Comprehensive Genotyping Research Articles

Distribution and Implications of Mycobacterium Tuberculosis Genotypes in Animals: A Comprehensive Review

This narrative review discusses the spread of Mycobacterium tuberculosis among animal populations, focusing on M. tuberculosis genotype distribution and frequency across different species. To develop comprehensive tuberculosis control strategies under the One Health approach, it is important to have an understanding of the genotypes of M. tuberculosis in animals. Animal infections are discussed, as well as the pathogenesis and epidemiology of M. tuberculosis. Various genotyping methods, including spoligotyping, MIRU-VNTR, and whole genome sequencing, are discussed in the review, emphasizing their role in understanding strain diversity and transmission dynamics. There are many factors influencing the distribution of M. tuberculosis genotypes in animals, including environmental conditions, host factors, human-animal interactions, and animal trade practices. It is clear from the review that the Euro-American lineage is widely distributed across animal species, with the Beijing genotype becoming increasingly prevalent in regions where it is prevalent in humans. The study highlights the need for more comprehensive genotyping studies as well as the development of better diagnostic tools. This will enhance our understanding of M. tuberculosis in animal populations. A conclusion to the review, emphasizes that standardizing genotyping protocols, utilizing whole genome sequencing, and fostering international collaboration are essential for improving our understanding of M. tuberculosis diversity in animals and developing targeted tuberculosis control strategies. The impact of Mycobacterium tuberculosis genotypes in animals is multi-faceted, affecting both public health and veterinary practices. Addressing these challenges requires a coordinated and comprehensive approach that considers the interconnectedness of human and animal health systems, fosters research, enhances surveillance, and implements effective control strategies.

Genomeprofile: Unveiling the genomic profile for livestock breeding through comprehensive SNP array-based genotyping.

In livestock breeding, single nucleotide polymorphism arrays have become a cornerstone of modern livestock breeding. SNP arrays facilitate the identification of genetic markers linked to economically important traits and provide a powerful tool for predicting breeding values. However, conventional breeding programs often overlook additional genomic features contained in the SNP array data that can provide valuable insights into the genetic diversity, copy number variation, inbreeding levels and potential challenges in breeding lines. Here we present genomeprofile, a tool using SNP array-based genomic data, offering a comprehensive profile of breeding animals including the identification of copy number variants and runs of homozygosity, and screening for aneuploidy. By integrating these features into the breeding landscape, genomeprofile enables a more comprehensive picture of genomic variation, ultimately enhancing precision breeding strategies. To illustrate the practicality and efficacy of genomeprofile, we applied the tool to a dataset of four pig breeding lines. The genomeprofile tool is a user-friendly tool that processes genotype data in finalreport or plink ped format efficiently into useful output. The output contains copy number variations, runs of homozygosity, selection signatures, aneuploidy and inbreeding per individual and across populations. This allows breeding companies and researchers to identify unique individuals or regions in the genome of interest based on routinely collected data.

Genetics of 21-OH Deficiency and Genotype-Phenotype Correlation: Experience of the Hellenic National Referral Center.

21-hydroxylase deficiency (21-OHD) represents the most common form of congenital adrenal hyperplasia (CAH) due to CYP21A2 gene pathogenic variants. Τhe aim of this study was the identification of CYP21A2 variants in 500 subjects of Greek origin with a suspicion of 21-OHD and, by using the existing hormonal assessment and genotypes of the 500 subjects tested, to identify a biomarker that could differentiate between the heterozygotes and the cases with no pathogenic variants identified. Five hundred subjects with clinical suspicion of 21-OHD underwent CYP21A2 gene sequencing and Multiplex Ligation Dependent Probe Amplification (MLPA). Genetic diagnosis was achieved in 27.4% of the subjects tested, most of which presented with the non-classic form (NC) of 21-OHD. Heterozygotes accounted for 42.6% of cases, whereas no pathogenic variants were identified in 27% of cases. De novo aberrations, duplications, and five novel variants were also identified. Statistical analysis revealed that the difference between the basal and 60' post-ACTH stimulation 17-hydroxyprogesterone concentrations (Δ17-OHP60-0) could be a potential biomarker (p < 0.05) distinguishing the heterozygotes from the cases with no pathogenic variants identified, although no clear cut-off value could be set. Further analysis revealed overlapping clinical manifestations among all the subjects tested. The presented phenotypic traits of the subjects tested and the inability to identify a discriminative biochemical marker highlight the importance of comprehensive CYP21A2 genotyping to ascertain the correct genetic diagnosis and proper genetic counselling.

Molecular detection and genotyping of HBV from HBsAg positive patients in a tertiary hospital in Nigeria.

Nigeria remains one of the countries with a high hepatitis B virus (HBV) burden in Africa. Reports have indicated the presence of mixed HBV genotypes in Nigeria; however, there is still paucity of data regarding mixed genotype infections particularly in the Southern part of the country. Our aim is to determine the HBV genotype distribution among HBsAg-positive gastroenterology patients at the University College Hospital Ibadan, Nigeria. Serum samples were screened for HBsAg by ELISA, and positive samples were genotyped by semi-nested multiplex PCR for HBV genotypes A, B, C, D, E and F. Data generated were analyzed in R-studio. A total of 81/90 (90%) of HBsAg-positive samples were successfully genotyped, and genotype A was most prevalent with 15.7%, while genotypes B and E were the least with 1.2% each. Genotypes A/C infection was the highest among mixed infections with 40% prevalence, while genotypes A/D were the least prevalent mixed infection with 4.8%. We advocate for a comprehensive genotype analysis in larger cohorts across Nigeria, to give a more comprehensive understanding of the distribution and prevalence of different HBV genotypes population wide.

Identification of GTF2I Polymorphisms as Potential Biomarkers for CKD in the Han Chinese Population : Multicentric Collaborative Cross-Sectional Cohort Study.

Chronic kidney disease (CKD) poses a global health challenge, but its molecular mechanisms are poorly understood. Genetic factors play a critical role, and phenome-wide association studies (PheWAS) and genome-wide association studies (GWAS) shed light on CKD's genetic architecture, shared variants, and biological pathways. Using data from the multicenter collaborative precision medicine cohort, we conducted a retrospective prospectively maintained cross-sectional study. Participants with comprehensive information and genotyping data were selected, and GWAS and PheWAS analyses were performed using the curated Taiwan Biobank version 2 array to identify CKD-associated genetic variants and explore their phenotypic associations. Among 58,091 volunteers, 8,420 participants were enrolled. Individuals with CKD exhibited higher prevalence of metabolic, cardiovascular, autoimmune, and nephritic disorders. Genetic analysis unveiled two closely linked SNPs, rs117026326 and rs73366469, both associated with GTF2I and CKD (r2=0.64). Further examination revealed significant associations between these SNPs and various kidney-related diseases. The CKD group showed a higher proportion of individuals with specific genotypes (CT/TT for rs117026326 and CT/CC for rs73366469), suggesting potential associations with CKD susceptibility(p<0.001). Furthermore, individuals with these genotypes developed CKD at an earlier age. Multiple logistic regression confirmed the independent association of these genetic variants with CKD. Subgroup analysis based on estimated glomerular filtration rate (eGFR) demonstrated an increased risk of CKD among carriers of the rs117026326 CT/TT genotypes (OR=1.15, 95% CI: 1.07-1.24, p<0.001; OR=1.32; 95% CI: 1.04-1.66, p=0.02, respectively) and carriers of the rs73366469 CT/CC genotypes (OR=1.13, 95% CI=1.05-1.21, p<0.001; OR=1.31, 95% CI: 1.08-1.58, p=0.0049, respectively). Additionally, men had a higher CKD risk than women at lower eGFR levels (OR=1.35, 95%: 1.13-1.61, p<0.001). Our study reveals important links between genetic variants GTF2I and susceptibility to CKD, advancing our understanding of CKD development in the Taiwanese population and suggesting potential for personalized prevention and management strategies. More research is needed to validate and explore these variants in diverse populations.

Electronic Medical Record-Based Nudge Intervention to Increase Comprehensive Molecular Genotyping in Patients With Metastatic Non-Small Cell Lung Cancer: Results From a Prospective Clinical Trial.

Less than half of the patients with newly diagnosed metastatic non-small cell lung cancer (NSCLC) undergo comprehensive molecular testing. We designed an electronic medical record (EMR)-based "nudge intervention" to prompt plasma-based molecular testing at the time of initial medical oncology consultation. A nonrandomized prospective trial was conducted at the University of Pennsylvania's academic practice and two affiliated community practices. Molecular genotyping was performed by tissue- and/or plasma-based next generation sequencing methods. Comprehensive testing was defined as testing for EGFR, ALK, BRAF, ROS1, MET, RET, KRAS, and NTRK. Guideline-concordant treatment was defined as the use of the appropriate first-line (1L) therapy as per the National Comprehensive Cancer Network (NCCN) guidelines. Proportion of patients with comprehensive molecular genotyping results available at any time, molecular results available before 1L therapy, and guideline-concordant 1L treatment were compared between the preintervention and postintervention cohorts using Fisher's exact test or Pearson's chi-squared test. Five hundred and thirty-three patients were included, 376 in the preintervention cohort and 157 in the postintervention cohort. After implementation of the EMR-based nudge, a higher proportion of patients underwent comprehensive molecular testing in the postintervention versus the preintervention cohort (100% v 88%, P = <.001), had results of comprehensive molecular testing available before initiating 1L treatment (97.3% v 91.6%, P = .026), and received NCCN guideline-concordant care (89.8% v 78.2%, P = .035). Across three practice sites in a large health system, implementation of a provider team-focused EMR-based nudge intervention was feasible, and led to a higher number of patients with NSCLC undergoing comprehensive molecular genotyping. These findings demonstrate that behavioral nudges can promote molecular testing and should be studied further as a tool to improve guideline-concordant care in both community and academic sites.

Genetic Insights into Skeletal Malocclusion: The Role of the FBN3 rs7351083 SNP in the Romanian Population.

Background and Objectives: irregularities in the growth and development of the jawbones can lead to misalignments of maxillary and mandibular structures, a complex condition known as skeletal malocclusion, one of the most common oral health problems. Skeletal malocclusions, particularly Class II and Class III, can significantly affect facial appearance, chewing efficiency, speech, and overall oral health, often requiring orthodontic treatment or surgery to correct. These dentofacial anomalies are influenced by genetic and environmental factors and exhibit diverse phenotypic expressions. Materials and Methods: in this study, we investigated the correlation between the rs7351083 SNP of the FBN3 gene that encodes a member of the fibrillin protein family and malocclusion risk in a group of 57 patients from Romania. Results: the results shed light on the relationship between the selected genetic marker and the investigated dentofacial disorder, revealing a positive association between the reference allele (A) and Class II and that the alternate allele (G) is associated with Class III. Conclusions: cephalometric analysis revealed no significant differences among genotypes, suggesting that while genetic factors are implicated in malocclusion, they may not directly affect cephalometric parameters or that the sample size was too small to detect these differences. The discovery of an A > T transversion in one individual with a Class II deformity underscores the genetic diversity within the population and the necessity of comprehensive genotyping to uncover rare genetic variants that might influence craniofacial development and the risk of malocclusion. This study highlights the need for larger studies to confirm these preliminary associations.

Genotype profile of HPV in ASC-H cytology and histologic follow-up-prevalence, distribution, and risk: A retrospective study of 1414 cases.

A cytologic diagnosis of atypical squamous cells, cannot exclude high-grade squamous lesion (ASC-H) poses a disproportionately high risk of cervical cancer development. The objective of this study was to analyze type-specific risks by mapping human papillomavirus (HPV) genotypes in ASC-H cytology. In total, 1,048,581 Papanicolaou tests that had ASC-H cytology were retrieved. Concurrent HPV genotyping using proprietary multiplex real-time (MRT) and polymerase chain reaction (PCR) HPV tests and histologic follow-up findings were analyzed. Among 1678 patients who had ASC-H findings (0.16%), 1414 (84.3%) underwent concurrent HPV genotyping (MRT, 857; HPV PCR test, 557). The overall high-risk HPV (hrHPV)-positive rate was 84.4%. Of the 857 MRT cases, 63.9% were infected with a single hrHPV, and 24.4% had multiple genotypes. The most prevalent HPV types were HPV16/52/58/33/31. Lesions that were identified as cervical intraepithelial neoplasia 2 or worse (CIN2+) were detected in 498 of 906 cases (55.0%), including 81 cervical carcinomas (8.9%). The risk of CIN2+ for the composite group of HPV16/52/58/33/31-positive cases was 62.7%, representing 90.7% (264 of 291) of total CIN2+ lesions in ASC-H/hrHPV-positive cases by MRT. CIN2+ lesions were detected in 108 of 142 (76.1%) HPV16-positive and/or HPV18-positive women by the PCR the HPV test. Among 128 hrHPV-negative ASC-H cases by both methods, CIN2+ lesions were identified in 21 of 128 (16.4%), including five cervical carcinomas (3.9%). The sensitivity, specificity, positive predictive value, and negative predictive value for patients in the composite group with HPV16/52/58/33/31 were 88.0%, 40.8%, 62.7%, and 75.0%, respectively. Papanicolaou tests classified as ASC-H are associated with a high CIN2+ rate and warrant colposcopy, regardless of HPV status. The extent to which the risk-stratification provided by comprehensive HPV genotyping can inform the management of ASC-H cytology remains to be explored.

INUDGE: Integration of liquid biopsy based next generation gene sequencing in newly diagnosed NSCLC—A stepped wedge cluster randomized clinical trial.

TPS8664 Background: The development of targeted therapies has changed the treatment paradigm for non–small cell lung cancer (NSCLC). With the growing number of FDA approved targeted therapies, current NCCN guidelines recommend comprehensive molecular genotyping prior to first line (1L) therapy for all newly diagnosed patients with metastatic non-squamous (mNSq) NSCLC to enable the delivery of personalized therapy. Based on prior work that found improved detection of clinically actionable alterations through concurrent plasma (P) based next-generation gene sequencing (NGS) and tissue NGS, we piloted a behavioral economics (BE) informed electronic health record (EHR)-based “nudge” intervention designed to facilitate P NGS at the new patient medical oncology visit. After implementing across 3 practices within the University of Pennsylvania Health System (UPHS), our findings demonstrated that EHR-based nudges are feasible and can promote guideline concordant diagnostic testing at both community and academic sites (1). Methods: Design: NCT05853887 is a prospective stepped wedge cluster randomized trial that includes 3 “clusters”, representing 6 community-based cancer centers. The primary objective of this trial is to test the effectiveness of a BE informed EHR nudge intervention to increase availability of comprehensive molecular test results before 1L therapy by incorporating concurrent tissue and plasma-based molecular testing. Secondary objectives include evaluation of contextual mechanisms contributing to the adoption, reach, and effectiveness of EHR nudge interventions with a lens for health equity. Intervention: An EHR-based nudge that allows for default order placement of P NGS testing at the time of the first medical oncology visit will be implemented. Subsequently, results detected on the default P NGS order will be conveyed to providers in the form of an electronic clinical decision support notification. Statistical Methods:Sample size is based on estimates of the proportion of patients undergoing comprehensive molecular testing prior to 1L. A sample of 3 clusters in a complete stepped-wedge cluster-randomized design with 4 time periods (including the baseline), 3 steps, 1 cluster(s) switching from control to treatment at each step, and an average of 120 subjects per cluster with an average of 30 subjects per cluster per time period (for a total sample size of 360 subjects) will provide 80% power to detect a difference between proportions of 0.21701. Patients with histological, or cytological diagnosis of Stage IV mNSq NSCLC who have not yet received systemic treatment for metastatic disease are eligible for inclusion. Enrollment is ongoing: Cluster 1 began enrollment in June 2023, Cluster 2 in October 2023, and Cluster 3 is planned for February 2024. 1. Aggarwal C et al, JCO, Vol 40, Num 16, Suppl Jun 2022. Clinical trial information: NCT05853887 .

Abstract PO5-20-11: Synchronous Breast Cancer and Pheochromocytoma: A Case Series

Abstract Background: Pheochromocytomas are rare, although literature suggests a rising incidence due to the widespread use of sensitive imaging modalities. Synchronous presentation of breast cancer with a pheochromocytoma is exceedingly rare with only one case documented in the literature. We present two cases of malignancy in the breast in which pheochromocytomas were incidentally detected during workup and management with vastly different presentations. Case Presentation: Two patients presented with breast cancer and were secondarily diagnosed with pheochromocytoma. Patient 1 is a 48-year-old postmenopausal female with a history of intraductal papilloma four years prior, who presented with right breast nipple discharge. Diagnostic mammogram showed a right breast mass and biopsy confirmed an intraductal papilloma with intermediate-grade DCIS, ER+/PR-. She had no family history of cancer or PGL/PPC. During a scheduled lumpectomy, she developed hypoxia, hypotension, and flash pulmonary edema requiring intubation and ECMO with prolonged ICU stay and long recovery with tracheostomy for four months. Contrast-enhanced CT of her abdomen showed a 9 cm right adrenal mass. Serum and urine metanephrines were markedly elevated, and it was determined that she had an adrenal crisis secondary to undiagnosed pheochromocytoma. She completed treatment for DCIS with lumpectomy and radiation and is currently on adjuvant anastrozole. She additionally had an adrenalectomy for pheochromocytoma, which was found to be stage PASS 2. Subsequent genetic testing confirmed the absence of germline mutations associated with hereditary PGL/PPC or breast cancer. Patient 2 is a 57-year-old female presenting with a history of left breast mass she first noted a year ago, with newly found nipple retraction and skin changes. Biopsy showed invasive ductal carcinoma of the breast, G3, Stage IIIA, pT3N2aM0, ER 95%/PR-/Her2- (IHC 0). Staging PET scan showed a 3.4 cm hypermetabolic left adrenal mass in addition to the breast mass and left axillary adenopathy. Serum and urine metanephrines were elevated, and pheochromocytoma was diagnosed. Family history was positive only for pancreatic cancer in a maternal aunt. Comprehensive genotype testing was negative for hereditary breast cancer and PGL/PCC syndromes. She was concomitantly treated with a radical mastectomy for breast cancer and a left adrenalectomy and tolerated the procedure well as she received perioperative alpha-adrenergic blockade with doxazosin. She has received chemotherapy and radiation for breast cancer treatment and is currently on adjuvant anastrozole. The adrenal mass was confirmed to be a pheochromocytoma PASS 2-3. Discussion: We present two unique cases of breast cancer and pheochromocytomas discovered in tandem with variable clinical manifestations. It is extremely uncommon to have a presentation of primary breast malignancy concomitantly with pheochromocytoma, and we demonstrate two cases that occurred within months of one another at our facility. During the first case, metastatic workup was not indicated for DCIS. However, during the seemingly benign lumpectomy procedure, the patient experienced significant complications, leading to intense ICU management, intubation, and tracheostomy. In the second case, a metastatic workup was completed due to locally advanced breast cancer where the adrenal mass was detected and a pheochromocytoma was diagnosed prior to surgery. This allowed both cancers to be resected simultaneously and safely. This series demonstrates the importance of keeping pheochromocytomas in the differential during the workup of adrenal masses prior to surgical interventions. Additionally, they demonstrate how the usual workup and management of breast cancer does not take into account these rare but significant diagnoses, and that can result in fatal consequences. Citation Format: Nikita Dahake, Abhimanyu Tushir, Aruna Padmanabhan. Synchronous Breast Cancer and Pheochromocytoma: A Case Series [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-20-11.

Unveiling Discrepant and Rare Dihydropyrimidine Dehydrogenase (DPYD) Results Using an In-House Genotyping Test: A Case Series.

Fluoropyrimidine chemotherapy is a primary component of many solid tumor treatment regimens, particularly those for gastrointestinal malignancies. Approximately one-third of patients receiving fluoropyrimidine-based chemotherapies experience serious adverse effects. This risk is substantially higher in patients carrying DPYD genetic variants, which cause reduced fluoropyrimidine metabolism and inactivation (ie, dihydropyridine dehydrogenase [DPD] deficiency). Despite the known relationship between DPD deficiency and severe toxicity risk, including drug-related fatalities, pretreatment DPYD testing is not standard of care in the United States. We developed an in-house DPYD genotyping test that detects 5 clinically actionable variants associated with DPD deficiency, and genotyped 827 patients receiving fluoropyrimidines, of which 49 (6%) were identified as heterozygous carriers. We highlight 3 unique cases: (1) a patient with a false-negative result from a commercial laboratory that only tested for the c.1905 + 1G>A (*2A) variant, (2) a White patient in whom the c.557A>G variant (typically observed in people of African ancestry) was detected, and (3) a patient with the rare c.1679T>G (*13) variant. Lastly, we evaluated which DPYD variants are detected by commercial laboratories offering DPYD genotyping in the United States and found 6 of 13 (46%) did not test for all 5 variants included on our panel. We estimated that 20.4% to 81.6% of DPYD heterozygous carriers identified on our panel would have had a false-negative result if tested by 1 of these 6 laboratories. The sensitivity and negative predictive value of the diagnostic tests from these laboratories ranged from 18.4% to 79.6% and 95.1% to 98.7%, respectively. These cases underscore the importance of comprehensive DPYD genotyping to accurately identify patients with DPD deficiency who may require lower fluoropyrimidine doses to mitigate severe toxicities and hospitalizations. Clinicians should be aware of test limitations and variability in variant detection by commercial laboratories, and seek assistance by pharmacogenetic experts or available resources for test selection and result interpretation.

Machine learning to optimize automated RH genotyping using whole-exome sequencing data

AbstractRh phenotype matching reduces but does not eliminate alloimmunization in patients with sickle cell disease (SCD) due to RH genetic diversity that is not distinguishable by serological typing. RH genotype matching can potentially mitigate Rh alloimmunization but comprehensive and accessible genotyping methods are needed. We developed RHtyper as an automated algorithm to predict RH genotypes using whole-genome sequencing (WGS) data with high accuracy. Here, we adapted RHtyper for whole-exome sequencing (WES) data, which are more affordable but challenged by uneven sequencing coverage and exacerbated sequencing read misalignment, resulting in uncertain predictions for (1) RHD zygosity and hybrid alleles, (2) RHCE∗C vs. RHCE∗c alleles, (3) RHD c.1136C>T zygosity, and (4) RHCE c.48G>C zygosity. We optimized RHtyper to accurately predict RHD and RHCE genotypes using WES data by leveraging machine learning models and improved the concordance of WES with WGS predictions from 90.8% to 97.2% for RHD and 96.3% to 98.2% for RHCE among 396 patients in the Sickle Cell Clinical Research and Intervention Program. In a second validation cohort of 3030 cancer survivors (15.2% Black or African Americans) from the St. Jude Lifetime Cohort Study, the optimized RHtyper reached concordance rates between WES and WGS predications to 96.3% for RHD and 94.6% for RHCE. Machine learning improved the accuracy of RH predication using WES data. RHtyper has the potential, once implemented, to provide a precision medicine-based approach to facilitate RH genotype–matched transfusion and improve transfusion safety for patients with SCD. This study used data from clinical trials registered at ClinicalTrials.gov as #NCT02098863 and NCT00760656.

Abstract 3888: TOP1 mutations mediate cross resistance to ADCs in metastatic breast cancer

Abstract Background: Antibody-drug conjugates (ADCs) have improved survival for patients with metastatic breast cancer (MBC). In patients with HER2 negative MBC, two ADCs have FDA approval: sacituzumab govitecan (SG) and trastuzumab deruxtecan (T-DXd), both with topoisomerase-I (topo-I) inhibitor payloads. Given the many other ADCs in development, there is great interest in using ADC after ADC to maximize treatment benefit for patients. However, there is limited understanding regarding resistance mechanisms to ADCs and impact on ADC sequencing. We conducted a translational study to address this, and here we report the incidence of TOP1 mutations and clinical impact on ADC sequencing. Methods: All patients with MBC treated with ADCs at a large academic medical institution (Massachusetts General Hospital) who had comprehensive plasma-based genotyping (500 gene GuardantOMNI panel) were included. Since both SG and T-DXd have topo-I inhibitor-based payloads, we particularly focused on TOP1 mutations, variant allele frequency (VAF), and germline/somatic characterization. Incidence of TOP1 mutations in the ADC cohort was compared to The Cancer Genome Atlas (TCGA). Clinical “cross-resistance” was defined as progressive disease (PD) as best response to second ADC (ADC2) or treatment time on ADC2 of less than 60 days. Results: Based on comprehensive plasma-based genotyping we identified 4 distinct TOP1 mutations: S57C, R364H, W401C, G359E, at a frequency of 6.0% (4/67) at the time of disease progression on ADC compared to a frequency of 0.5% described in primary breast cancer in TCGA. Two of the amino acids found to be mutated are known to form direct interactions with the DNA backbone (G359) or the topoisomerase inhibitor itself (R364). For clinical resistance, one patient was briefly on ADC2 but stopped after 1 dose for toxicity; among the other 3 patients, two had cross-resistance to ADC after ADC, with both ADCs containing topo-I inhibitor payloads. Median duration on first ADC was 455 days compared to a median of 52 days for ADC2. Finally, one patient treated sequentially with 3 ADCs (all with topo-I inhibitor payloads) was found to have rising TOP1 VAF with progressive ADC treatments. Conclusion: This is the first report describing emergence of TOP1 mutations under selective pressure from ADCs and the impact on mediating cross-resistance to ADC after ADC with topo-I inhibitor payloads. Novel ADCs with alternative payloads may potentially be more effective when used sequentially after an ADC with a topo-I inhibitor. Further biomarker research is needed to optimize ADC sequencing for patients with TOP1 mutant MBC. Citation Format: Rachel Occhiogrosso Abelman, Haley Barnes, Arielle J. Medford, Annika Putur, Bogang Wu, Caroline Weipert, Geoffrey Fell, Laura M. Spring, Seth A. Wander, Beverly Moy, Andreas Varkaris, Dejan Juric, Leif Ellisen, Ryan Corcoran, Aditya Bardia. TOP1 mutations mediate cross resistance to ADCs in metastatic breast cancer [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 3888.

Two-field resolution on-call HLA typing for deceased donors using nanopore sequencing.

The current practice of HLA genotyping in deceased donors poses challenges due to limited resolution within time constraints. Nevertheless, the assessment of compatibility between anti-HLA sensitized recipients and mismatched donors remains a critical medical need, particularly when dealing with allele-specific (second field genotyping level) donor-specific antibodies. In this study, we present a customized protocol based on the NanoTYPE® HLA typing kit, employing the MinION® sequencer, which enables rapid HLA typing of deceased donors within a short timeframe of 3.75 h on average at a three-field resolution with almost no residual ambiguities. Through a prospective real-time analysis of HLA typing in 18 donors, we demonstrated the efficacy and precision of our nanopore-based method in comparison to the conventional approach and without delaying organ allocation. Indeed, this duration was consistent with the deceased donor organ donation procedure leading to organ allocation via the French Biomedicine Agency. The improved resolution achieved with our protocol enhances the security of organ allocation, particularly benefiting highly sensitized recipients who often present intricate HLA antibody profiles. By overcoming technical challenges and providing comprehensive genotyping data, this approach holds the potential to significantly impact deceased donor HLA genotyping, thereby facilitating optimal organ allocation strategies.

Therapeutic Potential of Bacteroides fragilis SNBF-1 as a Next-Generation Probiotic: In Vitro Efficacy in Lipid and Carbohydrate Metabolism and Antioxidant Activity.

This study explores the potential of aerotolerant Bacteroides fragilis (B. fragilis) strains as next-generation probiotics (NGPs), focusing on their adaptability in the gastrointestinal environment, safety profile, and probiotic functions. From 23 healthy infant fecal samples, we successfully isolated 56 beneficial B. fragilis strains. Notably, the SNBF-1 strain demonstrated superior cholesterol removal efficiency in HepG2 cells, outshining all other strains by achieving a remarkable reduction in cholesterol by 55.38 ± 2.26%. Comprehensive genotype and phenotype analyses were conducted, including sugar utilization and antibiotic sensitivity tests, leading to the development of an optimized growth medium for SNBF-1. SNBF-1 also demonstrated robust and consistent antioxidant activity, particularly in cell-free extracts, as evidenced by an average oxygen radical absorbance capacity value of 1.061 and a 2,2-diphenyl-1-picrylhydrazyl scavenging ability of 94.53 ± 7.31%. The regulation of carbohydrate metabolism by SNBF-1 was assessed in the insulin-resistant HepG2 cell line. In enzyme inhibition assays, SNBF-1 showed significant α-amylase and α-glucosidase inhibition, with rates of 87.04 ± 2.03% and 37.82 ± 1.36%, respectively. Furthermore, the cell-free supernatant (CFS) of SNBF-1 enhanced glucose consumption and glycogen synthesis in insulin-resistant HepG2 cells, indicating improved cellular energy metabolism. This was consistent with the observation that the CFS of SNBF-1 increased the proliferation of HepG2 cells by 123.77 ± 0.82% compared to that of the control. Overall, this research significantly enhances our understanding of NGPs and their potential therapeutic applications in modulating the gut microbiome.

Comprehensive genotyping analysis of single nucleotide polymorphisms responsible for beef marbling in Japanese Black cattle.

Beef marbling is considered a desirable trait in the meat industry. Therefore, understanding the genetic factors that cause marbling is important. Previously, we performed a genome-wide association study to examine genetic factors associated with beef marbling in Japanese Black cattle and identified a candidate region between 10-30 Mbp on chromosome 7. We verified the effect of the SNPs in this region on beef marbling using linkage disequilibrium block analysis. We narrowed down the candidate region to a range of 15.8-16.1 Mbp. In this study, we comprehensively detected all of the SNPs in this region and verified their effects on beef marbling. Genome resequencing using four animals exhibiting high beef marbling standard (BMS) and four with low BMS revealed a total of 1,846 polymorphisms within the candidate region. Based on the annotation, we selected 13 SNPs exhibiting a moderate impact, as no high-impact SNPs were detected. All of the SNPs represented missense polymorphisms and were located in the following seven genes: RDH8, ANGPTL6, DNMT1, MRPL4, ICAM1, ICAM3, and ICAM5. Finally, we determined the effects of these SNPs on the BMS of a Japanese Black cattle population (n = 529). Analysis of variance revealed that the five SNPs were located in genes encoding the intercellular adhesion molecules (ICAM1, ICAM3, and ICAM5), and showed a highly significant association compared with the remainder (p < 0.01). The lowest p-value was observed for ICAM3_c.739G > A (p = 1.18E-04). Previous studies have suggested that intercellular adhesion molecules (ICAM) may be an upstream factor that regulates adipocyte differentiation. Therefore, considering the polymorphism and putative gene function, we suggest that ICAM1 is potentially responsible for beef marbling. c.470C > G and/or c.994G > A on ICAM1 may be responsible for this quantitative trait locus. Promising SNP candidates responsible for beef marbling were identified using extensive polymorphism verification in a previously reported QTL region. We aim to elucidate the mechanism of beef marbling in future studies by investigating how these polymorphisms alter protein structure and function.

Seq2Sat and SatAnalyzer toolkit: Towards comprehensive microsatellite genotyping from sequencing data.

Accurate and efficient microsatellite loci genotyping is an essential process in population genetics that is also used in various demographic analyses. Protocols for next-generation sequencing of microsatellite loci enable high-throughput and cross-compatible allele scoring, common issues that are not addressed by conventional capillary-based approaches. To improve this process, we have developed an all-in-one software, called Seq2Sat (sequence to microsatellite), in C++ to support automated microsatellite genotyping. It directly takes raw reads of microsatellite amplicons and conducts read quality control before inferring genotypes based on depth-of-read, read ratio, sequence composition and length. We have also developed a module for sex identification based on sex chromosome-specific locus amplicons. To allow for greater user access and complement autoscoring, we developed SatAnalyzer (microsatellite analyzer), a user-friendly web-based platform that conducts reads-to-report analyses by calling Seq2Sat for genotype autoscoring and produces interactive genotype graphs for manual editing. SatAnalyzer also allows users to troubleshoot multiplex optimization by analysing read quality and distribution across loci and samples in support of high-quality library preparation. To evaluate its performance, we benchmarked our toolkit Seq2Sat/SatAnalyzer against a conventional capillary gel method and existing microsatellite genotyping software, MEGASAT, using two datasets. Results showed that SatAnalyzer can achieve >99.70% genotyping accuracy and Seq2Sat is ~5 times faster than MEGASAT despite many more informative tables and figures being generated. Seq2Sat and SatAnalyzer are freely available on github (https://github.com/ecogenomicscanada/Seq2Sat) and dockerhub (https://hub.docker.com/r/rocpengliu/satanalyzer).

Genome-wide association studies for earliness, MYMIV resistance, and other associated traits in mungbean (Vigna radiata L. Wilczek) using genotyping by sequencing approach.

Yellow mosaic disease (YMD) remains a major constraint in mungbean (Vigna radiata (L.)) production; while short-duration genotypes offer multiple crop cycles per year and help in escaping terminal heat stress, especially during summer cultivation. A comprehensive genotyping by sequencing (GBS)-based genome-wide association studies (GWAS) analysis was conducted using 132 diverse mungbean genotypes for traits like flowering time, YMD resistance, soil plant analysis development (SPAD) value, trichome density, and leaf area. The frequency distribution revealed a wide range of values for all the traits. GBS studies identified 31,953 high-quality single nucleotide polymorphism (SNPs) across all 11 mungbean chromosomes and were used for GWAS. Structure analysis revealed the presence of two genetically distinct populations based on ΔK. The linkage disequilibrium (LD) varied throughout the chromosomes and at r2 = 0.2, the mean LD decay was estimated as 39.59 kb. Two statistical models, mixed linear model (MLM) and Bayesian-information and Linkage-disequilibrium Iteratively Nested Keyway (BLINK) identified 44 shared SNPs linked with various candidate genes. Notable candidate genes identified include FPA for flowering time (VRADI10G01470; chr. 10), TIR-NBS-LRR for mungbean yellow mosaic India virus (MYMIV) resistance (VRADI09G06940; chr. 9), E3 ubiquitin-protein ligase RIE1 for SPAD value (VRADI07G28100; chr. 11), WRKY family transcription factor for leaf area (VRADI03G06560; chr. 3), and LOB domain-containing protein 21 for trichomes (VRADI06G04290; chr. 6). In-silico validation of candidate genes was done through digital gene expression analysis using Arabidopsis orthologous (compared with Vigna radiata genome). The findings provided valuable insight for marker-assisted breeding aiming for the development of YMD-resistant and early-maturing mungbean varieties.

Mild behavioral impairment in early Alzheimer’s disease and its association with APOE and BDNF risk genetic polymorphisms

BackgroundMild behavioral impairment (MBI) has been commonly reported in early Alzheimer’s disease (AD) but rarely using biomarker-defined samples. It is also unclear whether genetic polymorphisms influence MBI in such individuals. We thus aimed to examine the association between the cognitive status of participants (amnestic mild cognitive impairment (aMCI-AD) vs cognitively normal (CN) older adults) and MBI severity. Within aMCI-AD, we further examined the association between APOE and BDNF risk genetic polymorphisms and MBI severity.MethodsWe included 62 aMCI-AD participants and 50 CN older adults from the Czech Brain Aging Study. The participants underwent neurological, comprehensive neuropsychological examination, APOE and BDNF genotyping, and magnetic resonance imaging. MBI was diagnosed with the Mild Behavioral Impairment Checklist (MBI-C), and the diagnosis was based on the MBI-C total score ≥ 7. Additionally, self-report instruments for anxiety (the Beck Anxiety Inventory) and depressive symptoms (the Geriatric Depression Scale-15) were administered. The participants were stratified based on the presence of at least one risk allele in genes for APOE (i.e., e4 carriers and non-carriers) and BDNF (i.e., Met carriers and non-carriers). We used linear regressions to examine the associations.ResultsMBI was present in 48.4% of the aMCI-AD individuals. Compared to the CN, aMCI-AD was associated with more affective, apathy, and impulse dyscontrol but not social inappropriateness or psychotic symptoms. Furthermore, aMCI-AD was related to more depressive but not anxiety symptoms on self-report measures. Within the aMCI-AD, there were no associations between APOE e4 and BDNF Met and MBI-C severity. However, a positive association between Met carriership and self-reported anxiety appeared.ConclusionsMBI is frequent in aMCI-AD and related to more severe affective, apathy, and impulse dyscontrol symptoms. APOE and BDNF polymorphisms were not associated with MBI severity separately; however, their combined effect warrants further investigation.

Miniaturization of CRISPR/Cas12-Based DNA Sensor Array by Non-Contact Printing.

DNA microarrays have been applied for comprehensive genotyping, but remain a drawback in complicated operations. As a solution, we previously reported the solid-phase collateral cleavage (SPCC) system based on the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 12 (CRISPR/Cas12). Surface-immobilized Cas12-CRISPR RNA (crRNA) can directly hybridize target double-stranded DNA (dsDNA) and subsequently produce a signal via the cleavage of single-stranded DNA (ssDNA) reporter immobilized on the same spot. Therefore, SPCC-based multiplex dsDNA detection can be performed easily. This study reports the miniaturization of SPCC-based spots patterned by a non-contact printer and its performance in comprehensive genotyping on a massively accumulated array. Initially, printing, immobilization, and washing processes of Cas12-crRNA were established to fabricate the non-contact-patterned SPCC-based sensor array. A target dsDNA concentration response was obtained based on the developed sensor array, even with a spot diameter of 0.64 ± 0.05 mm. Also, the limit of detection was 572 pM, 531 pM, and 3.04 nM with 40, 20, and 10 nL-printing of Cas12-crRNA, respectively. Furthermore, the sensor array specifically detected three dsDNA sequences in one-pot multiplexing; therefore, the feasibility of comprehensive genotyping was confirmed. These results demonstrate that our technology can be miniaturized as a CRISPR/Cas12-based microarray by using non-contact printing. In the future, the non-contact-patterned SPCC-based sensor array can be applied as an alternative tool to DNA microarrays.