulation method and the results obtained in the actual case. (2) In most cases, under the same conditions, the system effectiveness obtained by LR method was greater than that by IBS method. (3) According to the existing data, the number of genetic markers required for full-half siblings and half sibling identification could be obtained by curve fitting when the system effectiveness reached 0.95 or 0.99. CONCLUSIONS: When distinguishing half sibling from full sibling pairs or unrelated pairs, it is recommended to give preference to the LR method, and estimate the required number of markers according to the identification types and the population data, to ensure the identification effect.

Preliminary exploratory research on the application value of oral and intestinal meta-genomics in predicting subjects' occupations-A case study of the distinction between students and migrant workers.

Background: In the field of forensic science, accurately determining occupation of an individual can greatly assist in resolving cases such as criminal investigations or disaster victim identifications. However, estimating occupation can be challenging due to the intricate relationship between occupation and various factors, including gender, age, living environment, health status, medication use, and lifestyle habits such as alcohol consumption and smoking. All of these factors can impact the composition of oral or gut microbial community of an individual. Methods and results: In this study, we collected saliva and feces samples from individuals representing different occupational sectors, specifically students and manual laborers. We then performed metagenomic sequencing on the DNA extracted from these samples to obtain data that could be analyzed for taxonomic and functional annotations in five different databases. The correlation between occupation with microbial information was assisted from the perspective of α and ß diversity, showing that individuals belonging to the two occupations hold significantly different oral and gut microbial communities, and that this correlation is basically not affected by gender, drinking, and smoking in our datasets. Finally, random forest (RF) models were built with recursive feature elimination (RFE) processes. Models with 100% accuracy in both training and testing sets were constructed based on three species in saliva samples or on a single pathway annotated by the KEGG database in fecal samples, namely, "ko04145" or Phagosome. Conclusion: Although this study may have limited representativeness due to its small sample size, it provides preliminary evidence of the potential of using microbiome information for occupational inference.

The relationship between serum miR-21 levels and left atrium dilation in elderly patients with essential hypertension.

BACKGROUND: MicroRNA-21 (miR-21) is related to hypertension and cardiac remodelling. Left atrium (LA) dilation is highly sensitive to small haemodynamic changes in the left ventricle (LV) that are induced by hypertension. This study aimed to elucidate the relationship between miR-21 expression and LA dilation in elderly patients with essential hypertension (EH). METHODS: In this cross-sectional study, one hundred elderly patients with EH were recruited for the study. According to their left atrium diameters (LADs), the patients were divided into the LA dilation group [42 patients (42%)] and the no-LA dilation group [58 patients (58%)]. The serum levels of miR-21 and chemical biomarkers used in the clinic, such as creatinine, blood urea nitrogen, uric acid, fasting blood glucose, total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very-low-density lipoprotein cholesterol, Lp(a), apolipoprotein A1 (apoA1), and apolipoprotein B, were measured. All the patients underwent echocardiographic examination, and the LAD, interventricular septum (IVS), right atrium diameter (RAD), right ventricle diameter (RVD), left ventricular end-systolic diameter (LVESD), left ventricular end-systolic diameter (LVEDD) and left ventricular ejection fraction (LVEF) were measured. RESULTS: The levels of miR-21 [8.02 (5.21, 14.39) vs. 6.05 (3.81, 8.95), P = 0.011] and LVEF (67.02 ± 3.82 vs. 64.14 ± 4.43, P = 0.001) were higher in the LA dilation group. The levels of creatinine [70.40 (64.45, 80.15) vs. 63.9(60.1, 73.43)], P = 0.020] were higher in the no-LA dilation group. The levels of HDLC (r = - 0.209, P = 0.037), apoA1 (r = -0.269, P = 0.007) and RAD (r = 0.203, P = 0.043) were significantly correlated with miR-21 expression. The LAD was significantly correlated with the RAD (r = 0.287, P = 0.004), RVD (r = 0.450, P < 0.001), LVEDD (r = 0.248, P = 0.013) and LVEF (r = 0.232, P = 0.020). Multivariate logistic regression revealed that miR-21 significantly influenced LA dilation in elderly patients with EH (P < 0.05). CONCLUSIONS: Circulating serum levels of miR-21 are increased in elderly patients with EH with LA dilation. miR-21 levels are significantly correlated with LA dilation in elderly patients with EH, and miR-21 may be a factor related to the clinical pathophysiological occurrence of and treatment for the progression of hypertension-related early heart damage in EH patients.

Highly accurate mtGenome haplotypes from long-read SMRT sequencing can distinguish between monozygotic twins.

Discriminating between monozygotic twins (MZ) remains a challenge in the field of forensics globally. It is very difficult to find sequence variants within MZ twins, despite using ultra-deep next generation sequencing (NGS) for nuclear DNA. However, mitochondrial DNA might be a potential marker owing to its higher mutation rate and easier sequencing via NGS. Here, we aimed to introduce a long-read single molecule real-time sequencing (SMRT) strategy, with better continuity and fewer alignment errors, to obtain more accurate mitochondrial genome (mtGenome) sequence on the Sequel platform. Compared to Ion Torrent Personal Genome Machine (PGM), the long-read SMRT sequencing strategy generated highly accurate and mapped circular consensus sequence (CCS) reads and exhibited robust performance in terms of reliable repeatability, consistent coverage pattern, and balanced strands in mtGenome recovery. Moreover, the long-read SMRT strategy exhibited superior ability to not only identify accurate haplotypes but also discover a total of 785 low-level variants within 16 MZ twin pairs with threshold of 2% and 20 CCS reads with Q30 quality. Taken together, our findings suggested the long-read SMRT technology as an appreciable strategy for obtaining accurate mitotypes and providing a promising solution for distinguishing between MZ twin pairs in forensic genetics.

Development of an NGS panel containing 42 autosomal STR loci and the evaluation focusing on secondary kinship analysis.

High-throughput next-generation sequencing (NGS) is a feasible technique to detect considerably more markers and simultaneously obtain length and sequence information in a single reaction. In this study, we developed an NGS panel including 42 commonly used autosomal short tandem repeats (STRs) and amelogenin on the Illumina MiSeq FGx™. Sequencing accuracy was validated by the consistency of 2800M Control DNA detected using the ForenSeq™ DNA Signature Prep Kit and Sanger sequencing. Nomenclature incompatibility was found between NGS-STR and CE-STR typing at 9 loci (D3S3045, D6S477, D7S3048, D9S925, D14S608, D17S1290, D18S535, D21S1270, GATA198B05), despite the correct sequence. The difference was caused by the two different methods of identifying motif sequence and a one-to-one correspondence can be found. We evaluated the panel by investigating consistency, sequencing sensitivity and the effectiveness of the 2nd-degree relationship identification. Herein, we present sequencing results from 58 unrelated individuals of the Hebei Han population. The total discrimination power (TDP) and cumulative probability of exclusion for trio paternity testing (CPEtrio) of the 42 NGS-STR panels reached 1-2.84 × 10-57 and 1-9.87 × 10-21, respectively. By family simulation and likelihood ratio (LR) calculation, this panel was shown to have effectiveness for the 2nd-degree kinship identification similar to the ForenSeq™ DNA Signature Prep Kit and certain advantages compared with it due to the relatively small number of loci. As expected, it provides new data for the development of NGS-STR typing technology.

Circulating miRNA-21 as a diagnostic biomarker in elderly patients with type 2 cardiorenal syndrome.

Circulating miRNAs have attracted attention as serum biomarkers for several diseases. In this study, we aimed to evaluate the diagnostic value of circulating miRNA-21 (miR-21) as a novel biomarker for elderly patients with type 2 cardiorenal syndrome (CRS-2). A total of 157 elderly patients with chronic heart failure (CHF) were recruited for the study. According to an estimated glomerular filtration rate (eGFR) cut-off of 60 ml/min/1.73 m2, 84 patients (53.5%) and 73 patients (46.5%) were assigned to the CRS group and the CHF group, respectively. Expression levels of serum miR-21 and biomarkers for CRS, such as kidney injury factor-1 (KIM-1), neutrophil gelatinase-related apolipoprotein (NGAL), cystatin C (Cys C), amino-terminal pro-B-type natriuretic peptide (NT-proBNP), N-acetyl-κ-D-glucosaminidase (NAG), and heart-type fatty acid-binding protein (H-FABP), were detected. Serum miR-21, KIM-1, NGAL, Cys C, NT-proBNP and H-FABP levels were significantly higher in the CRS group than in the CHF group (P < 0.01), whereas NAG expression was not significantly different between the two groups (P > 0.05). Cys C, H-FABP and eGFR correlated significantly with miR-21 expression, but correlations with miR-21 were not significant for NT-proBNP, NGAL, NAG and KIM-1. Moreover, multivariate logistic regression found that serum miR-21, increased serum Cys C, serum KIM-1, hyperlipidaemia and ejection fraction (EF) were independent influencing factors for CRS (P < 0.05). The AUC of miR-21 based on the receiver operating characteristic (ROC) curve was 0.749, with a sensitivity of 55.95% and a specificity of 84.93%. Furthermore, combining miR-21 with Cys C enhanced the AUC to 0.902, with a sensitivity of 88.1% and a specificity of 83.6% (P < 0.001). Our findings suggest that circulating miR-21 has medium diagnostic value in CRS-2. The combined assessment of miR-21 and Cys C has good clinical value in elderly patients with CRS-2.

CpG methylation patterns of human mitochondrial DNA.

The epigenetic modification of mitochondrial DNA (mtDNA) is still in controversy. To clarify this point, we applied the gold standard method for DNA methylation, bisulfite pyrosequencing, to examine human mtDNA methylation status. Before bisulfite conversion, BamHI was used to digest DNA to open the loop of mtDNA. The results demonstrated that the linear mtDNA had significantly higher bisulfite conversion efficiency compared with circular mtDNA. Furthermore, the methylation values obtained from linear mtDNA were significantly lower than that of circular mtDNA, which was verified by SEQUENOM MassARRAY. The above impacts of circular structure were also observed in lung DNA samples but not in saliva DNA samples. Mitochondrial genome methylation of blood samples and saliva samples from 14 unrelated individuals was detected. The detected regions covered 83 CpG sites across mtDNA including D-loop, 12 S rRNA, 16 S rRNA, ND1, COXI, ND3, ND4, ND5, CYTB. We found that the average methylation levels of nine regions were all less than 2% for both sample types. In conclusion, our findings firstly show that the circular structure of mtDNA affects bisulfite conversion efficiency, which leads to overestimation of mtDNA methylation values. CpG methylation in human mtDNA is a very rare event at most DNA regions.

Expansion of a SNaPshot assay to a 55-SNP multiplex: Assay enhancements, validation, and power in forensic science.

A previously developed multiplex assay with 44 individual identification SNPs was expanded to a 55plex assay. Fifty-four highly informative SNPs and an amelogenin sex marker were amplified in one PCR reaction and then detected with two SNaPshot reactions using CE. PCR primers for four loci, 28 single-base extension primers, and the reaction conditions were altered to improve the robustness of the method. A detailed approach for allele calling was developed to guide analysis of the electropherogram. One hundred and eighty unrelated individuals and 100 father-child-mother trios of the Han population in Hebei, China were analyzed. No mutation was found in the SNP loci. The combined mean match probability and cumulative probability of exclusion were 1.327 × 10(-22) and 0.999932, respectively. Analysis of the 54 SNPs and 26 STRs (included in the AmpFLSTR Identifiler and Investigator HDplex kits) showed no significant linkage disequilibriums. Our research shows that the expanded SNP multiplex assay is an easily performed and valuable method to supplement STR analysis.

A Genome-Wide Scan of DNA Methylation Markers for Distinguishing Monozygotic Twins.

Identification of individuals within pairs of monozygotic (MZ) twins remains unresolved using common forensic DNA typing technology. For some criminal cases involving MZ twins as suspects, the twins had to be released due to inability to identify which of the pair was the perpetrator. In this study, we performed a genome-wide scan on whole blood-derived DNA from four pairs of healthy phenotypically concordant MZ twins using the methylated DNA immunoprecipitation sequencing technology to identify candidate DNA methylation markers with capacity to distinguish MZ twins within a pair. We identified 38 differential methylation regions showing within-pair methylation differences in all four MZ pairs. These are all located in CpG islands, 17 of which are promoter-associated, 17 are intergenic islands, and four are intragenic islands. Genes associated with these markers are related with cell proliferation, differentiation, and growth and development, including zinc finger proteins, PRRX2, RBBP9, or are involved in G-protein signaling, such as the regulator of G-protein signaling 16. Further validation studies on additional MZ twins are now required to evaluate the broader utility of these 38 markers for forensic use.

LINE-1 DNA methylation: A potential forensic marker for discriminating monozygotic twins.

Discriminating individuals within a pair of monozygotic (MZ) twins using genetic markers remains unresolved. This inability causes problems in criminal or paternity cases involving MZ twins as suspects or alleged fathers. Our previous study showed DNA methylation differences in interspersed repeat sequences such as Alu and LINE-1 within pairs of newborn MZ twins. To further evaluate the possible value of LINE-1 DNA methylation for discriminating MZ twins, this study investigated the LINE-1 DNA methylation of a large number of twins. We collected blood samples and buccal cell samples from 119 pairs of MZ and 57 pairs of dizygotic (DZ) twins. Genomic DNA was extracted and LINE-1 methylation level was detected using bisulfite pyrosequencing. The mean methylation level of the three CpG sites in the blood sample among the 176 unrelated individuals was 76.60% and 70.08% in buccal samples. This difference was significant, indicating the tissue specificity of LINE-1 DNA methylation. Among 119 pairs of MZ twins, 15 pairs could be discriminated according to the difference of CpG methylation level between them, which accounted for 12.61% of total number of MZ pairs. As for DZ twins, 10 pairs had significant differences between two individuals, which accounted for 17.54% of the total 57 DZ pairs. In conclusion, there are global DNA methylation differences within some healthy concordant monozygotic (MZ) twin pairs. LINE-1 DNA methylation might be a potential marker for helping to discriminate individuals within MZ twin pairs, and the tissue specificity must be considered in practice.

[Study of genetic polymorphisms of 12 short tandem repeats on X chromosome in ethnic Han population from Hebei Province].

OBJECTIVE: To investigate genetic polymorphisms of 12 X chromosome short tandem repeat (X-STR) loci in ethnic Hebei Han population using an Investigator Argus X-12 amplification kit. METHODS: DNA was extracted for 198 unrelated individuals (96 males and 102 females) and amplified with a fluorescence labeled multiplex PCR system. PCR products were separated and genotyped with capillary array electrophoresis. RESULTS: Only DXS10103 and DXS10101 showed significant linkage disequilibrium at the 12 X-STR loci. One hundred and forty-eight alleles, including 22 off-ladder (OL) alleles, were observed at the 12 X-STR loci in the population. The heterozygosity and polymorphic information content (PIC) were 0.5074-0.9143 and 0.4377-0.9079, respectively. The power of discrimination (PD) was 0.5074-0.9143 in males and 0.6876-0.9863 in females. The mean exclusion chance was 0.4377-0.9079 in the trios cases and 0.2984-0.8373 in the duo cases, respectively. CONCLUSION: The Investigator Argus X12 amplification system is highly polymorphic in ethnic Han population from Hebei and is useful for personal identification and paternity testing.