Genome-wide association studies combined with k-fold cross-validation identify rs17822931 as an ancestry-informative marker in Han Chinese population.

DNA-based ancestry inference has long been a research hot spot in forensic science. The differentiation of Han Chinese population, such as the northern-to-southern substructure, would benefit forensic practice. In the present study, we enrolled participants from northern and southern China, each participant was genotyped at ∼400 K single-nucleotide polymorphisms (SNPs) and data of CHB and CHS from 1000 Genomes Project were used to perform genome-wide association analyses. Meanwhile, a new method combining genome-wide association study (GWAS) analyses with k-fold cross-validation in a small sample size was introduced. As a result, one SNP rs17822931 emerged with a p-value of 7.51E - 6. We also simulated a huge dataset to verify whether k-fold cross-validation could reduce the false-negative rate of GWAS. The identified ABCC11 rs17822931 has been reported to have allele frequencies varied with the geographical gradient distribution in humans. We also found a great difference in the allele frequency distributions of rs17822931 among five different cohorts of the Chinese population. In conclusion, our study demonstrated that even small-scale GWAS can also have potential to identify effective loci with implemented k-fold cross-validation method and shed light on the potential maker of rs17822931 in differentiating the north-to-south substructure of the Han Chinese population.

Development of a simple and reliable method for α-amanitin detection in rat plasma and its application to a toxicokinetic study.

RATIONALE: α-Amanitin is a highly toxic peptide widely found in species of poisonous mushrooms. The matrix effect has been a major obstacle for accurate determination of α-amanitin in plasma samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS). In this study, the strategy to eliminate the matrix effect of α-amanitin with a one-step dilution approach after deproteinization was applied. METHODS: Rat plasma samples were processed by protein precipitation with methanol followed by a nine-fold dilution with pure water. The matrix effect value of α-amanitin was 19.7%-22.2% by protein precipitation and then changed to 87.5%-88.7% after dilution. α-Amanitin and the internal standard (roxithromycin) were analyzed on an ACQUITY UPLC® BEH C18 (50 mm × 2.1 mm, 1.7 µm) column within 3.0 min by gradient elution. RESULTS: The linear ranges were 0.90-600 ng/mL with a correlation coefficient r >0.9958. A lower limit of quantification (LLOQ) of 0.90 ng/mL was achieved using only 50 µL of rat plasma. The intra- and inter-day precisions for the analyte ranged from 3.2% to 7.5% and 3.1% to 7.1%, respectively, and the accuracy ranged from -5.3% to -8.0%. CONCLUSIONS: The matrix effect of α-amanitin was reduced by sample dilution after plasma deproteinization. A reliable LC/MS/MS method for the determination of α-amanitin in rat plasma was developed. This method was successfully applied for a toxicokinetic study of rats after intravenous injection of α-amanitin with a subacute toxicity dose at 0.10 mg/kg.

Identifying novel microhaplotypes for ancestry inference.

The use of DNA to determine the ancestry of an individual is becoming more and more important in the areas of forensics. Kidd et al. (Forensic Sci Int Genet 12:215-224, 2014) have been the first to identify and catalog haplotypes, termed as minihaplotypes (1-10-kilobase spans) and microhaplotypes (≤ 200 bp), with potential use in forensic analysis. In the present study, we selected 10 short ancestry informative microhaplotypes by calculating the informativeness (In) according to Rosenberg et al. (Am J Hum Genet 73(6):1402-1422, 2003). In total, 2504 individuals from 26 populations in 1000 Genomes Project database Phase 3 were enrolled. Among the studied microhaplotypes, eight of them are comprised of 3 SNPs while two microhaplotypes are made up of 4 SNPs. The size (molecular extent) range of 10 microhaplotypes is 5 to 48 bp with an average of 31.4 bp. The heterozygosity value ranges from 0.2235 to 0.8958 with an average of 0.6593. The average power of discrimination (PD) values is 0.7944 and ranges from 0.3786 to 0.9242. Analyses of this dataset provided clear differentiation of the populations from the Africa, East Asia, South Asia, and Europe biogeographic regions. However, individuals from American ancestry were not well separated. To conclude, our results revealed the significance of using microhaplotypes as an ancestry informative marker. The present panel could offer a valid complementary tool in forensic applications.

Plasma extracellular vesicles microRNA-208b-3p and microRNA-143-3p as novel biomarkers for sudden cardiac death prediction in acute coronary syndrome.

Acute coronary syndrome (ACS) occurs as a result of myocardial ischemia that can give rise to a variety of acute cardiovascular events, including arrhythmia, heart failure and sudden cardiac death (SCD). Currently, there are challenges and insufficient innovations regarding early diagnosis and therapeutic approaches within ACS patients experiencing SCD. Plasma extracellular vesicles (EVs) might serve as biomarkers of many diseases depending on the biological molecules of their cargo, such as miRNAs. This study aims to identify the plasma EVs containing miRNAs as novel biomarkers for the prediction of SCD in ACS patients. A total of 39 ACS patients experiencing SCD and 39 healthy control individuals (HC) were enrolled, among which 9 samples in each group were randomly selected as testing groups for miRNA sequencing in plasma EVs, and the remaining samples were assigned to the validation group. The top 10 significant expression miRNAs were verified by the real-time quantitative polymerase chain reaction. Upregulation of miR-208b-3p, miR-143-3p, miR-145-3p and miR-152-3p, and down-regulation of miR-183-5p were further validated in the validation group. Spearman's correlation analysis and the receiver operating characteristic (ROC) curve showed that both miR-208b-3p and miR-143-3p levels were positively correlated with myoglobin (MYO), and their predictive power for SCD was confirmed. In conclusion, our findings indicate that plasma EVs miR-208b-3p and miR-143-3p may serve as promising biomarkers in predicting SCD in patients with ACS, as well as postmortem forensic diagnosis of the cause of death due to ACS.

Whole-exome sequencing and electrophysiological study reveal a novel loss-of-function mutation of KCNA10 in epinephrine provoked long QT syndrome with familial history of sudden cardiac death.

Congenital long QT syndrome (LQTS) is one type of inherited fatal cardiac arrhythmia that may lead to sudden cardiac death (SCD). Mutations in more than 16 genes have been reported to be associated with LQTS, whereas the genetic causes of about 20% of cases remain unknown. In the present study, we investigated a four-generation pedigree with familial history of syncope and SCD. The proband was a 33-year-old young woman who experienced 3 episodes of syncope when walking at night. The electrocardiogram revealed a markedly epinephrine-provoked prolonged QT interval (QT = 468 ms, QTc = 651 ms) but no obvious arrhythmia in the resting state. Three family members have died of suspected SCD. Whole-exome sequencing and bioinformatic analysis based on pedigree revealed that a novel missense mutation KCNA10 (c.1397G＞A/Arg466Gln) was the potential genetic lesion. Sanger sequencing was performed to confirm the whole-exome sequencing results. This mutation resulted in the KV1.8 channel amino acid residue 466 changing from arginine to glutamine, and the electrophysiological experiments verified it as a loss-of-function mutation of KV1.8, which reduced the K+ currents of KV1.8 and might result in the prolonged QT interval. These findings suggested that KCNA10 (c.1397G＞A) mutation was possibly pathogenic in this enrolled LQTS family, and may provide a new potential genetic target for diagnosis and counseling of stress-related LQTS families as well as the postmortem diagnosis of SCD.

Evaluation of a SNP-STR haplotype panel for forensic genotype imputation.

Short tandem repeat polymorphism (STR)-based individual identification is a popular and reliable method in many forensic applications. However, STRs still frequently fail to find any matched records. In such cases, if known STRs could provide more information, it would be very helpful to solve specific problems. Genotype imputation has long been used in the study of single nucleotide polymorphisms (SNPs) and has recently been introduced into forensic fields. The idea is that, through a reference haplotype panel containing SNPs and STRs, we can obtain unknown genetic information through genotype imputation based on known STR or SNP genotypes. Several recent studies have already demonstrated this exciting idea, and a 1000 Genomes SNP-STR haplotype panel has also been released. To further study the performance of genotype imputation in forensic fields, we collected STR, microhaplotype (MH) and SNP array genotypes from Chinese Han population individuals and then performed genotype imputation analysis based on the released reference panel. As a result, the average locus imputation accuracy was ∼83 % (or ∼70 %) when SNPs in the SNP array (or MH SNPs) were imputed from STRs, and was ∼30 % when highly polymorphic markers (STRs and MHs) were imputed from each other. When STRs were imputed from SNP array, the average locus imputation accuracy increased to ∼48 %. After analyzing the match scores between real STRs and the STRs imputed from SNPs, ∼80 % of studied STR records can be connected to corresponding SNP records, which may help for individual identification. Our results indicate that genotype imputation has great potential for forensic applications.

Update on risk factors and biomarkers of sudden unexplained cardiac death.

Sudden cardiac death (SCD) accounts for approximately 15%-20% of all deaths worldwide, the causes of which are mainly structural heart diseases. However, SCD also occurs in patients without major cardiac structural abnormalities due to electrophysiological abnormalities or other unexplained causes which account for about 1%-15% cases of SCD, namely sudden unexplained cardiac death (SUCD). The causes of SUCD cannot be explained by routine forensic pathological examination. Hence, identifying the causes of SUCD remains a major challenge in forensic fields. Consequently, it is significant to discover the risk factors and molecular biomarkers for diagnosis and prediction of SUCD. SUCD can be at least partially explained by acute and chronic psychological stress, such as Takotsubo cardiomyopathy (TCM), which is increasingly recognized as a potentially modifiable risk factor for SUCD. Additionally, the inherited lethal cardiac arrhythmia due to ion channelopathies is one of the causes of SUCD. However, SCD resulted from psychological stress or lethal cardiac arrhythmia lacks postmortem observable morphological evidence. Therefore, the postmortem diagnosis of SUCD remains a primary challenge in forensic pathology because of the absence of recognized diagnostic biomarkers. Recent advances in knowledge give us additional possibility in understanding the cause of SUCD. In this review, we summarized the updated risk factors of SUCD including genetics and psychological stress, as well as the potential diagnostic biomarkers of SUCD, such as micro-RNAs, heat shock proteins, Galectin-3 and inflammation factors. Meanwhile, we concluded the ongoing researches on novel measurements including chemical analysis, machine learning and artificial intelligence for predicting the cause of SCD.

Rapid detection of α-amanitin and ß-amanitin in rat plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to the toxicokinetic study of Lepiota brunneoincarnata.

PURPOSE: Lepiota brunneoincarnata is a well-known poisonous mushroom and is responsible for fatal mushroom poisoning cases worldwide. α-Amanitin and ß-amanitin are the main amatoxin compounds of Lepiota brunneoincarnata. However, there are no published toxicokinetic studies of Lepiota brunneoincarnata. To study the toxicokinetics of Lepiota brunneoincarnata, we developed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of α-amanitin and ß-amanitin in rat plasma. METHODS: UPLC-MS/MS analyses were performed with a triple quadrupole mass spectrometer in positive-ion mode. The sensitivity of α-amanitin and ß-amanitin detection was increased by inhibiting the production of [M + Na]+ adducts. α-Amanitin and ß-amanitin were separated and quantified on an UPLC octadecyl silyl column in only 2.5 min. RESULTS: The linear ranges were 3.0-3000 ng/mL for α-amanitin and 1.8-1800 ng/mL for ß-amanitin with a correlation coefficient r > 0.99 for both analytes. The lower limit of quantification of 3.0 ng/mL for α-amanitin and 1.8 ng/mL for ß-amanitin was achieved using only 50 µL of rat plasma. The accuracy of α-amanitin and ß-amanitin was between - 9.5 and 7.0% with the precision ranged from 2.2 to 12.5%. The developed method was then applied for Lepiota brunneoincarnata toxicokinetic study after intravenous administration of Lepiota brunneoincarnata extracts. CONCLUSIONS: Establishing UPLC-MS/MS method for quantifying amanitines in rat plasma successfully enabled toxicokinetic study of Lepiota brunneoincarnata extracts.

Inferring bio-geographical ancestry with 35 microhaplotypes.

During the past decades, with the blooming of large-scale human genetic studies, we are beginning to understand how bio-geographical information could be reflected by genetic variations. And the technological advance in massively parallel sequencing gives advantages to some novel forensic markers such as microhaplotype (BIM). In the present study, we selected and characterized 35 novel bio-geographical informative BIMs based on the 1000 Genomes Project (1KG). All loci had short lengths less than 100 bp, high effective allele numbers (Ae) from 1.875 to 3.980 with an average of 2.798, and high informativeness (In) value from 0.701 to 0.865 with an average of 0.748, which indicates the 35 BIMs possessed great discriminating power. Using these 35 BIMs, the individuals from 1KG could be successfully differentiated into five supergroups defined by 1KG (AFR, AMR, EAS, EUR, and SAS). We also included some individuals from the Simons Genome Diversity Project (SGDP) for further validation. As a result, most individuals could be accurately predicted except for those from super-populations that do not exist in 1KG dataset. In conclusion, the present novel 35 BIMs could be a useful tool for bio-geographical ancestry inference.