Ion PGM Research Articles

Maple syrup urine disease diagnosis in Brazilian patients by massive parallel sequencing

Biallelic pathogenic variants cause maple syrup urine disease (MSUD) in one of the branched-chain α-keto acid dehydrogenase (BCKDH) complex genes (BCKDHA, BCKDHB, DBT, DLD, and PPM1K) leading to the accumulation of leucine, isoleucine, and valine. This study aimed to perform a molecular diagnosis of Brazilian patients with MSUD using gene panels and massive parallel sequencing. Eighteen Brazilian patients with a biochemical diagnosis of MSUD were analyzed by massive parallel sequencing in the Ion PGM Torrent Server using a gene panel with the BCKDHA, BCKDHB, and DBT genes. The American College of Medical Genetics and Genomics guidelines were used to determine variant pathogenicity. Thirteen patients had both variants found by massive parallel sequencing, whereas 3 patients had only one variant found. In 2 patients, the variants were not found by this analysis. These 5 patients required additional Sanger sequencing to confirm their genotype. Twenty-five pathogenic variants were identified in the 3 MSUD-related genes (BCKDHA, BCKDHB, and DBT). Most variants were present in the BCKDHB gene, and no common variants were found. Nine novel variants were observed: c.922 A > G, c.964C > A, and c.1237 T > C in the BCKDHA gene; and c.80_90dup, c.384delA, c.478 A > T, c.528C > G, c.977 T > C, and c.1039-2 A > G in the BCKDHB gene. All novel variants were classified as pathogenic. Molecular modeling of the novel variants indicated that the binding of monomers was affected in the BCKDH complex tetramer, which could lead to a change in the stability and activity of the enzyme. Massive parallel sequencing with targeted gene panels seems to be a cost-effective method that can provide a molecular diagnosis of MSUD.

Performance of 16S rRNA Gene Next-Generation Sequencing and the Culture Method in the Detection of Bacteria in Clinical Specimens.

Effective treatment of infectious diseases requires prompt and accurate bacterial identification and tailored antimicrobial treatments. Traditional culture methods are considered the gold standard, but their effectiveness diminishes for fastidious and hard-to-grow microorganisms. In recent years, molecular diagnostic tools such as 16S rRNA gene next-generation sequencing (16S NGS) have gained popularity in the field. We analysed data from samples submitted for 16S NGS between July 2022 and July 2023 at the Department of Advanced Translational Microbiology in Trieste, Italy. The study included samples submitted for both culture-based identification and 16S NGS. Conventional media were used for culture, and bacterial identification was performed using MALDI-TOF mass spectrometry. The V3 region of the 16S rRNA gene was sequenced using the Ion PGM platform. Among the 123 samples submitted, drainage fluids (38%) and blood (23%) were the most common, with requests predominantly from the Infectious Diseases (31.7%) and Orthopedic (21.13%) Units. In samples collected from patients with confirmed infections, 16S NGS demonstrated diagnostic utility in over 60% of cases, either by confirming culture results in 21% or providing enhanced detection in 40% of instances. Among the 71 patients who had received antibiotic therapies before sampling (mean 2.3 prior antibiotic days), pre-sampling antibiotic consumption did not significantly affect the sensitivity of 16S NGS. In routine microbiology laboratories, combining 16S NGS with culture method enhances the sensitivity of microbiological diagnostics, even when sampling is conducted during antibiotic therapy.

Abstract 7627: First NGS-based companion diagnostic to aid in selecting non-small cell lung cancer patients with ERBB2 (HER2) activating mutations for treatment with trastuzumab deruxtecan

Abstract Introduction The ERBB2 companion diagnostic program aims to expand the intended use of the Oncomine ™ Dx Target Test (ODxTT) to determine ERBB2 single nucleotide variants (SNVs) in exons 8, 17, 18, 19, 20, 21 and 22 and exon 20 insertions from formalin-fixed, paraffin-embedded (FFPE) tumor samples in non-small cell lung cancer (NSCLC) patients who may benefit from targeted drug therapy. Methods The ODxTT is a qualitative in vitro diagnostic test that uses targeted high throughput, parallel-sequencing technology to detect SNVs, deletions, insertions, and CNVs in genes from DNA and fusions from RNA isolated from FFPE tissue samples using the Ion PGM ™ Dx System. Analytical validation (AV) studies interrogated ODxTT’s ability to detect ERBB2 mutations. The clinical validation (CV) study involved a method comparison/concordance analysis of ERBB2 mutations between the ODxTT and the TruSight Tumor 170 (TST170) assay, as well as a series of clinical trial assays (CTAs). The CV study was conducted using clinical specimens from 91 patients enrolled in Destiny-Lung01 and 121 stage-matched commercially procured samples. The aim was to evaluate positive percent agreement (PPA), negative percent agreement (NPA) and overall percent agreement (OPA) for CTAs and TST170 assay. Results A total of 91 ERBB2 subjects were included in the full analysis of the Destiny-Lung01 clinical trial. 49 samples had valid ODxTT results available and 42 samples were unevaluable which included 13 unknowns and 29 test not performed. The 49 patient samples that were evaluable were used to assess the clinical efficacy based on ODxTT results. In the series of 121 samples that were used to assess clinical efficacy in the CV study, 109 had evaluable ODxTT results. The PPA, NPA and OPA of ODxTT to the CTAs when excluding unknowns were 98%, 100% and 99.4%, respectively, and based off the Clopper-Pearson method for binomial distribution a 95% lower bound confidence interval (CI) were 89.6%, 96.6% and 96.5%, respectively. There was one discordant sample with a negative ODxTT and a positive CTA result (false negative). The PPA, NPA and OPA of ODxTT to TST170 assay were 100%, 99.1% and 99.3% respectively with a 95% lower bound CI of 90.8%, 95.0% and 96.3%, respectively. There was one discordant sample with a negative TST170 and a positive ODxTT result (false positive). Conclusion AV and CV studies successfully verified and validated the ODxTT for the detection of ERBB2 mutations in NSCLC FFPE samples. The 6 AV studies met the necessary product requirements and acceptance criteria to detect clinical samples with ERBB2 mutations. Additionally, the CV study clinical accuracy and clinical efficacy results demonstrated the safety and effectiveness of the use of the ODxTT in FFPE samples as an aid to identify NSCLC patients eligible for treatment with the Daiichi Sankyo lung cancer therapeutic T-DXd. Citation Format: Thomas Ha. First NGS-based companion diagnostic to aid in selecting non-small cell lung cancer patients with ERBB2 (HER2) activating mutations for treatment with trastuzumab deruxtecan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7627.

#4317 CLINICAL DIVERSITY OF STEROID-RESISTANT NEPHROTIC SYNDROME CAUSED BY TRPC6 MUTATIONS

Abstract Background and Aims Steroid-resistant nephrotic syndrome (SRNS) is a clinically and genetically heterogeneous disorder caused by either genetic or immunological factors or their combination. Approximately 30–40% of patients with SRNS make a fast progression to ESRD. SRNS represents the second leading cause of end-stage renal disease in individuals under the age of 25 years worldwide. More than 60 podocyte-related gene mutations have thus far been reported in monogenic SRNS. Recent studies indicate that almost 30% of patients with childhood-onset SRNS have monogenic causative variants in one of the 27 SRNS genes. Among these, a mutation in Transient Receptor Potential Canonical 6 (TRPC6) gene, encoding non-selective cation channel, accounts for 6% of familial SRNS and approximately 2% of sporadic cases. We recently detected eight patients with TRPC6 mutations in our SRNS cohort (n = 39, 21%) and found inter- and intra-familial heterogeneity of clinical phenotypes in terms of disease onset and progression. Method A total of 39 Japanese SRNS patients were studied according to the protocol approved by the institutional review board of each affiliation. Subjects who manifested SRNS at age one to 14-year-old with biopsy-proven FSGS were enrolled. A total of 39 SRNS patients, whose average onset of proteinuria at median age 4 (range 0.9 to 17 yr) and ESRD at median age 7 (49% of total n = 19, range 3.0 to 16.0 yr) were studied. Family history was found in 44% (n = 17), including 13 autosomal recessive, 3 dominant, and one X-linked transmission. Sixteen percent (n = 10) of the patients who underwent renal transplantation win no recurrence. Sequence analysis revealed the pathogenic variants in 62% (n = 24) of all cases. Library for whole-exome sequencing or targeted panel sequencing were prepared from genomic DNA by use of SureSelect Human All Exon V5 (Agilent) or a HaloPlex target enrichment system kit (Agilent Technologies), or Ampli-Seq (Thermo), Prepared samples were run on a HiSeq 2000, or MiSeq (Illumina), or Ion PGM (Thermo). The sequence reads with 101-bp paired-end reads and 7-bp index reads were mapped to the human reference sequence (hg19, GRCh38.p7). After filtering the superfluous begin variants, only variants that fulfill the “pathogenic” criteria of ACMG and/or ClinVar, Varsome were selected. Results Sequence analysis revealed the pathogenic variants in 62% (n = 24) of all cases. TRPC6 mutations were the most frequent cause of SRNS (n = 8), which was followed by NUP107 (n = 5), PLCE1 (n = 3), COL4A3 (n = 2), COL4A5 (n = 1), and others were identified in only single case. The eight TRPC6 mutations clustered into the two distinctive cytoplasmic domains; N-terminal ANK (Y173D, R175W, R175G) and C-terminal coiled-coil domain (p.E875V, p.867_868Del, p.S893N, p.R895C). The p.R895C is deposited as the pathogenic in ClinVar and may be a gain-of-function. The remaining 7 variants are of unknown functions: six have not yet been deposited in ClinVar, whereas one is VUS. Four patients were de novo occurrence, three followed the dominant transmission, and one arose from gonadal mosaicism. Two index patients exhibited SRNS in early childhood (< age 3), four manifested it in childhood (age 3–12), while the remaining two developed it in adolescence (age >12). In familial cases, for example, affected ones harboring R175W or p.E875V showed a remarkable discordant age onset and disease severity. Conclusion TRPC6 channelopathy was found to be the most frequent cause in our SRNS cohort, of which age onset ranges from age 0.9 to 17 year. Heterogeneity of disease severity may reflect that channel activity is regulated by the mode of action (gain- or loss-of-function), the combination of channel subunit assembly, and interaction with other modifying factors. Mutations in these TRPCs are associated with relatively common kidney diseases as well as other pathologies. Further expression studies will improve our mechanistic understanding of TRP channel pathology and may help the development of novel therapeutic targets.

Oral microbiota analyses of Saudi sickle cell anemics with dental caries

The objectives of this study were to identify the composition of oral microbiota in a cohort of patients with sickle cell anemia (SCA) and a high mean number of decayed, missing, and filled permanent teeth (DMFT) and compare it to a cohort of patients with SCA and a low number of DMFT and elucidate the effect of fetal haemoglobin levels on the oral microbiota composition. Patients who had been diagnosed with SCA, who were homozygous for sickling β-globin mutation (βS/βS), who had Arab-Indian haplotype, and who ranged in age from 5 to 12 years were included in this study. Oral saliva from each participant (n=100) was collected in GeneFiX™ Saliva DNA Microbiome Collection tube and DNA was extracted using GeneFiX™ DNA Isolation Kits. The composition of oral 16S rRNA from patients with SCA and high dental caries (n=27, DMFT ≥5) and low dental caries (n=73, DMFT ≤4) was analysed. Sequencing was performed on an Ion Personal Genome Machine using, Ion PGM Hi-Q view Sequencing 400-bp kit. We observed an overall increase in abundance of Proteobacteria, Chloroflexi, and Bacteroidetes in the high DMFT index group compared to those with a low DMFT index. In addition, there was an overall increased abundance of microbiota from Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes in the patients with SCA with low fetal haemoglobin compared to those with high fetal haemoglobin (P < .05). Enterobacteriaceae species were the most significant abundant species of bacteria found in both the high DMFT index group and low fetal haemoglobin cohort (P < .05). Our data indicate that SCA in Saudi patients with high DMFT have a higher predominance of pathogenic bacteria compared to those with low DMFT. Furthermore, SCA in Saudi patients with low fetal haemoglobin have a higher predominance of pathogenic bacteria compared to those with higher fetal haemoglobin.

Synbiotic Supplementation Modulates Gut Microbiota, Regulates β-Catenin Expression and Prevents Weight Gain in ob/ob Mice: Preliminary Findings.

Background: Obesity is one of the main health problems in the world today, and dysbiosis seems to be one of the factors involved. The aim of this study was to examine the impact of synbiotic supplementation on obesity and the microbiota in ob/ob mice. Twenty animals were divided into four groups: obese treated (OT), obese control (OC), lean treated (LT) and lean control (LC). All animals received a standard diet for 8 weeks. The treated groups received a synbiotic (Simbioflora-Invictus Farmanutrição Ltd., Sao Paulo, Brazil) in water, while the nontreated groups received only water. After 8 weeks, all animals were sacrificed, and gut tissue and stool samples were collected for mRNA isolation and microbiota analysis, respectively. β-Catenin, occludin, cadherin and zonulin in the gut tissue were analyzed via RT-qPCR. Microbiome DNA was extracted from stool samples and sequenced using an Ion PGM Torrent platform. Results: Synbiotic supplementation reduced body weight gain in the OT group compared with the OC group (p = 0.0398) and was associated with an increase in Enterobacteriaceae (p = 0.005) and a decrease in Cyanobacteria (p = 0.047), Clostridiaceae (p = 0.026), Turicibacterales (p = 0.005) and Coprococcus (p = 0.047). On the other hand, a significant reduction in Sutterella (p = 0.009) and Turicibacter (p = 0.005) bacteria was observed in the LT group compared to the LC group. Alpha and beta diversities were different among all treated groups. β-Catenin gene expression was significantly decreased in the gut tissue of the OT group (p ≤ 0.0001) compared to the other groups. No changes were observed in occludin, cadherin or zonulin gene expression in the gut tissue. Conclusions: Synbiotic supplementation prevents excessive weight gain, modulates the gut microbiota, and reduces β-catenin expression in ob/ob mice.

Implementation and Evaluation of Preimplantation Genetic Testing at Vilnius University Hospital Santaros Klinikos.

The most effective treatment of infertility is in vitro fertilization (IVF). IVF with Preimplantation Genetic Testing (PGT) allows to identify embryos with a genetic abnormality associated with a specific medical disorder and to select the most optimal embryos for the transfer. PGT is divided into structural rearrangement testing (PGT-SR), monogenetic disorder testing (PGT-M), and aneuploidy testing (PGT-A). This study mostly analyzes PGT-SR, also describes a few cases of PGT-M. The aim of this study was to implement PGT procedure at Vilnius University Hospital Santaros Klinikos (VUHSK) Santaros Fertility Centre (SFC) and to perform retrospective analysis of PGT procedures after the implementation. A single-center retrospective analysis was carried out. The study population included infertile couples who underwent PGT at SFC, VUHSK from January 01st, 2017 to December 31st, 2020. Ion PGM platform (Life Technologies, USA) and Ion ReproSeq PGS View Kit (Life Technologies, USA) were used for the whole genome amplification. Results were assessed using descriptive statistics. PGT was successfully implemented in VUHSK in 2017. During the analyzed time period, thirty-four PGT procedures were performed for 26 couples. Two procedures were performed in 2017, 7 procedures - in 2018, 13 - in 2019, and 12 - in 2020. In comparison with all IVF procedures, 2.5% procedures were IVF with PGT, a highest percentage was in 2020 (3.8% of all procedures). The main indication for PGT was balanced chromosomal rearrangements (in 85.3% cases). In all 34 cases 515 oocytes were aspirated in total, 309 oocytes were fertilized, oocytes fertilization rate exceeded 60%. A normal diploid karyotype was found in 46 (16.8%) biopsied embryos. Out of all PGT procedures, 9 (26.5%) resulted in a clinical pregnancy. Six (66.7%) pregnancies were confirmed in 2019, and 3 (33.3%) - in 2020. Three (33.3%) pregnancies resulted in spontaneous abortion, 6 (66.7%) - in delivery. The implementation of PGT in VUHSK was successful. The most common indication for PGT was a reciprocal translocation. Oocytes fertilization rate exceeded 60%, a normal karyotype was found less than in one-fifth of biopsied embryos. A highest clinical pregnancy rate was achieved in 2019 when almost half of women conceived, which is probably related to the experience gained by the multidisciplinary team. This is the first study analyzing IVF with PGT in Lithuania, however, the results should be interpreted with caution due to a low number of total procedures performed.

Investigating the stability of the Properties of Vibrio cholerae strains – Producers of Active Components of the Chemical Cholera Vaccine

One of the key requirements to producer strains used in the manufacturing of immunobiological preparations is their stability, which consists in maintaining the main cultural, morphological, physiological, and productive properties in a series of generations. This paper describes a comprehensive methodological approach to testing strain stability using in vitro techniques.The purpose of this study was to conduct an integrated analysis of the stability in the strains that produce active components of the chemical cholera vaccine when preparing seed material and at the stage of cultivation.Materials and methods. Toxigenic strains of Vibrio сholerae 569B of the classical biovar, serovar Inaba and V. сholerae M-41 of the classical biovar, serovar Ogawa were used in the work. Cell morphology was monitored through light and transmission electron microscopy. Atomic force microscopy was applied to measure the main parameters of the bacterial cell. The strains were tested for the presence of ctxA gene in the chromosome using the “GenChol” test system with electrophoretic registration of results. Whole genome sequencing of the strains was performed on the Ion Torrent PGM platform using the Ion 318 Chip Kit and the Ion PGM Hi-Q View Chef 400 Kit. To determine the specific activity of cholera toxin and O-antigen, a DOT immunoassay with a conjugate based on staphylococcal protein A and colloidal gold nanoparticles was applied.Results and discussion. The stability of the main properties of industrial V. сholerae strains – producers of the active components of the chemical cholera vaccine has been confirmed using microbiological, immunochemical, molecular-genetic methods and microscopic analysis at all stages of cultivation, and the prospects for using the integrated methodological approach experimentally substantiated. Tailoring of these methods will make it possible to control the stability of producer strains, optimize cultivation conditions and, as a result, increase the yield of the necessary antigenic component of the vaccine.

Rh Blood Group D Antigen Genotyping Using a Portable Nanopore-based Sequencing Device: Proof of Principle.

Nanopore sequencing is direct sequencing of a single-stranded DNA molecule using biological pores. A portable nanopore-based sequencing device from Oxford Nanopore Technologies (MinION) depends on driving a DNA molecule through nanopores embedded in a membrane using a voltage. Changes in current are then measured by a sensor, thousands of times per second and translated to nucleobases. Genomic DNA (gDNA) samples (n = 13) were tested for Rh blood group D antigen (RHD) gene zygosity using droplet digital PCR. The RHD gene was amplified in 6 overlapping amplicons using long-range PCR. Amplicons were purified, and the sequencing library was prepared following the 1D Native barcoding gDNA protocol. Sequencing was carried out with 1D flow cells R9 version. Data analysis included basecalling, aligning to the RHD reference sequence, and calling variants. Variants detected were compared to the results acquired previously by the Ion Personal Genome Machine (Ion PGM). Up to 500× sequence coverage across the RHD gene allowed accurate variant calling. Exonic changes in the RHD gene allowed RHD allele determination for all samples sequenced except 1 RHD homozygous sample, where 2 heterozygous RHD variant alleles are suspected. There were 3 known variant RHD alleles (RHD*01W.02, RHD*11, and RHD*15) and 6 novel RHD variant alleles, as previously seen in Ion PGM sequencing data for these samples. MinION was effective in blood group genotyping, provided enough sequencing data to achieve high coverage of the RHD gene, and enabled confident calling of variants and RHD allele determination.

Spatial Structure of &lt;i&gt;Yersinia pestis&lt;/i&gt; Population Belonging to Altai Biovar, Subspecies central asiatica Acording to Genome-Wide Sequencing Data

The aim of the work was to conduct phylogenetic analysis of Y. pestis strains, biovar altaica ssp. central asiatica, isolated in Gorno-Altai high-mountain and Sailyugem natural plague foci on the territory of Russia and Mongolia in 1965–2020, by full-genome sequencing data.Materials and methods. To determine the population structure of the biovar altaica ssp. central asiatica, 34 whole genome sequences were used (including 20 Y. pestis strains of the biovar altaica, 18 of which were sequenced by us). To isolate DNA from the Y. pestis strains, a PureLink Genomic DNA Mini Kit (Invitrogen, USA) was applied. Sequencing of the nucleotide sequences of Y. pestis strains was carried out in Ion PGM system Lifetechnologies. The analysis and processing of the obtained data were performed with the help of Newblergs Assembler 2.6 and IonTorrent Suite software package, 3.4.2. The search for SNPs was performed using the Wombac 2.0 program. The Maximum Likelihood dendrogram was built in the PhyML 3.1. The dendrogram was visualized using the FigTree 1.4.3 software.Results and discussion. Based on the data of whole genome analysis, taking into account the 1871 revealed polymorphic nucleotides, the spatial structure of the biovar altaica ssp. central asiatica has been determined. It includes several phylogeographic branches: the Kurai-Tarkhatinskaya (cluster 0.PE4a-1) and the Ulandryk-Mongolian (0.PE4a-2), which is in agreement with the geographical regions of the isolation of strains forming these branches in the Altai Mountains. The Kurai-Tarkhatinskaya branch is further divided into the Kurai (sub-cluster 0.PE4a-1-1, formed by the strains of 2009–2018) and Tarkhatinskaya (subcluster 0.PE4a-1-2, formed by the strains of 2012–2020) sub-branches, while the Ulandryk-Mongolian branch of evolution is split into sub-branches represented by strains from the Ulandryk meso focus (sub-cluster 0.PE4a-2-2, strains 1965–2010) and the Sailyugem focus of Mongolia (sub-cluster 0.PE4a-2-1, strains 1964–1990).

Detection of Mosaic Variants in Mothers of MPS II Patients by Next Generation Sequencing.

Mucopolysaccharidosis type II is an X-linked lysosomal storage disorder caused by mutations in the IDS gene that encodes the iduronate-2-sulfatase enzyme. The IDS gene is located on the long arm of the X-chromosome, comprising 9 exons, spanning approximately 24 kb. The analysis of carriers, in addition to detecting mutations in patients, is essential for genetic counseling, since the risk of recurrence for male children is 50%. Mosaicism is a well-known phenomenon described in many genetic disorders caused by a variety of mechanisms that occur when a mutation arises in the early development of an embryo. Sanger sequencing is limited in detecting somatic mosaicism and sequence change levels of less than 20% may be missed. The Next Generation Sequencing (NGS) has been increasingly used in diagnosis. It is a sensitive and fast method for the detection of somatic mosaicism. Compared to Sanger sequencing, which represents a cumulative signal, NGS technology analyzes the sequence of each DNA read in a sample. NGS might therefore facilitate the detection of mosaicism in mothers of MPS II patients. The aim of this study was to reanalyze, by NGS, all MPS II mothers that showed to be non-carriers by Sanger analysis. Twelve non-carriers were selected for the reanalysis on the Ion PGM and Ion Torrent S5 platform, using a custom panel that includes the IDS gene. Results were visualized in the Integrative Genomics Viewer (IGV). We were able to detected the presence of the variant previously found in the index case in three of the mothers, with frequencies ranging between 13 and 49% of the reads. These results suggest the possibility of mosaicism in the mothers. The use of a more sensitive technology for detecting low-level mosaic mutations is essential for accurate recurrence-risk estimates. In our study, the NGS analysis showed to be an effective methodology to detect the mosaic event.

МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ УСТОЙЧИВОСТИ НОЗОКОМИАЛЬНЫХ ШТАММОВ KLEBSIELLA PNEUMONIAE К ПОЛИМИКСИНАМ И АНТИБИОТИКАМ ДРУГИХ ГРУПП ПО ДАННЫМ ПОЛНОГЕНОМНОГО СЕКВЕНИРОВАНИЯ

Identification of numerous mechanisms of resistance to colistin and other antibiotics is possible using whole genome sequencing. Objectives. To assess the molecular-genetic mechanisms of resistance to polymyxins and antibiotics of other groups in nosocomial Klebsiella pneumoniae strains. Material and methods. For 13 multidrug- and extensively drug-resistant K.pneumoniae strains semiconductor sequencing was performed in the Ion PGM System genomic sequencer (Thermo Fisher Scientific, USA). The assembly of genomic sequences and their annotation were carried out. The PROVEAN software tool was used to predict the influence of nucleotide replacements on the structure of amino acid sequences and functional activity of proteins. The identification of antibiotic resistance genes and the search for efflux mechanisms were performed by the ResFinder v.4.1 and CARD web resources. Results. Several types of β-lactamase genes were detected simultaneously in all strains, as well as genes of resistance to fosfomycin. Genes of resistance to aminoglycosides were identified in 11 strains, to chloramphenicol - in 10, to rifampicin - in 5, to macrolides - in 4. The mcr phosphoethanolamine transferase genes were absent in all strains. Functionally significant substitutions were revealed in the pmrB gene (D150Y, T157P, G207S) comparing the studied samples with the reference K. pneumoniae strain ATCC 700603. Changes in the mgrB gene were also found in colistin-resistant strains (W20R replacement, insertional inactivation of the gene by transposons of the IS1, IS4 and IS5 families). Conclusions. The results of whole genome sequencing represent the significant resistance of nosocomial Klebsiella pneumoniae strains to the majority of antibiotics including β-lactams, aminoglycosides, fluoroquinolones, fosfomycin, chloramphenicol, polymyxins. Genetic determinants of colistin resistance were revealed (insertional inactivation and deletion of the mgrB gene; D150Y, T157P and G207S substitutions in the pmrB gene) in strains with colistin MIC 64-128 mg/l and their absence in colistin-susceptible strains.

Coexistence of Two Rare Genetic Variants in Canonical and Non-canonical Exons of SCN5A: A Potential Source of Misinterpretation.

Primary cardiac channelopathies are a group of diseases wherein the role of DNA testing in aiding diagnosis and treatment-based decision-making is gaining increasing attention. However, in some cases, evaluating the pathogenicity of new variants is still challenging. We report an accurate multistage assessment of a rare genetic variant in the SCN5A gene using next-generation sequencing (NGS) techniques and Sanger sequencing. Female sportsman (14 years old) underwent genetic counseling and DNA testing due to QT interval prolongation registered during ECG Holter monitoring. Genetic testing of the proband was performed in two independent laboratories. Primary DNA testing was performed by WES using the Ion ProtonTM System. Target panel sequencing of 11 genes was performed using PGM Ion Torrent. Search for variants in non-canonical and canonical exons 6 was performed by Sanger sequencing. The cascade familial screening and control re-sequencing were provided for proband with identified genetic variant p.S216L (g.38655290G>A, NM_198056.2:c.647C>T, and rs41276525) in the canonical exon 6 of the SCN5A gene after receiving data from another laboratory. Control Sanger and NGS sequencing revealed the absence p.S216L in the canonical exon 6 and confirmed the presence of p.S216L (g.38655522G>A, c.647C>T, and rs201002736) in the non-canonical exon 6 of the SCN5A gene. The identified variant was re-interpreted. The non-canonical transcripts of the exon 6 of the SCN5A gene is poorly represented in cardiac tissue (gnomAD). The detected variant was found in proband’s healthy mother. The correct interpretation of genetic data requires close cooperation between clinicians and researchers. It can help to avoid financial costs and stress for proband’s and families.

Abstract 2976: Establishment of organoids derived from patients with advanced thoracic malignancies

Abstract Background: In the past decade, three-dimensional organoid culture derived from various tumors has been applied to cancer research. Compared to the conventional two-dimensional cell culture, organoid culture recapitulates in vivo system and furthermore, its high establishment rate may hold the potential to help decision-making for precision medicine. However, it remains challenging to establish organoids from advanced thoracic malignancies due to a limited amount of biopsy specimens. Here, we report the attempt to establish organoids from endoscopic biopsy specimens and pleural effusions in patients with advanced thoracic malignancies Methods: Organoid culture was attempted using biopsy specimens or pleural effusions from patients who underwent transbronchial biopsy, thoracoscopic biopsy or thoracentesis. After confirming the presence of malignant cells by rapid on-site evaluation, biopsy specimens were digested with collagenase and dispase at 37°C followed by being harvested and cultured in Matrigel. As for pleural effusions, mononuclear cells were isolated by ficoll density gradient centrifugation and CD45+ cells were depleted using anti-CD45 magnetic beads. Then, the resultant cells were cultured in Matrigel. Next-generation sequencing was performed to confirm cancer-related somatic mutations using Ion PGM system. Cell viability assay was performed by resazurin assay. Results:142 patients were registered in the study between May 2019 and October 2020 at Wakayama Medical University. Fifty-one cases were excluded because of negative diagnosis for malignancy, retraction of the consent or risk of bleeding associated with biopsy. We attempted to culture organoids using 98 samples from 91 patients. The types of malignancies were as follows: non-small cell lung cancers (NSCLC)/SCLC/others, 80/8/3. A total of 33 organoids were successfully cultured with sustainable proliferation and 16 were successfully cryopreserved. Established organoids were confirmed to harbor the same mutations as detected by clinical testing with tissue samples and to form tumors in a xenotransplantation model. HLCO-75, established from pleural effusion at disease progression after osimertinib treatment was genotyped and found to gain ERBB2 V777L mutation in exon 20 in addition to original EGFR L858R mutation. Marked growth-inhibitory effect by poziotinib, a HER2 tyrosine kinase inhibitor with IC50 value 6 nM was observed whereas not by TAS6417, another EGFR/HER2 exon 20 inhibitor, suggesting the potential of organoid culture for decision-making in cancer precision medicine. Conclusion: Our results suggest that organoid culture is feasible from small biopsy specimens and these organoids could be applied for personalized medicine. Citation Format: Jun Oyanagi, Yasuhiro Koh, Koichi Sato, Masanori Tanaka, Katsuyuki Furuta, Takeya Sugimoto, Ryota Shibaki, Shunsuke Teraoka, Daichi Fujimoto, Nahomi Tokudome, Hiroaki Akamatsu, Atsushi Hayata, Atsushi Hayata, Yuichi Ozawa, Hiroki Ueda, Nobuyuki Yamamoto. Establishment of organoids derived from patients with advanced thoracic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2976.

Malfeasance of KRAS mutations in carcinogenesis.

Activating mutations in the KRAS gene (Kirsten rat sarcoma 2 viral oncogene homolog gene) are commonly seen across the various solid organ and hematolymphoid neoplasms. With the likelihood of the mutation specific KRAS inhibitor entering clinical practice, the present studies profiled the landscape of these mutations in the Indian population to add to databases and posit the clinical utility of its emerging inhibitors. This study included 489 formalin fixed paraffin-embedded (FFPE) tissue samples from consecutive patients during a 5-year period (2015-2019). The clinical records were obtained from the medical record archives of the institution. Library preparation was done using the Oncomine Assay™. Sequencing was performed using the Ion PGM Hi-Q Sequencing Kit on the Ion Torrent Personal Genome Machine (Ion PGM) as well as on Ion Torrent S5 sequencer using the S5 sequencing kit. Ion Torrent Suite™Browser version 5.10 and Ion Reporter™version 5.10 were used for data analysis. A total of 50 cases with KRAS mutations were observed occurring most commonly in the codons 12 and 13. The G12D mutation was the most commonly encountered subtype in our cohort (21/50), whereas the G12C mutation was observed in 5 cases, and interestingly, this mutation was only seen in patients with non-small cell lung carcinoma (NSCLC). In the largest cohort from Indian subcontinent reporting spectrum of KRAS mutations in human cancers, an incidence of 11% was observed across all cancer types. Therapies targeting the G12C mutations can benefit up to 20% KRAS-mutated NSCLC. Building databases of spectrum of KRAS mutations in different populations across diverse cancer types is the anticipatory step to this end.

Abstract PS16-35: Microbiota and breast cancer in mexican women

Abstract Microbiota and breast cancer in Mexican womenStudy Type: ObservationalTime Perspective: Cross-SectionalThe human microbiome refers to the genes of the universe of microbes that inhabit our skinand mucosal surfaces. Epidemiological studies implicated that human microfloracontributes to 16% more of malignant neoplasms worldwide, either as a risk factor orcausative agent.Although hereditary and genetic factors represent 5% to 10% of breast cancer cases, 70%of them are due to a host of environmental factors. Migrant studies demonstrated that non-hereditary factors are the main drivers of international and inter-ethnic differences in theincidence and mortality of breast cancer. The environment contributes to the developmentof the disease; although, the factors involved are not well known, among the latter is theinfluence of microorganisms and, therefore, attention is recently being paid to themammary microbiota. This study hypothesizes that women with breast cancer havedifferences in the composition and functionality of breast microbiota compared to womenwithout breast disease.Inclusion and exclusion criteria. Women's age range of 25-70 years. Women with confirmedbreast cancer diagnosis scheduled for surgery as primary treatment (mastectomy orconservative surgery) surgically intervened with breast augmentation or reduction withoutbreast cancer and signed informed consent. Women with antecedents of cancer, or whohave received antibiotic treatment one month before recruitment, or any neoadjuvanttherapy, without breast surgery in the past for any reason. Immunocompromised patients.Pregnant patients or with the use of implants were excluded from the study.Sample size. Sixty women with confirmed breast cancer matched with 30 women withoutcancer, and 30 with benign breast disease. Three hospitals participated in the recruitment:Instituto Nacional de Cancerología, Centro Oncológico Estatal ISSEMYM and Centromedico ABC.Method: DNA total of tissue samples was extracted using the Quick-DNA Miniprep Plus Kit(Zymo research Cat. D4068). 5 uL of DNA isolated of tissue samples were amplified with16S™ Metagenomics Kit (Thermo Fisher Scientific, Cat. A26216) and were marked with IonXpress Barcode Adapter (Thermo Fisher Scientific cat. no. 4471250), purified with AMPureXP reagent (Beckman Coulter cat. no. A63881) and quantified with Ion Universal LibraryQuantitation Kit (Thermo Fisher Scientific cat. no. A26217). After that, emulsion PCR wasprepared in the Ion OneTouch 2 System (Thermo Fisher Scientific). For sequencing, we usedthe Ion PGM Hi-Q view Sequencing kit (REFA30044) with chips 318 in the Ion torrent PGMinstrument (Thermo Fisher Scientific). Results: In this study, we employed 16s rRNA sequencing to analyze the bacterial profiles ofnormal, benign, and breast tumor tissues. We observed that the beta diversity between thethree types of tissues is similar, although patients showed significant differences in theabundance of specific bacteria genera depending on the tissue of origin. We determinedthat bacteria from the genera Burkholderia had a lower relative abundance in benign tissuesthan normal tissues, although these genera had a higher relative abundance in tumortissues. We found a more significant difference in the amount of bacteria between benignand tumoral tissues; we observed that the seven genera (Aeromonas, Alcanivorax, Burkholderia, Corynebacterium, Finegoldia, Pseudomonas and Staphylococcus) are greaterintumortissueswhereasfivegenera: Cupriavidus, Microbacterium, Ralstonia, Renibacterium and Sphingomonas had alower relative abundance in tumoral tissues. This study suggests that bacterial beta-diversity does not change much among the analyzed tissues; however, it is specific bacterialgenera that change their relative abundance during tumor progression. Citation Format: Arely Berenice González Valdés, Juan Enrique Bargallo Rocha, Juan Manuel Medina Castro, Carlos Pérez Plasencia, Oliver Millan Catalan, Elvia Fernández Pérez, Antonio Daniel Martinez, David Cantú De León. Microbiota and breast cancer in mexican women [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-35.

Broad spectrum mutational analysis of chromophobe renal cell carcinoma using next-generation sequencing

BackgroundChromophobe renal cell carcinoma (ChRCC) is a rare subtype of non-clear cell renal cell carcinoma. Due to its rarity, its molecular characterization as well as therapeutic targets are still not fully understood. MethodsWe performed a next-generation sequencing analysis using the platform Ion PGM System on 20 retrospectively collected ChRCC cases with the aim of identify molecular biomarkers with potential prognostic value or that could have therapeutic implications. ResultsWe identified mutation onTP53, SMARCB1, RB1 and JAK3. The most frequently altered gene was TP53 (6/20, 30 % of cases). SMARCB1 mutation was found in 3 (15 %) patients and in all cases the mutational variant was p.T72 K, with known pathogenenic meaning. One (5%) patient presented a pathogenetic mutation of RB1. JAK3 was mutated in 1 (5%) patient and this mutation resulted to have uncertain pathogenetic significance. ConclusionChRCC is a rare disease still not fully molecularly characterized. Next-generation sequencing analysis could be useful to identify potential mutation with prognostic value or that could be potential therapeutic targets.

Wide diversity of parasites in Bombus terrestris (Linnaeus, 1758) revealed by a high-throughput sequencing approach.

Assessing the extent of parasite diversity requires the application of appropriate molecular tools, especially given the growing evidence of multiple parasite co-occurrence. Here, we compared the performance of a next-generation sequencing technology (Ion PGM ™ System) in 12 Bombus terrestris specimens that were PCR-identified as positive for trypanosomatids (Leishmaniinae) in a previous study. These bumblebees were also screened for the occurrence of Nosematidae and Neogregarinorida parasites using both classical protocols (either specific PCR amplification or amplification with broad-range primers plus Sanger sequencing) and Ion PGM sequencing. The latter revealed higher parasite diversity within individuals, especially among Leishmaniinae (which were present as a combination of Lotmaria passim, Crithidia mellificae and Crithidia bombi), and the occurrence of taxa never reported in these hosts: Crithidia acanthocephali and a novel neogregarinorida species. Furthermore, the complementary results produced by the different sets of primers highlighted the convenience of using multiple markers to minimize the chance of some target organisms going unnoticed. Altogether, the deep sequencing methodology offered a more comprehensive way to investigate parasite diversity than the usual identification methods and provided new insights whose importance for bumblebee health should be further analysed.

Allelic diversity and forensic estimations of the Beijing Hans: Comparative data on sequence-based and length-based STRs

Short tandem repeat (STR) profiling is routinely used in forensic genetics. At present, STR analysis is mainly performed by capillary electrophoresis (CE). However, due to limitations associated with the CE method, STR genotyping has been limited to length polymorphisms only. Because next generation sequencing (NGS) is capable of providing full resolution STR data at the sequence variation level, the individual identification capability of forensic STR loci could be significantly improved. Here we present sequence-based STR data for the Beijing Han population in which 291 individuals were screened for 23 commonly used forensic STRs using the SeqTypeR24 CASE kit on an Ion PGM platform. In total, 234 length-based alleles and 356 sequence-based alleles, which included 22 novel core repeat sequences, were observed. The sequence-based matching probability and power of discrimination were superior to the length-based numbers for 16 loci bearing micro-variant alleles. Combined matching probability reached 8.2 × 10−29 for 23 STR loci at the sequence level. This was two orders of magnitude higher than the parameters at length level and provides a data base for sequence-based STR casework applications.

Succinivibrionaceae is dominant family in fecal microbiota of Behçet's Syndrome patients with uveitis.

Behçet's Syndrome (BS) is a multisystem vasculitis with various clinical manifestations. Pathogenesis is unclear, but studies have shown genetic factors, innate immunity and autoinflammation to have an important role in the disease course. Diversity in the microbial community of gut microbiota may significantly contribute to the activation of the innate immune system. The clinical features of BS present themselves in clusters and each cluster may be a consequence of different disease mechanisms. For this reason we aimed to investigate the gut microbiota of BS patients with uveitis. In addition to healthy controls, we have aimed to compare the gut microbiota of BS with that of Familial Mediterranean Fever (FMF) and Crohn's Disease (CD) as both diseases have innate and autoinflammatory features in their pathogenesis. Seven patients with BS, 12 patients with FMF, 9 patients with CD and 16 healthy controls (HC) were included in the study. Total genomic DNAs were isolated from fecal samples of the patients. Partial 16S rRNA gene was sequenced using the PGM Ion Torrent (Thermo Fisher Scientific, Waltham, MA, USA) for microbiota analysis. Statistical analysis showed that significant differences were detected on the microbial community of four groups. Succinivibrionaceae is dominant and the signature family, whereas Bacteroides was absent in BS patients.