Genomics Laboratory Research Articles

Beyond the fever: shotgun metagenomic sequencing of stool unveils pathogenic players in HIV-infected children with non-malarial febrile illness

BackgroundNon-malarial febrile illnesses (NMFI) pose significant challenges in HIV-infected children, often leading to severe complications and increased morbidity. While traditional diagnostic approaches focus on specific pathogens, shotgun metagenomic sequencing offers a comprehensive tool to explore the microbial landscape underlying NMFI in this vulnerable population ensuring effective management.MethodsIn this study, we employed shotgun metagenomics to analyse stool samples from HIV-infected children at the Baylor Children’s Clinic Uganda presenting with non-malarial febrile illness. Samples were collected and subjected to DNA extraction at the Molecular and Genomics Laboratory, Makerere University followed by shotgun metagenomics sequencing at the Chan Zuckerberg Biohub San Francisco. Bioinformatics analysis was conducted to identify and characterise the microbial composition and potential pathogenic taxa associated with NMFI using the CZID pipeline.ResultsOur findings reveal a diverse array of microbial taxa in the stool samples of HIV-infected children with NMFI. Importantly, shotgun metagenomics revealed potentially pathogenic players including Trichomonas vaginalis, Candida albicans, Giardia, and Bacteroides in stool from this patient population. This sheds light on the complexities of microbial interactions that potentially underpin non-malarial febrile illness in this group. Taxonomic profiling identified recognised pathogens and comorbidities and revealed possible new correlations with NMFI, shedding light on the pathophysiology of fever in HIV-infected children.ConclusionShotgun metagenomics is a valuable method for understanding the gut microbial landscape of NMFI in HIV-infected children, providing a comprehensive approach to pathogen identification and characterisation. By revealing potential pathogenic actors beyond the fever, this work demonstrates how metagenomic sequencing may improve our knowledge of infectious illnesses in vulnerable groups and inspire targeted therapies for better clinical care and outcomes.

Identifying barriers and opportunities to facilitate the uptake of whole genome sequencing in paediatric haematology and oncology practice

BackgroundThe clinical utility of whole genome sequencing (WGS) in paediatric cancer has been demonstrated in recent years. WGS has been routinely available in the National Health Service (NHS) England for all children with cancer in England since 2021, but its uptake has been variable geographically. To explore the underlying barriers to routine use of WGS in this population across England and more widely in the United Kingdom (UK) and the Republic of Ireland (ROI), a one-day workshop was held in Cambridge, United Kingdom in October 2022.MethodsFollowing a series of talks, delegates participated in open, round-table discussions to outline local and broader challenges limiting routine WGS for diagnostic work-up for children with cancer in their Principal Treatment Centres (PTCs) and Genomic Laboratory Hubs (GLHs). Within smaller groups, delegates answered structured questions regarding clinical capability, education and training needs, and workforce competence and requirements. Data was recorded, centrally collated, and analysed following the event using thematic analysis.ResultsSixty participants attended the workshop with broad representation from the 20 PTCs across the UK and ROI and the seven GLHs in England. All healthcare professionals involved in the WGS pathway were represented, including paediatric oncologists, clinical geneticists, clinical scientists, and histopathologists. The main themes highlighted by the group in ensuring equitable access to WGS identified were: lack of knowledge equity between NHS trusts, with a perception of WGS being for research only; and perception of lack of financial support for the clinical process surrounding WGS, including lack of time to take informed consent from patients. The latter also included limited trained staff available for data interpretation, affecting the turnaround time for reporting. Finally, the need for an integrated digital pathway to order, track, and return data to clinicians was highlighted.ConclusionAt the workshop, the general motivation for including WGS in the diagnostic work up for children with cancer was high throughout the UK, however a perceived lack of resources and education opportunities limit the widespread use of this commissioned assay. This workshop has led to some recommendations to increase access to WGS in this population in England and more widely in the devolved national of the UK and the ROI.

The Value of Parental Karyotyping in Recurrent Pregnancy Loss Lies in Individual Risk Assessments.

Background and Objectives: Recurrent pregnancy loss (RPL) is a multifactorial condition, encompassing genetic, anatomical, immunological, endocrine, as well as infectious and environmental factors; however, the etiology remains elusive in a substantial number of cases. Genetic factors linked to RPL include parental karyotype abnormalities (e.g., translocations, inversions, copy number variants), an increase in sperm aneuploidy, fetal microchimerism, severe skewing of X chromosome inactivation, and various gene polymorphisms. Our study aims to explore the value of routine conventional parental karyotyping in couples with RPL. Materials and Methods: A total of 213 couples (426 individuals) with a history of RPL were enrolled in this retrospective study. The peripheral blood samples included in this study were referred to the Human Genomics Laboratory of the University of Medicine and Pharmacy in Craiova, Romania, for conventional cytogenetic analysis between January 2013 and December 2023, by the Outpatient Medical Genetics Clinic of the Emergency Clinical County Hospital of Craiova. Chromosome analysis was performed using standard protocols and karyotypes were reported according to ISCN. Results: Out of 426 patients provided with conventional G-banded chromosome analysis, 410 had a normal karyotype (96.2%) and 16 had chromosome abnormalities (3.8%). The most common chromosomal abnormalities were reciprocal and Robertsonian translocations, with chromosomes 8, 11, 14, and 21 being most frequently involved. A single numerical anomaly was detected (47,XYY). One or multiple chromosomal polymorphisms were identified in 104 subjects (24.4%). In addition, we conducted a stratified analysis of the unselected group and detected chromosome abnormalities in only four cases (0.94%). Conclusions: Our results are consistent with recommendations for paternal karyotyping after an individual risk assessment in instances such as a previous live birth with congenital anomalies and/or the detection of unbalanced chromosomes or a translocation in product of conception or chorionic villi/amniotic fluid samples. In the absence of a positive history, blindly karyotyping couples may prove too expensive and labor intensive, while providing no information on fertility status or live birth rates.

Correlation of COVID-19 vaccination and RT-PCR ct value among cases in Addis Ababa, Ethiopia: implication for future preparedness

BackgroundThe COVID-19 disease requires accurate diagnosis to effectively manage infection rates and disease progression. The study aims to assess the relationship between vaccination status and RT-PCR cycle threshold (Ct) values by comparing clinical, RDT and RT-PCR results.MethodsA total of 453 suspected COVID-19 cases were included in this study. Nasopharyngeal swabs were collected for both RDT and RT-PCR testing, with RDTs conducted on-site and RT-PCR at the Ethiopian Public Health Institute (EPHI) genomics laboratory. Detailed clinical, RDT, and RT-PCR results were analyzed. Data analysis included descriptive statistics, cross-tabulation, and Chi-Square tests to investigate the connections between diagnostic outcomes and vaccination status, with a focusing on Ct values.ResultsRDT results showed 34.0% negative and 65.8% positive, while RT-PCR results indicated 35.8% negative and 64.2% positive cases. The discrepancies between RDT and RT-PCR results emphasize the importance of thorough testing. No significant association was found between vaccination status and viral load, as indicated by Ct values. Among RT-PCR positive cases, 49.8% had been vaccinated, suggesting challenges in interpreting results among vaccinated individuals. Further analysis revealed that vaccination (first or second dose) had minimal impact on Ct values, indicating limited influence of vaccination status on viral load dynamics in infected individuals.ConclusionsThe study highlights the significant differences between RDT and RT-PCR outcomes, underscoring the need for a comprehensive testing approach. Additionally, the findings suggest that vaccination status does not significantly impact RT-PCR Ct values, complicating the interpretation of diagnostic results in vaccinated individuals, especially in breakthrough infections and potential false positives.

8486 Age at diagnosis in Prader Willi Syndrome (PWS)

Abstract Disclosure: R. Sritharan: None. J. Lodge: None. S. Tadros: None. L. Menzies: None. E.F. Gevers: None. PWS is a complex neurodevelopmental disorder characterised by neonatal hypotonia,failure to thrive, hypothalamic and endocrine dysfunction, behavioural and learning difficulties and hyperphagia. It is caused by absence of expression of paternally imprinted genes on chromosome 15q11-q13. PWS is confirmed by genetic methylation analysis of this region. Most patients with PWS are referred for genetic investigations in the first months of life due to severe hypotonia and/or failure to thrive. However, it is known that some patients are still only diagnosed in adulthood, with resulting missed opportunities for prompt early treatments for PWS, such as growth hormone therapy. Little information is available on age of diagnosis of PWS, and whether opportunities to request genetic investigation are missed in childhood. We therefore aimed to study age at diagnosis of PWS in the UK. To do so, we assessed data as reported to the UK patient association PWSA-UK from 1968-2023, and additionally audited PWS genetic testing by the North Thames Genomics Laboratory Hub from 2019-22. Of 1145 people registered at PWSA as having PWS, 945 had an age of diagnosis recorded. Of these, 254 (28%) were diagnosed over 1 year of age. From March 2021 to Sept 2023, 13 were diagnosed over 1 year of age with a mean age at diagnosis of 9.27 yr (95% CI 4.15 to 14.4) and with 8 patients below 5 years of age. We assessed PWS diagnoses made in blocks of 4 years from 1968, and the number of PWS diagnoses in people over age 1 year in those time blocks. The number of diagnoses in people over 1 year of age appeared to decrease since 1996 to 7 but from 2015 this has been fluctuating. North Thames Genomics Laboratory Hub provides testing for around 10 million people in and around London. Over a 3-year period between 2019-2022, there were 59 requests for PWS testing. 28 of these were positive (a 47% diagnostic yield). 20/28 (71%) of patients were diagnosed under 1 month of age, 7% were diagnosed at 1-24 months of age, 3.5% at 2-5 yrs and 18% at >5 yrs of age (one at age 9, two at age 18, one at age 29 and one at 33 yrs of age). Most tests were performed in children under1 month of age (91% yield), followed by those over 5 yrs of age (30% yield). For 10 individuals, microarray was requested, however methylation studies would have been more appropriate. All patients diagnosed over 5 years of age had hypotonia and were described as hyperphagic and having intellectual disability, and 75% were obese. Patients diagnosed after age 8were described as having neonatal hypotonia and may have been eligible for genetic testing soon after birth. These data suggest that a considerable percentage of patients are diagnosed with PWS after the first year of life in the UK. Further root analysis, interventions, and education is required to reduce age at diagnosis in patients with PWS to allow for optimal management starting in the first few months of life. Presentation: 6/1/2024

Establishing a Variant Allele Frequency Cutoff for Manual Curation of Medical Exome Sequencing Data

Medical exome sequencing pipelines consist of various preprocessing steps to prioritize credible causal variants before a pathologist or variant curation scientist manually interprets potential findings that are then reported to patients. The variant allele frequency (VAF), reported as the fraction of sequencing reads supporting a variant call, can be used to screen for technical artifacts, yet a specific filtering threshold has yet to be established. A total of 13,122 manually curated variants, sequenced from 289 patients using the Agilent SureSelect Focused Exome enrichment kit at the University of Kentucky Clinical Genomics laboratory from October 2019 to May 2023, were evaluated. Totals of 278 single-nucleotide polymorphisms (SNPs) and 3340 SNPs as technical artifacts are clinically reported. All reported variants had a VAF between 0.33 and 0.63, and 82% (2725/3340) of sequencing artifacts had a VAF of <0.33. It is proposed that removing SNPs in which the VAF is less than approximately 0.30 reduces manual curation time by approximately 20% while capturing all medically relevant variants in medical exome sequencing data sets.

Clinical and cost-effectiveness of clopidogrel resistance genotype testing after ischaemic stroke or transient ischaemic attack: a systematic review and economic model.

Stroke or transient ischaemic attack patients are at increased risk of secondary vascular events. Antiplatelet medications, most commonly clopidogrel, are prescribed to reduce this risk. Factors including CYP2C19 genetic variants can hinder clopidogrel metabolism. Laboratory-based or point-of-care tests can detect these variants, enabling targeted treatment. To assess the effectiveness of genetic testing to identify clopidogrel resistance in people with ischaemic stroke or transient ischaemic attack. Specific objectives: Do people tested for clopidogrel resistance, and treated accordingly, have a reduced risk of secondary vascular events? Do people with loss-of-function alleles associated with clopidogrel resistance have a reduced risk of secondary vascular events if treated with alternative interventions compared to clopidogrel? Do people with loss-of-function alleles associated with clopidogrel resistance have an increased risk of secondary vascular events when treated with clopidogrel? What is the accuracy of point-of-care tests for detecting variants associated with clopidogrel resistance? What is the technical performance and cost of CYP2C19 genetic tests? Is genetic testing for clopidogrel resistance cost-effective compared with no testing? Systematic review and economic model. Objective 1: Two studies assessed secondary vascular events in patients tested for loss-of-function alleles and treated accordingly. They found a reduced risk, but confidence intervals were wide (hazard ratio 0.50, 95% confidence interval 0.09 to 2.74 and hazard ratio 0.53, 95% confidence interval 0.24 to 1.18). Objective 2: Seven randomised controlled trials compared clopidogrel with alternative treatment in people with genetic variants. Ticagrelor was associated with a lower risk of secondary vascular events than clopidogrel (summary hazard ratio 0.76, 95% confidence interval 0.65 to 0.90; two studies). Objective 3: Twenty-five studies compared outcomes in people with and without genetic variants treated with clopidogrel. People with genetic variants were at an increased risk of secondary vascular events (hazard ratio 1.72, 95% confidence interval 1.43 to 2.08; 18 studies). There was no difference in bleeding risk (hazard ratio 0.98, 95% confidence interval 0.68 to 1.40; five studies). Objective 4: Eleven studies evaluated Genomadix Cube accuracy; no studies evaluated Genedrive. Summary sensitivity and specificity against laboratory reference standards were both 100% (95% confidence interval 94% to 100% and 99% to 100%). Objective 5: Seventeen studies evaluated technical performance of point-of-care tests. Test failure rate ranged from 0.4% to 19% for Genomadix Cube. A survey of 8/10 genomic laboratory hubs revealed variation in preferred technologies for testing, and cost per test ranging from £15 to £250. Most laboratories expected test failure rate to be < 1%. Additional resources could enhance testing capacity and expedite turnaround times. Objective 6: Laboratory and point-of-care CYP2C19 testing strategies were cost-saving and increase quality-adjusted life-years compared with no testing. Both strategies gave similar costs, quality-adjusted life-years and expected net monetary benefit. Our results suggest that CYP2C19 testing followed by tailored treatment is likely to be effective and cost-effective in both populations. Accuracy and technical performance of Genedrive. Test failure rate of Genomadix Cube in a NationalHealthServicesetting. Value of testing additional loss-of-function alleles. Appropriateness of treatment dichotomy based on loss-of-function alleles. Lack of data on Genedrive. No randomised 'test-and-treat' studies of dipyramidole plus aspirin. This study is registered as PROSPERO CRD42022357661. This award was funded by the National Institute for Health and Care Research (NIHR) Evidence Synthesis programme (NIHR award ref: NIHR135620) and is published in full in Health Technology Assessment; Vol. 28, No. 57. See the NIHR Funding and Awards website for further award information.

Extended panel testing in ovarian cancer reveals BRIP1 as the third most important predisposition gene

PurposeThe prevalence of germline pathogenic variants (PVs) in homologous recombination repair (HRR) and Lynch syndrome (LS) genes in ovarian cancer (OC) is uncertain. MethodsAn observational study reporting the detection rate of germline PVs in HRR and LS genes in all OC cases tested in the North West Genomic Laboratory Hub between September 1996 and May 2024. Effect sizes are reported using odds ratios (ORs) and 95% confidence intervals (95% CI) for unselected cases tested between April 2021 and May 2024 versus 50,703 controls from the Breast Cancer Risk after Diagnostic Gene Sequencing study. Results2934 women were tested for BRCA1/2 and 433 (14.8%) had a PV. In up to 1572 women tested for PVs in non-BRCA1/2 HRR genes, detection rates were PALB2 = 0.8%, BRIP1 = 1.1%, RAD51C = 0.4% and RAD51D = 0.4%. In 940 unselected cases, BRIP1 (OR = 8.7, 95% CI 4.6-15.8) was the third most common OC predisposition gene followed by RAD51C (OR = 8.3, 95% CI 3.1-23.1), RAD51D (OR = 6.5, 95% CI 2.1-19.7), and PALB2 (OR = 3.9, 95% CI 1.5-10.3). No PVs in LS genes were detected in unselected cases. ConclusionPanel testing in OC resulted in a detection rate of 2% to 3% for germline PVs in non-BRCA1/2 HRR genes, with the largest contributor being BRIP1. Screening for LS in unselected cases of OC is unnecessary.

Performance Evaluation of a Commercial Automated Library Preparation System for Clinical Microbial Whole-Genome Sequencing Assays

Next-generation sequencing (NGS) has proven clinical utility on disease management and serves as an important tool for genomic surveillance. Currently, hurdles surrounding its implementation, namely the complex and demanding analytical workflows, have impeded its widespread use in many laboratories. To address this challenge, the UCLA Molecular Microbiology and Pathogen Genomics Laboratory evaluated the performance of the Tecan MagicPrep NGS system, a commercial automated solution for library preparation for clinical whole-genome sequencing assays, against the Illumina Nextera DNA Flex Library Prep. Using 35 unique organisms (28 bacteria and 7 fungi) for various clinical applications, including microbial identification and genomic characterization, we compared the quantity and quality of the prepared libraries and the resulting sequences, and concordance of the overall results. We also assessed the impact of its implementation on laboratory workflow. The MagicPrep NGS produced higher library concentrations with smaller sizes, and correspondingly, higher molarity. Quality metrics of the sequences, however, demonstrated no significant impact on the overall results, producing 100% concordance with the reference method. Importantly, workflow analysis showed 5 hours less hands-on time per run with more flexibility. This evaluation study indicates that performance of the MagicPrep NGS is comparable to the Nextera DNA Flex with the added benefit of improving workflow efficiency and reducing labor for performing routine clinical microbial whole-genome sequencing tests.

CGP-based IO biomarker assessment and treatment utilization across a major US health system.

e23109 Background: Immune checkpoint inhibitors (IO) have become a powerful precision therapy option to treat advanced stage cancer with biomarkers such as PD-L1, tumor mutational burden (TMB), and microsatellite instability (MSI) associated with improved patient response rates. Despite this, many patients do not receive genomic testing for all IO biomarkers. Providence, a large US community health system, developed a pathologist-directed testing protocol where comprehensive genomic profiling (CGP) was routinely used at time of diagnosis for advanced cancer patients. Methods: Advanced cancer patients who received CGP (ProvSeq 523) and IHC testing for PD-L1 between 2019-2023 were included in the study. Patients were required to be treated at Providence and were assessed for presence of IO biomarkers and subsequent therapy selection. Real world data were curated from patient charts and genomic laboratory data by employing a novel natural-language processing (NLP) approach to accelerate abstraction. Results: The study included 2,502 patients (53% female, median age 68y, 83% white). Top 3 tumor types tested were lung (40%), colorectal (9%), and breast (8%). Overall, 58% (N = 1,455) of patients had presence of ≥1 IO biomarker, with 46% (N = 1,155) being PD-L1 positive, and 27% (N = 682) being TMB-H. In PD-L1 negative patients (N = 1,347; 54%), 300 (22%) were TMB-H and 18 (1%) were MSI-H. 53% (N = 767) of patients who harbored an actionable IO biomarker received IO-based therapy. Fewer patients with an IO biomarker received chemotherapy compared to patients without an IO biomarker (23% vs 35%, p &lt; 0.001). Patients who possessed ≥2 IO biomarkers received an IO precision therapy 68% of the time vs 47% in patients with 1 IO biomarker (p &lt; 0.001). In PD-L1 negative patients, 26% (N = 78) of TMB-H patients received IO monotherapy compared to 5% (N = 49) of TMB-L patients. Conclusions: IO biomarker presence is associated with increased precision therapy use. CGP identified many TMB-H patients for IO monotherapy that would have been missed with PD-L1 testing alone. More than half of patients eligible for IO therapy don’t receive it; ongoing analyses will evaluate the impact of pathology-directed reflex testing on IO as well as targeted therapy approaches. [Table: see text]

Multimodal data fusion using sparse canonical correlation analysis and cooperative learning: a COVID-19 cohort study

Through technological innovations, patient cohorts can be examined from multiple views with high-dimensional, multiscale biomedical data to classify clinical phenotypes and predict outcomes. Here, we aim to present our approach for analyzing multimodal data using unsupervised and supervised sparse linear methods in a COVID-19 patient cohort. This prospective cohort study of 149 adult patients was conducted in a tertiary care academic center. First, we used sparse canonical correlation analysis (CCA) to identify and quantify relationships across different data modalities, including viral genome sequencing, imaging, clinical data, and laboratory results. Then, we used cooperative learning to predict the clinical outcome of COVID-19 patients: Intensive care unit admission. We show that serum biomarkers representing severe disease and acute phase response correlate with original and wavelet radiomics features in the LLL frequency channel (cor(Xu1, Zv1) = 0.596, p value < 0.001). Among radiomics features, histogram-based first-order features reporting the skewness, kurtosis, and uniformity have the lowest negative, whereas entropy-related features have the highest positive coefficients. Moreover, unsupervised analysis of clinical data and laboratory results gives insights into distinct clinical phenotypes. Leveraging the availability of global viral genome databases, we demonstrate that the Word2Vec natural language processing model can be used for viral genome encoding. It not only separates major SARS-CoV-2 variants but also allows the preservation of phylogenetic relationships among them. Our quadruple model using Word2Vec encoding achieves better prediction results in the supervised task. The model yields area under the curve (AUC) and accuracy values of 0.87 and 0.77, respectively. Our study illustrates that sparse CCA analysis and cooperative learning are powerful techniques for handling high-dimensional, multimodal data to investigate multivariate associations in unsupervised and supervised tasks.

Abstract PO4-19-02: Genetic risk estimation in breast cancer and assessing health disparities

Abstract Background: Breast Cancer (BC) incidence and the distribution of BC risk factors vary between racial and ethnic groups within the United States. It has been demonstrated that providing education on individualized breast cancer risk will improve patient uptake to recommended BC screening and prevention strategies. Most clinical risk assessment tools, including the validated IBIS model were derived from data using non-Hispanic white women. Consortia have also now identified over 300 common genetic susceptibility loci for BC, summarized by the polygenic risk score (PRS). When combined with clinical risk assessment tools, such as the IBIS or BCRAT models, the PRS can further refine risk estimation for patients. To date, most studies on the use of PRS have been done in non-Hispanic white women. While these PRS still perform well in racial minorities, there has been a recognized need to improve racial and ethnic diversity in genomic research cohorts. Herein, we aim to combine clinical risk assessment models that are already used in routine clinical practice with information derived from PRS testing in women of racial minorities to determine if this can improve risk estimation, patient understanding, and uptake to recommended breast cancer screening and prevention strategies. Design: This is a minimal risk prospective study with a single arm incorporating the PRS into a standard breast cancer risk reduction consultation, followed by annual surveys over 10 years to determine if and how the information provided by the PRS influenced patient decisions regarding recommended BC screening and prevention. Patients will have IBIS and BCRAT risks calculated at baseline visit. A survey of patient perceptions/understanding of risk, intention to undergo screening, and use of preventive medicine is completed after baseline visit prior to receiving the PRS results. Blood sample is obtained at baseline and DNA samples are analyzed for approximately 300 SNPs on a custom-designed, targeted SNP panel from ThermoFisher Scientific by the Mayo Clinic Genomics Laboratory. This PRS result is then combined with the clinical risk assessments (IBIS and BCRAT scores) using the R package Individualized Coherent Absolute Risk Estimators (iCare) tool to provide a new estimate of breast cancer risk for 5year, 10year, and lifetime risk. Patients are seen for follow-up to review results and then complete a second survey to assess their understanding of the results, BC risk, and how the PRS impacted their decision to undergo screening/prevention strategies. Eligibility Criteria: Women who self-identify as African American/Black or Hispanic/Latinx between 30-75 years old with any of the following: 1.)IBIS score of ≥5% for the 10 year risk OR BCRAT score of ≥ 3 % for the 5 year risk. 2.) History of biopsy proven atypical hyperplasia 3.) History of biopsy proven lobular carcinoma in situ. Specific Aims: Aim 1: The primary aim of this study is to explore if the addition of PRS to the BCRAT and IBIS score will improve intentions to undergo recommended breast cancer screening strategies such as mammography, MRI, or molecular breast imaging in women of underserved racial minorities Aim 2: To explore if the addition of the PRS to the BCRAT and IBIS risk score will aid women of racial minorities in deciding whether to take preventative endocrine therapy. Aim 3: To understand how individualized risk assessment and information on PRS may alter perceived risk of breast cancer. Statistical Methods Analysis will be mostly descriptive. Continuous variables will be summarized as mean (standard deviation) or median (range) and categorical variables will be reported as frequency (percentage). Kaplan-Meier method will be used to estimate the long-term cumulative risk of cancer. Current Accrual: 3. Target Accrual: 50 Citation Format: Lauren Cornell, Sandhya Pruthi, Linda Hasadsri, Sarah McLaughlin. Genetic risk estimation in breast cancer and assessing health disparities [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 PO4-19-02.

Abstract 5377: Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia

Abstract Background: Aging, smoking and germline predisposition are known to accelerate leukemogenesis. However, the the ability for myeloid clonal pathogenic variants (MCPV) to elicit an antigen-driven host antileukemia immune response is not well characterized. A better understanding on how cellular immunity selectively discriminates leukemia-related “neopeptides” could uncover mechanisms supporting tumor genome evolution. Methods: 122 patients with acute myelogenous leukemia (AML) were included and 111 had available protein coding sequences. A neopeptide library was created using protein FASTA coding extracted for individual myeloid clonal mutations via National Center for Biotechnology Information (NCBI). Patient class 1 HLA typing was obtained through Tempus, and Genomic Testing Cooperative laboratories. Prediction of number and strength of AML neopeptide bindings to MHC class I molecules were obtained via NetMHCPan-1. Analysis was performed with K-means algorithm assigning age at diagnosis, detected MCPV, number of neopeptides binding (as continuous variable), overall survival (OS) and event-free survival (EFS) as variables. Unsupervised data input allowed 3 cluster partitions for differential survivals allowing HLA class 1 peptide binding aggregation with potential roles in leukemogenesis and patient outcome. Descriptive statistics were performed with SAS. Results: Sixty-six different MCPV were detected. Of 111 patients, 70 (63%) with available MCPV produced at least 1 MHC I neopeptide binding. Frequently observed variants were P53 (13%), FLT3 (9.2%), WT1 (7%), DNMT3A (6.5%), RAS (5%), ASXL1 (4.3%), RUNX1 (4.2%), NPM1 (4.2%) and IDH1/2 (2.6%). Cluster 2 demonstrated a lack of favorable and higher proportion of adverse ELN 2022 subgroups. Cluster 3 exhibited a higher of P53 [p=0.03] and MDS-related mutations. OS was 79, 239, and 1551 days for cluster 2,1 and 3, respectively [p=0.0001]. EFS was 134, 363 and 451 days for cluster 2,1 and 3, respectively [p=0.0001]. Interestingly, Cluster 2 and 3 assembled the highest probability for peptide binding [1 v 2 v 3 = 1.76% v 36.6% v 62%, p=0.01]. Lastly, albeit in low frequencies, higher diversity of DNA repairing enzymes was seen in cluster 2. Conclusions: Different MCPV arising in AML genome are variably immunogenic and leukemia-related neopeptide binding’s ability is differentially expressed in our 3 clusters. Frequent neopeptide bindings in clusters 2 and 3 may reflect higher AML immunogenicity resulting in genomic instability. It is possible that less “edited” genomes in cluster 1 preserve higher chemosensitivity resulting in better OS, while highly edited genomes in cluster 2 and 3 are less chemo-sensitive and circumvent host immunosurveillance. Further expansion of cluster 2 is necessary to better understand the role of DNA repairing enzymes in younger patients’ outcomes, especially when genomic instability is observed. Citation Format: Paul Sackstein, Sarah Mudra, Lacey Williams, Garrett Diltz, Rachel Zemel, Sravanti Teegavarapu, Martha Mims, Catherine Lai, Kimberley Doucette, Anne Renteria, Olga Timofeeva, Gustavo A. Rivero. Investigating human leukocyte antigen (HLA) class 1 leukemogenic pathways in acute myelogenous leukemia [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 5377.

ClinGen variant curation expert panel recommendations for classification of variants in GAMT, GATM and SLC6A8 for cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are inherited metabolic phenotypes of creatine synthesis and transport. There are two enzyme deficiencies, guanidinoacetate methyltransferase (GAMT), encoded by GAMT and arginine-glycine amidinotransferase (AGAT), encoded by GATM, which are involved in the synthesis of creatine. After synthesis, creatine is taken up by a sodium-dependent membrane bound creatine transporter (CRTR), encoded by SLC6A8, into all organs. Creatine uptake is very important especially in high energy demanding organs such as the brain, and muscle. To classify the pathogenicity of variants in GAMT, GATM, and SLC6A8, we developed the CCDS Variant Curation Expert Panel (VCEP) in 2018, supported by The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded resource. We developed disease-specific variant classification guidelines for GAMT-, GATM-, and SLC6A8-related CCDS, adapted from the American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant interpretation guidelines. We applied specific variant classification guidelines to 30 pilot variants in each of the three genes that have variants associated with CCDS. Our CCDS VCEP was approved by the ClinGen Sequence Variant Interpretation Working Group (SVI WG) and Clinical Domain Oversight Committee in July 2022. We curated 181 variants including 72 variants in GAMT, 45 variants in GATM, and 64 variants in SLC6A8 and submitted these classifications to ClinVar, a public variant database supported by the National Center for Biotechnology Information. Missense variants were the most common variant type in all three genes. We submitted 32 new variants and reclassified 34 variants with conflicting interpretations. We report specific phenotype (PP4) using a points system based on the urine and plasma guanidinoacetate and creatine levels, brain magnetic resonance spectroscopy (MRS) creatine level, and enzyme activity or creatine uptake in fibroblasts ranging from PP4, PP4_Moderate and PP4_Strong. Our CCDS VCEP is one of the first panels applying disease specific variant classification algorithms for an X-linked disease. The availability of these guidelines and classifications can guide molecular genetics and genomic laboratories and health care providers to assess the molecular diagnosis of individuals with a CCDS phenotype.

Immune interface interference vaccines: An evolution-informed approach to anti-bacterial vaccine design.

Developing protein-based vaccines against bacteria has proved much more challenging than producing similar immunisations against viruses. Currently, anti-bacterial vaccines are designed using methods based on reverse vaccinology. These identify broadly conserved, immunogenic proteins using a combination of genomic and high-throughput laboratory data. While this approach has successfully generated multiple rationally designed formulations that show promising immunogenicity in animal models, few have been licensed. The difficulty of inducing protective immunity in humans with such vaccines mirrors the ability of many bacteria to recolonise individuals despite recognition by natural polyvalent antibody repertoires. As bacteria express too many antigens to evade all adaptive immune responses through mutation, they must instead inhibit the efficacy of such host defences through expressing surface structures that interface with the immune system. Therefore, 'immune interface interference' (I3) vaccines that target these features should synergistically directly target bacteria and prevent them from inhibiting responses to other surface antigens. This approach may help us understand the efficacy of the two recently introduced immunisations against serotype B meningococci, which both target the Factor H-binding protein (fHbp) that inhibits complement deposition on the bacterial surface. Therefore, I3 vaccine designs may help overcome the current challenges of developing protein-based vaccines to prevent bacterial infections.

Microarray-Based DNA Methylation Profiling: Validation Considerations for Clinical Testing

Microarray-based methylation profiling has emerged as a valuable tool for refining diagnoses and revealing novel tumor subtypes, particularly in central nervous system tumors. Despite the increasing adoption of this technique in clinical genomic laboratories, no technical standards have been published in establishing minimum criteria for test validation. A working group with experience and expertise in DNA-based methylation profiling tests on CNS tumors collaborated to develop practical discussion points and focus on important considerations for validating this test in clinical laboratory settings. We summarize our experiences in validating this methodology in a clinical setting. Specifically, we highlight the advantages and challenges associated with utilizing an in-house classifier compared to a third-party classifier. Additionally, we share our experiences in demonstrating the assay's sensitivity and specificity, establishing minimum sample criteria, and implementing quality control metrics. As methylation profiling for tumor classification expands to other tumor types and continues to evolve for various other applications, the critical considerations described here are expected to serve as a guidance for future efforts in establishing professional guidelines for this assay.

Incidence, Phenotypes, and Genotypes of Neonatal Diabetes: A 16-Year Experience. The Rare Genetic Etiologies of Neonatal Diabetes Are Common in Sudan

Neonatal diabetes (ND) is a rare subtype of diabetes occurring in the first 6 months of life. High incidence has been reported among populations with high rates of consanguineous marriage. However, there is paucity of reported data from sub-Saharan African countries. We report the incidence, genotype, and phenotype of ND in a large cohort from Sudan and compare these findings to regional and international data. All infants with onset of diabetes in the first 6 months of life, attending one of the only two tertiary pediatric diabetes centers in Sudan, Gaafar Ibn Auf Pediatric Tertiary Hospital and Sudan Childhood Diabetes Center, during the period of January 2006 to December 2022 were included. Medical records were reviewed for demographic and clinical information. Genetic testing was performed for 48 patients by the Exeter Genomics laboratory in the UK and for one patient by the University of Cambridge, Metabolic Research Laboratories, UK. The estimated incidence was 4.8 per 100,000 live births. Forty-nine ND patients from 45 unrelated families were identified, and a genetic diagnosis was confirmed in 37 patients (75.5%) from 33 unrelated families. Consanguinity was reported in 34 families (75.6%). The commonest genetic cause for permanent neonatal diabetes was EIF2AK3 recessive variants causing Wolcott–Rallison syndrome (18.92%). Pathogenic variants in two recently identified genes, ZNF808 and NARS2, were found in three patients each (8.11%). Activating variants in KCNJ11 and ABCC8 were identified in four (10.81%) and two (5.41%) patients, respectively. Apart from hyperglycemia, the commonest clinical presentations included dehydration, failure to thrive, and diabetic ketoacidosis. ND in Sudan has a different pattern of etiologies compared to Western and Asian populations yet similar to some Arab countries with EIF2AK3 mutations being the commonest cause. Pathogenic variants in recently identified genes reflect the impact of genome sequencing on increasing the rate of genetic diagnosis.

Molecular speciation of Plasmodium and multiplicity of P. falciparum infection in the Central region of Ghana.

Malaria is endemic in the Central region of Ghana, however, the ecological and the seasonal variations of Plasmodium population structure and the intensity of malaria transmission in multiple sites in the region have not been explored. In this cross-sectional study, five districts in the region were involved. The districts were Agona Swedru, Assin Central and Gomoa East (representing the forest zone) and Abura-Asebu-Kwamankese and Cape Coast representing the coastal zone. Systematically, blood samples were collected from patients with malaria. The malaria status was screened with a rapid diagnostic test (RDT) kit (CareStart manufactured by Access Bio in Somerset, USA) and the positive ones confirmed microscopically. Approximately, 200 μL of blood was used to prepare four dried blood spots of 50μL from each microscopy positive sample. The Plasmodium genome was sequenced at the Malaria Genome Laboratory (MGL) of Wellcome Sanger Institute (WSI), Hinxton, UK. The single nucleotide polymorphisms (SNPs) in the parasite mitochondria (PfMIT:270) core genome aided the species identification of Plasmodium. Subsequently, the complexity of infection (COI) was determined using the complexity of infection likelihood (COIL) computational analysis. In all, 566 microscopy positive samples were sequenced. Of this number, Plasmodium genome was detected in 522 (92.2%). However, whole genome sequencing was successful in 409/522 (72.3%) samples. In total, 516/522 (98.8%) of the samples contained P. falciparum mono-infection while the rest (1.2%) were either P. falciparum/P. ovale (Pf/Po) (n = 4, 0.8%) or P. falciparum/P. malariae/P. vivax (Pf/Pm/Pv) mixed-infection (n = 2, 0.4%). All the four Pf/Po infections were identified in samples from the Assin Central municipality whilst the two Pf/Pm/Pv triple infections were identified in Abura-Asebu-Kwamankese district and Cape Coast metropolis. Analysis of the 409 successfully sequenced genome yielded between 1-6 P. falciparum clones per individual infection. The overall mean COI was 1.78±0.92 (95% CI: 1.55-2.00). Among the study districts, the differences in the mean COI between ecological zones (p = 0.0681) and seasons (p = 0.8034) were not significant. However, regression analysis indicated that the transmission of malaria was more than twice among study participants aged 15-19 years (OR = 2.16, p = 0.017) and almost twice among participants aged over 60 years (OR = 1.91, p = 0.021) compared to participants between 20-59 years. Between genders, mean COI was similar except in Gomoa East where females recorded higher values. In conclusion, the study reported, for the first time, P. vivax in Ghana. Additionally, intense malaria transmission was found to be higher in the 15-19 and > 60 years, compared to other age groups. Therefore, active surveillance for P. vivax in Ghana and enhanced malaria control measures in the 15-19 year group years and those over 60 years are recommended.

Investigation of Mutations in H19, IGF2, CDKN1C, KCNQ1, and NSD1 Genes in Iranian Children Suspected of Silver-Russell Syndrome (SRS) and Beckwith-Wiedemann Syndrome (BWS) with MS-MLPA

Background: Silver-Russell Syndrome (SRS) and Beckwith-Wiedemann Syndrome (BWS) are two syndromes that are poorly diagnosed in many affected people due to mild and subtle symptoms, genetic complexity, and lack of familiarity with the hallmarks. Objective: The present study was conducted with the aim of determining mutations in H19, IGF2, CDKN1C, KCNQ1, and NSD1 genes in Iranian children suspected of SRS and BWS by the MS-MLPA method. Methods: In this case series study, which was conducted in 2022 in Pars Genome Laboratory, Karaj, Iran, 10 children suspected of SRS or BWS syndrome were included. These 10 Iranian children were referred by pediatric endocrinologists. 5 ml of peripheral blood was taken per patient for DNA extraction. MS-MLPA method was undertaken for possible mutations (methylation and deletion) in H19, IGF2, CDKN1C, KCNQ1, and NSD1 genes. Results: The interpretation of MS-MLPA results showed that out of 10 children (4 boys and 6 girls) suspected of having SRS or BWS syndrome (based on the pediatric endocrinologist’s diagnosis), only 3 children were definitively diagnosed with SRS or BWS syndrome. Based on this, methylation changes in the promoter of ICR1 and ICR2, which are related to the genes H19, IGF2, CDKN1C, KCNQ1, and NSD1, lead to the development of SRS or BWS syndrome. Conclusion: The present findings showed that methylation changes in H19, IGF2, CDKN1C, KCNQ1, and NSD1 genes are associated with the occurrence of SRS or BWS syndrome. In this study, we show that MS-MLPA can serve as a rapid, inexpensive, and reliable method for the molecular diagnosis of these syndromes.

"A very big challenge": a qualitative study to explore the early barriers and enablers to implementing a national genomic medicine service in England.

Background: The Genomic Medicine Service (GMS) was launched in 2018 in England to create a step-change in the use of genomics in the NHS, including offering whole genome sequencing (WGS) as part of routine care. In this qualitative study on pediatric rare disease diagnosis, we used an implementation science framework to identify enablers and barriers which have influenced rollout. Methods: Semi-structured interviews were conducted with seven participants tasked with designing the GMS and 14 tasked with leading the implementation across the seven Genomic Medicine Service Alliances (GMSAs) and/or Genomic Laboratory Hubs (GLHs) between October 2021 and February 2022. Results: Overall, those involved in delivering the service strongly support its aims and ambitions. Challenges include: 1) concerns around the lack of trained and available workforce (clinicians and scientists) to seek consent from patients, interpret findings and communicate results; 2) the lack of a digital, coordinated infrastructure in place to support and standardize delivery with knock-on effects including onerous administrative aspects required to consent patients and order WGS tests; 3) that the "mainstreaming agenda", whilst considered important, encountered reluctance to become engaged from those who did not see it as a priority or viewed it as being politically rather than clinically driven; 4) the timelines and targets set for the GMS were perceived by some as too ambitious. Interviewees discussed local adaptations and strategies employed to address the various challenges they had encountered, including 1) capacity-building, 2) employing genomic associates and other support staff to support the consent and test ordering process, 3) having "genomic champions" embedded in mainstream services to impart knowledge and best practice, 4) enhancing collaboration between genetic and mainstream specialties, 5) building evaluation into the service and 6) co-creating services with patients and the public. Conclusion: Our findings highlight the challenges of implementing system-wide change within a complex healthcare system. Local as well as national solutions can undoubtedly address many of these barriers over time.