ResearchPad - mutation-detection https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[High prevalence of phenotypic pyrazinamide resistance and its association with <i>pncA</i> gene mutations in <i>Mycobacterium tuberculosis</i> isolates from Uganda]]> https://www.researchpad.co/article/elastic_article_14718 Susceptibility testing for pyrazinamide (PZA), a cornerstone anti-TB drug is not commonly done in Uganda because it is expensive and characterized with technical difficulties thus resistance to this drug is less studied. Resistance is commonly associated with mutations in the pncA gene and its promoter region. However, these mutations vary geographically and those conferring phenotypic resistance are unknown in Uganda. This study determined the prevalence of PZA resistance and its association with pncA mutations.Materials and methodsUsing a cross-sectional design, archived isolates collected during the Uganda national drug resistance survey between 2008–2011 were sub-cultured. PZA resistance was tested by BACTEC Mycobacterial Growth Indicator Tube (MGIT) 960 system. Sequence reads were downloaded from the NCBI Library and bioinformatics pipelines were used to screen for PZA resistance–conferring mutations.ResultsThe prevalence of phenotypic PZA resistance was found to be 21%. The sensitivity and specificity of pncA sequencing were 24% (95% CI, 9.36–45.13%) and 100% (73.54% - 100.0%) respectively. We identified four mutations associated with PZA phenotypic resistance in Uganda; K96R, T142R, R154G and V180F.ConclusionThere is a high prevalence of phenotypic PZA resistance among TB patients in Uganda. The low sensitivity of pncA gene sequencing confirms the already documented discordances suggesting other mechanisms of PZA resistance in Mycobacterium tuberculosis. ]]> <![CDATA[Myotonia congenita and periodic hypokalemia paralysis in a consanguineous marriage pedigree: Coexistence of a novel <i>CLCN1</i> mutation and an <i>SCN4A</i> mutation]]> https://www.researchpad.co/article/elastic_article_14559 Myotonia congenita and hypokalemic periodic paralysis type 2 are both rare genetic channelopathies caused by mutations in the CLCN1 gene encoding voltage-gated chloride channel CLC-1 and the SCN4A gene encoding voltage-gated sodium channel Nav1.4. The patients with concomitant mutations in both genes manifested different unique symptoms from mutations in these genes separately. Here, we describe a patient with myotonia and periodic paralysis in a consanguineous marriage pedigree. By using whole-exome sequencing, a novel F306S variant in the CLCN1 gene and a known R222W mutation in the SCN4A gene were identified in the pedigree. Patch clamp analysis revealed that the F306S mutant reduced the opening probability of CLC-1 and chloride conductance. Our study expanded the CLCN1 mutation database. We emphasized the value of whole-exome sequencing for differential diagnosis in atypical myotonic patients.

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<![CDATA[Identification and detection of a novel point mutation in the Chitin Synthase gene of <i>Culex pipiens</i> associated with diflubenzuron resistance]]> https://www.researchpad.co/article/elastic_article_14502 Diflubenzuron is one of the main larvicides used for the control of the West Nile Virus vector Culex pipiens in the Mediterranean. However, the efficiency of control is now under threat due to the selection of insecticide resistance. Two point mutations were previously identified at the Chitin synthase and shown to confer low and high levels of resistance and a diagnostic was developed to monitor the trait. This study reports the identification of a third mutation associated with high levels of diflubenzuron resistance in Italy. This mutation was also detected in France, whereas no resistance mutations were found in Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. The findings are of major concern for mosquito control programs in S. Europe, which rely on the use of a limited number of larvicides.

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<![CDATA[Presence, persistence and effects of pre-treatment HIV-1 drug resistance variants detected using next generation sequencing: A Retrospective longitudinal study from rural coastal Kenya]]> https://www.researchpad.co/article/5c6dc9f3d5eed0c48452a5bd

Background

The epidemiology of HIV-1 drug resistance (HIVDR) determined by Sanger capillary sequencing, has been widely studied. However, much less is known about HIVDR detected using next generation sequencing (NGS) methods. We aimed to determine the presence, persistence and effect of pre-treatment HIVDR variants detected using NGS in HIV-1 infected antiretroviral treatment (ART) naïve participants from rural Coastal Kenya.

Methods

In a retrospective longitudinal study, samples from HIV-1 infected participants collected prior [n = 2 time-points] and after [n = 1 time-point] ART initiation were considered. An ultra-deep amplicon-based NGS assay, calling for nucleotide variants at >2.0% frequency of viral population, was used. Suspected virologic failure (sVF) was defined as a one-off HIV-1 viral load of >1000 copies/ml whilst on ART.

Results

Of the 50 eligible participants, 12 (24.0% [95% CI: 13.1–38.2]) had at least one detectable pre-treatment HIVDR variant against Protease Inhibitors (PIs, n = 6 [12%]), Nucleoside Reverse Transcriptase Inhibitors (NRTIs, n = 4 [8.0%]) and Non-NRTIs (n = 3 [6.0%]). Overall, 15 pre-treatment resistance variants were detected (frequency, range: 2.3–92.0%). A positive correlation was observed between mutation frequency and absolute load for NRTI and/or NNRTI variants (r = 0.761 [p = 0.028]), but not for PI variants (r = -0.117 [p = 0.803]). Participants with pre-treatment NRTI and/or NNRTI resistance had increased odds of sVF (OR = 6.0; 95% CI = 1.0–36.9; p = 0.054).

Conclusions

Using NGS, pre-treatment resistance variants were common, though observed PI variants were unlikely transmitted, but rather probably generated de novo. Even when detected from a low frequency, pre-treatment NRTI and/or NNRTI resistance variants may adversely affect treatment outcomes.

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<![CDATA[Genotypes of 2579 patients with phenylketonuria reveal a high rate of BH4 non-responders in Russia]]> https://www.researchpad.co/article/5c50c46ad5eed0c4845e872a

Phenylalanine hydroxylase (PAH) deficiency is responsible for most cases of phenylketonuria (PKU). Furthermore, numerous studies on BH4-sensitive PAH deficiency have been conducted. To date, BH4, a cofactor of PAH, has not been used to treat PKU in Russia.Genotype data of patients with PKU can be used to predict their sensitivity to BH4 therapy. A cohort of 2579 patients with PKU from Russia was analyzed for 25 common PAH gene mutations using custom allele-specific multiplex ligation-dependent probe amplification-based technology. A mutation detection rate of 84.1% chromosomes was accomplished. Both pathogenic alleles were identified in 73.1% of patients. The most frequent pathogenic variants were p.Arg408Trp (50.9%), p.Arg261Gln (5.3%), p.Pro281Leu (3.5%), IVS12+1G>A (3.1%), IVS10-11G>A (2.6%), and p.Arg158Leu (2.4%). The exact boundaries of a PAH exon 5 deletion were defined as EX5del4154ins268 (c.442-2913_509+1173del4154ins268). Severe phenotypes prevailed in the cohort, and classical PKU was observed in 71.8% cases. Due to the genotype-based prediction, 55.9% of the probands were non-responders to the BH4-treatment, and 20.2% were potential responders. Analysis of genotype data is useful to predict BH4 response in PKU patients. The high rate of non-responders among Russian patients was due to the high allele frequency of severe PAH mutations.

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<![CDATA[Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing]]> https://www.researchpad.co/article/5c40f7d3d5eed0c484386a4b

Introduction/Objectives

An increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting.

Methods

Forty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced.

Results

Fifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2.

Conclusion

This study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.

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<![CDATA[Wagging the long tail of drivers of prostate cancer]]> https://www.researchpad.co/article/5c61b7c9d5eed0c484937fa7 ]]> <![CDATA[Evolution and mutations predisposing to daptomycin resistance in vancomycin-resistant Enterococcus faecium ST736 strains]]> https://www.researchpad.co/article/5c26972bd5eed0c48470ecf7

We recently identified a novel vancomycin-resistant Enterococcus faecium (VREfm) clone ST736 with reduced daptomycin susceptibility. The objectives of this study were to assess the population dynamics of local VREfm strains and genetic alterations predisposing to daptomycin resistance in VREfm ST736 strains. Multilocus sequence typing and single nucleotide variant data were derived from whole-genome sequencing of 250 E. faecium isolates from 1994–1995 (n = 43), 2009–2012 (n = 115) and 2013 (n = 92). A remarkable change was noticed in the clonality and antimicrobial resistance profiles of E. faecium strains between 1994–1995 and 2013. VREfm sequence type 17 (ST17), the prototype strain of clade A1, was the dominant clone (76.7%) recognized in 1994–1995. By contrast, clone ST736 accounted for 46.7% of VREfm isolates, followed by ST18 (26.1%) and ST412 (20.7%) in 2013. Bayesian evolutionary analysis suggested that clone ST736 emerged between 1996 and 2009. Co-mutations (liaR.W73C and liaS.T120A) of the liaFSR system were identified in all ST736 isolates (n = 111, 100%) examined. Thirty-eight (34.2%) ST736 isolates exhibited daptomycin-resistant phenotype, of which 13 isolates had mutations in both the liaFSR and cardiolipin synthase (cls) genes and showed high level of resistance with a daptomycin MIC50 of 32 μg/mL. The emergence of ST736 strains with mutations predisposing to daptomycin resistance and subsequent clonal spread among inpatients contributed to the observed high occurrence of daptomycin resistance in VREfm at our institution. The expanding geographic distribution of ST736 strains in other states and countries raises concerns about its global dissemination.

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<![CDATA[Mechanisms of acquired resistance to afatinib clarified with liquid biopsy]]> https://www.researchpad.co/article/5c1d5b8ed5eed0c4846ebefb

Although mechanisms of acquired resistance to 1st and 3rd generation EGFR-TKI continue to be elucidated, there have been few clinical investigations into the mechanisms of acquired resistance to the 2nd generation EGFR-TKI afatinib. We analyzed data from 20 patients with advanced lung adenocarcinoma who acquired resistance to afatinib, including resistance during EGFR-TKI re-challenge. We examined EGFR T790M and C797S mutations, BRAF V600E mutation, and MET amplification with the MBP-QP method and with droplet digital PCR using ctDNA and re-biopsy samples obtained before and after afatinib treatment. Just before afatinib treatment, 15 of the 20 patients were T790M negative and five were positive. Among the T790M negative patients, 40.0% (6/15) became positive at the time of PD under afatinib. In patients positive for T790M, changes in T790M allele frequency were correlated with afatinib treatment efficacy. C797S was not detected in any patients just before afatinib treatment, but it appeared after treatment in three patients, although with very low allele frequency. Two of these three patients, although positive for both C797S and T790M, achieved PR to osimertinib. However, PFS of these patients was somewhat shorter than that of patients positive for T790M only. BRAF V600E was detected in one patient at PD under afatinib. MET amplification was not detected in this study. T790M is associated with acquired resistance to afatinib, as with 1st generation EGFR-TKI, but with somewhat lower frequency. The influence of C797S on resistance to afatinib is less than that of T790M, but C797S might cause shorter PFS under osimertinib.

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<![CDATA[Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ]]> https://www.researchpad.co/article/5bfc6269d5eed0c484ec8fa8

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis.

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<![CDATA[New mutations found by Next-Generation Sequencing screening of Spanish patients with Nemaline Myopathy]]> https://www.researchpad.co/article/5c117b73d5eed0c484699464

Nemaline Myopathy (NM) is a rare genetic disorder that encompasses a large spectrum of myopathies characterized by hypotonia and generalized muscle weakness. To date, mutations in thirteen different genes have been associated with NM. The most frequently responsible genes are NEB (50% of cases) and ACTA1 (15–25% of cases). In this report all known NM related genes were screened by Next Generation Sequencing in five Spanish patients in order to genetically confirm the clinical and histological diagnosis of NM. Four mutations in NEB (c.17779_17780delTA, c.11086A>C, c.21076C>T and c.2310+5G>A) and one mutation in ACTA1 (c.871A>T) were found in four patients. Three of the four mutations in NEB were novel. A cDNA sequencing assay of the novel variants c.17779_17780delTA, c.11086A>C and c.2310+5G>A revealed that the intronic variant c.2310+5G>A affected the splicing process. Mutations reported here could help clinicians and geneticists in NM diagnosis.

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<![CDATA[Multiple configurations of EGFR exon 20 resistance mutations after first- and third-generation EGFR TKI treatment affect treatment options in NSCLC]]> https://www.researchpad.co/article/5c06f036d5eed0c484c6d3c8

After sequential treatment with first- and third-generation EGFR tyrosine kinase inhibitors (TKIs), EGFR-mutant non-small cell lung cancers frequently harbor multiple resistance mutations in exon 20 of EGFR including T790M, mediating resistance to first-generation TKIs, and at codons 792, 796, or 797 mediating resistance to third-generation TKIs. However, whether these resistance mutations are in cis or trans has therapeutic implications for patients. We analyzed a cohort of 29 patients with NSCLC harboring EGFR mutations at codons 792, 796, or 797 to establish the configuration of these mutations. We performed hybrid capture-based, next-generation sequencing on formalin-fixed paraffin-embedded biopsy tissue or liquid biopsy. 27 samples had both a T790M mutation and a mutation at codons 792, 796, or 797. In all of these cases, the mutations were found in the cis configuration; the trans configuration was not observed. Two patients’ samples harbored a mutation at codon 797 but no T790M mutation. In these two cases, longitudinal analysis showed earlier biopsies harbored EGFR T790M, which was undetectable following osimertinib treatment. Treatment of one these patients with both first- and third-generation EGFR TKIs resulted in a mixed response. Here we describe multiple configurations of EGFR T790M and third-generation TKI resistance mutations at codons 792, 796, and 797. These mutations are most commonly found in cis, which confers resistance to all current EGFR TKIs. We also describe two patients that exhibited T790M loss with acquisition of a mutation at codon 797. In addition, one of these patients, with an EGFR C797S in a lung biopsy was subsequently found to have EGFR C797N in a later biopsy of pleural fluid, highlighting the dynamic multiclonal nature of advanced NSCLC.

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<![CDATA[Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues]]> https://www.researchpad.co/article/5b28b870463d7e14181b184d

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

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<![CDATA[Exome Sequencing Identifies a Novel LMNA Splice-Site Mutation and Multigenic Heterozygosity of Potential Modifiers in a Family with Sick Sinus Syndrome, Dilated Cardiomyopathy, and Sudden Cardiac Death]]> https://www.researchpad.co/article/5989db3bab0ee8fa60bd4e06

The goals are to understand the primary genetic mechanisms that cause Sick Sinus Syndrome and to identify potential modifiers that may result in intrafamilial variability within a multigenerational family. The proband is a 63-year-old male with a family history of individuals (>10) with sinus node dysfunction, ventricular arrhythmia, cardiomyopathy, heart failure, and sudden death. We used exome sequencing of a single individual to identify a novel LMNA mutation and demonstrated the importance of Sanger validation and family studies when evaluating candidates. After initial single-gene studies were negative, we conducted exome sequencing for the proband which produced 9 gigabases of sequencing data. Bioinformatics analysis showed 94% of the reads mapped to the reference and identified 128,563 unique variants with 108,795 (85%) located in 16,319 genes of 19,056 target genes. We discovered multiple variants in known arrhythmia, cardiomyopathy, or ion channel associated genes that may serve as potential modifiers in disease expression. To identify candidate mutations, we focused on ~2,000 variants located in 237 genes of 283 known arrhythmia, cardiomyopathy, or ion channel associated genes. We filtered the candidates to 41 variants in 33 genes using zygosity, protein impact, database searches, and clinical association. Only 21 of 41 (51%) variants were validated by Sanger sequencing. We selected nine confirmed variants with minor allele frequencies <1% for family studies. The results identified LMNA c.357-2A>G, a novel heterozygous splice-site mutation as the primary mutation with rare or novel variants in HCN4, MYBPC3, PKP4, TMPO, TTN, DMPK and KCNJ10 as potential modifiers and a mechanism consistent with haploinsufficiency.

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<![CDATA[Clinical and Molecular Characterization of BSCL2 Mutations in a Taiwanese Cohort with Hereditary Neuropathy]]> https://www.researchpad.co/article/5989d9d4ab0ee8fa60b6563a

Background

A small group of patients with inherited neuropathy that has been shown to be caused by mutations in the BSCL2 gene. However, little information is available about the role of BSCL2 mutations in inherited neuropathies in Taiwan.

Methodology and Principal Findings

Utilizing targeted sequencing, 76 patients with molecularly unassigned Charcot-Marie-Tooth disease type 2 (CMT2) and 8 with distal hereditary motor neuropathy (dHMN), who were selected from 348 unrelated patients with inherited neuropathies, were screened for mutations in the coding regions of BSCL2. Two heterozygous BSCL2 mutations, p.S90L and p.R96H, were identified, of which the p.R96H mutation is novel. The p.S90L was identified in a pedigree with CMT2 while the p.R96H was identified in a patient with apparently sporadic dHMN. In vitro studies demonstrated that the p.R96H mutation results in a remarkably low seipin expression and reduced cell viability.

Conclusion

BSCL2 mutations account for a small number of patients with inherited neuropathies in Taiwan. The p.R96H mutation is associated with dHMN. This study expands the molecular spectrum of BSCL2 mutations and also emphasizes the pathogenic role of BSCL2 mutations in molecularly unassigned hereditary neuropathies.

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<![CDATA[Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi, India]]> https://www.researchpad.co/article/5989d9f5ab0ee8fa60b6fef5

Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011–2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011–2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.

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<![CDATA[Precision oncology: Charting a path forward to broader deployment of genomic profiling]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcde6

In this Perspective, David Hyman and coauthors discuss clinical research on the application of genomic information in oncology.

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<![CDATA[Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be02c9

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes.

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<![CDATA[Early mutation bursts in colorectal tumors]]> https://www.researchpad.co/article/5989db50ab0ee8fa60bdbd9d

Tumor growth is an evolutionary process involving accumulation of mutations, copy number alterations, and cancer stem cell (CSC) division and differentiation. As direct observation of this process is impossible, inference regarding when mutations occur and how stem cells divide is difficult. However, this ancestral information is encoded within the tumor itself, in the form of intratumoral heterogeneity of the tumor cell genomes. Here we present a framework that allows simulation of these processes and estimation of mutation rates at the various stages of tumor development and CSC division patterns for single-gland sequencing data from colorectal tumors. We parameterize the mutation rate and the CSC division pattern, and successfully retrieve their posterior distributions based on DNA sequence level data. Our approach exploits Approximate Bayesian Computation (ABC), a method that is becoming widely-used for problems of ancestral inference.

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<![CDATA[Development of Antibiotic Resistance during Simulated Treatment of Pseudomonas aeruginosa in Chemostats]]> https://www.researchpad.co/article/5989da61ab0ee8fa60b9106e

During treatment of infections with antibiotics in critically ill patients in the intensive care resistance often develops. This study aims to establish whether under those conditions this resistance can develop de novo or that genetic exchange between bacteria is by necessity involved. Chemostat cultures of Pseudomonas aeruginosa were exposed to treatment regimes with ceftazidime and meropenem that simulated conditions expected in patient plasma. Development of antibiotic resistance was monitored and mutations in resistance genes were searched for by sequencing PCR products. Even at the highest concentrations that can be expected in patients, sufficient bacteria survived in clumps of filamentous cells to recover and grow out after 3 to 5 days. At the end of a 7 days simulated treatment, the minimal inhibitory concentration (MIC) had increased by a factor between 10 and 10,000 depending on the antibiotic and the treatment protocol. The fitness costs of resistance were minimal. In the resistant strains, only three mutations were observed in genes associated with beta-lactam resistance. The development of resistance often observed during patient treatment can be explained by de novo acquisition of resistance and genetic exchange of resistance genes is not by necessity involved. As far as conclusions based on an in vitro study using P. aeruginosa and only two antibiotics can be generalized, it seems that development of resistance can be minimized by treating with antibiotics in the highest concentration the patient can endure for the shortest time needed to eliminate the infection.

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