The identification of causative mutations is important for treatment decisions and genetic counseling of patients with disorders of sex development (DSD). Here, we designed a new assay based on targeted next-generation sequencing (NGS) to diagnose these genetically heterogeneous disorders. All coding regions and flanking sequences of 219 genes implicated in DSD were designed to be included on a panel. A total of 45 samples were used for sex chromosome dosage validation by targeted sequencing using the NGS platform. Among these, 21 samples were processed to find the causative mutation. The sex chromosome dosages of all 45 samples in this assay were concordant with their corresponding karyotyping results. Among the 21 DSD patients, a total of 11 mutations in SRY, NR0B1, AR, CYP17A1, GK, CHD7, and SRD5A2 were identified, including five single nucleotide variants, three InDels, one in-frame duplication, one SRY-positive 46,XX, and one gross duplication with an estimated size of more than 427,038 bp containing NR0B1 and GK. We also identified six novel mutations: c.230_231insA in SRY, c.7389delA in CHD7, c.273C>G in NR0B1, and c.2158G>A, c.1825A>G, and c.2057_2065dupTGTGTGCTG in AR. Our assay was able to make a genetic diagnosis for eight DSD patients (38.1 %), and identified variants of uncertain clinical significance in the other three cases (14.3 %). Targeted NGS is therefore a comprehensive and efficient method to diagnose DSD. This work also expands the pathogenic mutation spectrum of DSD.

Background: Monogenic diabetes is a genetic disease often caused by mutations in genes involved in beta-cell function. Correct sub-categorization of the disease is a prerequisite for appropriate treatment and genetic counseling. Target-region capture sequencing is a combination of genomic region enrichment and next generation sequencing which might be used as an efficient way to diagnose various genetic disorders. We aimed to develop a target-region capture sequencing platform to screen 117 selected candidate genes involved in metabolism for mutations and to evaluate its performance using monogenic diabetes as a study-model. Results: The performance of the assay was evaluated in 70 patients carrying known disease causing mutations previously identified in HNF4A, GCK, HNF1A, HNF1B, INS, or KCNJ11. Target regions with a less than 20-fold sequencing depth were either introns or UTRs. When only considering translated regions, the coverage was 100% with a 50-fold minimum depth. Among the 70 analyzed samples, 63 small size single nucleotide polymorphisms and indels as well as 7 large deletions and duplications were identified as being the pathogenic variants. The mutations identified by the present technique were identical with those previously identified through Sanger sequencing and Multiplex Ligation-dependent Probe Amplification. Conclusions: We hereby demonstrated that the established platform as an accurate and high-throughput gene testing method which might be useful in the clinical diagnosis of monogenic diabetes.

Changes in physiological conditions could influence the perception of external odors, which is important for the reproduction and survival of insect. With the alteration of physiological conditions, such as age, feeding state, circadian rhythm and mating status, insect can modulate their olfactory systems accordingly. Gustatory, ionotropic and odorant receptors (GR, IR and ORs) are important elements of the insect chemosensory system, which enable insects to detect various external stimuli. In this study, we investigated the changes in these receptors at the mRNA level in Bactrocera dorsalis in different physiological states. We performed transcriptome analysis to identify chemosensory receptors: 21 IRs, 12 GRs and 43 ORs were identified from B.dorsalis antennae, including all previously known chemoreceptors in B. dorsalis and a few more. Quantitative real-time polymerase chain reaction analysis revealed the effects of feeding state, mating status and time of day on the expression of IR, GR and OR genes. The results showed that expression of chemosensory receptors changed in response to physiological states, and these changes were completely different for different types of receptors and between male and female flies. Our study suggests that the expression of chemosensory receptors changes to adapt to different physiological states, which may indicate the significant role of these receptors in such physiological processes.

Microbial degradation has been considered as a rapid, green, and cost-effective technique to reduce insecticide pollutions in a contaminated environment. However, the instability and low efficacy of non-indigenous microorganisms hampers their further exploitation when being introduced into a real environmental matrix. In order to overcome the restriction that these functional microorganisms are under, we investigated the optimal conditions to improve the pyrethroid-degrading ability of one previously isolated bacterium Bacillus cereus BCC01, where 9.6% of the culture suspension (with cell density adjusted to OD₆₀₀ = 0.6) was inoculated into 50 mL media and cultivated at pH 8 and 30 °C, and its metabolic pathway was illuminated by analyzing the main metabolites via gas chromatography mass spectrometry (GC-MS). Most importantly, a key pyrethroid-hydrolyzing carboxylesterase gene estA was identified from the genomic library of strain BCC01, and then expressed in Escherichia coli BL21 (DE3). After purification, the recombinant protein EstA remained soluble, displaying high degrading activity against different pyrethroids and favorable stability over a wide range of temperatures (from 15 °C to 50 °C) and pH values (6.5–9). Therefore, the EstA-associated biodegradation of pyrethroids was determined, which could provide novel insights to facilitate the practical application of B. cereus BCC01 in the microbial detoxification of pyrethroid contamination.