Genomic Mutations Within the Host Microbiome: Adaptive Evolution or Purifying Selection

Next-generation sequencing technology has transformed our ability to assess the taxonomic composition functions of host-associated microbiota and microbiomes. More human microbiome research projects—particularly those that explore genomic mutations within the microbiome—will be launched in the next decade. This review focuses on the coevolution of microbes within a microbiome, which shapes strain-level diversity both within and between host species. We also explore the correlation between microbial genomic mutations and common metabolic diseases, and the adaptive evolution of pathogens and probiotics during invasion and colonization. Finally, we discuss advances in methods and algorithms for annotating and analyzing microbial genomic mutations.

Remarkably different results between two studies from North America on genomic mutations and sensitivity to DNA demethylating agents for myelodysplastic syndromes

Sekeres et al. （1） conducted a multicenter randomized, controlled trial to compare whether azacitidine-based combinations with lenalidomide or vorinostat produce superior overall response rates to azacitidine in the treatment of myelodysplastic syndromes （MDS）. In that trial, 224 patients with higher-risk MDS and 53 with chronic myelomonocytic leukemia （CMML） were enrolled and randomly assigned to the ＂azacitidine＂ group, ＂azacitidine plus lenalidomide＂ group or ＂azacitidine plus vorinostat＂ group. The researchers found that patients with MDS treated with azacitidine-based combinations had similar response rate to azacitidine monotherapy. Using genomic mutation analysis, they found that the overall response rate to azacitidine-based treatment was higher for patients with mutations in DNMT3A and lower for those with mutations in SRSF2. Whereas in another study, Welch et al. enrolled 26 patients with MDS and 90 with acute myeloid leukemia （AML） who were treated with decitabine, and they found that patients with TP53 mutations had a higher response rate, but not those with DNMT3A mutations （2）. We propose that this big discrepancy in the conclusions between the two studies might have been caused by the presence of many co-interacting factors, e.g. study aims, DNA demethylating agents, treatment protocols, and patient sources.

Genetic Analysis of the PKHD1 Gene with Long-rang PCR Sequencing

PKHD1 gene mutations are found responsible for autosomal recessive polycystic kidney disease（ARPKD）. However, it is inconvenient to detect the mutations by common polymerase chain reaction（PCR） because the open reading frame of PKHD1 is very long. Recently, long-range（LR） PCR is demonstrated to be a more sensitive mutation screening method for PKHD1 by directly sequencing. In this study, the entire PKHD1 coding region was amplified by 29 reactions to avoid the specific PCR amplification of individual exons, which generated the size of 1 to 7 kb products by LR PCR. This method was compared to the screening method with standard direct sequencing of each individual exon of the gene by a reference laboratory in 15 patients with ARPKD. The results showed that a total of 37 genetic changes were detected with LR PCR sequencing, which included 33 variations identified by the reference laboratory with standard direct sequencing. LR PCR sequencing had 100% sensitivity, 96% specificity, and 97.0% accuracy, which were higher than those with standard direct sequencing method. In conclusion, LR PCR sequencing is a reliable method with high sensitivity, specificity and accuracy for detecting genetic variations. It also has more intronic coverage and lower cost, and is an applicable clinical method for complex genetic analyses.

Shared-probe system:An accurate,low-cost and general enzyme-assisted DNA probe system for detection of genetic mutation

Enzyme assisted DNA probes are powerful tools in molecular diagnostics for their simplicity,rapidity,and low detection limit.However,cost of probes,difficulty in optimization and disturbance of secondary structure hindered the wider application of enzyme assisted DNA probes.To solve the problems,we designed a new system named shared-probe system.By introducing two unlabeled single stranded DNA named Sh1 and Sh2 as the bridge between probe and the substrate,the same sequence of dually labeled probe with stable performance was shared for different mutations,thus sparing the expense and time cost on designing,synthesizing and optimizing corresponding probes.Besides,the hybridization between Sh1 and the substrate could overcome secondary structures,which guaranteed the detection of different substrates.The performance and generality of the design were tested by low abundance detection in synthetic single DNA samples and the limit of detection was 0.05%for PTENR130 Q,EGFR-L858 R and 0.02%for BRCA1-NM007294.3.In genomic DNA samples,the limit of detection of 0.1%can be achieved for EGFR-L858 R,demonstrating the potential of clinical application in our design.

Development and utilization of a new chemically-induced soybean library with a high mutation density

Mutagenized populations have provided important materials for introducing variation and identifying gene function in plants. In this study, an ethyl methanesulfonate（EMS）-induced soybean（Glycine max） population,consisting of 21,600 independent M_2 lines, was developed.Over 1,000 M_（4（5））families, with diverse abnormal phenotypes for seed composition, seed shape, plant morphology and maturity that are stably expressed across different environments and generations were identified. Phenotypic analysis of the population led to the identification of a yellow pigmentation mutant, gyl, that displayed significantly decreased chlorophyll（Chl） content and abnormal chloroplast development. Sequence analysis showed that gyl is allelic to Minn Gold, where a different single nucleotide polymorphism variation in the Mg-chelatase subunit gene（ChlI1a） results in golden yellow leaves. A cleaved amplified polymorphic sequence marker was developed and may be applied to marker-assisted selection for the golden yellow phenotype in soybean breeding. We show that the newly developed soybean EMS mutant population has potential for functional genomics research and genetic improvement in soybean.

IVS8 ＋ 1 DelG, a Novel Splice Site Mutation Causing DFNA5 Deafness in a Chinese Family

Background： Nonsyndrornic hearing loss （NSHL） is highly heterogeneous, in which more than 90 causative genes have currently been identified. DFNA5 is one of the deathess genes that known to cause autosomal dominant NSHL. Until date, only five DFN.45 mutations have been described in eight families worldwide. In this study, we reported the identification of a novel pathogenic mutation causing DFNA5 deafness in a five-generation Chinese family. Methods： Alter detailed clinical evaluations of this family, the genomic DNA of three affected individuals was selected for targeted exome sequencing of 101 known deafness genes, as well as mitochondrial DNA and microRNA regions. Co-segregation analysis between the hearing loss and the candidate variant was confirmed in available family members by direct polymerase chain reaction （PCR）-Sanger sequencing. Real-time PCR （RT-PCR） was pertormed to investigate the potential effect of the pathogenic mutation on messenger RNA splicing. Results： Clinical evaluations revealed a similar deafness phenotype in this family to that of previously reported DFNA5 families with autosomal dominant, late-onset bearing loss. Molecular analysis identified a novel splice site mutation in DFNA5 intron 8 （IVSS＋ 1 delG）. The mutation segregated with the hearing loss of the family and was absent in 120 unrelated control DNA samples of Chinese origin. RT-PCR showed skipping of exon 8 in the mutant transcript. Conclusions： We identified a novel DFNA5 mutation IVS8＋1 delG in a Chinese family which led to skipping ofexon 8. This is the sixth DFNA5 mutation relates to hearing loss and the second one in DFNA5 intron 8. Our findings provide further support to the hypothesis that the DFNA5-associated hearing loss represents a mechanism of gain-of-function.