Sanger Sequencing Method Research Articles

Bacterial microbiota of three commercially mass-reared predatory mite species (Mesostigmata: Phytoseiidae): pathogenic and beneficial interactions

Phytoseiidae predatory mites are used as biological control agents in glasshouse environments to control pestiferous mites and insects. The influence of the microbiomes on these mites within mass-reared facilities is largely unknown; this study aims to improve our understanding of the Phytoseiidae microbiome. We used Sanger sequencing and traditional culturing methods to characterise the dominant species within the external and internal bacterial microbiotas of three commercially reared predatory mites: Phytoseiulus persimilis, Typhlodromips (=Amblyseius) swirskii, and Neoseiulus (=Amblyseius) cucumeris. The effects of selected bacterial taxa on the survival, voracity, and oviposition of P. persimilis under lab conditions were also examined. A total of 34 different OTUs were found from three phyla (Actinobacteria, Proteobacteria, and Firmicutes), 12 families, and 22 genera. N. cucumeris had 17 OTUs, compared to 15 from P. persimilis and 12 from T. swirskii. Significant differences were found between internal and external microbiota for each mite species, suggesting functional differences. Principal component analysis (PCA) of bacterial diversity between mite species showed that T. swirskii and N. cucumeris microbiotas were more similar, compared to P. persimilis. This could be due to the characteristics of the environment in which N. cucumeris and T. swirskii are reared, and the use of factitious Astigmata prey. Phytoseiidae microbiotas were found to contain bacterial taxa previously isolated from Astigmata mites. Also, Acaricomes phytoseiuli was isolated from the integument of P. persimilis and T. swirskii; this bacterium has previously been suggested as a pathogen of P. persimilis. Bacteria from gut extracts and body surfaces were cultured and selected taxa were inoculated onto P. persimilis within controlled leaf disc experiments. Of the seven bacterial species tested, Serratia marcescens significantly reduced mite survival, voracity, and oviposition; however, a closely related species, Serratia odorifera, significantly increased mite voracity. Mites showed the highest survival rates and highest mean ages at death in the presence of Elizabethkingia sp. and Staphylococcus kloosii, but these results were not significantly different to the control. The implications of these findings are discussed in the context of benefits to mass rearing and the health of predatory mites in these systems.

Portuguese Neonatal Screening Programme: A Retrospective Cohort Study of 18 Years of MS/MS.

The Portuguese Neonatal Screening Programme (PNSP) identifies patients with rare diseases through nationwide screening. Currently, 27 diseases are diagnosed, amongst which are 24 Inborn Errors of Metabolism (IEM), covering approximately 100% of neonates (1). In 2004, the national laboratory implemented a new screening method, tandem mass spectrometry (MS/MS) to test for amino acids and acylcarnitines. This new protocol revolutionized the PNSP and allowed for the analysis of an increased number of IEM, with clear improvements in treatment timings and clinical outcomes (2). From 2004 to 2022, 1 764 830 neonates were screened with MS/MS technology. Those who displayed biochemical profiles indicating an IEM were subjected to molecular characterization via genomic DNA extraction, PCR amplification, and direct Sanger sequencing method of dried blood spot samples. A cohort of 681 newborns were diagnosed with an IEM. MCAD deficiency is the most frequent, with 233 confirmed diagnoses, showing predominantly c.985A>G (p.K329E) mutation of the ACADM gene in homozygosity. Approximately 1/3 of the 33 confirmed cases of Glutaric Aciduria type I present homozygous for the c.1204C>T (p.Arg402Trp) mutation in GCDH. Around 60% of cases of MAT II/III deficiency display the dominant mutation of the MAT1A gene, c.791G>A (p.Arg264His). These genetic profiles and others were determined as diagnostic confirmation for 24 of the IEM screened. This data shows the molecular epidemiology of patients with confirmed IEM diagnosis identified by neonatal screening. Some diseases out of the scope of the PNSP were also detected as a differential diagnosis after biochemical suspicion in the dried blood spot sample. The retrospective analysis of the PNSP allows for an overview of 18 years of achievements accomplished by the national screening for IEM since MS/MS was implemented. For some pathologies with low incidence, it's difficult to trace a discernible pattern. However, presenting de novo mutations for these diseases might provide insights on how to approach different phenotypes. The aim of this work is to establish the molecular epidemiology of metabolic diseases screened.

Arg4810Lys mutation in RNF213 among Eastern Indian non-MMD ischemic stroke patients: a genotype-phenotype correlation.

RNF213 mutations have been reported mostly in moyamoya disease (MMD) with varying frequencies across different ethnicities. However, its prevalence in non-MMD adult-onset ischemic stroke is still not well explored. This present study thus aims to screen the most common RNF213 variant (Arg4810Lys, among East Asians) in the Eastern Indian non-MMD ischemic stroke patients and correlate it with long-term progression and prognosis of the patients. The subjects were analyzed for this variant using PCR-RFLP and confirmed using Sanger sequencing method. We have identified Arg4810Lys variant among eleven young-onset familial ischemic stroke patients in heterozygous manner. A positive correlation of the variant with positive family history (P = 0.001), earlier age at onset (P = 0.002), and history of recurrent stroke (P = 0.015) was observed. However, the carriers showed better cognitive performances in memory (P = 0.042) and executive function (P = 0.004). Therefore, we can conclude that Arg4810Lys/RNF213 - a pathogenic variant for young-onset familial ischemic stroke with higher incidence of recurrent events unlike in MMD cases, have no additional impact on cognition among Eastern Indians.

RB1 gene mutations and genetic spectrum in retinoblastoma cases.

The aim of the study was to investigate the frequency and types of mutations on the retinoblastoma gene (RB1 gene) in Turkish population. RB1 gene mutation analysis was performed in a total of 219 individuals (122 probands with retinoblastoma, 14 family members with retinoblastoma and 83 clinically healthy family members). All 27 exons and close intronic regions of the RB1 gene were sequenced for small deletions and insertions using both the Sanger sequencing or NGS methods, and the large deletions and duplications were investigated using the MLPA analysis and CNV algorithm. The bilateral/trilateral retinoblastoma rate was 66% in the study population. The general frequency of RB1 gene mutation in the germline of the patients with retinoblastoma was 41.9%. Approximately 51.5% of the patients were diagnosed earlier than 12 months old, and de novo mutation was found in 32.4% of the patients. Germline small genetic rearrangement mutations were detected in 78.9% of patients and LGRs were detected in 21.1% of patients. An association was detected between the eye color of the RB patients and RB1 mutations. 8 of the mutations detected in the RB1 gene were novel in the study.

Closing the Gap - Detection of 5q-Spinal Muscular Atrophy by Short-Read Next-Generation Sequencing and Unexpected Results in a Diagnostic Patient Cohort.

The importance of early diagnosis of 5q-Spinal muscular atrophy (5q-SMA) has heightened as early intervention can significantly improve clinical outcomes. In 96% of cases, 5q-SMA is caused by a homozygous deletion of SMN1. Around 4 % of patients carry a SMN1 deletion and a single-nucleotide variant (SNV) on the other allele. Traditionally, diagnosis is based on multiplex ligation probe amplification (MLPA) to detect homozygous or heterozygous exon 7 deletions in SMN1. Due to high homologies within the SMN1/SMN2 locus, sequence analysis to identify SNVs of the SMN1 gene is unreliable by standard Sanger or short-read next-generation sequencing (srNGS) methods. The objective was to overcome the limitations in high-throughput srNGS with the aim of providing SMA patients with a fast and reliable diagnosis to enable their timely therapy. A bioinformatics workflow to detect homozygous SMN1 deletions and SMN1 SNVs on srNGS analysis was applied to diagnostic whole exome and panel testing for suggested neuromuscular disorders (1684 patients) and to fetal samples in prenatal diagnostics (260 patients). SNVs were detected by aligning sequencing reads from SMN1 and SMN2 to an SMN1 reference sequence. Homozygous SMN1 deletions were identified by filtering sequence reads for the ,, gene-determining variant" (GDV). 10 patients were diagnosed with 5q-SMA based on (i) SMN1 deletion and hemizygous SNV (2 patients), (ii) homozygous SMN1 deletion (6 patients), and (iii) compound heterozygous SNVs in SMN1 (2 patients). Applying our workflow in srNGS-based panel and whole exome sequencing (WES) is crucial in a clinical laboratory, as otherwise patients with an atypical clinical presentation initially not suspected to suffer from SMA remain undiagnosed.

Targeted Sanger Sequencing of a Cluster of COVID-19 Cases in the Surgical ICU of a Non-COVID Hospital: Lessons Learned.

Small clusters of infection due to SARS-CoV-2 in a non-COVID-19 healthcare facility can disrupt services. Here, we investigated a cluster of SARS-CoV-2 cases by targeted Sanger sequencing and clinical epidemiological methods in a non-COVID-19 super-specialty hospital. Epidemiological data were collected in a blinded manner using a proforma to find the risk factors associated with infection. Targeted Sanger sequencing of the spike protein receptor binding domain (RBD) coding region was performed on all the available real-time reverse transcription polymerase chain reaction (RT-PCR)-positive samples that included a patient, his mother, and 11 healthcare workers (HCWs) to determine any genomic variations in the samples from the cluster. All positive cases were due to the Delta variant. However, it detected a unique mutation, N501I, in the RBD region of the SARS-CoV-2 strains. The viral genome extracted from the mother's sample lacked the mutation, thus excluding her from the cluster and pointing out that the outbreak was nosocomial, leading to a focus on infection control measures. Though whole genome sequencing is more universally accepted, in this study, targeted sanger sequencing provided a rapid and cost-effective solution to correctly delineate between the actual cases that form the cluster and other community cases in a pandemic situation.

Absence of the influence of the APOE gene on the incidence of type 2 diabetes mellitus in a cohort of workers: Effect of diet and shift work

BackgroundAPOE gene encoded a multifunctional protein in lipid metabolism, also associated with inflammatory markers. Type 2 diabetes (T2D) is a complex metabolic disease related to increased blood glucose, triglycerides and VLDL and associated with different dyslipidaemias. The aim of this study was to analyze whether the APOE genotype could determining the risk of developing T2D in a large cohort of workers. Material and methodsData from the Aragon Workers Health Study (AWHS) (n=4895) were used to investigate the relationship between glycemic levels and APOE genotype. All patients in the AWHS cohort had their blood drawn after an overnight fast and laboratory tests were performed on the same day as the blood drawn. Dietary and physical assessment was assessed by face-to-face interview. APOE genotype was determined by the Sanger sequencing method. ResultsThe relationship between APOE genotype and glycemic profile showed that glucose, Hb1Ac, insulin and HOMA levels did not seem to be associated with the APOE genotype (p=0.563, p=0.605, p=0.333 and p=0.276, respectively). In addition, the T2D prevalence did not show an association with the APOE genotype (p=0.354). Along the same lines, blood glucose levels and T2D prevalence did not show association with the APOE allele. Shift work had some effect on the glycaemic profile, showing that night shift workers have significantly lower levels of glucose, insulin and HOMA (p<0.001). However, the APOE genotype did not show difference in the concentration of glycaemic parameters adjusting by sex, age and BMI, work shift and dietary parameters. ConclusionGlycemic profile and T2D prevalence did not show any significant association with the APOE genotype. Besides, individuals, who worked in non-rotating night shift showed significantly lower glycemic levels, while workers in the morning-afternoon-night shift showed significantly higher values.

Screening of IL-22 first and second introns and FOXP3 second exon for SNPs and mutations with potential role in the susceptibility of SLE in selected population.

The main objective of performing this study was the mutational analysis of Forkhead box family member (FoxP3) and Interleukin-22 (IL-22) genes and their associations with systemic lupus erythematosus (SLE). A total of sixty blood samples were collected from SLE patients from different hospitals in Lahore. Proforma was based on American College of Rheumatology (ACR) criteria. The total time for this research was one year (2018-2019). DNA was extracted, and FoxP3 and IL-22 genes were polymerized through PCR and further sequenced through the Sanger Sequencing method. Chromas version 2.6.6 was used for the similarity index of sequences. NG_060763 and NG_007392.1 were used as Reference Sequences of IL-22 and FoxP3 genes, respectively. Three already identified Single Nucleotide Polymorphisms (SNPs) in the IL-22 gene i.e., rs2227491, rs2227485, and rs2227513, were confirmed in the sequencing results of SLE patients. Results showed that there were nine novel mutations (27.27%) in the case of the IL-22 gene in the studied genotyped samples. These SNPs had remarkably increased allele T frequency in rs2227485 and allele C frequency in rs2227491 and rs2227513. On the other hand, in the case of FoxP3 gene exon 2, there was an addition of T at position 10 in the intronic portion, thus not involved in the progression of the disease. The importance of cytokine-mediated signaling pathways, such as the IL-22 gene, is thus established. Novel variants in the IL-22 gene likely contributed significantly to the development of this autoimmune disorder. The current study found that the dysregulation of the inflammatory markers in SLE is not related to the FoxP3 gene, even though FoxP3 is implicated in the tolerance process.

Deletions of SMNI gene exon 7 and NAIP gene exon 5 in spinal muscular atrophy patients in selected population.

Spinal muscular atrophy (SMA) is common among various populations because the genetic makeup is monogamous due to consanguineous marriages. Two genes, i.e., survival motor neuron (SMN1) and neuronal apoptosis inhibitory protein (NAIP) are mapped to the SMA vicinity of chromosome 5q13. The main objective of the study was to develop a solitary advanced genetic tool for the diagnosis of SMA by using SMN1 gene exon 7 and NAIP gene exon 5. This study involved SMA patients (n=84) belonging to different clinical features and socio-economic status. The identity of the intact NAIP gene is primarily based on the amplification of exon 5 only in those SMA patients that have a deletion of SMN1 gene exon 7. Healthy controls (n=84) were also included in this study. The mutational analysis was observed through the Sanger sequencing method, where chromatograms were observed by using Chromas version 2.6.0. This study showed a higher prevalence of SMA in females than in males. NAIP gene is considered a phenotype modifier as most SMA patients (94.90%) have SMN1 exon 7 deletion along with a deletion in exon 5 of the NAIP gene. Single nucleotide conversion C-T in exon 7 of SMN1 gene leads to its complete deletion. Mutated proteins encoded by SMN1 and NAIP genes also result in degeneration and muscle weakness in SMA patients. These SMA-associated gene deletions can be used as a molecular evaluation tool for pre- and postnatal diagnosis of SMA. This will be valuable when there is a need for precise and consistent results with a strong focus on quantification.

Polymorphism of CSN2 Gene Exon 7 in Indonesian Dairy Goat Breeds

Milk production and composition are the main factors in the dairy industry. However, these traits are influenced by several factors, such as genetic factors. The CSN2 gene exon 7 is one of the genes that can affect the protein composition of goat milk. Several studies on the CSN2 gene exon seven have been conducted on Italian and Indian goat breeds, and their genetic variations have been found. Studies about CSN2 gene exon seven polymorphisms in Indonesia have been completed but found no variations. Therefore, this study aims to find genetic variations of CSN2 gene exon 7 using the Sanger Sequencing Method. Ninety-five blood samples of 66 Sapera, 15 Saanen, and 14 Etawa Grade goats were collected from the Livestock Research Center, Ciawi. Genetic diversity was calculated using PopGene32 programs. The results found two polymorphic SNPs in all three dairy goat breeds, namely g.8946C&gt;T and g.8956G&gt;A. Three genotypes found in SNP g.8946C&gt;T are CC, CT, and TT, while a new SNP g.8956G&gt;A found two genotypes (GG and GA). In conclusion, SNPs g.8946C&gt;T and g.8956G&gt;A are polymorphic. Additional research should be conducted to determine whether there is any association between these 2 SNPs and milk production and quality.

Development, Identification and Validation of a Novel SSR Molecular Marker for Heat Resistance of Grapes Based on miRNA

The adverse effects of high temperatures on fruit quality and yield have been reported in many studies of grapevines. MiRNAs play crucial roles in plant growth and development and also fulfill functions in regulating the high-temperature response. In this research, miRNA-based SSR (simple sequence repeat) polymorphisms were analyzed according to the grape genome sequence and then combined with polymerase chain reaction (PCR) amplification and Sanger sequencing methods to analyze the miRNA-SSR diversity of different heat-resistant grape varieties. A statistical analysis of SSR sequences showed that 391 and 12 SSRs were specific to pri-miRNA and pre-miRNA, respectively. In pri-miRNA containing SSR, hexa-nucleotide repeats were the most abundant (52.69%), followed by tetra-nucleotide (13.04%) and minimum penta-nucleotide (4.09%), which were also observed in pre-miRNA sequences. On the other hand, differences in heat resistance among grape varieties were observed. Based on the results of leaf Fv/Fm images, values and phenotypic changes under high-temperature stress, 20 heat-resistant (e.g., Niagara Rosada and Grand Noir) and 20 heat-sensitive (e.g., Shine Muscat and Jumeigui) grape varieties were identified. Further, PCR-Sanger sequencing was used to screen SSR differences in four thermos-tolerant and four thermos-sensitive grape varieties, and finally, eight SSR differential primers were found to be able to distinguish these varieties. Of these, four pairs of primers were selected for validation in 40 grape germplasm resources (20 thermo-tolerance and 20 thermo-sensitivity). The VMIRSSR167c3 highlights that the ratio of SSR (CT) repeat values greater than 17 in heat-resistant varieties was 90%. In conclusion, the VMIRSSR167c3 marker can accurately distinguish between heat-resistant and heat-sensitive grape varieties. The results provide a novel molecular marker for the genetic improvement of grape germplasm resources and will be beneficial to the breeding of heat-resistant varieties in the future.

Long-Read Sequencing with Hierarchical Clustering for Antiretroviral Resistance Profiling of Mixed Human Immunodeficiency Virus Quasispecies.

HIV infections often develop drug resistance mutations (DRMs), which can increase the risk of virological failure. However, it has been difficult to determine if minor mutations occur in the same genome or in different virions using Sanger sequencing and short-read sequencing methods. Oxford Nanopore Technologies (ONT) sequencing may improve antiretroviral resistance profiling by allowing for long-read clustering. A new ONT sequencing-based method for profiling DRMs in HIV quasispecies was developed and validated. The method used hierarchical clustering of long amplicons that cover regions associated with different types of antiretroviral drugs. A gradient series of an HIV plasmid and 2 plasma samples was prepared to validate the clustering performance. The ONT results were compared to those obtained with Sanger sequencing and Illumina sequencing in 77 HIV-positive plasma samples to evaluate the diagnostic performance. In the validation study, the abundance of detected quasispecies was concordant with the predicted result with the R2 of > 0.99. During the diagnostic evaluation, 59/77 samples were successfully sequenced for DRMs. Among 18 failed samples, 17 were below the limit of detection of 303.9 copies/μL. Based on the receiver operating characteristic analysis, the ONT workflow achieved an F1 score of 0.96 with a cutoff of 0.4 variant allele frequency. Four cases were found to have quasispecies with DRMs, in which 2 harbored quasispecies with more than one class of DRMs. Treatment modifications were recommended for these cases. Long-read sequencing coupled with hierarchical clustering could differentiate the quasispecies resistance profiles in HIV-infected samples, providing a clearer picture for medical care.

Analysis of CSF3R Gene Mutations and Clinical Characteristics in Patients with t(8;21) Acute Myeloid Leukemia

To investigate the occurrence of CSF3R mutation in patients with t(8;21) acute myeloid leukemia (AML) and its correlation with some clinical parameters. The clinical and laboratory data of 167 newly diagnosed AML patients with t(8;21) translocation were analyzed retrospectively. High-throughput DNA sequencing technology combined with Sanger sequencing method was used to detect 112 gene mutations. The occurrence of CSF3R gene mutation and its influence on the remission rate after chemotherapy were analyzed. Among 167 patients with t(8;21) AML, 15 patients (9.0%) carried CSF3R mutations, including 6 cases of membrane proximal region mutations and 9 cases of truncation mutations in the cytoplasmic tail. The most common coexisting mutations of CSF3R were KIT (40.0%), TET2 (33.3%), DNMT3A (26.7%), FLT3 (20.0%), CBL (20.0%), IDH1 (13.3%), etc. Compared with the wild type, the CSF3R mutant group had a higher mutation rate of DNA methylation-related genes（P <0.001）. The median peripheral white blood cell (WBC) count of patients with CSF3R gene mutation was 5.80 (3.20-8.56)×109/L at initial diagnosis, which was significantly lower than 8.80 (5.26-19.92)×109/L of the CSF3R wild-type patients (P =0.017). There was no significant difference between the two groups in sex, median age, FAB classification, hemoglobin level, platelet count, etc. (P >0.05). The CR rate of the CSF3R gene mutation group (100%) was significantly higher than that of the wild-type group (86.8%), but the difference was not statistically significant (P >0.05). The CSF3R gene mutation group had a significantly higher CD19 positive rate and a higher -X rate than the wild group (86.7% vs 47.4%, P =0.004; 33.3% vs 13.2%, P =0.037). There is a high incidence of CSF3R mutation in t (8;21) AML patients. The clinical characteristics and coexisting mutation genes of CSF3R mutation-positive patients are different from those of wild-type patients.

HIV-1 Low-Frequency Variants Identified in Antiretroviral-Naïve Subjects with Virologic Failure after 12 Months of Follow-Up in Panama.

Low-frequency mutations associated with drug resistance have been related to virologic failure in subjects with no history of pre-treatment and recent HIV diagnosis. In total, 78 antiretroviral treatment (ART)-naïve subjects with a recent HIV diagnosis were selected and followed by CD4+ T lymphocytes and viral load tests to detect virologic failure. We sequenced the basal samples retrospectively using next-generation sequencing (NGS), looking for low-frequency mutations that had not been detected before using the Sanger sequencing method (SSM) and describing the response to ART. Twenty-two subjects developed virologic failure (VF), and thirteen of them had at least one drug-resistance mutation associated with Reverse Transcriptase Inhibitors (RTI) and Protease Inhibitors (PIs) at frequency levels ≤ 1%, not detected previously in their basal genotyping test. No resistance mutations were observed to Integrase Strand Transfer Inhibitors (INSTIs). We identified a possible cause of VF in ART-naïve subjects with low-frequency mutations detected. To our knowledge, this is the first evaluation of pre-existing drug resistance for HIV-1 minority variants carried out on ART-naïve people living with HIV/AIDS (PLWHA) by analyzing the HIV-1 pol gene using NGS in the country.

Molecular evidence and phylogenetic delineation of spotted fever group Rickettsia species in Amblyomma ticks from cattle in Gauteng and Limpopo Provinces, South Africa

Objective: To determine the prevalence of tick-borne pathogens with a particular focus on Rickettsia spp. in ticks collected from cattle in Gauteng and Limpopo Provinces, South Africa. Methods: A total of 200 ticks were collected from cattle within the Madala livestock, Pretoria, Gauteng Province and in Mankweng Township, Polokwane, Limpopo Province in 2019. The ticks were morphologically identified and processed individually for a total genomic DNA extraction. Specific primers targetting ompA, ompB, and the 17KDa genes were used for a molecular screening and delineation of Rickettsia from the extracted genetic materials using polymerase chain reaction (PCR) technique. PCR amplicons of positive samples were sequenced bidirectionally using the Sanger sequencing method. Sequences generated were processed and analysed using appropriate bioinformatics software. Results: The ticks were morphologically identified as Amblyomma spp. PCR profiling of the genomic DNA samples revealed the presence of the Rickettsia pathogen in 42 (21%) of the ticks collected from both Provinces. Out of the genes profiled, 14 (7%) were positive for 17KDa, 42 (21%) for ompA and 32 (16%) were positive for ompB genes respectively. The nucleotide blast of the sequenced genomes showed high similarity, as high as 100% with other reference Rickettsia (R.) africae in the GenBank. The phylogenetic analysis of the sequences further validated them as R. africae with their characteristic clustering pattern with related reference sequences. Conclusions: There is an abundance of R. africae in Amblyomma ticks collected from cattle in the study areas. This has serious public health implications as individuals who accidentally get infested with the ticks could acquire R. africae. Hence, adequate precautions in terms of sensitization of farmers about the risk and mass mobilization drive to control the vectors in the areas are highly recommended to safeguard public health.

Comparative analysis of onychomycosis in Puerto Rico using molecular and conventional approaches.

Onychomycosis is the most prevalent nail ailment in adults, accounting for 50% of all nail infections. Dermatophyte fungi are the primary cause, but non-dermatophyte molds (NDM) and yeasts can also cause onychomycosis. It remains important to precisely determine the fungal cause of onychomycosis since the response to current treatments may vary between fungal classes. Real-time polymerase chain reaction (qPCR) has become a widespread tool for detecting fungal organisms for diagnosis due to its sensitivity and ability to detect down to the species level. This retrospective study aims to evaluate the qPCR Onycho+ test for dermatophyte detection using remnants of toenails from a cohort of patients from Puerto Rico. Two hundred forty-two toenail samples submitted for histological examination via Periodic acid Schiff (PAS) staining for suspected onychomycosis were analyzed by the Onycho+ test and Sanger sequencing of the internal transcribed spacer (ITS-2). Compared to the gold standard Sanger sequencing method, the Onycho+ test reported an agreement of 91.39%, a sensitivity of 100% and a specificity of 84.5% in detecting dermatophytes, superior to the histology method which had a 69.53% agreement, 85.1% sensitivity and 57.1% specificity. The distribution of fungal organisms detected in this cohort shows a dermatophyte majority but a higher-than-expected proportion of NDMs. Nails negative for the Onycho+ test and positive for histology were mostly NDMs. This study demonstrates that the clinical performance of the Onycho+ test is superior to histology in detecting dermatophytes and that a combination of Onycho+ and histology can result in a higher clinical accuracy.

Phenotypic and Genetic Features that Differ Between Hereditary and Sporadic Melanoma: Results of a Preliminary Study from a Single Center from Turkey.

Most melanoma patients under our supervision lack characteristic phenotypic features for melanoma. In contrast, history of cancers other than melanoma and early age at onset were common. This observation was in favor of hereditary melanoma. To search for the phenotypic and genetic features that differ between sporadic and hereditary melanomas. In order to reveal phenotypic features, detailed physical exam was conducted to all melanoma patients (N = 43) and for genetic features. CDKN2A and MC1R mutations were detected with Sanger sequencing method. Assignment to hereditary and sporadic groups was done according to the "melanoma cancer syndrome assessment tool". Patients who were diagnosed before the age of 50 were also assigned to the hereditary melanoma group. Thirty-one patients were assigned to the hereditary group and 12 to the sporadic group. Fair eye color was statistically significantly higher in the sporadic group (P = 0.000). CDKN2A was detected in only 1 patient in the hereditary group. MC1R mutations were found in 12 out of 13 (92.3%) in the hereditary group with a score =3 points, 13 out of 18 (72.2%) in the early age at onset group and 5 out of 12 (41.7%) in the sporadic group (P = 0.024). Incidence of CDKN2A mutations in our hereditary group is in accordance with the reported incidences from Mediterranean countries. The difference between the hereditary and sporadic groups in terms of MC1R mutations supports the idea that MC1R genetic testing might help to determine patients with higher risk for hereditary melanoma.

Cdh23 affects congenital hearing loss through regulating purine metabolism.

The pathogenic gene CDH23 plays a pivotal role in tip links, which is indispensable for mechanoelectrical transduction in the hair cells. However, the underlying molecular mechanism and signal regulatory networks that influence deafness is still largely unknown. In this study, a congenital deafness family, whole exome sequencing revealed a new mutation in the pathogenic gene CDH23, subsequently; the mutation has been validated using Sanger sequencing method. Then CRISPR/Cas9 technology was employed to knockout zebrafish cdh23 gene. Startle response experiment was used to compare with wide-type, the response to sound stimulation between wide-type and cdh23-/-. To further illustrate the molecular mechanisms underlying congenital deafness, comparative transcriptomic profiling and multiple bioinformatics analyses were performed. The YO-PRO-1 assay result showed that in cdh23 deficient embryos, the YO-PRO-1 signal in inner ear and lateral line neuromast hair cells were completely lost. Startle response experiment showed that compared with wide-type, the response to sound stimulation decreased significantly in cdh23 mutant larvae. Comparative transcriptomic showed that the candidate genes such as atp1b2b and myof could affect hearing by regulating ATP production and purine metabolism in a synergetic way with cdh23. RT-qPCR results further confirmed the transcriptomics results. Further compensatory experiment showed that ATP treated cdh23-/- embryos can partially recover the mutant phenotype. In conclusion, our study may shed light on deciphering the principal mechanism and provide a potential therapeutic method for congenital hearing loss under the condition of CDH23 mutation.

Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

A novel mutation on OCA2 gene detection and preimplantationgenetic diagnosis for oculocutaneous albinism type 2 (OCA2)

Oculocutaneous albinism (OCA) is a group of recessive genetic congenital disorders characterised by a disorder of melanin synthesis. OCA is currently classified into seven different types, of which OCA type 2 (OCA2 - OMIM 203200) ranks as the second most common type of albinism in the world and accounts for nearly 30% of albinism cases. Nowadays, there is currently no known way to treat OCA. Preimplantation genetic diagnosis (PGD) is the most effective method of disease prevention, which helps families carrying gene mutations to have healthy children. Objectives: To develop a procedure and conduct PGD for embryos of a family carrying the OCA2 mutation causing OCA2. Materials and methods: Research subjects are blood samples of four members of an OCA family, including father, mother, and two daughters; and 6 embryo biopsies on day 5 of this parents. Next-generation sequencing (NGS) was carried out on family members participating in the study. Based on the identified mutation results, primers for PCR amplification of the segments spanning the detected mutation were designed. Then, Sanger sequencing method is combined with linkage genetic analysis to detect pathogenic mutations on embryo biopsies after whole-genome amplification (WGA). Results: A novel mutation NM_000275.3:c.2323G&gt;A (NP_000266.2: p.Gly775Ser) was detected on exon 22 of the OCA2 gene. The outcomes of PGD for 6 embryos of a family carrying the OCA2 mutant gene causing OCA2 were 3 affected embryos, 2 normal embryos, and 1 carriers.