Tandem Repeat Analysis Research Articles

Genetic structure and diversity of Mycoplasma hyopneumoniae based on a MLVA typing scheme

BackgroundSeveral epidemiological studies have been carried out using Multiple-Locus Variable-number tandem repeat Analysis (MLVA) for M. hyopneumoniae typing. However, a global perspective on the implications of the genetic diversity of this pathogen is lacking.ObjectiveThis study aimed to determine and to analyze the genetic structure of M. hyopneumoniae based on the p97R1-p146R3 MLVA typing scheme and to characterize, analyze and compare MLVA types among countries where the information was publicly available.MethodsA set of 797 publicly available data of M. hyopneumoniaep97R1-p146R3 MLVA genetic types from six different countries were analyzed using Genalex 6.41 software to characterize loci polymorphism and using Structure 2.3.4 software in order to identify the genetic structure.ResultsA total of 185 MLVA types were identified among the analyzed data. For the p97R1 and p146R3 loci, most of the molecular variation in M. hyopneumoniae populations was identified within countries. Three genetic clusters and their recombinations were identified globally.ConclusionM. hyopneumoniae is a genetically diverse pathogen with limited clonality and three well-defined clusters and their combinations were identified in this investigation. The greatest genetic variation of M. hyopneumoniae was observed within countries.

Genetic diversity atlas of Brucella melitensis strains from Sichuan Province, China

Human brucellosis is a re-emerging disease in Sichuan Province, China. In this study, bacteriology, conventional bio-typing, multi-locus sequence typing (MLST), and multiple locus variable-number tandem repeat analysis (MLVA) were applied to preliminarily characterize the strains in terms of genetic diversity and epidemiological links. A total of 101 Brucella strains were isolated from 16 cities (autonomous prefectures) from 2014 to 2021, and all of the strains were identified as Brucella melitensis bv. 3, suggesting that surveillance should focus on ruminants. MLST analysis identified four STs, namely, ST8 (n = 93), ST39 (n = 6), ST101 (n = 1), and ST118 (n = 1). The latter were new STs, indicating that strains displayed high population diversity. Six MLVA-8, namely, 42, 43, 45, 63, 83, and 114, and eight MLVA-11, namely, 111, 115, 116, 125, 180, 291, 298, and 342, genotypes were identified, demonstrating that all of the strains were from the Eastern Mediterranean lineage, and these strains exhibited a high genotype diversity. MLVA-16 analysis revealed that there was a co-existing transmission pattern, where sporadic cases and multiple outbreak events had a common origin. The dominant STs and MLVA genotypes of strains were epidemic in Northern, China, and 36 MLVA-16 genotypes were shared among strains (n = 51, 50.4%, 51/101) from Sichuan and strains from 22 other provinces. The findings imply that infected animals were introduced from outside the province. The surveillance and control of the disease have become public health challenges. Animal quarantines should be strengthened to prevent the spread of B. melitensis species among adjacent regions.

Establishment and characterization of a new intestinal-type ampullary carcinoma cell line, DPC-X3

Ampullary carcinoma (AC) of the intestinal type represents a distinct variant within the broader category of ampullary neoplasms. The scarcity of pertinent cellular models has constrained investigations centered on this particular malignancy. This research effectively generated a cell line (CL) of intestinal-type AC (DPC-X3). This newly developed CL has been continuously cultured for 1 year and has demonstrated stable passaging exceeding 60 generations. Morphologically, DPC-X3 exhibited characteristic attributes of an epithelial tumor. The cell proliferation rate of DPC-X3 exhibited a doubling interval of 79 h. Short tandem repeat (STR) analysis validated the high consistency between DPC-X3 and the patient’s primary tumor. Characteristically, DPC-X3 displayed sub diploid karyotypes, primarily featuring 44, XY inv (9), -18, -20, -22, and + mar. Under suspension culture conditions, DPC-X3 could efficiently form organoids, and DPC-X3 cells inoculated subcutaneously into NXG mice could form transplanted tumors. Drug susceptibility assays demonstrated that DPC-X3 resisted paclitaxel, oxaliplatin, 5-fluorouracil(5-FU), and gemcitabine. Immunohistochemical (IHC) evaluation revealed affirmative reactivity for CK7 and CK20 within DPC-X3 cells, while CDX2 exhibited no detectable expression. E-cadherin and Vimentin demonstrated positive immunoreactivity, whereas CEA and CA19-9 displayed faint positivity. The Ki-67 proliferation index was determined to be approximately 40%. DPC-X3 presents a valuable experimental platform for elucidating the pathogenesis of intestinal-type AC and can serve as a driver for drug development efforts.

Automatic Single-Cell Harvesting for Fetal Nucleated Red Blood Cell Isolation on a Self-Assemble Cell Array (SACA) Chip.

(1) Background: Fetal chromosomal examination is a critical component of modern prenatal testing. Traditionally, maternal serum biomarkers such as free β-human chorionic gonadotropin (Free β-HCG) and pregnancy-associated plasma protein A (PAPPA) have been employed for screening, achieving a detection rate of approximately 90% for fetuses with Down syndrome, albeit with a false positive rate of 5%. While amniocentesis remains the gold standard for the prenatal diagnosis of chromosomal abnormalities, including Down syndrome and Edwards syndrome, its invasive nature carries a significant risk of complications, such as infection, preterm labor, or miscarriage, occurring at a rate of 7 per 1000 procedures. Beyond Down syndrome and Edwards syndrome, other chromosomal abnormalities, such as trisomy of chromosomes 9, 16, or Barr bodies, pose additional diagnostic challenges. Non-invasive prenatal testing (NIPT) has emerged as a powerful alternative for fetal genetic screening by leveraging maternal blood sampling. However, due to the extremely low abundance of fetal cells in maternal circulation, NIPT based on fetal cells faces substantial technical challenges. (2) Methods: Fetal nucleated red blood cells (FnRBCs) were first identified in maternal circulation in a landmark study published in The Lancet in 1959. Due to their fetal origin and presence in maternal peripheral blood, FnRBCs represent an ideal target for non-invasive prenatal testing (NIPT). In this study, we introduce a novel self-assembled cell array (SACA) chip system, a microfluidic-based platform designed to efficiently settle and align cells into a monolayer at the chip's base within five minutes using lateral flow dynamics and gravity. This system is integrated with a fully automated, multi-channel fluorescence scanning module, enabling the real-time imaging and molecular profiling of fetal cells through fluorescence-tagged antibodies. By employing a combination of Hoechst+/CD71+/HbF+/CD45- markers, the platform achieves the precise enrichment and isolation of FnRBCs at the single-cell level from maternal peripheral blood. (3) Results: The SACA chip system effectively reduces the displacement of non-target cells by 31.2%, achieving a single-cell capture accuracy of 97.85%. This isolation and enrichment system for single cells is well suited for subsequent genetic analysis. Furthermore, the platform achieves a high purity of isolated cells, overcoming the concentration detection limit of short tandem repeat (STR) analysis, demonstrating its capability for reliable non-invasive prenatal testing. (4) Conclusions: This study demonstrates that the SACA chip, combined with an automated image positioning system, can efficiently isolate single fetal nucleated red blood cells (FnRBCs) from 50 million PBMCs in 2 mL of maternal blood, completing STR analysis within 120 min. With higher purification efficiency compared to existing NIPT methods, this platform shows great promise for prenatal diagnostics and potential applications in other clinical fields.

Uniparental disomy (UPD) exclusion in embryos following Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR).

Uniparental disomy (UPD) is a genetic condition which both copies of a chromosome are inherited from a single parent, potentially leading to imprinting disorders. This study aimed to assess the integration of Short Tandem Repeat (STR) analysis into Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) to assess UPD risk and its impact on selecting euploid embryos for embryo transfer in couples with chromosomal translocations involving imprinted chromosomes. This study evaluated three couples carrying balanced chromosomal translocations: 45,XX,der(13;14)(q10;q10), 46,XX,t(10;11)(q22;q13), and 45,XY,der(14;15)(q10;q10). STR analysis was performed on trophectoderm (TE) biopsies after Whole Genome Amplification (WGA) after PGT-SR analysis using parental blood samples to assess UPD risk in euploid embryos. Haplotyping was conducted with five to six STR markers specific to each rearranged chromosome to detect UPD in euploid embryos. Of the four embryos analyzed across the three families, two couples had euploid embryos that tested negative for UPD. These embryos were successfully transferred, resulting in the birth of two healthy children. In the third family, the euploid embryo also tested negative for UPD but failed to implant after transfer, resulting in no pregnancy. Despite its rarity, UPD involving imprinted chromosomes poses significant clinical risks, as seen in disorders such as Prader-Willi syndrome and Angelman syndrome. This study highlights the importance of integrating UPD screening into PGT-SR protocols, to detect both heterodisomic and isodisomic UPD events minimizing the risk of severe genetic disorders. Integrating STR-based UPD screening within PGT-SR workflows is a reliable and cost-effective strategy that enhances embryo selection and mitigates the risk of imprinting disorders. This approach improves reproductive outcomes for families with chromosomal rearrangements, offering a practical advancement in assisted reproduction.

Post-transplant transient abnormal myelopoiesis evolving from a GATA1 mutant clone in umbilical cord blood.

Transient abnormal myelopoiesis (TAM) generally affects newborns with Down syndrome and is associated with constitutional trisomy 21 and a somatic GATA1 mutation. Here we describe a case of TAM which evolved after umbilical cord blood transplantation (UCBT), whose origin was identified as a GATA1 mutation-harboring clone in umbilical cord blood (UCB) by detailed genetic analyses. A 58-year-old male who received UCBT for peripheral T-cell lymphoma presented progressive anemia and thrombocytopenia, and leukocytosis with blast cells in the peripheral blood (PB). Bone marrow (BM) aspiration showed granulocytic and megakaryocytic dysplasia with excess blasts whose karyotype was trisomy 21. Short tandem repeat analysis showed complete donor chimerism. He was initially diagnosed as donor-derived myelodysplastic syndrome (MDS) and treated with azacitidine, followed by secondary transplantation using unrelated BM, providing durable complete remission. Retrospective targeted-capture sequencing analysis of PB/BM samples collected at multiple timepoints identified trisomy 21 and a GATA1 mutation, suggestive of a diagnosis of donor cell-derived TAM (DC-TAM). Importantly, a minor clone with the same GATA1 mutation was detected in UCB by droplet digital PCR. DC-TAM is a rare UCBT-related complication which resembles MDS, but the identification of GATA1 mutation may be useful for its diagnosis. Our genetic analyses revealed that a pre-existing clone in UCB may contribute to the development of donor cell-derived hematologic neoplasms, highlighting the potential relevance of genetic screening of donor UCB.

Genomic surveillance of multidrug-resistant organisms based on long-read sequencing

BackgroundMultidrug-resistant organisms (MDRO) pose a significant threat to public health worldwide. The ability to identify antimicrobial resistance determinants, to assess changes in molecular types, and to detect transmission are essential for surveillance and infection prevention of MDRO. Molecular characterization based on long-read sequencing has emerged as a promising alternative to short-read sequencing. The aim of this study was to characterize MDRO for surveillance and transmission studies based on long-read sequencing only.MethodsGenomic DNA of 356 MDRO was automatically extracted using the Maxwell-RSC48. The MDRO included 106 Klebsiella pneumoniae isolates, 85 Escherichia coli, 15 Enterobacter cloacae complex, 10 Citrobacter freundii, 34 Pseudomonas aeruginosa, 16 Acinetobacter baumannii, and 69 methicillin-resistant Staphylococcus aureus (MRSA), of which 24 were from an outbreak. MDRO were sequenced using both short-read (Illumina NextSeq 550) and long-read (Nanopore Rapid Barcoding Kit-24-V14, R10.4.1) whole-genome sequencing (WGS). Basecalling was performed for two distinct models using Dorado-0.3.2 duplex mode. Long-read data was assembled using Flye, Canu, Miniasm, Unicycler, Necat, Raven, and Redbean assemblers. Long-read WGS data with > 40 × coverage was used for multi-locus sequence typing (MLST), whole-genome MLST (wgMLST), whole-genome single-nucleotide polymorphisms (wgSNP), in silico multiple locus variable-number of tandem repeat analysis (iMLVA) for MRSA, and identification of resistance genes (ABRicate).ResultsComparison of wgMLST profiles based on long-read and short-read WGS data revealed > 95% of wgMLST profiles within the species-specific cluster cut-off, except for P. aeruginosa. The wgMLST profiles obtained by long-read and short-read WGS differed only one to nine wgMLST alleles or SNPs for K. pneumoniae, E. coli, E. cloacae complex, C. freundii, A. baumannii complex, and MRSA. For P. aeruginosa, differences were up to 27 wgMLST alleles between long-read and short-read wgMLST and 0–10 SNPs. MLST sequence types and iMLVA types were concordant between long-read and short-read WGS data and conventional MLVA typing. Antimicrobial resistance genes were detected in long-read sequencing data with high sensitivity/specificity (92–100%/99–100%). Long-read sequencing enabled analysis of an MRSA outbreak.ConclusionsWe demonstrate that molecular characterization of automatically extracted DNA followed by long-read sequencing is as accurate compared to short-read sequencing and suitable for typing and outbreak analysis as part of genomic surveillance of MDRO. However, the analysis of P. aeruginosa requires further improvement which may be obtained by other basecalling algorithms. The low implementation costs and rapid library preparation for long-read sequencing of MDRO extends its applicability to resource-constrained settings and low-income countries worldwide.

Impact of population size on population genetic analysis of Short Tandem Repeat (STR) allelic data, forensic and paternity parameters and its effect on forensic DNA analysis.

The selection of an appropriate STR allelic frequency database is the prerequisite for assessing the evidentiary value of DNA evidence. Four data sets comprising 50, 100, 200, and 500 samples were evaluated in 21 autosomal STR markers in the Indian and the Bahrain population. Allelic richness showed an increasing trend with the increase in sample size i.e., 193 and 201 (50 samples), 217 and 221 (100 samples), 255 and 238 (200 samples), and 292 and 285 (500 samples) in both the populations. TPOX and D13S317 markers did not show any increase in allele number, whereas SE33 markers showed the highest increase in both populations. With the increase in sample size, 70 (Bahrain population) and 100 (Indian population) alleles having < MAF were detected. Similarly, 37 and 47 previously undetected alleles could be detected when the sample size was increased from 50 to 500 in the Indian and Bahrain populations respectively. In the Indian population, Match probability, decreased with a 500-sample size, whereas, the PIC, PE, Heterozygosity, and PI increased with the increase in sample size. Further, database size did not show any statistical difference in the outcome of the Paternity Index value in the 50 paternity trio cases studied.

Unravelling Antimicrobial Resistance in Mycoplasma hyopneumoniae: Genetic Mechanisms and Future Directions.

Antimicrobial resistance (AMR) in Mycoplasma hyopneumoniae, the causative agent of Enzootic Pneumonia in swine, poses a significant challenge to the swine industry. This review focuses on the genetic foundations of AMR in M. hyopneumoniae, highlighting the complexity of resistance mechanisms, including mutations, horizontal gene transfer, and adaptive evolutionary processes. Techniques such as Whole Genome Sequencing (WGS) and multiple-locus variable number tandem repeats analysis (MLVA) have provided insights into the genetic diversity and resistance mechanisms of M. hyopneumoniae. The study underscores the role of selective pressures from antimicrobial use in driving genomic variations that enhance resistance. Additionally, bioinformatic tools utilizing machine learning algorithms, such as CARD and PATRIC, can predict resistance traits, with PATRIC predicting 7 to 12 AMR genes and CARD predicting 0 to 3 AMR genes in 24 whole genome sequences available on NCBI. The review advocates for a multidisciplinary approach integrating genomic, phenotypic, and bioinformatics data to combat AMR effectively. It also elaborates on the need for refining genotyping methods, enhancing resistance prediction accuracy, and developing standardized antimicrobial susceptibility testing procedures specific to M. hyopneumoniae as a fastidious microorganism. By leveraging contemporary genomic technologies and bioinformatics resources, the scientific community can better manage AMR in M. hyopneumoniae, ultimately safeguarding animal health and agricultural productivity. This comprehensive understanding of AMR mechanisms will be beneficial in the adaptation of more effective treatment and management strategies for Enzootic Pneumonia in swine.

Uniparental IsoDisomy: a case study on a new mechanism of Friedreich ataxia.

Friedreich's Ataxia (FRDA) is the most common hereditary ataxia and is mainly caused by biallelic GAA repeat expansion in the FXN gene. Rare patients carrying FXN point mutations or intragenic deletions are reported. We describe the first FRDA patient with a chromosome 9 segmental Uniparental isoDisomy (UPiD) unmasking a homozygous FXN expansion initially undetected by TP-PCR. The child presented with a progressive proprioceptive ataxia associated with peripheral sensory neuronopathy and severe scoliosis. Whole genome sequencing (WGS) identified a maternal segmental Uniparental Isodisomy (UPiD) encompassing FXN. Short tandem repeats analysis on WGS showed a biallelic FXN expansion. The identification of a deletion in the primer-annealing region of the TP-PCR explained the initial TP-PCR failure. This is the first documented case of FRDA caused by segmental UPiD. This case highlights the complexity of the molecular diagnosis of FRDA, and emphasises the importance of integrating results from various technical diagnostic approaches.

Analysis of Tandem Repeats in Short-Read Sequencing Data: From Genotyping Known Pathogenic Repeats to Discovering Novel Expansions.

Short tandem repeats (STRs) and variable-number tandem repeats (VNTRs) are repetitive genomic sequences seen widely throughout the genome. These repeat expansions are currently known to cause ∼60 diseases, with expansions in new loci linked to rare diseases continuing to be discovered. Genome sequencing is an important tool for detecting disease-causing variants and several computational tools have been developed to analyze tandem repeats from genomic data, enabling the genotyping and the identification of expanded alleles. However, guidelines for conducting the analysis of these repeats and, more importantly, for assessing the findings are lacking. Understanding the tools and their technical limitations is important for accurately interpreting the results. This article provides detailed, step-by-step instructions for three key use cases in STR analysis from short-read genome sequencing data, which are also applicable to smaller VNTRs. First, it demonstrates an approach for genotyping known pathogenic loci and the identification of clinically significant expansions. Second, we offer guidance on defining tandem repeat loci and conducting genome-wide genotyping studies, which is also applicable to diploid organisms other than humans. Third, instructions are provided on how to find novel expansions at loci not previously known to be associated with disease, aiding in the discovery of new pathogenic loci. Moreover, we introduce the use of newly-developed helper tools that enable a complete and streamlined tandem repeat analysis protocol by addressing the gaps in current methods. All three protocols are compatible with human hg19, hg38, and the latest telomere-to-telomere (hs1) reference genomes. Additionally, this protocol provides an overview and discussion on how to interpret genotyping results. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Genotyping known pathogenic tandem repeat loci Alternate Protocol: Genotyping known pathogenic tandem repeat loci with STRipy Support Protocol 1: Installation of tools and ExpansionHunter catalog modification Basic Protocol 2: Performing genome-wide genotyping of tandem repeats Basic Protocol 3: Discovering de novo tandem repeat expansions Support Protocol 2: Compiling ExpansionHunter Denovo from source code and generating STR profiles.

Follow the Path: Unveiling an Azole Resistant Candida parapsilosis Outbreak by FTIR Spectroscopy and STR Analysis.

Accurate identification and rapid genotyping of Candida parapsilosis, a significant opportunistic pathogen in healthcare settings, is crucial for managing outbreaks, timely intervention, and effective infection control measures. This study includes 24 clinical samples and 2 positive environmental surveillance swabs collected during a fluconazole-resistant Candida parapsilosis outbreak at the Tuscany Rehabilitation Clinic (Clinica di Riabilitazione Toscana, CRT), located in the province of Arezzo, Italy. Fourier-transform infrared (FTIR) spectroscopy, genetic sequencing of the ERG11 gene, and short tandem repeat (STR) analysis was applied to track the fluconazole-resistant C. parapsilosis outbreak at the CRT facility. FTIR analysis clustered the isolates into two major groups, correlating with resistance-associated ERG11 mutations (Y132F and R398I), azole resistance levels, and year of isolation. The combined use of FTIR spectroscopy and STR typing provided a comprehensive approach to identify and track fluconazole-resistant C. parapsilosis isolates, which identified specific clusters of genetically similar isolates. By comparison with feasible molecular techniques, we conclude that FTIR spectroscopy applied in real time can inform targeted infection control strategies and aid in the effective management of nosocomial infections.

A single-center analysis of clonal transmission of carbapenem-resistant Acinetobacter baumannii among intensive care unit patients during the COVID-19 pandemic

Carbapenem-Resistant Acinetobacter baumannii (CRAB) outbreak in intensive care units (ICUs) is a significant problem for healthcare facilities. In this study, we aimed to investigate the occurrence of CRAB isolates among ICU-admitted patients during the three waves of the COVID-19 pandemic in Iran using Multiple-Locus Variable Number Tandem-Repeat Analysis (MLVA). We obtained 50 (A) baumannii isolates from tracheal aspirate and blood culture samples. In the disc diffusion method, all isolates were cefotaxime, ceftriaxone, and cefepime-resistant, while 98% (49/50) of isolates were resistant to piperacillin, piperacillin-tazobactam, ceftazidime, and ciprofloxacin. Levofloxacin and tobramycin resistance was found in 76% (38/50) of isolates. In the microbroth dilution test all isolates were resistant to imipenem, 98% (49/50) to meropenem, 68% (34/50) to colistin, and 20% (10/50) to polymyxin (B) Based on the PCR findings, all isolates harbored blaOXA−40, ISAba-1, and int-2 genes. There were no isolates found that have the blaOXA−58, blaOXA−143, blaVEB−1, blaVIM, and int-3 genes. Among Extended-spectrum beta-lactamases (ESBL) genes, blaCTX−M, blaTEM, blaSHV, blaGES, and blaPER−1 have a prevalence of 42% (21/50), 84% (42/50), 58% (29/50), 78% (39/50), and 54% (27/50), respectively. 74% (37/50) of the isolates had the blaOXA−23 gene, while all of the isolates carried the blaOXA−40 gene. Among MBL genes, blaIPM, blaGIM, blaSIM, and blaNDM−1 have a prevalence of 20% (10/50), 8% (4/50), 22% (11/50), and 60% (30/50), respectively. The prevalence of int-1 was documented as 74% (37/50). Accordingly, all isolates were identified as CRAB. The co-existence of blaOXA−23/int-2 and blaOXA−23/isaba-1 was 74% (37/50). The co-existence of blaNDM−1/ISAba-1 was observed in 30 (60%) isolates. Using an 80% similarity threshold on the dendrogram constructed through MLVA typing, all isolates were grouped into two clusters: cluster A with 9 isolates from wave 3, and cluster B with 41 isolates from waves 3, 4, and 5. Our study confirms a clonal transmission of CRAB during the study period and suggests using molecular typing methods like MLVA in healthcare settings to identify dominant clones, antibiotic resistance patterns, and transmission routes. This will help to better manage the emergence and spread of antibiotic-resistant strains in future outbreaks.

"CLADE-FINDER": Candida auris Lineage Analysis Determination by Fourier Transform Infrared Spectroscopy and Artificial Neural Networks.

In 2019, Candida auris became the first fungal pathogen included in the list of the urgent antimicrobial threats by the Centers for Disease Control (CDC). Short tandem repeat (STR) analysis and whole-genome sequencing (WGS) are considered the gold standard, and can be complemented by other molecular methods, for the genomic surveillance and clade classification of this multidrug-resistant yeast. However, these methods can be expensive and require time and expertise that are not always available. The long turnaround time is especially not compatible with the speed needed to manage clonal transmission in healthcare settings. Fourier transform infrared (FTIR) spectroscopy, a biochemical fingerprint approach, has been applied in this study to a set of 74 C. auris isolates belonging to the five clades of C. auris (I-V) in combination with an artificial neural network (ANN) algorithm to create and validate "CLADE-FINDER", a tool for C. auris clade determination. The CLADE-FINDER classifier allowed us to discriminate the four primary C. auris clades (I-IV) with a correct classification for 96% of the samples in the validation set. This newly developed genotyping scheme can be reasonably applied for the effective epidemiological monitoring and management of C. auris cases in real time.

Molecular Typing of Pseudomonas aeruginosa Isolates Collected in Abidjan Hospitals (Côte d'Ivoire) Using the Multiple-Locus Variable Number of Tandem Repeats Method.

Background/objectives:Pseudomonas aeruginosa can cause community-acquired infections affecting various body sites. The present retrospective study investigated the genetic diversity of 173 isolates (166 clinical, 7 environmental) of P. aeruginosa collected from clinical pathology laboratories in Abidjan, Côte d'Ivoire (2001-2011). Methods: Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) using 13 loci was applied to all isolates and compared to published MLVA data. The antibiotics status of the isolates was compiled when available and compared to published profiles. Results: Among 95 isolates analyzed for their antibiotics status, 14 displayed concerning resistance profiles: five multidrug-resistant (MDR) and nine extensively drug-resistant (XDR). MLVA typing revealed a high genetic diversity (>130 genotypes), with many genotypes represented by a single strain. Notably, thirteen clusters (≥4 related isolates) were observed. Some clusters displayed close genetic relatedness to isolates from France, Korea, and well-studied strains (ST560, LES and PA14). Comparative analysis suggested the presence of international high-risk MDR clones (CC233, CC111) in Côte d'Ivoire. Importantly, MLVA clustering revealed a close relationship of CC235-MDR strains with a locally identified cluster (group 9). Conclusions: These findings support MLVA as a reliable and cost-effective tool for low-resource settings, allowing the selection of relevant strains for future whole genome sequence analyses. This approach can improve outbreak investigations and public health interventions aimed at curbing MDR P. aeruginosa transmission within hospitals and at the national level.

Forensic STR and SNP Genotyping of Formalin-Fixed Skeleton Samples With Illumina's ForenSeq System.

Formalin fixatives are widely used in forensics to preserve tissues. However, extracting high-quality genomic DNA from formalin-fixed samples is challenging. Traditional short tandem repeat (STR) analysis using capillary electrophoresis (CE) for forensic DNA typing frequently results in failure. Massively parallel sequencing (MPS) can handle many samples and thousands of genetic markers, usually single-nucleotide polymorphisms (SNPs) and STRs, in a single test. Thus, it is useful for assessing highly deteriorated forensic evidence. Few studies have examined the effectiveness of STRs and SNPs genotyping of formalin-fixed skeletons using MPS. In this study, 55 skeletal samples from 5 individuals that were treated under different formalin fixation times (5-75 days) were examined and sequenced using the ForenSeq DNA Signature Prep Kit on the Illumina MiSeq FGX platform. The results showed that as the duration of formalin fixation increased, the detection rates of STRs and SNPs gradually decreased. After 75 days of fixation, the average detection rates for STRs and SNPs were 4% and 10%, respectively. The cumulative discrimination power (CDP) of individual identification SNPs (iiSNPs) was >0.9999 on the 45th day. However, the CDP of STRs was 0.9930 on the 22nd day. Low detection rates were observed for six STRs (D1S1656, PentaE, D22S1045, PentaD, DX8378 and DX10103) and five SNPs (rs2920816, rs354439, rs1736442, rs338882 and rs1031825). In conclusion, DNA extracted from formalin-fixed skeletons decomposes rapidly over time, and MPS technology can be a useful tool for detecting forensic genetic markers in such samples.

9194 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C&amp;lt;A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

7151 Generation of a Human-Induced Pluripotent Stem Cell Line From a Patient With Hypopituitarism and a Novel Nonsense Variant in FOXA2

Abstract Disclosure: M.A. Camilletti: None. G.T. Chirino Felker: None. G. Amin: None. S. Castañeda: None. F. Zabalegui: None. C. Romano Florit: None. A. Waisman: None. M. Ciaccio: None. M.I. Di Palma: None. E. Vaiani: None. A. Belgorosky: None. S.G. Miriuka: None. L.N. Moro: None. M.I. Perez-Millan: None. Congenital Hypopituitarism (CH) is a genetically complex disease characterized by one or more pituitary hormone deficiencies. Forkhead Box A2 (FOXA2, 600288) is a pioneer transcription factor associated with development of multiple tissues in humans and mice. Heterozygous loss of function mutations in FOXA2 were recently shown to cause CH with hyperinsulinemic hypoglycemia, but little is known about the molecular mechanism of FOXA2 action during pituitary development. We identified a novel, heterozygous nonsense variant in FOXA2 (c.686C&amp;lt;A; p.S229X) in a patient diagnosed with GH deficiency at 1.7 years of age, with anterior lobe hypoplasia and absent pituitary stalk, asymptomatic hypoglycemia, and craniofacial malformations. To discover the mechanism of FOXA2 regulation of pituitary development we generated an induced pluripotent stem cell line (FOXA2mut-iPSC) from the patient’s polymorphonuclear blood cells (PBMCs) using an EF1a-hSTEMCCA-loxP vector containing the pluripotency genes OCT-4, SOX-2, c-MYC and KLF4. FOXA2mut-iPSC clone R1 had a normal karyotype and embryonic stem cell-like morphology. These cells express endogenous pluripotency-associated markers NANOG, SOX2, and OCT4, have alkaline phosphatase activity, and can differentiate into all three developmental layers, indicating that these cells are pluripotent. Short tandem repeat (STR) analysis confirmed that FOXA2mut-iPSC R1 clone profiles matched those of the donor PBMCs. Next, we compared pituitary differentiation of FOXA2mut-iPSC vs. control-iPSCs in vitro. Cells were cultured in a differentiation medium containing BMP4, the smoothened agonist SAG, and fibroblast growth factor (FGF), as described in Zimmer et al., 2016 (1). Gene expression analysis (by qRT-PCR) of samples collected on days 0, 4, 7, and 15 of the differentiation protocol showed an increased expression of genes associated with anterior pituitary development including OTX2, PITX1/2, and SIX1 in both cell lines (n=2). These preliminary data suggest that pituitary cell fate can be induced in the heterozygous FOXA2mut-iPSC clone. Ongoing studies will assess pituitary hormone-producing cell differentiation after longer periods of FOXA2mut-iPSC cell culture. In sum, patient iPSC differentiation is a promising tool for assessment of normal and variant FOXA2 function in human pituitary development, and for enlightening the mechanisms of FOXA2 action in human pituitary development. (1) Zimmer et al., Stem Cell Reports. 2016 Jun 14;6(6):858-872. Presentation: 6/1/2024

Generation and Characterization of hiPS Lines from Three Patients Affected by Different Forms of HPDL-Related Neurological Disorders.

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene, leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear, though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here, we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations, using episomal reprogramming. The patients' clinical characteristics are carefully listed. The hiPS lines were meticulously characterized, demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4, TRA1-60, NANOG, and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition, HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation, similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83, offering insights into its molecular mechanisms and potential for developing targeted therapies.

NASTRA: accurate analysis of short tandem repeat markers by nanopore sequencing with repeat-structure-aware algorithm.

Short-tandem repeats (STRs) are the type of genetic markers extensively utilized in biomedical and forensic applications. Due to sequencing noise in nanopore sequencing, accurate analysis methods are lacking. We developed NASTRA, an innovative tool for Nanopore Autosomal Short Tandem Repeat Analysis, which overcomes traditional database-based methods' limitations and provides a precise germline analysis of STR genetic markers without the need for allele sequence reference. Demonstrating high accuracy in cell line authentication testing and paternity testing, NASTRA significantly surpasses existing methods in both speed and accuracy. This advancement makes it a promising solution for rapid cell line authentication and kinship testing, highlighting the potential of nanopore sequencing for in-field applications.