Regarding several cytotoxic agents, it was evidenced that genetic polymorphisms in genes encoding enzymes involved in their metabolism are associated with higher risk of toxicity. Genotyping these ...genes before treatment is a valuable strategy to prevent side effects and to predict individual response to drug therapy. This pharmacogenetic approach is recommended for chemotherapies such as thiopurines (azathioprine, 6-mercaptopurine, thioguanine), irinotecan, and fluoropyrimidines (capecitabine and 5-fluorouracil). In this study, we aimed at developing and validating a fast, cost-effective, and easily implementable multiplex genotyping method suitable for analyzing a panel of nine variants involved in the pharmacogenetics of widely prescribed anticancer drugs. We designed a multiplex-specific PCR assay where fragments were labeled by two different fluorescent dye markers (HEX/FAM) identifiable by fragment analysis. These two labels were used to discriminate bi-allelic variants, while the size of the fragment allowed the identification of a particular polymorphism location. Variants of interest were
(rs1800462, rs1142345, rs1800460),
(rs116855232),
(rs55886062, rs3918290, rs67376798, rs75017182), and
(rs8175347). The assay was repeatable, and genotypes could be determined when DNA sample amounts ranged from 25 to 100 ng. Primers and dye remained stable in a ready-to-use mixture solution after five freeze-thaw cycles. Accuracy was evidenced by the consistency of 187 genotyping results obtained with our multiplex assay and a reference method. The developed method is fast and cost-effective in simultaneously identifying nine variants involved in the pharmacological response of anticancer drugs. This assay can be easily implemented in laboratories for widespread access to pharmacogenetics in clinical practice.
Single-nucleotide variants (SNVs) within gene coding sequences can significantly impact pre-mRNA splicing, bearing profound implications for pathogenic mechanisms and precision medicine. In this ...study, we aim to harness the well-established full-length gene splicing assay (FLGSA) in conjunction with SpliceAI to prospectively interpret the splicing effects of all potential coding SNVs within the four-exon SPINK1 gene, a gene associated with chronic pancreatitis.
Our study began with a retrospective analysis of 27 SPINK1 coding SNVs previously assessed using FLGSA, proceeded with a prospective analysis of 35 new FLGSA-tested SPINK1 coding SNVs, followed by data extrapolation, and ended with further validation. In total, we analyzed 67 SPINK1 coding SNVs, which account for 9.3% of the 720 possible coding SNVs. Among these 67 FLGSA-analyzed SNVs, 12 were found to impact splicing. Through detailed comparison of FLGSA results and SpliceAI predictions, we inferred that the remaining 653 untested coding SNVs in the SPINK1 gene are unlikely to significantly affect splicing. Of the 12 splice-altering events, nine produced both normally spliced and aberrantly spliced transcripts, while the remaining three only generated aberrantly spliced transcripts. These splice-impacting SNVs were found solely in exons 1 and 2, notably at the first and/or last coding nucleotides of these exons. Among the 12 splice-altering events, 11 were missense variants (2.17% of 506 potential missense variants), and one was synonymous (0.61% of 164 potential synonymous variants). Notably, adjusting the SpliceAI cut-off to 0.30 instead of the conventional 0.20 would improve specificity without reducing sensitivity.
By integrating FLGSA with SpliceAI, we have determined that less than 2% (1.67%) of all possible coding SNVs in SPINK1 significantly influence splicing outcomes. Our findings emphasize the critical importance of conducting splicing analysis within the broader genomic sequence context of the study gene and highlight the inherent uncertainties associated with intermediate SpliceAI scores (0.20 to 0.80). This study contributes to the field by being the first to prospectively interpret all potential coding SNVs in a disease-associated gene with a high degree of accuracy, representing a meaningful attempt at shifting from retrospective to prospective variant analysis in the era of exome and genome sequencing.
Multiple osteochondromas (MO), the most common type of benign bone tumor, is an autosomal dominant skeletal disorder characterized by multiple cartilage-capped bony protuberances. In most cases,
and
..., which encode glycosyltransferases involved in the biosynthesis of heparan sulfate, are the genes responsible. Here we describe the clinical, phenotypic and genetic characterization of MO in 22 unrelated Chinese families involving a total of 60 patients. Variant detection was performed by means of a battery of different techniques including Sanger sequencing and whole-exome sequencing (WES). The pathogenicity of the missense and splicing variants was explored by means of
prediction algorithms. Sixteen unique pathogenic variants, including 10 in the
gene and 6 in the
gene, were identified in 18 (82%) of the 22 families. Fourteen (88%) of the 16 variants were predicted to give rise to truncated proteins whereas the remaining two were missense. Seven variants were newly described here, further expanding the spectrum of MO-causing variants in the
and
genes. More importantly, the identification of causative variants allowed us to provide genetic counseling to 8 MO patients in terms either of preimplantation genetic testing (PGT) or prenatal diagnosis, thereby preventing the reoccurrence of MO in the corresponding families. This study is the first to report the successful implementation of PGT in MO families and describes the largest number of subjects undergoing prenatal diagnosis to date.
Roscovitine (Seliciclib), a new protein kinase inhibitor, was administered orally to adult patients with cystic fibrosis for the first time in the ROSCO-CF trial, a dose-escalation, phase IIa, ...randomized, controlled trial. Extensive pharmacokinetic sampling was performed up to 12 h after the first oral dose. Roscovitine and its main metabolite M3 were quantified by liquid chromatography coupled with tandem mass spectrometry. The pharmacokinetics analyses were performed by non-linear mixed effects modelling. Monte Carlo simulations were performed to assess the impact of dose on the pharmacokinetics of oral roscovitine. Twenty-three patients received oral doses ranging from 200 to 800 mg of roscovitine and 138 data points were available for both roscovitine and M3 concentrations. The pharmacokinetics was best described by a two-compartment parent-metabolite model, with a complex saturable absorption process modelled as the sum of Gaussian inverse density functions. The Monte Carlo simulations showed a dose-dependent and saturable first-pass effect leading to pre-systemic formation of M3. The treatment with proton-pump inhibitors reduced the rate of absorption of oral roscovitine. The pharmacokinetics of oral roscovitine in adult patients with cystic fibrosis was non-linear and showed significant inter-individual variability. A repeat-dose study will be required to assess the inter-occasional variability of its pharmacokinetics.
Breast reduction surgery for hypertrophy is one of the most commonly performed procedures in plastic surgery. This surgery exposes patients to complications that are well-documented in the ...literature. The objective of this study is to identify risk factors to establish an estimate of the risk of developing complications. The authors propose the first predictive score of postoperative complications, including continuous preoperative variables such as body mass index (BMI) and suprasternal notch-to-nipple distance (SSN:N).
An analytic observational retrospective cohort study was conducted including 1306 patients who underwent superior pedicle reduction mammaplasty at the Rennes University Hospital (France) between January 1, 2011, and December 31, 2016. The primary endpoint was to study the association between known preoperative risk factors and occurrence of any complications using multivariable logistic regression to identify independent risk factors. A secondary endpoint was to establish a score to estimate a probability of occurrence of complications.
A total of 1306 patients were analyzed. Multivariable logistic regression showed three independent risk factors: active smoking OR, 6.10 (95% CI: 4.23, 8.78); P < 0.0001, BMI OR, 1.16 (95% CI: 1.11, 1.22); P < 0.0001, and SSN:N OR, 1.14 (95% CI: 1.08, 1.21); P < 0.0001. The Rennes Plastic Surgery Score estimating the occurrence of postoperative complications was determined, integrating regression coefficients of each risk factor.
Active smoking, BMI, and SSN:N distance are independent preoperative risk factors for the occurrence of breast reduction complications. The Rennes Plastic Surgery Score including the continuous values of BMI and SSN:N allows us to provide our patients with a reliable estimation of the risk of occurrence of these complications.
Risk, III.
INTRODUCTION: Breast reduction surgery for hypertrophy is one of the most commonly performed procedures in plastic surgery. This surgery exposes patients to complications well documented in the ...literature. The objective of this study is therefore to identify the risk factors in order to establish an estimate of the risk of developing complications. We propose the first predictive score of postoperative complications including continuous preoperative variables like Body Mass Index (BMI) and Supra Sternal Notch – Nipple Distance (SSN:N). MATERIALS AND METHODS: An analytic observational retrospective cohort study was conducted including 1306 patients who underwent superior pedicle reduction mammoplasty at the Rennes University Hospital (France) between 2011/01/01 and 2016/31/12. Primary endpoint was to study association between known preoperative risk factors and occurrence of any complications using multivariable logistic regression in order to identify independent risk factors. Secondary endpoint was to establish a score to estimate a probability of occurrence of complication. RESULTS: 1306 patients were analyzed. Multivariable logistic regression showed three independent risk factors : active smoking (OR 6.10 4.23; 8.78 p < 0.0001), BMI (OR 1.16 1.11; 1.22 p < 0.0001), SSN:N (OR 1.14 1.08; 1.21 p < 0.0001). The Rennes Plastic Surgery Score estimating occurrence of postoperative complications was determined, integrating regression coefficient of each risk factor. CONCLUSION: Active smoking, BMI and SSN:N distance are independent preoperative risk factors for the occurrence of breast reduction complications. The Rennes Plastic Surgery Score including the continuous values of BMI and SSN:N allows us to provide to our patients a reliable estimate of the risk of occurrence of these complications. Evidence Based Medicine Level II: Lesser-quality prospective cohort or comparative study; retrospective cohort or comparative study; or untreated controls from a randomized controlled trial
Abstract
Objectives
To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France.
Methods
The genome of 15-307.1 was entirely ...sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A.
Results
As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization.
Conclusions
Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).
Abstract
Objectives
To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports ...and environments.
Methods
All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments.
Results
Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids.
Conclusions
The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use.
Alpha‐mannosidosis is a rare autosomal recessive lysosomal storage disorder caused by biallelic mutations in the MAN2B1 gene and characterized by a wide clinical heterogeneity. Diagnosis for this ...multisystemic disorder is confirmed by the presence of either a deficiency in the lysosomal enzyme acid alpha‐mannosidase or biallelic mutations in the MAN2B1 gene. This diagnosis confirmation is crucial for both clinical management and genetic counseling purposes. Here we describe a late diagnosis of alpha‐mannosidosis in a patient presenting with syndromic intellectual disability, and a rare retinopathy, where reverse phenotyping played a pivotal role in interpreting the exome sequencing result. While a first missense variant was classified as a variant of uncertain significance, the phenotype‐guided analysis helped us detect and interpret an in‐trans apparent alu‐element insertion, which appeared to be a copy number variant (CNV) not identified by the CNV caller. A biochemical analysis showing abnormal excretion of urinary mannosyloligosaccharide and an enzyme assay permitted the re‐classification of the missense variant to likely pathogenic, establishing the diagnosis of alpha‐mannosidosis. This work emphasizes the importance of reverse phenotyping in the context of exome sequencing.
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