Mosaic Gene Research Articles

Genetic Variability in Neisseria meningitidis Strains Isolated in A Japanese Hospital

Neisseria meningitidis is a significant pathogen causing invasive meningococcal disease, posing clinical and public health concerns worldwide. This study aimed to investigate the genetic characteristics of N. meningitidis clinical isolates at Okayama University Hospital in Japan. Between 2018 and 2023, five clinical strains were isolated, of which three were subjected to the antimicrobial susceptibility testing and whole genetic analysis using MiSeq platform (Illumina, San Diego, CA, USA). One non-groupable isolate, belonging to sequence types (STs)-11026 (ST-32 complex), exhibited non-susceptibility to penicillin G, with a five-mutation pattern (F504L, A510V, I515V, H541N, I566V) in the penA amino acid sequence and additional mutations (XXXIV, N513Y) characteristic of a mosaic penA gene. The other two isolates, ST-1655 (ST-23 complex) with serogroup Y and ST-2057 with serogroup B, were susceptible to penicillin G, neither of which contained the five-mutation pattern. Levofloxacin resistance was observed in two isolates carrying T91I mutation in the gyrA protein. Our findings suggest the presence of antimicrobial-resistant N. meningitidis in Japan, underscoring the necessity for continuous local surveillance. Additional research is crucial for clarifying the ongoing spread of resistance mechanisms and for establishing effective countermeasures to reduce the clinical burden of invasive meningococcal disease.

A deep-learning-based histopathology classifier for focal cortical dysplasia (FCD) unravels a complex scenario of comorbid FCD subtypes.

Recently, we developed a first artificial intelligence (AI)-based digital pathology classifier for focal cortical dysplasia (FCD) as defined by the ILAE classification. Herein, we tested the usefulness of the classifier in a retrospective histopathology workup scenario. Eighty-six new cases with histopathologically confirmed FCD ILAE type Ia (FCDIa), FCDIIa, FCDIIb, mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE), or mild malformations of cortical development were selected, 20 of which had confirmed gene mosaicism. The classifier always recognized the correct histopathology diagnosis in four or more 1000 × 1000-μm digital tiles in all cases. Furthermore, the final diagnosis overlapped with the largest batch of tiles assigned by the algorithm to one diagnostic entity in 80.2% of all cases. However, 86.2% of all cases revealed more than one diagnostic category. As an example, FCDIIb was identified in all of the 23 patients with histopathologically assigned FCDIIb, whereas the classifier correctly recognized FCDIIa tiles in 19 of these cases (83%), that is, dysmorphic neurons but no balloon cells. In contrast, the classifier misdiagnosed FCDIIb tiles in seven of 23 cases histopathologically assigned to FCDIIa (33%). This mandates a second look by the signing histopathologist to either confirm balloon cells or differentiate from reactive astrocytes. The algorithm also recognized coexisting architectural dysplasia, for example, vertically oriented microcolumns as in FCDIa, in 22% of cases classified as FCDII and in 62% of cases with MOGHE. Microscopic review confirmed microcolumns in the majority of tiles, suggesting that vertically oriented architectural abnormalities are more common than previously anticipated. An AI-based diagnostic classifier will become a helpful tool in our future histopathology laboratory, in particular when large anatomical resections from epilepsy surgery require extensive resources. We also provide an open access web application allowing the histopathologist to virtually review digital tiles obtained from epilepsy surgery to corroborate their final diagnosis.

A Simple Model to Study Mosaic Gene Expression in 3D Endothelial Spheroids.

The goal of this study was to establish a simple model of 3D endothelial spheroids with mosaic gene expression using adeno-associated virus (AAV) transduction, with a future aim being to study the activity of post-zygotic mutations common to vascular malformations. In this study, 96-well U-bottom plates coated with a commercial repellent were seeded with two immortalized human endothelial cell lines and aggregation monitored using standard microscopy or live-cell analysis. The eGFP expression was used to monitor the AAV transduction. HUVEC-TERT2 could not form spheroids spontaneously. The inclusion of collagen I in the growth medium could stimulate cell aggregation; however, these spheroids were not stable. In contrast, the hCMEC/D3 cells aggregated spontaneously and formed reproducible, robust 3D spheroids within 3 days, growing steadily for at least 4 weeks without the need for media refreshment. The hCMEC/D3 spheroids spontaneously developed a basement membrane, including collagen I, and expressed endothelial-specific CD31 at the spheroid surface. Serotypes AAV1 and AAV2QUADYF transduced these spheroids without toxicity and established sustained, mosaic eGFP expression. In the future, this simple approach to endothelial spheroid formation combined with live-cell imaging could be used to rapidly assess the 3D phenotypes and drug and radiation sensitivities arising from mosaic mutations common to brain vascular malformations.

Transgenic expression of artificial microRNA targeting soybean mosaic virus P1 gene confers virus resistance in plant.

RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.

An analysis of semaphorin-mediated cellular interactions in the Caenorhabditis elegans epidermis using the IR-LEGO single-cell gene induction system.

One of the major functions of the semaphorin signaling system is the regulation of cell shape. In the nematode Caenorhabditis elegans, membrane-bound semaphorins SMP-1/2 (SMPs) regulate the morphology of epidermal cells via their receptor plexin, PLX-1. In the larval male tail of the SMP-PLX-1 signaling mutants, the border between two epidermal cells, R1.p and R2.p, is displaced anteriorly, resulting in the anterior displacement of the anterior-most ray, ray 1, in the adult male. To elucidate how the intercellular signaling mediated by SMPs regulates the position of the intercellular border, we performed mosaic gene expression analyses by using infrared laser-evoked gene operator (IR-LEGO). We show that PLX-1 expressed in R1.p and SMP-1 expressed in R2.p are required for the proper positioning of ray 1. The result suggests that SMP signaling promotes extension, rather than retraction, of R1.p. This is in contrast to a previous finding that SMPs mediate inhibition of cell extension of vulval precursor cells, another group of epidermal cells of C. elegans, indicating the context dependence of cell shape control via the semaphorin signaling system.

An enzyme-free and ratiometric biosensor for single nucleotide polymorphism with portable electrochemical system

Portable, low-cost and efficient biosensing systems are valuable for single nucleotide polymorphisms (SNPs) analysis, particularly in resource-limited fields. Herein, a rapid, enzyme-free and ratiometric SNPs biosensor with portable electrochemical system enabling simultaneous recognition and signal amplification is proposed. To demonstrate its versatility and effectiveness, we have selected the human cancer-related KRAS gene and the soybean mosaic virus resistance Rsv3 gene as our analysis models. Our biosensor is constructed based on a combination of strand displacement reactions and hybridization chain reactions (HCR). This unique design enables to differentiate between mutant (MT) and wild (WD) sequences based on the differential release of HCR products on the electrode surfaces. Importantly, this enzyme-free SNPs biosensor concurrently achieves both SNPs recognition and signal amplification, significantly reducing the reaction time to just 40 minutes. Furthermore, we have developed a miniaturized biosensor system that incorporates screen printed gold electrodes (SPGE) and smartphones. This system is integrated with a ratiometric strategy with an limit of detection as low as 1.2 fM, enhancing accuracy and eliminating external interference. This simple SNPs biosensing system possesses high reliability, positioning it as a versatile biosensor for the determination of various SNPs mutations.

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BackgroundCampylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide.MethodsWe systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids.ResultsOverall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3’)-IIIa, aph(2’’)-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter.ConclusionsThe results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Easy-to-Use CRISPR-Cas9 Genome Editing in the Cultured Pacific Abalone (Haliotis discus hannai).

The Pacific abalone is an important aquaculture shellfish and serves as an important model in basic biology study. However, the study of abalone is limited by lack of highly efficient and easy-to-use gene-editing tools. In this paper, we demonstrate efficient gene knockout in Pacific abalone using CRISPR-Cas9. We developed a highly effective microinjection method by nesting fertilized eggs in a low-concentration agarose gel. We identified the cilia developmental gene β-tubulin and light-sensitive transmembrane protein r-opsin as target genes and designed highly specific sgRNAs for modifying their genomic sequences. Sanger sequencing of the genomic regions of β-tubulin and r-opsin genes from injected larvae identified various genomic long-fragment deletions. In situ hybridization showed gene expression patterns of β-tubulin and r-opsin were significantly altered in the mosaic mutants. Knocking out β-tubulin in abalone embryos efficiently affected cilia development. Scanning electron microscopy and swimming behavior assay showed defecting cilia and decreased motility. Moreover, knocking out of r-opsin in abalone embryos effectively affected the expression and development of eyespots. Overall, this work developed an easy-to-use mosaic gene knockout protocol for abalone, which will allow researchers to utilize CRISPR-Cas9 approaches to study unexploited abalone biology and will lead to novel breeding methods for this aquaculture species.

Comparative genomic analysis of uropathogenic Escherichia coli strains from women with recurrent urinary tract infection.

Recurrent urinary tract infections (RUTIs) caused by uropathogenic Escherichia coli are costly public health problems impacting patients' quality of life. In this work, a comparative genomics analysis of three clinical RUTI strains isolated from bladder biopsy specimens was performed. One hundred seventy-two whole genomes of urinary tract E. coli strains were selected from the NCBI database. The search for virulence factors, fitness genes, regions of interest, and genetic elements associated with resistance was manually carried out. The phenotypic characterization of antibiotic resistance, haemolysis, motility, and biofilm formation was performed. Moreover, adherence and invasion assays with human bladder HTB-5 cells, and transmission electron microscopy (TEM) were performed. The UTI-1_774U and UTI-3_455U/ST1193 strains were associated with the extraintestinal pathotypes, and the UTI-2_245U/ST295 strain was associated with the intestinal pathotype, according to a phylogenetic analysis of 172 E. coli urinary strains. The three RUTI strains were of clinical, epidemiological, and zoonotic relevance. Several resistance genes were found within the plasmids of these strains, and a multidrug resistance phenotype was revealed. Other virulence genes associated with CFT073 were not identified in the three RUTI strains (genes for type 1 and P fimbriae, haemolysin hlyA, and sat toxin). Quantitative adherence analysis showed that UTI-1_774U was significantly (p < 0.0001) more adherent to human bladder HTB-5 cells. Quantitative invasion analysis showed that UTI-2_245U was significantly more invasive than the control strains. No haemolysis or biofilm activity was detected in the three RUTI strains. The TEM micrographs showed the presence of short and thin fimbriae only in the UTI-2_245U strain. The high variability and genetic diversity of the RUTI strains indicate that are a mosaic of virulence, resistance, and fitness genes that could promote recurrence in susceptible patients.

Neisseria meningitidis with decreased susceptibility to penicillin G and molecular characterization of the penA gene in strains isolated at University Hospital Centers of Casablanca and Marrakech (Morocco).

the laboratory diagnosis of meningococcal meningitis relies on conventional techniques. This study aims to evaluate the correlation between the reduced sensitivity to penicillin G of Neisseria meningitidis (N.m) strains and the expression of the altered PBP 2 gene. out of 190 strains of N.m isolated between 2010 and 2021 at the bacteriology laboratories of Ibn Rochd University Hospital Centre (IR-UHC) in Casablanca and the UHC Mohammed VI in Marrakech, 23 isolates were part of our study. We first determined their state of sensitivity to penicillin G by E-Test strips and searched for the expression of the penA gene by PCR followed by Sanger sequencing. of all the confirmed cases of N.m, 93.15% (n=177) are of serogroup B, 75.2% (n = 143) are sensitive to penicillin G and 24.73% (n = 47) are of intermediate sensitivity. No resistance to penicillin G was observed. Reduced sensitivity to penicillin G in N.m is characterized by mutations namely F504 L, A510 V, I515 V, G541 N and I566 V located in the C-terminal region of the penA gene encoding the penicillin-binding protein 2 (PBP2) (mosaic gene). our study presents useful data for the phenotypic and genotypic monitoring of resistance to penicillin G in N.m and can contribute to the analysis of genetic exchanges between different Neisseria species.

Resistant Gene Analogue Marker(S) Screening against Yellow Mosaic Disease in Mungbean [Vigna radiata (L.) Wilczek

During Rabi 2021–2022, 100 genotypes of mungbean were tested for resistance to yellow mosaic disease in the field using the disease grading system. Out of the 100 mungbean genotypes, 49 were resistant to the yellow mosaic disease, 22 as Moderately resistant, 11 as Moderately susceptible, 3 as susceptible, and 15 were highly susceptible. The sequenced virus shares 98.94% similarity with the whole coding sequence of the MYMIV-Mb02 coat protein (AV1) gene from the mungbean yellow mosaic India virus strain, accession number GQ387502.1., as per the results of the BLAST program. As a result, the variation was designated mungbean yellow mosaic India virus isolates NAU-RJ coat protein gene segment and assigned the accession number ON622515.1. The current study examined Six resistant (40 C, NMS-21-01, NMS-21-06, NMS-21-22, NMS-21-49, NMS-21-95) and six highly susceptible (GM 4, NMS-21-23, NMS-21-24, NMS-21-40, NMS-21-68, NMS-21-69). In order to identify the six susceptible and six resistant mungbean genotypes, nine pairs of RGA primers were employed. Five pairs of RGA primers were successfully amplified in all resistant genotypes as a consequence, but not in all highly susceptible genotypes. The results of this study suggest that five pairs of RGA markers can effectively distinguish between the genotypes of mungbean that are highly susceptible and resistant. The long-lasting YMV resistance of these RGA markers makes them useful for mapping resistance genes and marker validation studies.

Antimicrobial susceptibility profile of Neisseria gonorrhoeae from patients attending a medical laboratory, Institut Pasteur de Madagascar between 2014 and 2020: phenotypical and genomic characterisation in a subset of Neisseria gonorrhoeae isolates

ObjectivesAntimicrobial-resistant Neisseria gonorrhoeae (NG) is a concern. Little is known about antimicrobial susceptibility profiles and associated genetic resistance mechanisms of NG in Madagascar. We report susceptibility data of NG isolates...

Virulence and Molecular Detection of Cassava Mosaic and Brown Streak Virus Genes on Elite Mutant Cassava Lines

Unavailability of germplasm with Cassava Brown Streak Disease (CBSD) and Cassava Mosaic Disease (CMD) tolerance genes remains the main bottleneck towards management of the two diseases in tropic and sub-tropic regions in Africa. We assessed the pathogenicity of two viruses and established the presence of genes linked to CBSD and CMD among some elite cassava lines using field techniques and PCR methods. The screen-house experiment was arranged in split plot in completely randomized block design replicated thrice. Severity data was done on a scale of 1 - 5 at an interval of 14 days post inoculation. Symptomatic fresh leaves were used in the extraction of genomic deoxyribonucleic acid and amplification done using markers linked to African cassava mosaic virus (ACMV), separated bands scored as present (+) or absent (-) and visualized under UV trans-illuminator. Virulence and pathogenicity results showed higher tolerance to CBSD and CMD under single inoculation. In contrast, dual inoculation synergistically increased severity expression leading defoliation at early stages of growth. DNA amplification found no CBSD and CMD alleles in CAS1. However, some traces of the alleles were amplified on CAS2, CAS3, KME3 and KME4. Mutant cassava lines exhibited low severity compared to parental lines which were genetically different in terms of viral genes detected by the ACMV linked markers.

Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration

Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human postmortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.

Identification of ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone and isolates harboring a novel mosaic penA gene in Chengdu in 2019–2020

BackgroundAntimicrobial resistance in gonorrhea has become a growing global public health burden. Neisseria gonorrhoeae isolates with resistance to ceftriaxone, the last remaining first-line option, represent an emerging threat of untreatable gonorrhea.MethodsA total of ten ceftriaxone-resistant N. gonorrhoeae FC428 isolates and two isolates harboring a novel mosaic penA-232.001 allele from 160 gonococcal isolates in Chengdu in 2019–2020 was described in the present study. Multilocus sequence typing (MLST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were performed to characterize the isolates. Whole genome sequencing and maximum-likelihood method were performed to infer how the genetic phylogenetic tree of these isolates looks like. Recombination analysis was performed using the RDP4 software. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2100048771, registration date: 20210716).ResultsThe genetic phylogeny showed that the ten FC428 isolates sporadically clustered into different phylogenetic clades, suggesting different introductions and local transmission of FC428. Two isolates showed close genetic relatedness to ceftriaxone-resistant clone A8806, which was only reported from Australia in 2013. Homologous recombination events were detected in penA between Neisseria gonorrhoeae and commensal Neisseria species (N. perflava and N. polysaccharea), providing evidence of commensal Neisseria species might serve as reservoirs of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains.ConclusionsOur results demonstrate further dissemination of FC428 in China and resurgence risks of sporadic ceftriaxone-resistant A8806 to become the next clone to spread.

Screening of Resistance Gene Analogue Marker(S) in Indian Bean [Lablab purpureus (L.) Sweet] against Yellow Mosaic Disease

In this study, 104 genotypes of Indian beans were evaluated against the yellow mosaic disease at College Farm, N. M. College of Agriculture, Navsari Agricultural University, Navsari during Rabi 2021–2022 (South Gujarat Heavy Rainfall Zone-II ), based on the disease rating scale (1–9). Out of the 104 genotypes of Indian beans, 33 genotypes showed resistance to the yellow mosaic disease, 14 genotypes showed moderate resistance, 17 genotypes showed moderate susceptibility, 29 genotypes showed susceptibility, and 11 genotypes showed highly susceptibility. The obtained sequence has a greatest identity of 95.05 percent with the full sequence of the dolichos yellow mosaic virus isolate AG segment DNA A with accession number MZ821026.1. As a result, the variant was given the accession number ON461886 and named dolichos yellow mosaic virus isolate NAU-RA coat protein gene segment DNA -A. In present investigation six resistant (P-19-169, V-19-15, V-19-35, GNIB-22, W-19-05, V-19-24) and six highly susceptible (P-19-176, W-19-60, P-19-127, V-19-11, W-19-58, V-19-106) Indian bean genotypes were screened by nine pairs of RGA primers. The amplicons were obtained in seven pairs of RGA primers, where five pairs of RGA primersRGA1FCG &amp;RGA1R,VMYR1F &amp; VMYR1R,YR4F &amp; YR4R, RGAIB3 &amp; RGAIB4, RGAIB5 &amp; RGAIB6 amplified single band size of approximately 450 bp, 450 bp, 450 bp, 450 bp and 1050 bp respectively in resistant genotypes, while only two pairs of RGA primers CYR1F &amp; CYR1R and RGAIB1 &amp; RGAIB2 were found with prominent band of approximately 950bp and 350bp. According to the data from the current investigation, seven pairs of RGA markers have proven successful in differentiating between the resistant and highly susceptible genotypes of Indian beans. These RGA markers can be employed in investigations mapping resistance genes and validating markers with long-lasting YMV resistance.

Jansen de Vries syndrome: Report of four new patients and review of the literature

Jansen de Vries syndrome (JDVS, OMIM: 617450) is a rare neurodevelopmental disorder associated with hypotonia, behavioral features, high threshold to pain, short stature, ophthalmological abnormalities, dysmorphism and occasionally a structural cardiac condition. It is caused by truncating variants of the last and penultimate exons of PPM1D. So far, 21 patients with JVDS have been reported in the literature.Here, we describe four novel cases of JVDS and review the current literature. Notably, our patients 1, 3 and 4 do not have intellectual disability albeit they have significant developmental difficulties. Thus, the phenotype may span from a classic intellectual disability syndrome to a milder neurodevelopmental disorder. Interestingly, two of our patients have received successful growth hormone treatment. Considering the phenotype of all the known JDVS patients, a cardiological consultation is recommended, as at least 7/25 patients showed a structural cardiac defect. Episodic fever and vomiting may associate with hypoglycemia and may even mimic a metabolic disorder. We also report the first JDVS patient with a mosaic gene defect and a mild neurodevelopmental phenotype.

A mosaic pathogenic variant in MSH6 causes MSH6-deficient colorectal and endometrial cancer in a patient classified as suspected Lynch syndrome: a case report

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. However, mosaic variants in the MMR genes have been rarely described. We identified a likely de novo mosaic MSH6:c.1135_1139del p.Arg379* pathogenic variant in a patient diagnosed with suspected Lynch syndrome/Lynch-like syndrome. The patient developed MSH6-deficient EC and CRC at 54 and 58 years of age, respectively, without a detectable germline MMR pathogenic variant. Multigene panel sequencing of tumor and blood-derived DNA identified an MSH6 somatic mutation (MSH6:c.1135_1139del p.Arg379*) common to both the EC and CRC, raising suspicion of mosaicism. A droplet digital polymerase chain reaction (ddPCR) assay detected the MSH6 variant at 5.34% frequency in normal colonic tissue, 3.49% in saliva and 1.64% in blood DNA, demonstrating the presence of the MSH6 variant in all three germ layers. This study highlights the utility of tumor sequencing to guide sensitive ddPCR testing to detect low-level mosaicism in the MMR genes. Further investigation of the prevalence of MMR mosaicism is needed to inform routine diagnostic approaches and genetic counselling.

Nrl:CreERT2 mouse model to induce mosaic gene expression in rod photoreceptors.

Photoreceptors are sensory neurons that capture light within their outer segment, a narrow cylindrical organelle stacked with disc-shaped membranes housing the visual pigment. Photoreceptors are the most abundant neurons in the retina and are tightly packed to maximize the capture of incoming light. As a result, it is challenging to visualize an individual cell within a crowded photoreceptor population. To address this limitation, we developed a rod-specific mouse model that expresses tamoxifen-inducible cre recombinase under the control of the Nrl promoter. We characterized this mouse using a farnyslated GFP (GFPf) reporter mouse and found mosaic rod expression throughout the retina. The number of GFPf-expressing rods stabilized within 3 days post tamoxifen injection. At that time, the GFPf reporter began to accumulate in basal disc membranes. Using this new reporter mouse, we attempted to quantify the time course of photoreceptor disc renewal in WT and Rd9 mice, a model of X-linked retinitis pigmentosa previously proposed to have a reduced disc renewal rate. We measured GFPf accumulation in individual outer segments at 3 and 6 days post-induction and found that basal accumulation of the GFPf reporter was unchanged between WT and Rd9 mice. However, rates of renewal based on the GFPf measurements were inconsistent with historical calculations from radiolabeled pulse-chase experiments. By extending GFPf reporter accumulation to 10 and 13 days we found that this reporter had an unexpected distribution pattern that preferentially labeled the basal region of the outer segment. For these reasons the GFPf reporter cannot be used for measuring rates of disc renewal. Therefore, we used an alternative method that labels newly forming discs with a fluorescent dye to measure disc renewal rates directly in the Rd9 model and found it was not significantly different from WT. Our study finds that the Rd9 mouse has normal rates of disc renewal and introduces a novel Nrl:CreERT2 mouse for gene manipulation of individual rods.

Ancient Rapid Radiation Explains Most Conflicts Among Gene Trees and Well-Supported Phylogenomic Trees of Nostocalean Cyanobacteria.

Prokaryotic genomes are often considered to be mosaics of genes that do not necessarily share the same evolutionary history due to widespread horizontal gene transfers (HGTs). Consequently, representing evolutionary relationships of prokaryotes as bifurcating trees has long been controversial. However, studies reporting conflicts among gene trees derived from phylogenomic data sets have shown that these conflicts can be the result of artifacts or evolutionary processes other than HGT, such as incomplete lineage sorting, low phylogenetic signal, and systematic errors due to substitution model misspecification. Here, we present the results of an extensive exploration of phylogenetic conflicts in the cyanobacterial order Nostocales, for which previous studies have inferred strongly supported conflicting relationships when using different concatenated phylogenomic data sets. We found that most of these conflicts are concentrated in deep clusters of short internodes of the Nostocales phylogeny, where the great majority of individual genes have low resolving power. We then inferred phylogenetic networks to detect HGT events while also accounting for incomplete lineage sorting. Our results indicate that most conflicts among gene trees are likely due to incomplete lineage sorting linked to an ancient rapid radiation, rather than to HGTs. Moreover, the short internodes of this radiation fit the expectations of the anomaly zone, i.e., a region of the tree parameter space where a species tree is discordant with its most likely gene tree. We demonstrated that concatenation of different sets of loci can recover up to 17 distinct and well-supported relationships within the putative anomaly zone of Nostocales, corresponding to the observed conflicts among well-supported trees based on concatenated data sets from previous studies. Our findings highlight the important role of rapid radiations as a potential cause of strongly conflicting phylogenetic relationships when using phylogenomic data sets of bacteria. We propose that polytomies may be the most appropriate phylogenetic representation of these rapid radiations that are part of anomaly zones, especially when all possible genomic markers have been considered to infer these phylogenies. [Anomaly zone; bacteria; horizontal gene transfer; incomplete lineage sorting; Nostocales; phylogenomic conflict; rapid radiation; Rhizonema.].