Whole-genome sequencing of six dog breeds from continuous altitudes reveals adaptation to high-altitude hypoxia.

The hypoxic environment imposes severe selective pressure on species living at high altitude. To understand the genetic bases of adaptation to high altitude in dogs, we performed whole-genome sequencing of 60 dogs including five breeds living at continuous altitudes along the Tibetan Plateau from 800 to 5100 m as well as one European breed. More than 150× sequencing coverage for each breed provides us with a comprehensive assessment of the genetic polymorphisms of the dogs, including Tibetan Mastiffs. Comparison of the breeds from different altitudes reveals strong signals of population differentiation at the locus of hypoxia-related genes including endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) and beta hemoglobin cluster. Notably, four novel nonsynonymous mutations specific to high-altitude dogs are identified at EPAS1, one of which occurred at a quite conserved site in the PAS domain. The association testing between EPAS1 genotypes and blood-related phenotypes on additional high-altitude dogs reveals that the homozygous mutation is associated with decreased blood flow resistance, which may help to improve hemorheologic fitness. Interestingly, EPAS1 was also identified as a selective target in Tibetan highlanders, though no amino acid changes were found. Thus, our results not only indicate parallel evolution of humans and dogs in adaptation to high-altitude hypoxia, but also provide a new opportunity to study the role of EPAS1 in the adaptive processes.

Noninvasive diagnosis and monitoring of mutations by deep sequencing of circulating tumor DNA in esophageal squamous cell carcinoma.

Circulating tumor DNA (ctDNA) is becoming an important biomarker in noninvasive diagnosis and monitoring of tumor dynamics. This study tested the feasibility of plasma ctDNA for the non-invasive analysis of tumor mutations in esophageal squamous cell carcinoma (ESCC) by sequencing of tumor, tumor-adjacent, and normal tissue, as well as pre-surgery and post-surgery plasma. Exome sequencing of eight patients identified between 29 and 134 somatic mutations in ESCCs, many of which were also determined in ctDNA. Comparison of pre-surgery and post-surgery plasma has shown that mutations had reduced frequency or disappeared after surgery treatment. We further evaluated the TruSight Cancer sequencing panel by using it to detect mutations in the plasma of three patients. Tumor mutations were only found in one of them. To design a sequencing panel with improved targeting, we identified significantly mutated genes by meta-analysis of 532 ESCC genomes. Our results confirmed the well-known driver genes and found several uncharacterized genes. The new panel consisted of 90 recurrent genes, which theoretically achieved 94% and 75% of sensitivity when detecting at least 1 and 2 mutant genes in ESCC patients, respectively. Our results demonstrate the feasibility of using ctDNA to detect ESCCs and monitor treatment effect. The low-cost and sensitive target panel could facilitate clinical usage of ctDNA as a noninvasive biomarker.

Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme.

A feather-degrading bacterium was isolated from poultry decomposition feathers in China. The strain, named L1, showed significant feather-degrading activity because it grew and reproduced quickly on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. According to the phenotypic characteristics and 16S rRNA profile, the isolate belongs to Stenotrophomonas maltophilia. Keratinase activity of the isolate was determined during cultivation on raw feathers at different temperatures and initial pH. Maximum growth and feather-degrading activity of the bacterium were observed at 40 degrees C and initial pH ranging from 7.5 to 8.0. The crude enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 chromatographic and ceramic hydroxyapatite (CHT) chromatographic. Its molecular mass estimated as 35.2 kDa in SDS-PAGE. The enzyme had an optimum activity at the pH was 7.8 and the temperature was 40 degrees C. The keratinase was wholly inhibited by a serine protease inhibitor, PMSF. Its activity was activated or inhibited by different metal ions. The keratinase activity of enzyme from strain L1 functioned on different keratins, such as feather, hair, wool, horn, and so on.

MiR-222-3p in Platelets Serves as a Distinguishing Marker for Early Recognition of Kawasaki Disease.

Kawasaki disease (KD) is an acute vasculitis, which leads to 20% of sufferers developing coronary artery aneurysm in children if not appropriately treated. Therefore, the early diagnosis of KD is essential for alleviating the risk of developing heart disease. MicroRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate gene expression and have been shown to play critical roles in numerous biological processes and diseases. In this study, we used high-throughput miRNA sequencing and found dozens of miRNAs are highly expressed in platelets. By comparing the miRNA expression profile of platelets of acute KD patients and other febrile patients, miR-222-3p is validated to be significantly upregulated in platelets of acute KD patients. Furthermore, KEGG pathway analysis shows that targets of miR-222-3p are enriched in immune-related signaling pathways. Our study uncovers the potential of miR-222-3p in platelets as biomarker for early diagnosis of Kawasaki disease.

A multiplex ligation­dependent probe amplification­based next­generation sequencing approach for the detection of copy number variations in the human genome.

The aim of the present study was to describe a multiplex ligation­dependent probe amplification (MLPA)­based next­generation sequencing (NGS) assay that exhibited a significantly higher efficiency in detecting copy number variations (CNVs) and known single­nucleotide variants, compared with traditional MLPA. MLPA polymerase chain reaction products were used to construct a library with indexed adapters, which was subsequently tested on an NGS platform, and the resulting data were analyzed by a series of analytical software. The reads from each probe reflected genetic variations in the target regions, and fragment differentiation was based on the specific base composition of the sequences, rather than fragment length, which was determined by capillary electrophoresis. The results of this approach were not only consistent with the MLPA results following capillary electrophoresis, but also coincided with the CNV results from the single­nucleotide polymorphism array chip. This method allowed high­throughput screening for the number of fragments and samples by integrating additional indices for detection. Furthermore, this technology precisely and accurately performed large­scale detection and quantification of DNA variations, thereby serving as an effective and sensitive method for diagnosing genetic disorders caused by CNVs and known single­nucleotide variations. Notably, MLPA­NGS circumvents the problems associated with the inaccuracies of NGS in CNV detection due to the use of target sequence capture.

Integrative analysis of DNA methylation and gene expression reveals hepatocellular carcinoma-specific diagnostic biomarkers.

BACKGROUND: Hepatocellular carcinoma (HCC) is the one of the most common cancers and lethal diseases in the world. DNA methylation alteration is frequently observed in HCC and may play important roles in carcinogenesis and diagnosis. METHODS: Using the TCGA HCC dataset, we classified HCC patients into different methylation subtypes, identified differentially methylated and expressed genes, and analyzed cis- and trans-regulation of DNA methylation and gene expression. To find potential diagnostic biomarkers for HCC, we screened HCC-specific CpGs by comparing the methylation profiles of 375 samples from HCC patients, 50 normal liver samples, 184 normal blood samples, and 3780 samples from patients with other cancers. A logistic regression model was constructed to distinguish HCC patients from normal controls. Model performance was evaluated using three independent datasets (including 327 HCC samples and 122 normal samples) and ten newly collected biopsies. RESULTS: We identified a group of patients with a CpG island methylator phenotype (CIMP) and found that the overall survival of CIMP patients was poorer than that of non-CIMP patients. Our analyses showed that the cis-regulation of DNA methylation and gene expression was dominated by the negative correlation, while the trans-regulation was more complex. More importantly, we identified six HCC-specific hypermethylated sites as potential diagnostic biomarkers. The combination of six sites achieved ~ 92% sensitivity in predicting HCC, ~ 98% specificity in excluding normal livers, and ~ 98% specificity in excluding other cancers. Compared with previously published methylation markers, our markers are the only ones that can distinguish HCC from other cancers. CONCLUSIONS: Overall, our study systematically describes the DNA methylation characteristics of HCC and provides promising biomarkers for the diagnosis of HCC.

Whole-genome sequences of 89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization.

Background: Animal domestication has been extensively studied, but the process of feralization remains poorly understood. Results: Here, we performed whole-genome sequencing of 99 sheep and identified a primary genetic divergence between 2 heterogeneous populations in the Tibetan Plateau, including 1 semi-feral lineage. Selective sweep and candidate gene analysis revealed local adaptations of these sheep associated with sensory perception, muscle strength, eating habit, mating process, and aggressive behavior. In particular, a horn-related gene, RXFP2, showed signs of rapid evolution specifically in the semi-feral breeds. A unique haplotype and repressed horn-related tissue expression of RXFP2 were correlated with higher horn length, as well as spiral and horizontally extended horn shape. Conclusions: Semi-feralization has an extensive impact on diverse phenotypic traits of sheep. By acquiring features like those of their wild ancestors, semi-feral sheep were able to regain fitness while in frequent contact with wild surroundings and rare human interventions. This study provides a new insight into the evolution of domestic animals when human interventions are no longer dominant.