Including Next-generation Sequencing Research Articles

Population genomics and demographic modeling enhance our understanding of trophic level interactions in biological control

In this paper, we focus on the application of population genomics, including next-generation sequencing and demographic modeling, to enhance the understanding of historical and contemporary trophic level interactions that are a fundamental basis of biological control. We outline how the integration of ecological approaches with population genomics and demographic modeling can provide additional insights into existing trophic interactions and how natural enemies, their parasitoids, and pest species evolve through these interactions, potentially affecting future levels of biological control. We briefly review and summarize our recent population genomic studies of predatory ladybird beetles and discuss how our results provide guidelines for the use of population genomics techniques and population demographic modeling to enhance our understanding of trophic level interactions. Our intent is to demonstrate as a proof of concept, how these methods can supplement currently used techniques and add a complementary genomic aspect to ecological understandings of trophic level interactions, including estimates of effective population sizes of natural enemies and pest species, examination of correlations between these effective population sizes, and evolutionary changes in natural enemies and pest species which may influence levels of biological control. This in combination with experiments and simulations provide a well-rounded framework for using population genomics to inform multi-tier trophic level interactions.

Fine-needle aspiration technique under endoscopic ultrasound guidance: A technical approach for RNA profiling of pancreatic neoplasms.

Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) has been a longstanding challenge. The prognosis of patients with PDAC depends on the stage at diagnosis. It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis. Most molecular studies are done using paraffin-embedded blocks; however, the integrity of DNA and RNA is often compromised in this format. Moreover, RNA isolated from human pancreatic tissue samples is generally of low quality, in part, because of the high concentration of endogenous pancreatic RNAse activity present. To assess the potential of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling, including next-generation sequencing (NGS). Thirty-four EUS-FNA samples were included in this study: PDAC (n = 15), chronic pancreatitis (n = 5), pancreatic cysts (n = 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia) n = 7, serous cystic neoplasms n = 5, and pseudocysts n = 2. Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy (TTNB). Samples were stored at -80 °C until analysis. RNA purity (A260/230, A260/280 ratios), concentration, and integrity (RIN) were assessed. Real-time polymerase chain reaction was conducted on all samples, and small RNA libraries were prepared from solid mass samples. RNA was successfully extracted from 29/34 (85%) EUS-FNA samples: 100% pancreatic adenocarcinoma samples, 100% chronic pancreatitis samples, 70% pancreatic fluid cyst samples, and 50% TTNB samples. The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples (n = 29) including low-quality RNA specimens. Low concentration and nonoptimal RIN values (no less than 3) of RNA extracted from EUS-FNA samples did not prevent NGS library preparation. The suitability of cyst fluid samples for RNA profiling varied. The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7 (5.0-8.2), which was compatible with that from solid neoplasms [6.2 (0-7.8)], whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0. The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens.

Genomic Approaches for Enhancing Yield and Quality Traits in Mustard (Brassica spp.): A Review of Breeding Strategies

Mustard, a vital oilseed crop, plays a significant role in global agriculture due to its versatile applications in food, feed, and biofuel industries. However, meeting the increasing demands for yield and quality traits poses a substantial challenge to mustard breeders. In response, genomic approaches have emerged as powerful tools to expedite mustard breeding programs by unraveling the genetic basis of key agronomic traits. This review provides a comprehensive overview of genomic strategies aimed at enhancing yield and quality traits in mustard. Beginning with an exploration of traditional breeding methods and their limitations, we delve into the advancements in genomics, including next-generation sequencing technologies, marker-assisted selection (MAS), and genome editing techniques. We discuss how these tools are leveraged to identify yield-related genes, quantitative trait loci (QTLs), and markers for efficient trait selection. Furthermore, we examine genomic approaches for improving oil content, nutritional profiles, and phytochemical composition, crucial for enhancing mustard quality. Case studies demonstrating the successful integration of genomics into breeding programs are highlighted, along with discussions on challenges such as regulatory concerns and technical hurdles. Finally, we outline future directions and the potential of genomic approaches to revolutionize mustard breeding, paving the way for sustainable crop improvement. This study offers valuable insights into the application of genomics in mustard breeding and underscores its importance in addressing the evolving needs of agriculture in the 21st century.

Surfing the Waves of SARS-CoV-2: Analysis of Viral Genome Variants Using an NGS Survey in Verona, Italy.

The availability of new technologies for deep sequencing, including next-generation sequencing (NGS), allows for the detection of viral genome variations. The epidemiological determination of SARS-CoV-2 viral genome changes during the pandemic waves displayed the genome evolution and subsequent onset of variants over time. These variants were often associated with a different impact on viral transmission and disease severity. We investigated, in a retrospective study, the trend of SARS-CoV-2-positive samples collected from the start of the Italian pandemic (January 2020) to June 2023. In addition, viral RNAs extracted from 938 nasopharyngeal swab samples were analyzed using NGS between February 2022 and June 2023. Sequences were analyzed with bioinformatic tools to identify lineages and mutations and for phylogenetic studies. Six pandemic waves were detected. In our samples, we predominantly detected BA.2, BQ.1, BA.5.1, BA.5.2, and, more recently, XBB.1 and its subvariants. The data describe the SARS-CoV-2 genome evolution involved in viral interactions with the host and the dynamics of specific genome mutations and deletions.

A phase II/III randomized clinical trial of CisPlatin plUs Gemcitabine and Nabpaclitaxel (GAP) as pReoperative chemotherapy versus immediate resection in patIents with resecTable BiliarY Tract Cancers (BTC) at high risk for recurrence: PURITY study

BackgroundBiliary tract cancers (BTCs) are rare and lethal cancers, with a 5-year survival inferior to 20%(1–3). The only potential curative treatment is surgical resection. However, despite complex surgical procedures that have a remarkable risk of postoperative morbidity and mortality, the 5-year survival rate after radical surgery (R0) is 20–40% and recurrence rates are up to ~ 75%(4–6). Up to ~ 40% of patients relapse within 12 months after resection, and half of these patient will recur systemically(4–6). There is no standard of care for neoadjuvant chemotherapy (NAC) in resectable BTC, but retrospective reports suggest its potential benefit (7, 8).MethodsPURITY is a no-profit, multicentre, randomized phase II/III trial aimed at evaluating the efficacy of the combination of gemcitabine, cisplatin and nabpaclitaxel (GAP) as neoadjuvant treatment in patients with resectable BTC at high risk for recurrence. Primary objective of this study is to evaluate the efficacy of neoadjuvant GAP followed by surgery as compared to upfront surgery, in terms of 12-month progression-free survival for the phase II part and of progression free survival (PFS) for the phase III study.Key Secondary objectives are event free survival (EFS), relapse-free survival, (RFS), overall survival (OS), R0/R1/R2 resection rate, quality of life (QoL), overall response rate (ORR), resectability. Safety analyses will include toxicity rate and perioperative morbidity and mortality rate.Exploratory studies including Next-Generation Sequencing (NGS) in archival tumor tissues and longitudinal ctDNA analysis are planned to identify potential biomarkers of primary resistance and prognosis.DiscussionConsidering the poor prognosis of resected BTC experiencing early tumor recurrence and the negative prognostic impact of R1/R2 resections, PURITY study is based on the rationale that NAC may improve R0 resection rates and ultimately patients’ outcomes. Furthermore, NAC should allow early eradication of microscopic distant metastases, undetectable by imaging but already present at the time of diagnosis and avoid mortality and morbidity associated with resection for patients with rapid progression or worsening general condition during neoadjuvant therapy. The randomized PURITY study will evaluate whether patients affected by BTC at high risk from recurrence benefit from a neoadjuvant therapy with GAP regimen as compared to immediate surgery.Trial registrationPURITY is registered at ClinicalTrials.gov (NCT06037980) and EuCT(2023–503295-25–00).

Advancements and Applications of Preimplantation Genetic Testing in In Vitro Fertilization: A Comprehensive Review.

Preimplantation genetic testing (PGT) has become an integral component of assisted reproductive technology (ART), offering couples the opportunity to screen embryos for genetic abnormalities before implantation during in vitro fertilization (IVF). This comprehensive review explores the advancements and applications of PGT in IVF, covering its various types, technological developments, clinical applications, efficacy, challenges, regulatory aspects, and future directions. The evolution of PGT techniques, including next-generation sequencing (NGS) and comparative genomic hybridization (CGH), has significantly enhanced the accuracy and reliability of genetic testing in embryos. PGT holds profound implications for the future of ART by improving IVF success rates, reducing the incidence of genetic disorders, and mitigating the emotional and financial burdens associated with failed pregnancies and genetic diseases. Recommendations for clinicians, researchers, and policymakers include staying updated on the latest PGT techniques and guidelines, exploring innovative technologies, establishing clear regulatory frameworks, and fostering collaboration to maximize the potential benefits of PGT in assisted reproduction. Overall, this review provides valuable insights into the current state of PGT and its implications for the field of reproductive medicine.

Comprehensive Analysis of PKD1 and PKD2 by Long-Read Sequencing in Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by heterogeneous variants in the PKD1 and PKD2 genes. Genetic analysis of PKD1 has been challenging due to homology with 6 PKD1 pseudogenes and high GC content. A single-tube multiplex long-range-PCR and long-read sequencing-based assay termed "comprehensive analysis of ADPKD" (CAPKD) was developed and evaluated in 170 unrelated patients by comparing to control methods including next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification. CAPKD achieved highly specific analysis of PKD1 with a residual noise ratio of 0.05% for the 6 pseudogenes combined. CAPKD identified PKD1 and PKD2 variants (ranging from variants of uncertain significance to pathogenic) in 160 out of the 170 patients, including 151 single-nucleotide variants (SNVs) and insertion-deletion variants (indels), 6 large deletions, and one large duplication. Compared to NGS, CAPKD additionally identified 2 PKD1 variants (c.78_96dup and c.10729_10732dup). Overall, CAPKD increased the rate of variant detection from 92.9% (158/170) to 94.1% (160/170), and the rate of diagnosis with pathogenic or likely pathogenic variants from 82.4% (140/170) to 83.5% (142/170). CAPKD also directly determined the cis-/trans-configurations in 11 samples with 2 or 3 SNVs/indels, and the breakpoints of 6 large deletions and one large duplication, including 2 breakpoints in the intron 21 AG-repeat of PKD1, which could only be correctly characterized by aligning to T2T-CHM13. CAPKD represents a comprehensive and specific assay toward full characterization of PKD1 and PKD2 variants, and improves the genetic diagnosis for ADPKD.

In vivo detection of DNA secondary structures using permanganate/S1 footprinting with direct adapter ligation and sequencing (PDAL-Seq).

DNA secondary structures are essential elements of the genomic landscape, playing a critical role in regulating various cellular processes. These structures refer to G-quadruplexes, cruciforms, Z-DNA or H-DNA structures, amongst others (collectively called 'non-B DNA'), which DNA molecules can adopt beyond the B conformation. DNA secondary structures have significant biological roles, and their landscape is dynamic and can rearrange due to various factors, including changes in cellular conditions, temperature, and DNA-binding proteins. Understanding this dynamic nature is crucial for unraveling their functions in cellular processes. Detecting DNA secondary structures remains a challenge. Conventional methods, such as gel electrophoresis and chemical probing, have limitations in terms of sensitivity and specificity. Emerging techniques, including next-generation sequencing and single-molecule approaches, offer promise but face challenges since these techniques are mostly limited to only one type of secondary structure. Here we describe an updated version of a technique permanganate/S1 nuclease footprinting, which uses potassium permanganate to trap single-stranded DNA regions as found in many non-B structures, in combination with S1 nuclease digest and adapter ligation to detect genome-wide non-B formation. To overcome technical hurdles, we combined this method with direct adapter ligation and sequencing (PDAL-Seq). Furthermore, we established a user-friendly pipeline available on Galaxy to standardize PDAL-Seq data analysis. This optimized method allows the analysis of many types of DNA secondary structures that form in a living cell and will advance our knowledge of their roles in health and disease.

Reclassification of RAS/BRAF allele mutations predicts the survival benefit of triplet chemotherapy in metastatic colorectal cancer.

Different RAS/BRAF allele mutations imply distinct biological properties in various solid tumors. Recently, several studies have focused on the predictive and prognostic roles of various RAS/BRAF allele mutations in colorectal cancer (CRC) but the results remain controversial. Between March 2017 and September 2022, the patients diagnosed as stages I-IV CRC with detailed medical records including next-generation sequencing (NGS) data and clinicopathological follow-up information available at our center were enrolled. Survival data were estimated using the Kaplan-Meier method, and the difference was tested in a log-rank test. Multivariate tests were carried out using Cox models. A total of 1029 CRC patients were included, and the incidence of RAS/BRAF mutation was 58.4%. The hypermutated cohort was defined as patients with microsatellite instability-H or POLE/D mutation. In the non-hypermutational cohort, only KRAS G13D mutation was associated with a higher incidence and inferior disease-free survival in patients with stage I-III CRC. In the cohort of patients with non-hypermutated metastatic colorectal cancer (mCRC), we assessed the risk of various RAS/BRAF allele mutations and subsequently reclassified patients into four groups based on first-line median progression-free survival: wild type (group 1), low-risk RAS/BRAF mutation (group 2, RAS/BRAF mutations other than KRAS G13D/G12V/G12C or BRAF V600E), high-risk RAS mutation (group 3, KRAS G13D/G12V/G12C), and BRAF V600E mutation (group 4). mCRC patients with high-risk RAS mutation could significantly benefit from intensive triplet chemotherapy (hazard ratio, 2.54; 95% confidence interval, 1.36-5.12; p = 0.0091). In the non-hypermutated CRC cohort, the prognostic risk of various RAS/BRAF allele mutations varied between local and metastatic CRC. KRAS G13D mutation tended to be the only prognostic marker for stages I-III CRC; however, KRAS G13D/G12V/G12C mutations collectively defined a high-risk subgroup of mCRC patients with poor prognosis, who would benefit from intensive triplet chemotherapy.

Eosinophil disorders: an update on diagnosis and management

Abstract Eosinophilia can be seen in almost all medical subspecialty patients. Delay in diagnostic workup and treatment is associated with significant morbidity and mortality. Clinical vigilance and timely referral for diagnostic evaluation are critical. Causes of hypereosinophilia (HE) are diverse and can be grouped under 3 categories: primary (neoplastic), secondary (reactive), and idiopathic. Advances in molecular genetic diagnostics have led to elucidation of the genetic basis for many neoplastic hypereosinophilic disorders. One common molecular feature is formation of a fusion gene, resulting in the expression of an aberrantly activated tyrosine kinase (TK). The World Health Organization endorsed a biologically oriented classification scheme and created a new major disease category, namely, myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions. Rearrangement of other TK genes and activating somatic mutation(s) in TK genes have also been reported in eosinophilic neoplasms. Diagnostic evaluation of HE involves a combination of clinical, histopathologic, and immunophenotypic analyses, as well as molecular genetic testing, including next-generation sequencing–based mutation panels. The management of primary HE is largely guided by the underlying molecular genetic abnormalities. Good knowledge of recent advances in HE is necessary to ensure timely and accurate diagnosis and to help optimize patient care.

Bioinspired Squid Peptides─A Tale of Curiosity-Driven Research Leading to Unforeseen Biomedical Applications.

The molecular design of many peptide-based materials originates from structural proteins identified in living organisms. Prominent examples that have garnered broad interdisciplinary research interest (chemistry, materials science, bioengineering, etc.) include elastin, silk, or mussel adhesive proteins. The critical first steps in this type of research are to identify a convenient model system of interest followed by sequencing the prevailing proteins from which these biological structures are assembled. In our laboratory, the main model systems for many years have been the hard biotools of cephalopods, particularly their parrot-like tough beak and their sucker ring teeth (SRT) embedded within the sucker cuptions that line the interior surfaces of their arms and tentacles. Unlike the majority of biological hard tissues, these structures are devoid of biominerals and consist of protein/polysaccharide biomolecular composites (the beak) or, in the case of SRT, are entirely made of proteins that are assembled by supramolecular interactions.In this Account, we chronicle our journey into the discovery of these intriguing biological materials. We initially focus on their excellent mechanical robustness followed by the identification and sequencing of the structural proteins from which they are built, using the latest "omics" techniques including next-generation sequencing and high-throughput proteomics. A common feature of these proteins is their modular architecture at the molecular level consisting of short peptide repeats. We describe the molecular design of these peptide building blocks, highlighting the consensus motifs identified to play a key role in biofabrication and in regulating the mechanical properties of the macroscopic biological material. Structure/property relationships unveiled through advanced spectroscopic and scattering techniques, including Raman, infrared, circular dichroism, and NMR spectroscopies as well as wide-angle and small-angle X-ray scattering, are also discussed.We then present recent developments in exploiting the discovered molecular designs to engineer peptides and their conjugates for promising biomedical applications. One example includes short peptide hydrogels that self-assemble entirely under aqueous conditions and simultaneously encapsulate large macromolecules during the gelation process. A second example involves peptide coacervate microdroplets produced by liquid-liquid phase separation. These microdroplets are capable of recruiting and delivering large macromolecular therapeutics (genes, mRNA, proteins, peptides, CRISPR/Cas 9 modalities, etc.) into mammalian cells, which introduces exciting prospects in cancer, gene, and immune therapies.This Account also serves as a testament to how curiosity-driven explorations, which may lack an obvious practical goal initially, can lead to discoveries with unexpected and promising translational potential.

Molecular markers as possible efficacy predictors of targeted therapy for myelofibrosis: single-center study

Background. Targeted therapy is the most promising in the treatment of myelofibrosis, but it is necessary to search for the reasons limiting its effectiveness. There are known factors negatively affecting the development of myelofibrosis, but data on their negative impact in the context of targeted therapy are scarce.Aim. Assessing the impact of cytogenetic and genetic abnormalities on the course and therapy results for primary and secondary myelofibrosis during ruxolitinib therapy.Materials and methods. The prospective study included 106 patients with myelofibrosis in the chronic phase (53 (50 %) men and 53 (50 %) women) who received ruxolitinib at the Moscow City Hematology Center, S.P. Botkin City Clinical Hospital. The median age of patients was 62 (18–84) years. The median disease duration before initiation of ruxolitinib therapy was 79 (1–432) months. Before therapy, genetic studies were performed, including next-generation sequencing. The median duration of ruxolitinib therapy was 33 (1–111) months. The influence of the cytogenetic landscape, driver mutations, allele burden of JAK2 (over time) and CALR, additional mutations on the dynamics of symptoms, spleen size, achievement of hematological response, overall survival, progression-free survival, survival without blast crisis and without progression of myelofibrosis with targeted therapy was assessed.Results. The studied genetic factors did not have a significant correlation with hemogram parameters. The hematological response in patients with JAK2 and CALR mutations compared favorably with the response in the groups with the MPL mutation and triple negative status (TNS). Higher hematological response rate was obtained in the group with initially low allele burden <50 % of JAK2 or CALR. Significant differences in 5-year overall survival were found between groups of patients with TNS and JAK2 and CALR mutations (p <0.05); with CALR allele burden <50 % and ³50 % before initiation of ruxolitinib therapy (p = 0.01); the presence or absence of positive dynamics of the JAK2 allele burden during treatment (p <0.05); additional mutations assigned to different pathogenicity groups (p <0.05); with different number of pathogenic mutations (1 or ³2), the presence or absence of pathogenic mutations in the ASXL1 (p = 0.002) and SETBP1 (p = 0.00001) genes. The 5-year progression-free survival was significantly different in cohorts of patients with or without positive dynamics of the JAK2 allelic load during treatment (p <0.05); additional mutations assigned to different pathogenicity groups (p <0.05); with a different number of pathogenic mutations (1 or ³2), the presence or absence of a pathogenic mutation of the SETBP1 gene (p = 0.003). Progression-free survival did not correlate with the type of driver mutation or its absence; however, all patients with TNS died from myelofibrosis progression. Significant differences in 5-year blast crisis-free survival were observed between groups with JAK2 and MPL mutations (p = 0.001), JAK2 and TNS (p = 0.002); difference in 5-year survival without progression of fibrosis – between groups with pathogenic and benign (p = 0.031); uncertain and benign (p = 0.001) mutations.Conclusion. The study identified genetic markers associated with decreased efficacy of ruxolitinib therapy.

Biological and genetic characterization of a newly established human external auditory canal carcinoma cell line, SCEACono2

Squamous cell carcinoma of the external auditory canal (EACSCC) is an extraordinarily rare and aggressive malignant disease. Establishment of EACSCC cell line with robust molecular characteristics is essential for the basic and translational research of EACSCC. Here, we show the newly established EACSCC cell line SCEACono2, derived from a patient with well-to-moderately differentiated EACSCC. We analyzed histologic and genetic features of SCEACono2 hiring multiple experiments, including next-generation sequencing (NGS). Immunocytochemical staining of SCEACono2 showed positivity of p53 and SCC1/2. Furthermore, SCEACono2 exhibited a unique characteristic that cytokeratin, vimentin as well as cancer stem cell markers (CD44, CD133, ALP and Oct3/4) were positive. SCEACono2 had an ability to form tumors at the temporal lesion xenograft nude mice model. NGS revealed that SCEACono2 harbored the somatic mutations of TP53 (p.G245S) and NOTCH1 (p.A465T). RNA-seq and downstream bioinformatics analysis revealed significant enrichment of genes involved in inflammation and cell adhesion in SCEACono2 compared to SCC-9 and HSC-4. STR profiling indicated no evidence of cross-contamination. In conclusion, SCEACono2 could serves as a promising and robust research resource of EACSCC in vitro and in vivo.

Immunogenicity of a Plasma-Derived Von Willebrand Factor/Factor VIII Concentrate during the Wil-31 Prophylaxis Study in Patients with Von Willebrand Disease

Background: Long-term prophylaxis with a von Willebrand factor (VWF) concentrate is recommended in people with von Willebrand disease (VWD) with a history of frequent and severe bleeds. The WIL-31 study demonstrated the efficacy of prophylaxis with a plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrate containing VWF and FVIII in a 1:1 activity ratio (wilate ®) in adults and children with VWD of all types. The primary endpoint was met, showing a decrease in mean total annualized bleeding rate (ABR) of 84% compared with prior on-demand treatment. Aim: To assess the immunogenicity of pdVWF/FVIII when used during the WIL-31 study in children and adults with VWD of all types. Methods: WIL-31 (NCT04052698) was a prospective, non-controlled, international, multicenter phase 3 trial that enrolled male/female patients aged ≥6 years with VWD type 1 (VWF: RCo <30 IU/dL), type 2 (except 2N) or type 3. Prior to entering the WIL-31 study, all patients had received on-demand treatment with a VWF-containing product during a 6-month, prospective, observational, run-in study (WIL-29); patients who experienced at least 6 bleeding episodes (BEs), excluding menstrual bleeds, of which ≥2 were treated with a VWF-containing product, were eligible to enter WIL-31. During WIL-31, patients received regular pdVWF/FVIII (containing VWF and FVIII in a 1:1 activity ratio) prophylaxis 2-3 times per week at a dose of 20-40 IU/kg for 12 months. FVIII (chromogenic and one-stage assays) and VWF (VWF:RCo assay) activities were assessed at baseline and throughout the study. Retention samples were collected at screening and baseline visits for possible later inhibitor testing (to be performed at a central laboratory using the Nijmegen-Bethesda assay for FVIII inhibitors and the mixing study based on VWF:RCo for VWF inhibitors). Inhibitor testing was also performed if inhibitor development was suspected. Results: Inhibitor testing was performed in 2 of 33 patients because of unusual pharmacokinetic results at the 6-month visits, even though neither patient had any clinical signs of an inhibitor. The VWF inhibitors were detected in samples from two brothers taken at their 6-month visits. However, when retained blood samples from the screening visits of both boys were tested, both samples tested inhibitor-positive, demonstrating that both boys had developed the inhibitors prior to starting prophylaxis during WIL-31. During and before the WIL-29 run-in study, both boys had been treated on demand with another pdVWF/FVIII concentrate. The boys had both been diagnosed with VWD shortly after birth, and it was suspected that they had type 2A based on family history and were 15 (patient A) and 11 years old (patient B) at the start of WIL-31. They had a family history of VWD (both parents were diagnosed with VWD), but no family history of inhibitors. After the completion of WIL-31, both patients were reclassified to type 3 VWD following further investigation including next-generation sequencing. Baseline VWF activity indicated low VWF:RCo levels (Patient A: 0.092 IU/mL and Patient B: 0.65 IU/mL; Figure 1). The boys received 38 (patient A) and 34 IU/kg (patient B) pdVWF/FVIII respectively, twice weekly during WIL-31, and their mean total ABRs decreased by 85% and 91%, respectively, during WIL-31 compared with WIL-29. All bleeding episodes during WIL-31 were spontaneous nose bleeds. Both boys continued the same prophylactic regimen until the end of WIL-31. In one patient the inhibitor titer was negative by the end of the study. Conclusion: VWF inhibitors were detected in 2 of the 33 patients on prophylaxis with pdVWF/FVIII in the WIL-31 study and subsequent investigation revealed that both patients were already positive for VWF inhibitors prior to the study start. In one of the patients, the inhibitor resolved during continued prophylaxis. Despite the inhibitors, prophylaxis was effective in both patients with an ≥85% reduction in ABR compared to on demand treatment.

Novel cercozoan and heterolobosean protists from the rhizosphere and phyllosphere of two endemic cacti from the Atacama Desert

Cercozoans and heterolobose amoebae are found across terrestrial habitats where they feed on other unicellular microbes, including bacteria, fungi and microalgae. They constitute a significant fraction of soil ecosystems and are integral members of plant microbiota. Here, we present the results on the isolation of protozoans from the rhizosphere and phyllosphere of Browningia candelaris (Meyen) in the Andean Altiplano and Eulychnia taltalensis (F. Ritter) from the Coastal Cordillera of the Atacama Desert, both endemic to this ancient desert. We identified a new heterolobose amoeba species of the genus Allovahlkampfia isolated from cactus soil, three new species of the different glissomonad genera Allapsa, Neoheteromita, Neocercomonas and one new thecofilosean amoeba of the genus Rhogostoma isolated from the phyllosphere of one studied cactus. In addition, one bacterivorous flagellate was isolated from cactus spines and identified as a member of the non-scaled imbricatean family Spongomonadidae (Spongomonas). The isolation of protists from cactus spines extends the knowledge on the habitat ranges of taxa typically found on plant leaves or soils. The molecular data presented here is a prerequisite for further investigations on the ecology and diversity of protists including next-generation sequencing of microhabitats in plants and the rhizosphere, allowing for deeper taxonomic classification.

604 Impact of including next-generation sequencing in cystic fibrosis newborn screening in Pennsylvania

604 Impact of including next-generation sequencing in cystic fibrosis newborn screening in Pennsylvania

Prospect of genetic disorders in Saudi Arabia.

Introduction: Rare diseases (RDs) create a massive burden for governments and families because sufferers of these diseases are required to undergo long-term treatment or rehabilitation to maintain a normal life. In Saudi Arabia (SA), the prevalence of RDs is high as a result of cultural and socio-economic factors. This study, however, aims to shed light on the genetic component of the prevalence of RDs in SA. Methodology: A retrospective study was conducted between September 2020 and December 2021at King Saud Medical City, a tertiary hospital of the Ministry of Health (MOH), SA. A total of 1080 individuals with 544 potentially relevant variants were included. The index was 738, and the samples were tested in a commercialized laboratory using different molecular techniques, including next-generation sequencing. Result: A total of 867 molecular genetics tests were conducted on 738 probands. These tests included 610 exome sequencing (ES) tests, four genome sequencing (GS) tests, 82 molecular panels, 106 single nucleotide polymorphism (SNP) array, four methylation studies, 58 single-gene studies and three mitochondrial genome sequencing tests. The diagnostic yield among molecular genetics studies was 41.8% in ES, 24% in panels, 12% in SNP array and 24% in single gene studies. The majority of the identified potential variants (68%) were single nucleotide variants (SNV). Other ascertained variants included frameshift (11%), deletion (10%), duplication (5%), splicing (9%), in-frame deletion (3%) and indels (1%). The rate of positive consanguinity was 56%, and the autosomal recessive accounted for 54%. We found a significant correlation between the ES detection rate and positive consanguinity. We illustrated the presence of rare treatable conditions in DNAJC12, SLC19A3, and ALDH7A1, and the presence of the founder effect variant in SKIC2. Neurodevelopmental disorders were the main phenotype for which genetics studies were required (35.7%). Conclusion: This is the sixth-largest local study reporting next-generation sequencing. The results indicate the influence of consanguineous marriages on genetic disease and the burden it causes for the Kingdom of SA. This study highlights the need to enrich our society's knowledge of genetic disorders. We recommend utilising ES as a first-tier test to establish genetic diagnosis in a highly consanguineous population.

The Natural History of Observed SDHx-Related Head and Neck Paragangliomas Using Three-Dimensional Volumetric Tumor Analysis.

Characterize the natural history and clinical behavior of head and neck paragangliomas (HNPGLs) in subjects with succinate dehydrogenase (SDHx) pathogenic variants using volumetric tumor measurements. Cohort study. Tertiary academic referral center. Subjects with SDHx HNPGLs under observation for at least 6 months with 2 or more magnetic resonance imaging or computed tomography scans. Diagnostic interventions include next-generation sequencing, magnetic resonance imaging, and computed tomography. Therapeutic interventions include microsurgical resection or stereotactic radiosurgery. Radiographic progression was defined as a 20% or greater increase in volume. Cranial nerve (CN) functional outcomes were assessed using clinical documentation. A total of 19 subjects with 32 tumors met the inclusion criteria. Median radiographic follow-up was 2.2 years, and the median volumetric growth rate was 0.47 cm3/yr. Kaplan-Meier estimated rates of survival free of radiographic progression for all SDHx tumors at 1, 2, and 3 years were 69, 50, and 22%, respectively. No tumors developed new CN palsies during the period of observation. Over intermediate-term follow-up, observation of treatment-naive SDHx-related HNPGLs did not result in new cranial neuropathy. Although indefinite observation is only appropriate for select cases, these data support an interval of observation to characterize growth rate in asymptomatic to minimally symptomatic patients, who are at high risk of treatment-related morbidity. Given the early age at diagnosis and high risk of bilateral multifocal phenotypes in SDHx HNPGL mutation carriers, these data may aid in optimizing patient tumor control and CN functional preservation. Further studies are necessary to determine whether pretreatment growth rate is correlated with clinical outcomes.

Microbial Composition of Natural, Agricultural, and Technogenic Soils of Both Forest and Forest-Tundra of the Russian North

Technogenic processes and agrodevelopment of the soil cover lead to significant transformations of soil chemical and biological properties. New methods of soil microbiology, including next-generation sequencing, allows us to investigate soil microbial composition in detail, including the taxonomy and ecological functions of soil bacteria. This study presents data on the taxonomic diversity of mature and anthropogenically disturbed soils in various ecosystems of Russia. Natural soils in the southern taiga (Leningrad region and Novgorod region), northern taiga (Komi republic), forest-tundra, and tundra (Nadym city and Salekhard city) were investigated using next-generation sequencing (16S rDNA amplicon sequencing). In each natural bioclimatic zone, anthropogenically disturbed quarry soils or agriculturally transformed soils were also investigated. It was found that Proteobacteria, Actinobateriota, Acidobateriota, Bacteroidota, Chroloflexi, Planctomycetota, Verrucomicrobiota and Firmicutes phyla were dominant in natural soils, with minor differences between agrosoils and mature soils. In the soils of quarries, there were revealed processes of declining diversity of microbiome communities and the replacement of them by bacterial communities, different from natural and agrogenic soils. Thus, the microbial community is the most sensitive indicator of anthropogenic soil amendments and can serve to assess the success of soil self-restoration after human intervention.

Exploring the human microbiome - A step forward for precision medicine in breast cancer.

The second most frequent cancer in the world and the most common malignancy in women is breast cancer. Breast cancer is a significant health concern in India with a high mortality-to-incidence ratio and presentation at a younger age. Recent studies have identified gut microbiota as a significant factor that can have an influence on the development, treatment, and prognosis of breast cancer. This review article aims to describe the influence of microbial dysbiosis on breast cancer occurrence and the possible interactions between oncobiome and specific breast cancer molecular subtypes. The review further also discusses the role of epigenetics and diet/nutrition in the regulation of the gut and breast microbiome and its association with breast cancer prevention, therapy, and recurrence. Additionally, the recent technological advances in microbiome research, including next-generation sequencing (NGS) technologies, genome sequencing, single-cell sequencing, and microbial metabolomics along with recent advances in artificial intelligence (AI) have also been reviewed. This is an attempt to present a comprehensive status of the microbiome as a key cancer biomarker. We believe that correlating microbiome and carcinogenesis is important as it can provide insights into the mechanisms by which microbial dysbiosis can influence cancer development and progression, leading to the potential use of the microbiome as a tool for prognostication and personalized therapy.