Repeat Analysis Research Articles

Genetic variability of abundant littoral species of mesostigmatic mites (Acari, Mesostigmata) with different distributions from the seashores of Eurasia

The marine littoral acarofauna is of interest both in relation to the history of seas and as biological indicators of water quality, but it remains poorly studied. For the first time, we characterize the molecular genetic variability of four widespread, common and abundant species of littoral mesostigmatic mites: Phorytocarpais kempersi, Parasitidae; Halolaelaps celticus and Halolaelaps orientalis, Halolaelapidae; and Thinoseius spinosus, Eviphididae. Based on the nucleotide sequence analysis of nuclear ribosomal repeats (ITS), we studied mite collections from the coasts of the Caspian, Azov, Black, Baltic, White, Barents, Norwegian, Bering, Okhotsk, and Japan seas. The molecular identification of the species studied, based on DNA barcoding using ITS sequences, appears to be clear. We obtained genetic evidence for the separation of Halolaelaps celticus and H. orientalis as two distinct species, based both on clustering their ITS genotypes (separated by 6 parsimony-informative substitutions) and the geographic distributions of their genotypes. The high genetic similarity of the western and eastern Palaearctic populations in both arctic-adapted species, Phorytocarpais kempersi and Thinoseius spinosus, is explicable in terms of their rather recent contact on the northern coast of Eurasia during the last interglacial or Holocene climatic optimum. The high genetic similarity of Halolaelaps orientalis, a relatively thermophilous species, from the Mediterranean region (the Azov, Black, and Caspian seas) with a sample from the very distant Sea of Japan suggests a recent connection between the European and Pacific populations via the Indian Ocean.

The genome of the cryopelagic Antarctic bald notothen, Trematomus borchgrevinki.

The Antarctic bald notothen, Trematomus borchgrevinki (family Nototheniidae) occupies a high latitude, ice-laden environment and represents an extreme example of cold-specialization among fishes. We present the first, high quality, chromosome-scale genome of a female T. borchgrevinki individual comprised of 23 putative chromosomes, the largest of which is 65 megabasepairs (Mbp) in length. The total length of the genome 935.13 Mbp, composed of 2,094 scaffolds, with a scaffold N50 of 42.67 Mbp. Annotation yielded 22,192 protein coding genes while 54.75% of the genome was occupied by repetitive elements; an analysis of repeats demonstrated that an expansion occurred in recent time. Conserved synteny analysis revealed that the genome architecture of T. borchgrevinki is largely maintained with other members of the notothenioid clade, although several significant translocations and inversions are present, including the fusion of orthologous chromosomes 8 and 11 into a single element. This genome will serve as a cold-specialized model for comparisons to other members of the notothenioid adaptive radiation.

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.

ESTIMATION OF DOSE ESTIMATION USING THE SPECIFIC DOSE SIZE METHOD OF RADIOGRAPH IMAGE REPEAT ANALYSIS AS A FORM OF RADIATION SAFETY IN THE RADIOLOGY INSTALLATION OF KLATEN ISLAMIC HOSPITAL

Repeat analyses are radiographs that are not clinically acceptable and are requested to be retaken. The primary purpose of the repeat analyses program is to take corrective action, record the repeated radiographic images, and determine the cause of the repetition so that it can be minimized or even eliminated. Klaten Islamic Hospital needs to keep its radiology services optimal and safe for patients as a referral hospital. One indicator of the quality of service is the low repetition rate of radiographic images. This study aims to determine the percentage of factors causing the repetition of radiographic images and suggested solutions to minimize repeat radiographic images in the Radiology Department. Survey research with a quantitative approach to repeating radiographic images at the Radiology Department. The study was conducted in February-May 2024 using observation, survey, and documentation. The percentage of repetition of radiographic images at the Radiology Installation of Klaten Islamic Hospital in the first month was 2.72%, in the second month was 3.04%, in the third month was 4.05%, and in the fourth month was 3.43%. Factors causing the repetition of radiographic images include miscollimation (clipped), artifacts, exposure factors, tools, and patient movement. Suggested solutions to minimize the factors causing the repetition of radiographic images in the Radiology Installation of Klaten Islamic Hospital are good communication between radiographers and patients, double checking before exposure, clear instructions to patients, ensuring there are no objects that can become artifacts, using appropriate exposure parameters, calibrating and testing suitability regularly, reporting if there is equipment damage, and using immobilization techniques. Keywords: Repeat Analyses, Digital Radiography, radiation safety for patient.

The expression of shelterin genes and telomere repeat analysis and their effect on Alzheimer's disease.

Alzheimer's disease (AD) is an age-related dementia disorder characterized by memory loss and behavioral changes. Maintaining the integrity of telomere shortening in AD is important for cellular survival and homeostasis in all cells, especially glial cells. The shelterin protein complex provides telomere integrity. Measuring the expression levels of shelterin genes and determining the telomere lengths regulated by this complex will reveal their effects on AD progression and adult neurogenesis and will allow the detection of the disease or the determination of the progression process from an accessible tissue. The study population included 111 patients and 91 healthy controls (male and female, < 50 age). The clinical histories (age, gender, hypertension, diabetes mellitus, obesity, cardiovascular disease, MMSE, medication use, family history, sleep disorders, seizure), covariates (HGB, ESR, Na, P, Cl, BUN, CRP, B12, TSH, Glucose, and MRI findings) and the expressional changes of shelterin genes (TERF1, TERF2, TINF2, POT1, TPP1, and RAP1) between the patient and control groups were evaluated relatively. ROC analyses determined the diagnostic power of telomere repeats and gene expressions. In conclusion, upregulation of expression of shleterin complex genes was detected in AD, where telomeres are significantly shorter than in controls (P < 0.05). However, only TERF2 and RAP1 were significant (P < 0.05). A positive relationship was detected between telomere repeats and these genes (P < 0.05). Telomere repeats may be a strong diagnostic criterion to distinguish AD individuals from healthy individuals (AUC = 1.000). The upregulation of TERF2 and RAP1 core genes required for telomere integrity results in the instability of excessively shortened telomeres. Expression silencing of these genes may increase telomerase activity and maintain cellular survival. Also, the detection of telomere repeats has potential in the early diagnosis of AD patients.

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.

Repeat-based holocentromeres of the woodrush Luzula sylvatica reveal insights into the evolutionary transition to holocentricity

In most studied eukaryotes, chromosomes are monocentric, with centromere activity confined to a single region. However, the rush family (Juncaceae) includes species with both monocentric (Juncus) and holocentric (Luzula) chromosomes, where centromere activity is distributed along the entire chromosome length. Here, we combine chromosome-scale genome assembly, epigenetic analysis, immuno-FISH and super-resolution microscopy to study the transition to holocentricity in Luzula sylvatica. We report repeat-based holocentromeres with an irregular distribution of features along the chromosomes. Luzula sylvatica holocentromeres are predominantly associated with two satellite DNA repeats (Lusy1 and Lusy2), while CENH3 also binds satellite-free gene-poor regions. Comparative repeat analysis suggests that Lusy1 plays a crucial role in centromere function across most Luzula species. Furthermore, synteny analysis between L. sylvatica (n = 6) and Juncus effusus (n = 21) suggests that holocentric chromosomes in Luzula could have arisen from chromosome fusions of ancestral monocentric chromosomes, accompanied by the expansion of CENH3-associated satellite repeats.

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.

The complete chloroplast genome of Erodium stephanianum (Geraniaceae)

Erodium stephanianum (Erodium stephanianum Willd. Sp. Pl. 1800) has not had its complete chloroplast genome reported, which limits our understanding of its genetics and evolution. In this study, we assembled and annotated its chloroplast genome, revealing a quadripartite structure with 76 protein-coding genes. Repeat analysis indicated the presence of simple sequence repeats. Phylogenetic analysis confirmed E. stephanianum's placement within the genus Erodium of the Geraniaceae family. These findings offer valuable genomic resources for comparative studies in Erodium and Geraniaceae, aiding genetic diversity and phylogenetic analyses.

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.

Duration of sinus rhythm after direct current cardioversion does not predict recurrence of atrial fibrillation after subsequent catheter ablation: a multicentre study

Abstract Background Catheter ablation (CA) for persistent atrial fibrillation (PsAF) is associated with high rates of AF recurrence. DC cardioversion (DCCV) is often performed before CA, with wide variation in duration of maintenance of sinus rhythm (SR) post-DCCV. Factors associated with short duration of SR after DCCV, such as raised body mass index, prior duration of AF, prolonged p wave duration on the 12-lead ECG, and indexed left atrial volume, are either associated with or indicative of atrial electrical and structural remodelling, suggesting that shorter duration of SR after DCCV reflects a more advanced substrate for AF, which could in turn also influence the likelihood of success after CA. Objective To determine if post-DCCV SR duration is associated with AF recurrence after subsequent CA. Methods We performed a multicentre retrospective study of patients undergoing first-time CA for PsAF between 2015-2018 with prior DCCV. SR duration after the last DCCV prior to CA was recorded. The primary outcome was the first recurrence of atrial arrhythmia post-CA (after a 90-day post-CA blanking period). We also performed a pre-specified sensitivity analysis for amiodarone use at DCCV, by performing repeat analysis after excluding all such patients across the three groups. Results A total of 331 patients were identified across five centres and categorised by post-DCCV SR duration: &amp;lt;7 days (group 1); 7-31 days (group 2); &amp;gt;31 days (group 3; Table 1). Over median post-CA follow-up of 591 days, 207 patients (62.5%) met the primary outcome. There was no significant between-group difference in the time to primary outcome (log rank p=0.82; Figure 1). There were significantly more blanking period arrhythmias in groups 1 and 2 than group 3 after post hoc Bonferroni correction (pairwise p=0.001, 0.002 respectively; Table 1). After exclusion of patients on amiodarone at time of DCCV, there remained no significant between-group difference in the time to primary outcome (log rank p=0.85). Conclusion Shorter duration of SR post-DCCV is associated with blanking period arrhythmia after CA, but crucially does not predict long-term AF recurrence. Early AF recurrence after DCCV should not influence decisions on CA suitability or be used to predict long-term CA success.

Discordant Microsatellite Instability Status in Metachronous Primary Rectal Cancer Detected During IdyllaTM Microsatellite Instability Test Validation: A Case Report

Abstract Introduction/Objective Defects in DNA mismatch repair (MMR) can be identified by immunohistochemistry (IHC) MMR, Microsatellite Instability (MSI) status testing by PCR or by MSI testing by NGS. Discrepancies may occur between the different test methods. We describe a case of a discrepancy discovered during validation of an in-house IdyllaTM MSI test (PCR method), in which repeat testing by the external reference lab led to a definitive deficient MMR/MSI-High (MSI-H) status. Methods/Case Report A 67-year-old male presented with a new rectal mass on a follow-up colonoscopy. His past surgical history included a colorectal anastomosis secondary to a low anterior resection for pT3N0 upper rectal cancer ten years ago. He had a strong family history of colorectal cancer with his father, 3 brothers, 3 paternal uncles and 2 cousins affected. Colonoscopy revealed an adenocarcinoma 3 cm distal to the anastomosis. MRI showed a plaque-like mucosal thickening along the remaining rectum below the anastomosis. He underwent a complicated redo low anterior resection. Histopathology revealed a pT1N0 invasive moderately differentiated midrectal adenocarcinoma. Test results for an external PCR-based MSI test were microsatellite stable (MSS). However, repeat testing (requested after MSI-H status was detected by the in-house IdyllaTM MSI assay) revealed a deficient MMR status (loss of expression of MSH2 and MSH6 proteins) and MSI-H by PCR analysis. These discrepant results may have been due to low tumor cellularity and tumor heterogeneity (ie, heterogeneous pattern of MSH2/MSH6 loss on IHC). Results (if a Case Study enter NA) NA Conclusion Discrepancies in deficient MMR/MSI status between and within different test methods may be attributable to intratumoral heterogeneity and low tumor cellularity. In discordant cases, pathologists must not hesitate to perform a repeat analysis using alternate methods, as a deficient MMR/MSI-H diagnosis has crucial diagnostic, prognostic and predictive implications.

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.

A-337 The Utility of Urinary Human Chorionic Gonadotropin Testing in the Emergency Department

Abstract Background Human chorionic gonadotropin (hCG) levels are measured in both urine and blood as an indication of suspected pregnancy, and assessment of progress of conception. It is also measured in the investigation of patients presenting with acute abdomen and suspected of ectopic or molar pregnancy. This study reviewed the utility of urine pregnancy test in a large university teaching hospital. Methods Results of all urinary hCG tests (using hCG Combo Pregnancy kit, MEDLINE, IL) performed as point of care in the emergency department during the year 2023 were obtained. Corresponding plasma hcG levels performed (Total β-HCG Alinity immunoassay, Abbott Diagnostics, IL) were also obtained. Analysis of hCG testing outcomes were assessed. Results A total of 2848 urinary hCG tests were performed during the study period. The majority 2113 (74.2%) of samples were negative for hCG whereas 835 samples (29.3) were positive. Among the positive hCG urine samples, 401 patients (48%) had plasma hCG requested and performed. Plasma hCG values ranged from &amp;lt; 2.4 (26.5) to 219,462.5 mIU/mL (median 16,872.35 mIU/mL). One plasma sample with corresponding positive urinary hCG result had plasma hCG level &amp;lt;2.4 mIU/mL which is below the detection limit of the assay suggesting a false positive urinary hCG result. Among the 13 patients with serial plasma hCG values, only one patient exhibited doubling (48 hours) hCG values when less than 13,000 mIU/mL. One patient had a declining serial hCG plasma values suggesting failing conception. The remaining patients with consecutive plasma hCG values cannot be assessed as either the time gap was excessive or hCG values were markedly elevated (&amp;gt; 30,000 mIU/mL). Conclusions Although the utility of urinary hCG testing in the emergency department appeared appropriate, unnecessary repeat analysis with appropriate plasma request represented 3.2%. An electronic test request duplicate notification may help resolve this unnecessary repeat.

421 - Reject and repeat analysis in digital radiography in a Kenyan radiology department

421 - Reject and repeat analysis in digital radiography in a Kenyan radiology department