Mosaic Allele Research Articles

Emergence and evolution of mosaic penA-60 and penA-237 alleles in a Neisseria gonorrhoeae core genogroup that was historically susceptible to extended spectrum cephalosporins.

Neisseria gonorrhoeae (Ng) has successively developed resistance to all previously recommended antimicrobial therapies, with ceftriaxone being the last option for monotherapy of gonorrhea. Global emergence and international spread of the FC428 clone derived mosaic penA-60 allele, associated with highlevel ceftriaxone minimum inhibitory concentrations (MICs) in non FC428 clone Ng lineages, has become an increasing concern. The penA-60 allele carrying Ng was first identified in the U.S. in Las Vegas, Nevada (2019; GCWGS-102723), with a multi-locus sequence type (MLST)-1901 strain, in a non FC428 clone Ng lineage, which is associated with a historically ceftriaxone susceptible core genogroup. Later in 2022, an allele genetically similar to penA-60, mosaic penA-237, was identified in the UK (H22-722) and France (F92) with high-level ceftriaxone MICs and both belonged to MLST-1901. In this study, we assessed phylogenomic relatedness and antimicrobial resistance (AMR) determinant profiles of these three isolates with high-level ceftriaxone MICs among a global collection of 2,104 genomes belonging to the MLST-1901 core genome cluster group 31, which includes strains separated by a locus threshold of 200 or fewer differences (Ng_cgc_200). Recombination events in and around the penA coding region were catalogued and potential sources of inter species recombinant DNA were also inferred. The global population structure of MLST-1901 core genogroup falls into 4 major lineages. Isolates GCWGS-10723, F92, and H22-722 clustered within Lineage 1, which was dominated by non-mosaic penA-5 alleles. These three isolates formed a clade within Lineage 1 that consisted of isolates from North America and southeast Asia. Neisseria subflava and Neisseria sicca were identified as likely progenitors of two independent recombination events that may have led to the generation of mosaic penA-60 and penA-237, within a possible non-mosaic penA-5 background. Our study suggests that there are multiple evolutionary pathways that could generate concerning mosaic penA alleles via homologous recombination of historically susceptible Ng lineages with Neisseria commensals. Enhanced surveillance of gonococcal strains and Neisseria commensals is crucial for understanding of the evolution of AMR, particularly in less-studied regions (e.g., Asia), where high-level ceftriaxone MICs and multi-drug resistance are more prevalent.

Genome-wide detection of somatic mosaicism at short tandem repeats.

Somatic mosaicism has been implicated in several developmental disorders, cancers, and other diseases. Short tandem repeats (STRs) consist of repeated sequences of 1-6bp and comprise >1 million loci in the human genome. Somatic mosaicism at STRs is known to play a key role in the pathogenicity of loci implicated in repeat expansion disorders and is highly prevalent in cancers exhibiting microsatellite instability. While a variety of tools have been developed to genotype germline variation at STRs, a method for systematically identifying mosaic STRs is lacking. We introduce prancSTR, a novel method for detecting mosaic STRs from individual high-throughput sequencing datasets. prancSTR is designed to detect loci characterized by a single high-frequency mosaic allele, but can also detect loci with multiple mosaic alleles. Unlike many existing mosaicism detection methods for other variant types, prancSTR does not require a matched control sample as input. We show that prancSTR accurately identifies mosaic STRs in simulated data, demonstrate its feasibility by identifying candidate mosaic STRs in Illumina whole genome sequencing data derived from lymphoblastoid cell lines for individuals sequenced by the 1000 Genomes Project, and evaluate the use of prancSTR on Element and PacBio data. In addition to prancSTR, we present simTR, a novel simulation framework which simulates raw sequencing reads with realistic error profiles at STRs. prancSTR and simTR are freely available at https://github.com/gymrek-lab/trtools. Detailed documentation is available at https://trtools.readthedocs.io/.

Contributions of Germline and Somatic Mosaic Genetics to Thoracic Aortic Aneurysms in Nonsyndromic Individuals.

Thoracic aortic aneurysm (TAA) is associated with significant morbidity and mortality. Although individuals with family histories of TAA often undergo clinical molecular genetic testing, adults with nonsyndromic TAA are not typically evaluated for genetic causes. We sought to understand the genetic contribution of both germline and somatic mosaic variants in a cohort of adult individuals with nonsyndromic TAA at a single center. One hundred eighty-one consecutive patients <60 years who presented with nonsyndromic TAA at the Massachusetts General Hospital underwent deep (>500×) targeted sequencing across 114 candidate genes associated with TAA and its related functional pathways. Samples from 354 age- and sex-matched individuals without TAA were also sequenced, with a 2:1 matching. We found significant enrichments for germline (odds ratio [OR], 2.44, P=4.6×10-6 [95% CI, 1.67-3.58]) and also somatic mosaic variants (OR, 4.71, P=0.026 [95% CI, 1.20-18.43]) between individuals with and without TAA. Likely genetic causes were present in 24% with nonsyndromic TAA, of which 21% arose from germline variants and 3% from somatic mosaic alleles. The 3 most frequently mutated genes in our cohort were FLNA (encoding Filamin A), NOTCH3 (encoding Notch receptor 3), and FBN1 (encoding Fibrillin-1). There was increased frequency of both missense and loss of function variants in TAA individuals. Likely contributory dominant acting genetic variants were found in almost one quarter of nonsyndromic adults with TAA. Our findings suggest a more extensive genetic architecture to TAA than expected and that genetic testing may improve the care and clinical management of adults with nonsyndromic TAA.

Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy in Patients Clinically Suspected of FSHD Using Optical Genome Mapping.

Facioscapulohumeral muscular dystrophy (FSHD) represents the third most common muscular dystrophy in the general population and is characterized by progressive and often asymmetric muscle weakness of the face, upper extremities, arms, lower leg, and hip girdle. In FSHD type 1, contraction of the number of D4Z4 repeats to 1-10 on the chromosome 4-permissive allele (4qA) results in abnormal epigenetic derepression of the DUX4 gene in skeletal muscle. In FSHD type 2, epigenetic derepression of the DUX4 gene on the permissive allele (4qA) with normal-sized D4Z4 repeats (mostly 8-20) is caused by heterozygous pathogenic variants in chromatin modifier genes such as SMCHD1, DNMT3B, or LRIF1. We present validation of the optical genome mapping (OGM) platform for accurate mapping of the D4Z4 repeat size, followed by diagnostic testing of 547 cases with a suspected clinical diagnosis of FSHD and next-generation sequencing (NGS) of the SMCHD1 gene to identify cases with FSHD2. OGM with Bionano Genomics Saphyr and EnFocus FSHD analysis software was used to identify FSHD haplotypes and D4Z4 repeat number and compared with the gold standard of Southern blot-based diagnosis. A custom Agilent SureSelect enrichment kit was used to enrich SMCHD1, followed by NGS on an Illumina system with 100-bp paired-end reads. Copy number variants were assessed using NxClinical software. We performed OGM for the diagnosis of FSHD in 547 patients suspected of FSHD between December 2019 and December 2022, including 301 male (55%) and 246 female patients (45%). Overall, 308 of the referred patients were positive for D4Z4 contraction on a permissive haplotype, resulting in a diagnosis of FSHD1. A total of 252 of 547 patients were referred for concurrent testing for FSHD1 and FSHD2. This resulted in the identification of FSHD2 in 9/252 (3.6%) patients. In our FSHD2 cohort, the 4qA allele size ranged from 8 to 18 repeats. Among FSHD1-positive cases, 2 patients had biallelic contraction and 4 patients had homozygous contraction and showed early onset of clinical features. Nine of the 308 patients (3%) positive for 4qA contraction had mosaic 4q alleles with contraction on at least one 4qA allele. The overall diagnostic yield in our cohort was 58%. A combination of OGM to identify the FSHD haplotype and D4Z4 repeat number and NGS to identify sequence and copy number variants in the SMCHD1 gene is a practical and cost-effective option with increased precision for accurate diagnosis of FSHD types 1 and 2.

Evaluating the clinical utility of a long-read sequencing-based approach in genetic testing of fragile-X syndrome

BackgroundFragile X syndrome (FXS) arises from the FMR1 CGG expansion. Comprehensive genetic testing for FMR1 CGG expansions, AGG interruptions, and microdeletions is essential to provide genetic counseling for females carrying premutation alleles. However, conventional PCR-based FMR1 assays mainly focus on CGG repeats, and could detect AGG interruption only in males. MethodsThe clinical utility of a long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was evaluated in 238 high-risk samples by comparing to conventional PCR assays. ResultsPCR assays identified five premuation and three full mutation categories alleles in all the samples, and CAFXS successfully called all the FMR1 CGG expansion. CAFXS identified 24-bp microdeletions upstream to the trinucleotide region with 30 CGG repeats, which was miscalled by the length-based PCR methods. CAFXS also identified a 187-bp deletion in about 1/7 of the sequencing reads in a male patient with mosaic full mutation alleles. CAFXS allowed for precise constructing the FMR1 CGG repeat and AGG interruption pattern in all the samples, and identified a novel and alternative CGA interruption in one normal female sample. ConclusionsCAFXS represents a more comprehensive and accurate approach for FXS genetic testing that potentially enables more informed genetic counseling compared to PCR-based methods.

Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a neurogenetic disorder due to loss-of-function TSC1 or TSC2 variants, characterized by tumors affecting multiple organs, including skin, brain, heart, lung, and kidney. Mosaicism for TSC1 or TSC2 variants occurs in 10%-15% of individuals diagnosed with TSC. Here, we report comprehensive characterization of TSC mosaicism by using massively parallel sequencing (MPS) of 330 TSC samples from a variety of tissues and fluids from a cohort of 95 individuals with mosaic TSC. TSC1 variants in individuals with mosaic TSC are much less common (9%) than in germline TSC overall (26%) (p<0.0001). The mosaic variant allele frequency (VAF) is significantly higher in TSC1 than in TSC2, in both blood and saliva (median VAF: TSC1, 4.91%; TSC2, 1.93%; p= 0.036) and facial angiofibromas (median VAF: TSC1, 7.7%; TSC2 3.7%; p= 0.004), while the number of TSC clinical features in individuals with TSC1 and TSC2 mosaicism was similar. The distribution of mosaic variants across TSC1 and TSC2 is similar to that for pathogenic germline variants in general TSC. The systemic mosaic variant was not present in blood in 14 of 76 (18%) individuals with TSC, highlighting the value of analysis of multiple samples from each individual. A detailed comparison revealed that nearly all TSC clinical features are less common in individuals with mosaic versus germline TSC. A large number of previously unreported TSC1 and TSC2 variants, including intronic and large rearrangements (n= 11), were also identified.

Azithromycin Susceptibility Testing and Molecular Investigation of Neisseria gonorrhoeae Isolates Collected in Russia, 2020-2021.

The aim of this work was to study the resistance to macrolides (azithromycin) in the modern Russian population of N. gonorrhoeae with the analysis of genetic resistance determinants. Azithromycin is not used to treat gonococcal infection in Russia. However, among 162 isolates collected in 2020-2021, 22 isolates (13.6%) were phenotypically resistant to azithromycin. Mutations in 23S rRNA genes were found only in two isolates; erm and mefA genes were absent. Azithromycin resistance was shown to be predominantly associated with mutations in the mtrR and mtrD genes of the MtrCDE efflux pump and their mosaic alleles which may have formed due to a horizontal transfer from N. meningitidis. A total of 30 types of mtrR alleles and 10 types of mtrD alleles were identified including mosaic variants. Matching between the mtrR and mtrD alleles was revealed to indicate the cooperative molecular evolution of these genes. A link between the mtrR and mtrD alleles and NG-MAST types was found only for NG-MAST 228 and 807, typical of N. gonorrhoeae in Russia. The high level of resistance to azithromycin in Russia may be related to the spread of multiple transferable resistance to antimicrobials regardless of their use in the treatment of gonococcal infection.

Comprehensive Analysis of Fragile X Syndrome: Full Characterization of the FMR1 Locus by Long-Read Sequencing.

Fragile X syndrome (FXS) is the most frequent cause of inherited X-linked intellectual disability. Conventional FXS genetic testing methods mainly focus on FMR1 CGG expansions and fail to identify AGG interruptions, rare intragenic variants, and large gene deletions. A long-range PCR and long-read sequencing-based assay termed comprehensive analysis of FXS (CAFXS) was developed and evaluated in Coriell and clinical samples by comparing to Southern blot analysis and triplet repeat-primed PCR (TP-PCR). CAFXS accurately detected the number of CGG repeats in the range of 93 to at least 940 with mass fraction of 0.5% to 1% in the background of normal alleles, which was 2-4-fold analytically more sensitive than TP-PCR. All categories of mutations detected by control methods, including full mutations in 30 samples, were identified by CAFXS for all 62 clinical samples. CAFXS accurately determined AGG interruptions in all 133 alleles identified, even in mosaic alleles. CAFXS successfully identified 2 rare intragenic variants including the c.879A > C variant in exon 9 and a 697-bp microdeletion flanking upstream of CGG repeats, which disrupted primer annealing in TP-PCR assay. In addition, CAFXS directly determined the breakpoints of a 237.1-kb deletion and a 774.0-kb deletion encompassing the entire FMR1 gene in 2 samples. Long-read sequencing-based CAFXS represents a comprehensive assay for identifying FMR1 CGG expansions, AGG interruptions, rare intragenic variants, and large gene deletions, which greatly improves the genetic screening and diagnosis for FXS.

Origins of biallelic inactivation of NF2 in neurofibromatosis type 2.

Elucidating the mechanism by which biallelic inactivation evolved could provide a mechanistic understanding for NF2 tumorigenesis and also a rationale for clinical management. A cohort of 60 NF2 patients was recruited. Next-generation sequencing of tumor and paired control samples was used to explore how NF2 mutations evolve in determining the clinical phenotypes. In total, 60 blood samples (one from each patient) and 61 (from 35 patients) NF2-associated tumors were collected. Next-generation sequencing of the blood samples detected "first hit" NF2 mutation in 35/60 donors (58.3%), 82.9% of which (29/35) bear heterozygous germline mutations, and 17.1% (6/35) of which are mosaics with variable allelic frequency (VAF). While a number of NF2 patients were found without germline mutation, most (57/61, 93.4%) NF2-associated tumors were identified with NF2 somatic mutation. We calculated the correlation between the onset latency of mosaic and germline NF2 allele carriers with the mosaicism VAF. The mosaicism VAF is negatively and linearly correlated to clinical symptom onset latency (R2 = 0.3677, P = .00351), suggesting biallelic inactivation probability is a linear function of "first hit" prevalence in the body. The second NF2 somatic mutation occurrence time positively correlates with the onset of clinical symptoms (R2 = 0.4151, P = .02633), suggesting tumor growth is linearly proportional to the time after biallelic inactivation. Our results suggested that biallelic inactivation of NF2 evolved through neutral drift and preexisting first hit NF2 allele determines certain aspects of the clinical symptoms. Genetic diagnosis should be included in the diagnostic criteria and treatment consideration of NF2.

The emergence of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone by transfer of resistance from an oral Neisseria subflava reservoir of resistance.

The ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone was first discovered in Japan in 2015. We investigated the possibility of horizontal gene transfer from Neisseria subflava harbouring the mosaic-like PBP-2 in the emergence of the FC428 clone. We also analysed whether there were fitness costs associated with the sustained international dissemination of the clone. Sequencing of the penA gene in ceftriaxone-resistant N. subflava strains was performed. For transformation experiments between donor N. subflava and ciprofloxacin-resistant wild-type penA N. gonorrhoeae recipient, the full-length PCR amplification product of the penA gene, including DUS regions, was used as the donor DNA. Biological fitness of the transformants was measured by growth competition assays. The impact of QRDR and mtrR mutations, which have been reported as compensatory mutations, on fitness was also assessed. The penA mosaic allele of the FC428 clone showed 100%, 91.8%, and 89.8% homology, respectively, with penA genes of three ceftriaxone-resistant N. subflava strains, No. 30, No. 9 and No. 14. Results were consistent with homologous recombination with the donated penA mosaic allele. In co-cultures with the parent strain, transformants showed comparable growth indicating that a gyrA mutation compensates for the fitness cost of mosaic penA alleles. Our findings support the hypothesis that the FC428 clone was generated by transformation of the mosaic penA allele from oropharyngeal N. subflava to N. gonorrhoeae. Furthermore, it suggests that mutations in the gyrA QRDR region compensate for fitness costs and contribute to the continued transmission of the FC428 clone.

Phylogenomic analysis reveals persistence of gonococcal strains with reduced-susceptibility to extended-spectrum cephalosporins and mosaic penA-34

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935–1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally.

Prognostic Information for Known Genetic Carriers of RB1 Pathogenic Variants (Germline and Mosaic)

To compare the number of tumors per eye for mosaic carriers of RB1 pathogenic variants with full germline variants and the conversion from unilateral to bilateral disease. Retrospective cohort study comparing patients with retinoblastoma and different genetic subtypes: high penetrance (HP), low penetrance (LP), and mosaicism. Data were analyzed between 1992 and 2018 at the Retinoblastoma Unit, Royal London Hospital, London, United Kingdom. All familial patients had a parent with a known pathogenic variant even if the parent did not manifest the disease. Number of tumors per eye in children who developed retinoblastoma in that eye. Other outcomes included total number of tumors per patient, age at diagnosis, laterality at presentation and later, sex, and stage according to International Intraocular Retinoblastoma Classification. A total of 111 patients were included: 64 full germline, familial patients (53 HP and 11 LP) and 47 mosaic patients. Twelve HP patients (23%) were unilateral, and 8 of 12 patients (67%) developed tumors in their previously unaffected eye. A total of 34 mosaic patients (72%) were unilateral, and only 2 (6%) developed tumors in their unaffected eye. Age at diagnosis was higher in mosaic patients (median, 22 months) than in HP patients (median 7) (P < 0.00002). The number of tumors per eye was fewer in patients with mosaic alleles (median, 1.0; range, 1-6) compared with patients with HP alleles (median, 3.0; range, 1-8) (P < 0.0003). All 3 children (4 eyes) with mosaicism and more than 2 tumors per eye had high levels of mosaicism. Children with mosaic alleles have fewer tumors per eye compared with those with known high-penetrant pathogenic variants and are more likely to remain unilateral. The level of mosaicism has an impact on laterality and number of tumors.

Highly Sensitive Blocker Displacement Amplification and Droplet Digital PCR Reveal Low-Level Parental FOXF1 Somatic Mosaicism in Families with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins

Detection of low-level somatic mosaicism [alternate allele fraction (AAF) ≤ 10%] in parents of affected individuals with the apparent de novo pathogenic variants enables more accurate estimate of recurrence risk. To date, only a few systematic analyses of low-level parental somatic mosaicism have been performed. Herein, highly sensitive blocker displacement amplification, droplet digital PCR, quantitative PCR, long-range PCR, and array comparative genomic hybridization were applied in families with alveolar capillary dysplasia with misalignment of pulmonary veins. We screened 18 unrelated families with the FOXF1 variant previously determined to be apparent de novo (n=14), of unknown parental origin (n=1), or inherited from a parent suspected to be somatic and/or germline mosaic (n=3). We identified four (22%) families with FOXF1 parental somatic mosaic single-nucleotide variants (n=3) and copy number variant deletion (n=1) detected in parental blood samples and an AAF ranging between 0.03% and 19%. In one family, mosaic allele ratio in tissues originating from three germ layers ranged between <0.03% and 0.65%. Because the ratio of parental somatic mosaicism have significant implications for the recurrence risk, this study further implies the importance of a systematic screening of parental samples for low-level and very-low-level (AAF ≤ 1%) somatic mosaicism using methods that are more sensitive than those routinely applied in diagnostics.

Emergence of ceftriaxone-resistant Neisseria gonorrhoeae strains harbouring a novel mosaic penA gene in China.

The continuous emergence of ceftriaxone-resistant Neisseria gonorrhoeae strains threatens the effectiveness of current treatment regimens for gonorrhoea. The objective of the present study was to characterize three ceftriaxone-resistant N. gonorrhoeae strains with a novel mosaic penA allele isolated in China. Three ceftriaxone-resistant Neisseria gonorrhoeae strains (GC150, GC161 and GC208) isolated in 2017 were characterized by N. gonorrhoeae multiantigen sequence typing (NG-MAST), MLST and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR). Recombination analyses were performed using the SimPlot software. Three strains had the same antibiotic resistance profiles, with resistance to ceftriaxone (MIC 0.5 mg/L), ciprofloxacin (MIC 8.0 mg/L), penicillin (MIC 2.0 mg/L) and tetracycline (MIC 2.0-8.0 mg/L). STs were assigned as MLST7360, NG-MAST14292 and NG-STAR1611/NG-STAR1612. The penA gene of these three strains differed from previous ceftriaxone-resistant gonococcal strains and harboured a novel mosaic allele (penA-121.001). Like N. gonorrhoeae FC428, a widely disseminated ceftriaxone-resistant strain that was initially described in Japan in 2015, all strains also possessed substitutions A311V and T483S in PBP2, which are associated with resistance to ceftriaxone. Potential recombination events were detected in penA between N. gonorrhoeae strain FC428 and commensal Neisseria species. Our results provide further evidence that the commensal Neisseria species (Neisseria cinerea and Neisseria perflava) can serve as a reservoir of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains. The emergence of such strains may be the result of the interspecies recombination of penA genes between N. gonorrhoeae strain FC428 and commensal Neisseria species.

Antimicrobial resistance (AMR) and molecular characterization of Neisseria gonorrhoeae in Ghana, 2012-2015

Neisseria gonorrhoeae antimicrobial resistance (AMR) surveillance is essential for tracking the emergence and spread of AMR strains in local, national and international populations. This is crucial for developing or refining treatment guidelines. N. gonorrhoeae multiantigen sequence typing (NG-MAST) is beneficial for describing the molecular epidemiology of gonococci at national and international levels. Elucidation of AMR determinants to β-lactam drugs, is a means of monitoring the development of resistance. In Ghana, little is known about the current gonococcal AMR prevalence and no characterization of gonococcal isolates has been previously performed. In this study, gonococcal isolates (n = 44) collected from five health facilities in Ghana from 2012 to 2015, were examined using AMR testing, NG-MAST and sequencing of penA. High rates of resistance were identified to tetracycline (100%), benzylpenicillin (90.9%), and ciprofloxacin (81.8%). One isolate had a high cefixime MIC (0.75 μg/ml). Twenty-eight NG-MAST sequence types (STs) were identified, seventeen of which were novel. The isolate with the high cefixime MIC contained a mosaic penA-34 allele and belonged to NG-MAST ST1407, an internationally spreading multidrug-resistant clone that has accounted for most cefixime resistance in many countries. In conclusion, AMR testing, NG-MAST, and sequencing of the AMR determinant penA, revealed high rates of resistance to tetracycline, benzylpenicillin, and ciprofloxacin; as well as a highly diverse population of N. gonorrhoeae in Ghana. It is imperative to continue with enhanced AMR surveillance and to understand the molecular epidemiology of gonococcal strains circulating in Ghana and other African countries.

High-resolution melting analysis for rapid detection of the internationally spreading ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone.

Increased awareness of the international spread of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone, which threatens recommended dual therapy, is essential. The objective of the present study was to develop and evaluate a rapid, simple and cost-effective method based on high-resolution melting (HRM) analysis for direct detection of the FC428 clone from clinical isolates and specimens. The singleplex HRM assay was designed to identify the FC428 clone by using specific primers, which flank the alteration A311V in the penA-60.001 allele. Analytical performance was initially evaluated by testing 623 isolates and a panel of non-gonococcal strains. To ensure the method can be directly applied in clinical samples, two internal control targets (opa and porA) were also designed and included in the final multiplex HRM assay. Two hundred and eighty-two clinical samples (94 urine and 188 urethral/genital swabs) were then analysed using this multiplex HRM assay. The FC428 clone was easily differentiated from the non-mosaic alleles and other mosaic alleles without A311 mutations by comparing the differences in melt curves. Cross-reactivity was not observed for the penA-60.001 allele when testing 15 non-gonococcal Neisseria strains. When applied to the 623 isolates, the HRM assay successfully characterized one isolate as an FC428 clone (MLST1903, NG-MAST3435, NG-STAR233). Our data show that the multiplex HRM assay with high specificity can be directly applied in clinical samples. This method can generate results within 90 min at a cost of less than US$0.5 per isolate or sample, making this assay an ideal tool for large epidemiological studies to enhance surveillance of the internationally transmitted ceftriaxone-resistant N. gonorrhoeae FC428 clone.

Genetic Characterization and Enhanced Surveillance of Ceftriaxone-Resistant Neisseria gonorrhoeae Strain, Alberta, Canada, 2018.

In July 2018, a case of Neisseria gonorrhoeae associated with ceftriaxone treatment failure was identified in Alberta, Canada. We identified the isolate and nucleic acid amplification testing (NAAT) specimen as the ceftriaxone-resistant strain multilocus sequence type 1903/NG-MAST 3435/NG-STAR 233, originally identified in Japan (FC428), with the same penA 60.001 mosaic allele and genetic resistance determinants. Core single-nucleotide variant (SNV) analysis identified 13 SNVs between this isolate and FC428. Culture-independent surveillance by PCR for the A311V mutation in the penA allele and N. gonorrhoeae multiantigen sequence typing directly from NAAT transport media positive for N. gonorrhoeae by NAAT did not detect spread of the strain. We identified multiple sequence types not previously detected in Alberta by routine surveillance. This case demonstrates the benefit of using culture-independent methods to enhance detection, public health investigations, and surveillance to address this global threat.

A clinical survey of mosaic single nucleotide variants in disease-causing genes detected by exome sequencing

BackgroundAlthough mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed.MethodsTo investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics.ResultsWe found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes.ConclusionsIn sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.

Evaluation of Susceptibilities to Carbapenems and Faropenem Against Cephalosporin-Resistant Neisseria gonorrhoeae Clinical Isolates with penA Mosaic Alleles.

Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with β-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5-2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.

A Whole-genome Sequencing Analysis of Neisseria gonorrhoeae Isolates in China: An Observational Study

BackgroundTracking the spread of the Neisseria gonorrhoeae strains with decreased susceptibility or resistance to cephalosporins is a major priority for global surveillance programmes. Whole-genome sequencing (WGS) has been widely used by increasing countries in North America, Europe, and Pacific to determine the decreased susceptible or resistance determinants of Neisseria gonorrhoeae, track the spread of these determinants throughout the gonococcal population at national or regional level. However, no studies to date have examined the genomic epidemiology of gonorrhea in Asia where the antimicrobial resistant strains of Neisseria gonorrhoeae appears to have emerged before disseminating the strains globally. MethodsWe obtained clinical isolates and data from the China Gonococcal Resistance Surveillance Programme (China-GRSP) from 2012 to 2013. We sequenced the genomes of 435 clinical isolates of Neisseria gonorrhoeae, including 112 (25.6%) isolates with decreased susceptibility to ceftriaxone (Cfx-DS). We assessed the association between antimicrobial resistance genotype and phenotype. We also compared our data with the whole genome data of the isolates from the USA and the UK in the GenBank. FindingsThe most prevalent MLST STs in our gonococcal population were MLST ST7827 (n = 74), followed by ST7365 (n = 58), ST1600 (n = 38), ST7367 (n = 35), and ST7363 (n = 29). MLST ST1901 which was reported as the predominant ST in the US was not found in our population. A total of 2512 strains, including additional 2077 published NG strains, were further included for phylogenetic analysis. It generated two distinct lineages - lineage 1 and lineage 2. Analysis of MLST ST1901 in the database indicate that most of MLST ST1901 isolates in the lineage2.6 were Cfx-DS isolates while all isolates in the lineage 2.1 were sensitive to ceftriaxone (77/110 vs. 0/13; p < 0.001). ST1901/lineage 2.6 is a ceftriaxone resistant clone which cannot distinguished by MLST genotyping. In the isolates from our study, the MICs of ceftriaxone for ST7363/lineage 2.6 isolates ranged from 0.008–0.125 mg/L (mean ± SD; 0.054 ± 0.043 mg/L) while those for ST7363/lineage 2.8 isolates ranged from 0.032–0.250 mg/L (0.134 ± 0.085 mg/L). All ST7363/lineage 2.8 isolates contained penA mosaic alleles. InterpretationTo our knowledge, current study is the first WGS-based analysis of gonococcal population at national level in Asia. China harbors the different predominant clones associated with decreased susceptibility to ceftriaxone from those clones circulated in other regions. The findings from the study can be not only used as baseline data for future studies in China but also contributable to our understanding on spread of N. gonorrhoeae and its resistant strains at regional and global levels. FundingThe Chinese Academy Medical Sciences (CAMS) Initiative for Innovative Medicine.