Compound Heterozygous RYR1 Variants in a Patient with Severe Congenital Myopathy: Case Report and Comparison with Additional Cases of Recessive RYR1-Related Myopathy.

Pathogenic variants in the ryanodine receptor 1 (RYR1) gene are causative for a wide spectrum of muscular phenotypes, ranging from malignant hyperthermia over mild, non-progressive to severe congenital myopathy. Both autosomal dominant and recessive inheritance can occur, with the more severe forms usually showing recessive inheritance. However, genotype-phenotype correlations are complicated due to the large size of the gene and heterogeneous phenotypes. We present a 6-year-old patient with severe congenital myopathy, carrying a heterozygous pathogenic RYR1 variant inherited from the healthy mother. Through whole genome sequencing we identified a second, deep intronic RYR1 variant that has recently been described in another patient with severe congenital myopathy and shown to affect splicing. Segregation analyses confirmed the variants to be compound heterozygous. We compared our patient's phenotype to that of the patient from the literature as well as five additional patients with compound heterozygous RYR1 variants from our center. The main overlapping features comprised congenital onset, predominant muscular hypotonia, and normal creatine kinase (CK) levels, while overall clinical expression varied substantially. Interestingly, both patients carrying the new intronic splice variant showed a very severe disease course. More widespread use of genome sequencing will open the way for better genotype-phenotype correlations.

Complex structural variation is prevalent and highly pathogenic in pediatric solid tumors.

In pediatric cancer, structural variants (SVs) and copy-number alterations contribute to cancer initiation as well as progression, thereby aiding diagnosis and treatment stratification. Although suggested to be of importance, the prevalence and biological relevance of complex genomic rearrangements (CGRs) across pediatric solid tumors is largely unexplored. In a cohort of 120 primary tumors, we systematically characterized patterns of extrachromosomal DNA, chromoplexy, and chromothripsis across five pediatric solid cancer types. CGRs were identified in 56 tumors (47%), and in 42 of these tumors, CGRs affect cancer driver genes or result in unfavorable chromosomal alterations. This demonstrates that CGRs are prevalent and pathogenic in pediatric solid tumors and suggests that selection likely contributes to the structural variation landscape. Moreover, carrying CGRs is associated with more adverse clinical events. Our study highlights the potential for CGRs to be incorporated in risk stratification or exploited for targeted treatments.

Identification of novel genomic hotspots and tumor-relevant genes via comprehensive analysis of HPV integration in Chinese patients of cervical cancer.

Cervical cancer accounts for 10-15% of cancer-related mortality among women globally. Infection with high-risk human papillomavirus (HPV) types constitutes a significant etiological factor in the development of cervical carcinoma. The integration of HPV DNA into the host genome is considered a pivotal event in cervical carcinogenesis. Nevertheless, the precise mechanisms underlying HPV integration and its role in promoting cancer progression remain inadequately understood. Therefore, this study aims to identify potential common denominators at HPV DNA integration sites and to analyze the adjacent cellular sequences. We conducted whole-genome sequencing on 13 primary cervical cancer samples, employing the chromosomal coordinates of 537 breakpoints to assess the statistical overrepresentation of integration sites in relation to various chromatin features. Our analysis, which encompassed all chromosomes, identified several integration hotspots within the human genome, notably at 14q32.2, 10p15, and 2q37. Additionally, our findings indicated a preferential integration of HPV DNA into intragenic and gene-dense regions of human chromosomes. A substantial number of host cellular genes impacted by the integration sites were associated with cancer, including IKZF2, IL26, AHRR, and PDCD6. Furthermore, the cellular genes targeted by integration were enriched in tumor-related terms and pathways, as demonstrated by gene ontology and KEGG analysis. In conclusion, these findings enhance our understanding of HPV integration sites and provide deeper insights into the molecular mechanisms underlying the pathogenesis of cervical carcinoma.

Detecting pulmonary malignancy against benign nodules using noninvasive cell-free DNA fragmentomics assay.

BACKGROUND: Early screening using low-dose computed tomography (LDCT) can reduce mortality caused by non-small-cell lung cancer. However, ∼25% of the 'suspicious' pulmonary nodules identified by LDCT are later confirmed benign through resection surgery, adding to patients' discomfort and the burden on the healthcare system. In this study, we aim to develop a noninvasive liquid biopsy assay for distinguishing pulmonary malignancy from benign yet 'suspicious' lung nodules using cell-free DNA (cfDNA) fragmentomics profiling. METHODS: An independent training cohort consisting of 193 patients with malignant nodules and 44 patients with benign nodules was used to construct a machine learning model. Base models using four different fragmentomics profiles were optimized using an automated machine learning approach before being stacked into the final predictive model. An independent validation cohort, including 96 malignant nodules and 22 benign nodules, and an external test cohort, including 58 malignant nodules and 41 benign nodules, were used to assess the performance of the stacked ensemble model. RESULTS: Our machine learning models demonstrated excellent performance in detecting patients with malignant nodules. The area under the curves reached 0.857 and 0.860 in the independent validation cohort and the external test cohort, respectively. The validation cohort achieved an excellent specificity (68.2%) at the targeted 90% sensitivity (89.6%). An equivalently good performance was observed while applying the cut-off to the external cohort, which reached a specificity of 63.4% at 89.7% sensitivity. A subgroup analysis for the independent validation cohort showed that the sensitivities for detecting various subgroups of nodule size (<1 cm: 91.7%; 1-3 cm: 88.1%; >3 cm: 100%; unknown: 100%) and smoking history (yes: 88.2%; no: 89.9%) all remained high among the lung cancer group. CONCLUSIONS: Our cfDNA fragmentomics assay can provide a noninvasive approach to distinguishing malignant nodules from radiographically suspicious but pathologically benign ones, amending LDCT false positives.

Developing a Paired Whole Genome Sequencing Service for Children With Cancer.

BACKGROUND: The uniqueness of paired (tumor and germline) whole genome sequencing (PWGS) in cancer diagnosis and management lies in not just its ability to uncover oncogenic drivers and potential treatment targets but also on the identification of underlying cancer predisposition syndromes, which has significant implications for the patient and their family. AIMS: This is a descriptive article highlighting the processes taken by our team to incorporate PWGS into routine National Health Service (NHS) clinical care for children with cancer. The main aim of this article is to share our experience with other centers that may wish to set up similar services and set the stage for future quantitative/qualitative research. METHODS: This article is further supported by an audit focusing on children in whom an underlying cancer predisposition was confirmed. RESULTS: The audit highlights the success of the program to date, with 100% of families identified as being at risk of a cancer predisposition syndrome being offered referral to clinical genetics and 100% of at-risk first-degree relatives being offered predictive counseling and testing. Areas requiring improvement included discussion of reproductive options as only six out of nine families (67%) had a documented discussion. CONCLUSIONS: Incorporation of the audit recommendations will improve our service, and sharing of our experience will hopefully encourage more pediatric oncology services to introduce PWGS into routine clinical care and reduce inequity of access. Further work is required to assess the long-term cancer risk reduction and establish the psychosocial impact of PWGS for the child and family.

Genomic Landscape of Branchio-Oto-Renal Syndrome through Whole-Genome Sequencing: A Single Rare Disease Center Experience in South Korea.

Branchio-oto-renal (BOR) and branchio-otic (BO) syndromes are characterized by anomalies affecting the ears, often accompanied by hearing loss, as well as abnormalities in the branchial arches and renal system. These syndromes exhibit a broad spectrum of phenotypes and a complex genomic landscape, with significant contributions from the EYA1 gene and the SIX gene family, including SIX1 and SIX5. Due to their diverse phenotypic presentations, which can overlap with other genetic syndromes, molecular genetic confirmation is essential. As sequencing technologies advance, whole-genome sequencing (WGS) is increasingly used in rare disease diagnostics. We explored the genomic landscape of 23 unrelated Korean families with typical or atypical BOR/BO syndrome using a stepwise approach: targeted panel sequencing and exome sequencing (Step 1), multiplex ligation-dependent probe amplification (MLPA) with copy number variation screening (Step 2), and WGS (Step 3). Integrating WGS into our diagnostic pipeline detected structure variations, including cryptic inversion and complex genomic rearrangement, eventually enhancing the diagnostic yield to 91%. Our findings expand the genomic architecture of BOR/BO syndrome and highlight the need for WGS to address the genetic diagnosis of clinically heterogeneous rare diseases.

A rare homozygous ALX4 mutation in a Bangladeshi girl with frontonasal dysplasia type-2 (FND2).

Background: Frontonasal dysplasia type-2(FND2), a rare phenotypically variable and heterogeneous developmental anomaly resulting from mutation of the ALX4 gene, is primarily characterized by malformation of the skull and facial skeleton. This study was designed to showcase a clinical, imaging, and genetic analysis of FND2 in a consanguineous family of Bangladeshi origin. Methodology: Clinical imaging and whole genome sequencing of mother, father and patient was done by using Nextera DNA flex library preparation kit (Illumina, USA) using Novaseq 6000 next generation sequencer to find out ALX4 mutation which causes FND2 in patient. Result: We report the clinical as well as molecular findings in an 8-year-old girl with FND2. The child presented with various characteristic features of skull and facial anomalies associated with FND 2 along with numerous other features many of which have not been described in previous literature. The parents also showed some key clinical, radiological, and genetic features of FND 2. The whole genome sequencing (WGS) revealed homozygosity for a 793C-T transition in the ALX4 gene, which resulted in premature termination at codon 265 (p.Arg265Ter). Both of her parents were heterozygous carriers of ALX4 mutation. Conclusions: This is the first report that associates clinical, imaging, and genomics analyses in a Bangladeshi patient for better understanding the disease FND2. These results will facilitate diagnosis and genetic counseling of the future FND2 patients.

Plasmid-Mediated Spread of Carbapenem Resistance in Enterobacterales: A Three-Year Genome-Based Survey.

The worldwide emergence and dissemination of carbapenem-resistant Gram-negative bacteria (CRGNB) is a challenging problem of antimicrobial resistance today. Outbreaks with CRGNB have severe consequences for both the affected healthcare settings as well as the patients with infection. Thus, bloodstream infections caused by metallo-ß-lactamase-producing Enterobacterales can often have clinical implications, resulting in high mortality rates due to delays in administering effective treatment and the limited availability of treatment options. The overall threat of CRGNB is substantial because carbapenems are used to treat infections caused by ESBL-producing Enterobacterales which also exist with high frequency within the same geographical regions. A genome-based surveillance of 589 CRGNB from 61 hospitals across the federal state Hesse in Germany was implemented using next-generation sequencing (NGS) technology to obtain a high-resolution landscape of carbapenem-resistant isolates over a three-year period (2017-2019). The study examined all reportable CRGNB isolates submitted by participating hospitals. This included isolates carrying known carbapenemases (435) together with carbapenem-resistant non-carbapenemase producers (154). Predominant carbapenemase producers included Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii and Acinetobacter baumannii. Over 80% of 375 carbapenem-resistant determinants including KPC-, NDM-, VIM- and OXA-48-like ones detected in 520 Enterobacterales were plasmid-encoded, and half of these were dominated by a few incompatibility (Inc) types, viz., IncN, IncL/M, IncFII and IncF(K). Our results revealed that plasmids play an extraordinary role in the dissemination of carbapenem resistance in the heterogeneous CRGNB population. The plasmids were also associated with several multispecies dissemination events and local outbreaks throughout the study period, indicating the substantial role of horizontal gene transfer in carbapenemase spread. Furthermore, due to vertical and horizontal plasmid transfer, this can have an impact on implant-associated infections and is therefore important for antibiotic-loaded bone cement and drug-containing devices in orthopedic surgery. Future genomic surveillance projects should increase their focus on plasmid characterization.

Comparative genomic analysis of Bacillus atrophaeus HAB-5 reveals genes associated with antimicrobial and plant growth-promoting activities.

Bacillus atrophaeus HAB-5 is a plant growth-promoting rhizobacterium (PGPR) that exhibits several biotechnological traits, such as enhancing plant growth, colonizing the rhizosphere, and engaging in biocontrol activities. In this study, we conducted whole-genome sequencing of B. atrophaeus HAB-5 using the single-molecule real-time (SMRT) sequencing platform by Pacific Biosciences (PacBio; United States), which has a circular chromosome with a total length of 4,083,597 bp and a G + C content of 44.21%. The comparative genomic analysis of B. atrophaeus HAB-5 with other strains, Bacillus amyloliquefaciens DSM7, B. atrophaeus SRCM101359, Bacillus velezensis FZB42, B. velezensis HAB-2, and Bacillus subtilis 168, revealed that these strains share 2,465 CDSs, while 599 CDSs are exclusive to the B. atrophaeus HAB-5 strain. Many gene clusters in the B. atrophaeus HAB-5 genome are associated with the production of antimicrobial lipopeptides and polypeptides. These gene clusters comprise distinct enzymes that encode three NRPs, two Transat-Pks, one terpene, one lanthipeptide, one T3PKS, one Ripp, and one thiopeptide. In addition to the likely IAA-producing genes (trpA, trpB, trpC, trpD, trpE, trpS, ywkB, miaA, and nadE), there are probable genes that produce volatile chemicals (acoA, acoB, acoR, acuB, and acuC). Moreover, HAB-5 contained genes linked to iron transportation (fbpA, fetB, feuC, feuB, feuA, and fecD), sulfur metabolism (cysC, sat, cysK, cysS, and sulP), phosphorus solubilization (ispH, pstA, pstC, pstS, pstB, gltP, and phoH), and nitrogen fixation (nif3-like, gltP, gltX, glnR, glnA, nadR, nirB, nirD, nasD, narl, narH, narJ, and nark). In conclusion, this study provides a comprehensive genomic analysis of B. atrophaeus HAB-5, pinpointing the genes and genomic regions linked to the antimicrobial properties of the strain. These findings advance our knowledge of the genetic basis of the antimicrobial properties of B. atrophaeus and imply that HAB-5 may employ a variety of commercial biopesticides and biofertilizers as a substitute strategy to increase agricultural output and manage a variety of plant diseases.

EDA Missense Variant in a Cat with X-Linked Hypohidrotic Ectodermal Dysplasia.

Hypohidrotic ectodermal dysplasia is a developmental defect characterized by sparse or absent hair, missing or malformed teeth and defects in eccrine glands. Loss-of-function variants in the X-chromosomal EDA gene have been reported to cause hypohidrotic ectodermal dysplasia in humans, mice, dogs and cattle. We investigated a male cat exhibiting diffuse truncal alopecia with a completely absent undercoat. The cat lacked several teeth, and the remaining teeth had an abnormal conical shape. Whole-genome sequencing revealed a hemizygous missense variant in the EDA gene, XM_011291781.3:c.1042G>A or XP_011290083.1:p.(Ala348Thr). The predicted amino acid exchange is located in the C-terminal TNF signaling domain of the encoded ectodysplasin. The corresponding missense variant in the human EDA gene, p.Ala349Thr, has been reported as a recurring pathogenic variant in several human patients with X-linked hypohidrotic ectodermal dysplasia. The identified feline variant therefore represents the likely cause of the hypohidrotic ectodermal dysplasia in the investigated cat, and the genetic investigation confirmed the suspected clinical diagnosis. This is the first report of an EDA-related hypohidrotic ectodermal dysplasia in cats.

A novel method addressing NGS-based mappability bias for sensitive detection of DNA alterations.

A turning point in cancer research is the introduction of massively parallel sequencing technology which greatly reduced the cost and time for genome sequencing. This enhanced the scope for detecting and analyzing the role of structural alterations in cancer. However, certain bias exists in NGS-based approaches, which badly affects the CNV identification process. Moreover, DNA repeats existing in CNV regions need special attention as they will degrade the performance of majority of the existing CNV detection tools, even after applying generalized bias correction method. This motivated this work, where a novel method has been designed to address the issue of DNA repeats and thereby mappability bias existing in regions of CNV. The method consists of three phases, where the first phase computes the alignment information of uniquely mapped DNA reads, considering the base quality and base mismatch parameters at nucleotide level precision. The second and the third phase use a novel approach to allocate the non-uniquely mapped reads to an optimal region of the DNA repeats based on a probabilistic membership model. The proposed method is capable of identifying CNVs present in coding, as well as non-coding region of the DNA, and is also capable of detecting CNVs existing in DNA repeat regions. The methodology achieves a sensitivity greater than [Formula: see text] during the performed simulations, and on real data, the detected variants are validated with the database of genomic variants, where the percentage overlap is also greater than 95%, and has achieved much better breakpoint prediction, as compared with other popular bias correction CNV detection methods.

Deciphering the pathogenic role of rare RAF1 heterozygous missense mutation in the late-presenting DDH.

Background: Developmental Dysplasia of the Hip (DDH) is a skeletal disorder where late-presenting forms often escape early diagnosis, leading to limb and pain in adults. The genetic basis of DDH is not fully understood despite known genetic predispositions. Methods: We employed Whole Genome Sequencing (WGS) to explore the genetic factors in late-presenting DDH in two unrelated families, supported by phenotypic analyses and in vitro validation. Results: In both cases, a novel de novo heterozygous missense mutation in RAF1 (c.193A>G [p.Lys65Glu]) was identified. This mutation impacted RAF1 protein structure and function, altering downstream signaling in the Ras/ERK pathway, as demonstrated by bioinformatics, molecular dynamics simulations, and in vitro validations. Conclusion: This study contributes to our understanding of the genetic factors involved in DDH by identifying a novel mutation in RAF1. The identification of the RAF1 mutation suggests a possible involvement of the Ras/ERK pathway in the pathogenesis of late-presenting DDH, indicating its potential role in skeletal development.

Novel melatonin-producing Bacillus safensis EH143 mitigates salt and cadmium stress in soybean.

Recently, microorganism and exogenous melatonin application has been recognized as an efficient biological tool for enhancing salt tolerance and heavy metal detoxification in agriculture crops. Thus, the goal of this study was to isolate and evaluate a novel melatonin-producing plant growth promoting bacterium. With high-throughput whole genome sequencing, phytohormone measurements, expression profiling, and biochemical analysis, we can identify a novel PGPB that produces melatonin and unravel how it promotes soybean growth and development and protects against salt and Cd stress. We identify the melatonin synthesis pathway (tryptophan→tryptamine→serotonin melatonin) of the halotolerant (NaCl > 800 mM) and heavy metal-resistant (Cd >3 mM) rhizobacterium Bacillus safensis EH143 and use it to treat soybean plants subjected to Cd and NaCl stresses. Results show that EH143 will highly bioaccumulate heavy metals and significantly improve P and Ca2+ uptake and the K+/Na+ (93%↑under salt stress) ratio while reducing Cd uptake (49% under Cd stress) in shoots. This activity was supported by the expression of the ion regulator HKT1, MYPB67, and the calcium sensors CDPK5 and CaMK1 which ultimately led to increased plant growth. EH143 significantly decreased ABA content in shoots by 13%, 20%, and 34% and increased SA biosynthesis in shoots by 14.8%, 31%, and 48.2% in control, salt, and Cd-treated plants, upregulating CYP707A1 and CYP707A2 and PAL1 and ICS, respectively. The melatonin content significantly decreased along with a reduced expression of ASMT3 following treatment with EH143; moreover, reduced expression of peroxidase (POD) and superoxide dismutase (SOD) by 134.5% and 39% under salt+Cd stress, respectively and increased level of total amino acids were observed. Whole-genome sequencing and annotation of EH143 revealed the presence of the melatonin precursor tryptophan synthase (trpA, trpB, trpS), metal and other ion regulators (Cd: cadA, potassium: KtrA and KtrB, phosphate: glpT, calcium: yloB, the sodium/glucose cotransporter: sgIT, and the magnesium transporter: mgtE), and enzyme activators (including the siderophore transport proteins yfiZ and yfhA, the SOD sodA, the catalase katA1, and the glutathione regulator KefG) that may be involved in programming the plant metabolic system. As a consequence, EH143 treatment significantly reduced the contents of lipid peroxidation (O2-, MDA, and H2O2) up to 69%, 46%, and 29% in plants under salt+Cd stress, respectively. These findings suggest that EH143 could be a potent biofertilizer to alleviate NaCl and Cd toxicity in crops and serve as an alternative substitute for exogenous melatonin application.

Whole genome sequencing identifies elusive variants in genetically unsolved Italian inherited retinal disease patients.

Inherited retinal diseases (IRDs) are a group of rare monogenic diseases with high genetic heterogeneity (pathogenic variants identified in over 280 causative genes). The genetic diagnostic rate for IRDs is around 60%, mainly thanks to the routine application of next-generation sequencing (NGS) approaches such as extensive gene panels or whole exome analyses. Whole-genome sequencing (WGS) has been reported to improve this diagnostic rate by revealing elusive variants, such as structural variants (SVs) and deep intronic variants (DIVs). We performed WGS on 33 unsolved cases with suspected autosomal recessive IRD, aiming to identify causative genetic variants in non-coding regions or to detect SVs that were unexplored in the initial screening. Most of the selected cases (30 of 33, 90.9%) carried monoallelic pathogenic variants in genes associated with their clinical presentation, hence we first analyzed the non-coding regions of these candidate genes. Whenever additional pathogenic variants were not identified with this approach, we extended the search for SVs and DIVs to all IRD-associated genes. Overall, we identified the missing causative variants in 11 patients (11 of 33, 33.3%). These included three DIVs in ABCA4, CEP290 and RPGRIP1; one non-canonical splice site (NCSS) variant in PROM1 and three SVs (large deletions) in EYS, PCDH15 and USH2A. For the previously unreported DIV in CEP290 and for the NCCS variant in PROM1, we confirmed the effect on splicing by reverse transcription (RT)-PCR on patient-derived RNA. This study demonstrates the power and clinical utility of WGS as an all-in-one test to identify disease-causing variants missed by standard NGS diagnostic methodologies.

Phylogenomic evaluation of Mangrovimicrobium sediminis gen. nov. sp. nov., the first nitrogen fixing member of the family Halieaceae adapted to mangrove habitat and reclassification of Halioglobus pacificus to Pseudohaliglobus pacificus comb. nov.

The taxonomic position and genomic characteristics of a nitrogen fixing and polymer degrading marine bacterium, strain SAOS 164 isolated from a mangrove sediment sample was investigated. Sequence analysis based on 16S rRNA gene identified it as a member of family Halieaceae with closest similarity to Haliea salexigens DSM 19537T (96.3 %), H. alexandrii LZ-16-2T (96.2 %) and Parahaliea maris HSLHS9T (96.0 %) but was distantly related to the genera Haliea, Parahaliea and Halioglobus in phylogenetic trees. In order to ascertain the exact taxonomic position, phylogeny based on RpoBC proteins, whole genome, core and orthologous genes, and comparative analysis of metabolic potential retrieved the strain in an independent lineage clustering along with the genera Halioglobus, Pseudohalioglobus and Seongchinamella. Further, various genome based delimitation parameters represented by mol % GC content, percentage of conserved proteins (POCP), and amino acid identity (AAI) along with chemotaxonomic markers (i.e. fatty acids and polar lipids) supported the inferences of genome based phylogeny and indicated that the strain SAOS 164 belongs to a novel genus. The genome was mapped to 4.8 Mb in size with 65.1 % DNA mol% G + C content. In-silico genomic investigation and phenotyping revealed diverse metabolite genes/pathways related to polymer hydrolysis, nitrogen fixation, light induced growth, carbohydrate, sulfur, phosphorus and amino acid metabolism, virulence factors, defense mechanism, and stress-responsive elements facilitating survival in the mangrove habitat. Based on polyphasic taxonomic approach including genome analyses, a novel genus Mangrovimicrobium sediminis gen. nov. sp. nov. (=SAOS 164T = MTCC 12907T = KCTC 52755T = JCM 32136T) is proposed. Additionally, the reclassification of Halioglobus pacificus (=DSM 27932T = KCTC 23430T = S1-72T) to Pseudhalioglobus pacificus comb. nov. is also proposed.

Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009 to 2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome sequencing and WGS and, latterly, WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. In summary, 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100 000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment, leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre and has benefitted from the use of WGS, particularly access to the raw data. However, almost one-quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.

Characterization of a Novel Species of Legionella Isolated from a Healthcare Facility: Legionella resiliens sp. nov.

Two Legionella-like isolates, 8cVS16T and 9fVS26, were isolated from a water distribution system (WDS) in a healthcare facility. Cells were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, motile, and exhibited a blue-white fluorescence under Wood's lamp at 365 nm. The strains grew in a range of 32-37 °C on BCYE with L-cysteine (Cys+), GVPC, and MWY agar medium, with a positive reaction for oxidase, catalase, and gelatinase. The dominant fatty acids were summed features 3 (C16:1ω7c/C16:1ω6c) (27.7%), C16:0 iso (17.5%), and C16:0 (16.3%), and Q13 as the major ubiquinone. The mip and rpoB gene sequences showed a similarity of 96.7% and 92.4%, with L. anisa (ATCC 35292T). The whole genomes sequencing (WGS) performed displayed a GC content of 38.21 mol% for both. The digital DNA-DNA hybridization (dDDH) analysis demonstrated the separation of the two strains from the phylogenetically most related L. anisa (ATCC 35292T), with ≤43% DNA-DNA relatedness. The Average Nucleotide Identity (ANI) between the two strains and L. anisa (ATCC 35292T) was 90.74%, confirming that the two isolates represent a novel species of the genus Legionella. The name proposed for this species is Legionella resiliens sp. nov., with 8cVS16T (=DSM 114356T = CCUG 76627T) as the type strain.

Prediction of methylation status using WGS data of plasma cfDNA for multi-cancer early detection (MCED).

BACKGROUND: Cell-free DNA (cfDNA) contains a large amount of molecular information that can be used for multi-cancer early detection (MCED), including changes in epigenetic status of cfDNA, such as cfDNA fragmentation profile. The fragmentation of cfDNA is non-random and may be related to cfDNA methylation. This study provides clinical evidence for the feasibility of inferring cfDNA methylation levels based on cfDNA fragmentation patterns. We performed whole-genome bisulfite sequencing and whole-genome sequencing (WGS) on both healthy individuals and cancer patients. Using the information of whole-genome methylation levels, we investigated cytosine-phosphate-guanine (CpG) cleavage profile and validated the method of predicting the methylation level of individual CpG sites using WGS data. RESULTS: We conducted CpG cleavage profile biomarker analysis on data from both healthy individuals and cancer patients. We obtained unique or shared potential biomarkers for each group and built models accordingly. The modeling results proved the feasibility to predict the methylation status of single CpG sites in cfDNA using cleavage profile model from WGS data. CONCLUSION: By combining cfDNA cleavage profile of CpG sites with machine learning algorithms, we have identified specific CpG cleavage profile as biomarkers to predict the methylation status of individual CpG sites. Therefore, methylation profile, a widely used epigenetic biomarker, can be obtained from a single WGS assay for MCED.

Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing.

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.