Conventional Sanger Sequencing Research Articles

Next generation sequencing improves the resolution of detecting mixed host blood meal sources in field collected arboviral mosquito vectors.

Accurate knowledge of blood meal hosts of different mosquito species is critical for identifying potential vectors and establishing the risk of pathogen transmission. We compared the performance of Miseq next generation sequencing approach relative to conventional Sanger sequencing approach in identification of mosquito blood meals using genetic markers targeting the 12S rRNA and cytochrome oxidase I (COI) genes. We analysed the blood meals of three mosquito vector species (Aedes aegypti, Aedes simpsoni s.l. and Culex pipiens s.l.) collected outdoors, and compared the frequency of single- versus multiple-blood feeding. Single host blood meals were mostly recovered for Sanger-based sequencing of the mitochondrial 12S rRNA gene, whereas Miseq sequencing employing this marker and the COI marker detected both single and multiple blood meal hosts in individual mosquitoes. Multiple blood meals (two or more hosts) which mostly included humans were detected in 19%-22.7% of Ae. aegypti samples. Most single host blood meals for this mosquito species were from humans (47.7%-57.1%) and dogs (9.1%-19.0%), with livestock, reptile and rodent hosts collectively accounting for 4.7%-28.9% of single host blood meals. The frequency of two or more host blood meals in Ae. simpsoni s.l. was 26.3%-45.5% mostly including humans, while single host blood meals were predominantly from humans (31.8%-47.4%) with representation of rodent, reptile and livestock blood meals (18.2%-68.2%). Single host blood meals from Cx. pipiens s.l. were mostly from humans (27.0%-39.4%) and cows (11.5%-27.36%). Multiple blood meal hosts that mostly included humans occurred in 21.2%-24.4% of Cx. pipiens s.l. samples. Estimated human blood indices ranged from 53%-76% for Ae. aegypti, 32%-82% for Ae. simpsoni s.l. and 26%-61% for Cx. pipiens s.l. and were consistently lower for Sanger-based sequencing approach compared to Miseq-based sequencing approach. These findings demonstrate that Miseq sequencing approach is superior to Sanger sequencing approach as it can reliably identify mixed host blood meals in a single mosquito, improving our ability to understand the transmission dynamics of mosquito-borne pathogens.

Molecular Detection and Genotyping of Dengue Viruses: Current Techniques and Future Prospect

The Dengue virus is a prevalent mosquito-borne disease, and its global incidence has been steadily increasing due to the favorable environmental conditions that promote mosquito breeding, primarily influenced by rising temperatures. Bangladesh has been particularly hard-hit by an intense and ongoing outbreak, resulting in a surge of cases and fatalities. Effective management of this disease necessitates the implementation of robust public health measures, including rigorous surveillance and early diagnosis. While serological tests are commonly employed in clinical diagnosis, molecular methods hold a critical role in identifying the specific Dengue virus strain, thereby contributing to a more comprehensive understanding of disease severity. This article serves as an extensive review, delving into various molecular testing techniques employed for both surveillance and clinical diagnosis. It offers valuable insights for research and clinical laboratories engaged in the detection of Dengue virus RNA in mosquitoes, environmental samples, and clinical specimens. The methods covered encompass a spectrum of approaches, including conventional PCR, isothermal amplification, real-time RT-PCR, Sanger sequencing, and whole-genome sequencing, providing a holistic overview of the available techniques. These methods play pivotal roles in clinical diagnosis, outbreak analysis, vector surveillance, and vaccine development. Furthermore, the article underscores the importance of integrating these techniques into existing healthcare systems, emphasizing their significance in ensuring precise dengue diagnostics to enhance the efficiency of disease management. These molecular methods are indispensable tools that contribute to accurate diagnosis, enable effective outbreak investigation, facilitate vector surveillance, and support vaccine preparation, thereby enhancing the overall management and control of dengue, ultimately working toward mitigating its impact on public health.
 Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 41-49

Comparison of four different human papillomavirus genotyping methods in cervical samples: Addressing method-specific advantages and limitations

Since human papillomavirus (HPV) is recognized as the causative agent of cervical cancer and associated with anogenital non-cervical and oropharyngeal cancers, the characterization of the HPV types circulating in different geographic regions is an important tool in screening and prevention. In this context, this study compared four methodologies for HPV detection and genotyping: real-time PCR (Cobas® HPV test), nested PCR followed by conventional Sanger sequencing, reverse hybridization (High + Low PapillomaStrip® kit) and next-generation sequencing (NGS) at an Illumina HiSeq2500 platform. Cervical samples from patients followed at the Family Health Strategy from Juiz de Fora, Minas Gerais, Brazil, were collected and subjected to the real-time PCR. Of those, 114 were included in this study according to the results obtained with the real-time PCR, considered herein as the gold standard method. For the 110 samples tested by at least one methodology in addition to real-time PCR, NGS showed the lowest concordance rates of HPV and high-risk HPV identification compared to the other three methods (67–75 %). Real-time PCR and Sanger sequencing showed the highest rates of concordance (97–100 %). All methods differed in their sensitivity and specificity. HPV genotyping contributes to individual risk stratification, therapeutic decisions, epidemiological studies and vaccine development, supporting approaches in prevention, healthcare and management of HPV infection.

Hospital-based norovirus surveillance in children

After the introduction of the rotavirus vaccine into the Universal Immunization Program in India in 2016, relatively few studies have assessed the prevalence and epidemiological patterns of acute gastroenteritis (AGE) among hospitalized children ≤5 years of age. We used a uniform protocol to recruit children with AGE as well as standardized testing and typing protocols. Stool specimens from children with AGE younger than 5 years of age admitted to six hospitals in three cities in India were collected from January 2017 through December 2019. Norovirus was detected by real-time reverse transcription-polymerase chain reaction (RT-qPCR) followed by typing positive specimens by conventional RT-PCR and Sanger sequencing. Norovirus was detected in 322 (14.8%) of 2182 specimens with the highest rate in 2018 (17.6%, 146/829), followed by 2019 (14.4%, 122/849)and 2017 (10.7%, 54/504). Rotavirus vaccine status was known for 91.6% of the children of which 70.4% were vaccinated and 29.6% not. Norovirus positivity in rotavirus-vaccinated children was 16.3% and 12% in unvaccinated children. GII.4 Sydney[P16] (39.3%), GII.4 Sydney[P31] (18.7%), GII.2[P16] (10%), GI.3[P13] (6.8%), GII.3[P16] (5.9%), and GII.13[P16] (5%) accounted for 85.8% (188/219) of the typed strains. Our data highlight the importance of norovirus in Indian children hospitalized with AGE.

Full genome analysis of a novel genotype of Decapod hepandensovirus 1 (DHPV) infecting Pacific whiteleg shrimp, Penaeus vannamei

Decapod hepandensovirus 1 (DHPV) is a viral pathogen that causes growth retardation and decreased feed conversion efficiency in penaeid shrimp. The virus exhibits high genetic variation between different hosts and geographical locations. It has been primarily reported in cultured Penaeus monodon, P. chinensis, and P. merguiensis, and a new type of DHPV has recently been reported in P. vannamei cultured in Korea and Taiwan. In this study, the genome of a recently reported new type of DHPV was fully sequenced by conventional Sanger sequencing combined with a next-generation sequencing approach. Similar to the other types of DHPVs infecting several shrimp species other than P. vannamei, a new type of DHPV contained a linear, single-stranded DNA genome of 6173 bp comprising three major open reading frames. However, by detailed comparative genome and phylogenetic analysis, the virus was not clustered with the pre-existing genotypes, suggesting the emergence of a possible novel genotype of DHPV in cultured shrimps. This study is the first to report the complete genomic sequence of DHPV identified in P. vannamei.

Mutation of <em>PAX3</em> and <em>MITF</em> genes in a family with type 1 Waardenburg syndrome: a case series

Waardenburg syndrome (WS) is a rare genetical disorder, characterized with pigmentary abnormalities of the eyes, skin, hair, dystopia canthorum, and sensorineural deafness. In Majene, West Sulawesi, 12 members of a 4-generation family presented manifestations of WS. We examined the presence of mutations in 5 family members with type 1 WS and the other 5 normal phenotype family members to identify mutations of PAX3 and MITF genes. Ophthalmic examination and peripheral blood test were done. Conventional polymerase chain reaction and direct Sanger sequencing were then performed to detect the mutation. 26 mutations of PAX3 gene were only identified in patients with major and minor criteria, including 7 missense mutations (substitutions) and 2 insertions in exons 1, 2, and 6, as well as 17 intronic changes in intron 8. No mutations were detected in MITF gene.

Co-Occurrence of Wing Deformity and Impaired Mobility of Alates with Deformed Wing Virus in Solenopsis invicta Buren (Hymenoptera: Formicidae).

Deformed wing virus (DWV), a major honey bee pathogen, is a generalist insect virus detected in diverse insect phyla, including numerous ant genera. Its clinical symptoms have only been reported in honey bees, bumble bees, and wasps. DWV is a quasispecies virus with three main variants, which, in association with the ectoparasitic mite, Varroa destructor, causes wing deformity, shortened abdomens, neurological impairments, and colony mortality in honey bees. The red imported fire ant, Solenopsis invicta Buren, is one of the most-invasive and detrimental pests in the world. In this study, we report the co-occurrence of DWV-like symptoms in S. invicta and DWV for the first time and provide molecular evidence of viral replication in S. invicta. Some alates in 17 of 23 (74%) lab colonies and 9 of 14 (64%) field colonies displayed deformed wings (DWs), ranging from a single crumpled wing tip to twisted, shriveled wings. Numerous symptomatic alates also exhibited altered locomotion ranging from an altered gait to the inability to walk. Deformed wings may prevent S. invicta alates from reproducing since mating only occurs during a nuptial flight. The results from conventional RT-PCR and Sanger sequencing confirmed the presence of DWV-A, and viral replication of DWV was confirmed using a modified strand-specific RT-PCR. Our results suggest that S. invicta can potentially be an alternative and reservoir host for DWV. However, further research is needed to determine whether DWV is the infectious agent that causes the DW syndrome in S. invicta.

Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study.

Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack® IGH assay, and LymphoTrack® IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack® FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.

Identification of two novel COL3A1 variants in patients with vascular Ehlers-Danlos syndrome.

Vascular Ehlers-Danlos syndrome (vEDS) is an autosomal dominant disease caused by aberrations in COL3A1, which encodes type III collagen. Sanger sequencing has limitations for diagnosis since exon deletion/duplication and splicing alterations are not uncommon in COL3A1. We report 2 patients with vEDS who were not diagnosed by conventional Sanger sequencing. We performed either targeted panel or whole-genome sequencing. Complementary DNA (cDNA) sequencing was performed using cultured skin fibroblasts. Sanger sequencing of DNA was performed for the confirmation of breakpoints in the case of exon deletion. We also evaluated the sensitivity of the splicing prediction tool, SpliceAI. An exon 27 deletion was suspected on targeted panel sequencing of 1 patient. The deletion was confirmed using cDNA sequencing (r.1870_1923del) and breakpoints were confirmed (c.1870-109_1923+10del). On targeted panel sequencing in the other patient, we found a novel intronic variant of c.1149+6T>C that leads to skipping of exon 16 (r.1051_1149del) by cDNA sequencing. SpliceAI showed 98.8% sensitivity for known splicing variants in COL3A1. Our study highlights the necessity of a comprehensive approach to the genetic diagnosis of vEDS. In addition, cDNA sequencing was useful as an auxiliary method, especially considering the limited sensitivity of the splicing prediction tool.

Value of next-generation sequencing in inherited arrhythmia syndromes

BackgroundGenetic studies are clinically recommended in some cases of inherited arrhythmia syndromes. Next-generation sequencing (NGS) would be helpful because of its high analytical throughput and relative speed. This study aimed to assess the mutation-detection yield obtained by NGS compared with conventional Sanger sequencing method.MethodsPatients with aborted sudden cardiac death and their families who underwent gene sequencing tests for inherited arrhythmia syndromes were retrospectively and enrolled in this study from 2017 to 2022 at Chonnam National University Hospital. We evaluated NGS study results of 17 patients (NGS group) and Sanger study results of 19 patients (Sanger group).Results64.7% of NGS and 94.7% of Sanger group were probands. Type 1 Brugada pattern ECG was more frequent in NGS group (64.7% vs. 21.1%; p = 0.007). BrS was the most common disorder in NGS group (76.5%), and idiopathic ventricular fibrillation was the most common one in Sanger group (63.2%). Mutations with uncertain significance were the most common ones in NGS group (89.5%), and pathogenic or likely pathogenic mutations were the most common ones in Sanger group (45.7%). When positive yield was defined as the ratio of pathogenic or likely pathogenic mutations that were detected by sequencing, the yields were 10.5% and 45.7% in NGS and Sanger groups, respectively. The NGS arrhythmia panel did not cover two inherited arrhythmia-related mutations (RYR1, APOA5) that were detected by the Sanger method. The extended NGS arrhythmia panel was able to detect 84.8% of inherited arrhythmia-related mutations that were detected in Sanger group.ConclusionsNGS study has some limitations in obtaining the full genetic data of probands. Well-designed NGS panels are needed to increase the efficiency of the NGS study. With the well-designed panels, large-scale gene sequencing can efficiently and rapidly be applied in real clinical practices, especially in inherited fatal arrhythmia syndromes that have a high detection yield in genetic analyses.

Long-read sequencing: An effective method for genetic analysis of CYP21A2 variation in congenital adrenal hyperplasia

BackgroundThe sequence similarity between CYP21A2 gene and its inactive pseudogene CYP21A1P, and copy number variation (CNV) caused by unequal crossover, make it challenging to characterize the CYP21A2 gene through traditional methods. This study aimed to evaluate the clinical utility of the long-read sequencing (LRS) method in carrier screening and genetic diagnosis of congenital adrenal hyperplasia (CAH) by comparing the efficiency of the LRS method with the conventional multiplex ligation-dependent probe amplification (MLPA) plus Sanger sequencing approaches in CYP21A2 analysis. MethodsIn a retrospective study, full sequence analysis of the CYP21A2 and CYP21A1P was performed for three pedigrees through long-range locus-specific PCR followed by LRS based on the Pacific Biosciences (PacBio, California, USA) single-molecule real-time (SMRT) platform, and the results were compared with those obtained from next-generation sequencing (NGS)-based whole exome sequencing (WES) and the traditional methods of MLPA plus Sanger sequencing. ResultsThe LRS method successfully identified seven CYP21A2 variants, including three single nucleotide variants (NM_000500.9:c.1451G > C p.(Arg484Pro), c.293-13A/C > G (IVS2-13A/C > G), c.518 T > A p.(Ile173Asn)), one 111-bp polynucleotide insertion, one set of 3′URT variants (NM_000500.9:c.*368 T > C, c.*390A > G, c.*440C > T, c.*443 T > C) and two types of chimeric genes and straightforwardly depicted the inheritance patterns of these variants within families. Moreover, the LRS method enabled us to determine the cis–trans configuration of multiple variants in one assay, without the need to analyze additional family samples. Compared with traditional methods, this LRS method can achieve a precise, comprehensive and intuitive result in the genetic diagnosis of 21-hydroxylase deficiency (21-OHD). ConclusionThe LRS method is comprehensive in CYP21A2 analysis and intuitive in result presentation, which holds substantial promise in clinical application as a crucial tool for carrier screening and genetic diagnosis of CAH.

Zoonotic helminths of dogs and risk factors associated with polyparasitism in Grenada, West Indies.

Canine soil-transmitted helminths (STHs) cause important zoonoses in the tropics, with varying degrees of intensity of infection in humans and dogs. This study aimed to investigate the prevalence and associated risk factors for STHs in community dogs residing in Grenada, West Indies. In May 2021, 232 canine fecal samples were examined for zoonotic helminths by microscopy (following flotation), and genomic DNA from a subset of 211 of these samples were subjected to multiplex qPCR for the detection and specific identification of hookworms, Toxocara spp. and Strongyloides. Microscopic examination revealed that 46.5% (108/232, 95% CI 40–52.9), 9% (21/232, 95% CI 5.35–12.7) and 5.2% (12/232, 95% CI 2.3–8) of the samples contained eggs of Ancylostoma spp., Toxocara spp. and Trichuris vulpis, respectively. Multiplex qPCR revealed that, 42.2% (89/211, 95% CI 35.5–48.8) were positive for at least 1 zoonotic parasite. Of these, 40.8% (86/211, 95% CI 34.1–47.3) of samples tested positive for Ancylostoma spp., 36% (76/211, 95% CI 29.5–42.9) were positive for A. caninum, 13.3% (28/211, 95% CI 9–18.6) for A. ceylanicum, 5.7% for T. canis (12/211, 95% CI 2.97–8.81) and 1% (2/211, 95% CI 0–2.26) for Strongyloides spp. (identified as S. stercoralis and S. papillosus by conventional PCR-based Sanger sequencing). Using a multiple logistic regression model, a low body score and free-roaming behaviour were significant predictors of test-positivity for these parasitic nematodes in dogs (P < 0.05). Further studies of zoonotic STHs in humans should help elucidate the public health relevance of these parasites in Grenada.

Development and evaluation of a centrifugal disk system for the rapid detection of multiple pathogens and their antibiotic resistance genes in urinary tract infection.

Urinary tract infections (UTIs) are some of the most common bacterial infections in the world. Nevertheless, as uncomplicated UTIs are treated empirically without culturing the urine, adequate knowledge of the resistance pattern of uropathogens is essential. Conventional urine culture and identification take at least 2 days. Here, we developed a platform based on LAMP and centrifugal disk system (LCD) to simultaneously detect the main pathogens and antibiotic resistant genes (ARGs) of urgent concern multidrug-resistant among UTIs. We designed specific primers to detect the target genes above and evaluated their sensitivity and specificity. We also assessed the result of our preload LCD platform on 645 urine specimens with a conventional culturing method and Sanger sequencing. The results obtained with the 645 clinical samples indicated that the platform has high specificity (0.988-1) and sensitivity (0.904-1) for the studied pathogens and ARGs. Moreover, the kappa value of all pathogens was more than 0.75, revealing an excellent agreement between the LCD and culture method. Compared to phenotypic tests, the LCD platform is a practical and fast detection approach for methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter, carbapenem-resistant Pseudomonas aeruginosa (kappa value of all >0.75), and non-extended-spectrum β-lactamase producers. We developed a detection platform that has high accuracy and that meets the need for rapid diagnosis, which can be completed within 1.5 h from specimen collection. It may be a powerful tool for evidence-based UTIs diagnosis, which has essential support for the rational use of antibiotics. More high-quality clinical studies are required to prove the effectiveness of our platform.

The assessment on cross immunity with smallpox virus and antiviral drug sensitivity of the isolated mpox virus strain WIBP-MPXV-001 in China

ABSTRACT Since May 2022, human mpox cases have increased unexpectedly in non-endemic countries. The first imported case of human mpox in Hong Kong was reported in September 2022. Here we report the isolation and identification of MPXV from the vesicle swabs of this patient. In this research, the vesicle swabs were inoculated in Vero and Vero E6 cells. In addition to observing cytopathic effects (CPEs) in Vero or Vero E6 cells, the isolated virus was identified as mpox virus (MPXV) using quantitative Real-Time PCR (RT–PCR), transmission electron microscopy, and high-throughput sequencing. The experiment also assessed the cross-protective efficacy of sera from the smallpox vaccinated population and preliminarily assessed the inhibitory effect of anti-smallpox virus drugs against MPXV. CPEs can be observed on Vero E6 cells at 24 h and Vero cells at 48 h. The virus particles could be observed by transmission electron microscope, showing typical orthopoxvirus morphology. In addition, F3L and ATI genes which from MPXV A39R, B2R, HA genes which from orthopoxvirus were confirmed by conventional PCR and Sanger sequencing. The next generation sequencing (NGS) suggests that the MPXV strain belongs to B.1 branch of the West African linage, and has a high identity with the sequence of the 2022 ongoing outbreak. PRNT50 results showed that 26.7% of sera from individuals born before 1981 who had been immunized with smallpox were positive, but no MPXV-neutralizing antibodies were found in sera from individuals born later. All four anti-smallpox virus drugs evaluated demonstrated inhibition of mpox virus.

Distribution characteristics and clinical phenotype analyses of hemoglobin variants in the Z region of Central Guangxi, Southern China

ABSTRACTObjectiveTo investigate the molecular diagnosis of hemoglobin variants in Z region by Capillary electrophoresis in Central Guangxi, Southern China, and analyze their distribution and phenotypic characteristics, to provide a reference for clinical consultation and prenatal diagnosis for couples.MethodsA total of 23,709 subjects were collected for blood routine analysis, hemoglobin analysis, and common α- and β-globin gene loci in Chinese population. The hemoglobin electrophoresis components were divided into Zone 1-Zone 15 (Z1-Z15) by Capillary zone electrophoresis (CE). For samples not clearly detected by the conventional technology, Sanger sequencing, and multiplex ligation-dependent probe amplification (MLPA) were used. Single-molecule real-time (SMRT) sequencing technology was used to analyze rare-type genes in a sample with a structural variation.ResultsTen rare hemoglobin variants distributed in Z region were detected in 23,709 samples, including Hb Cibeles, which was reported for the first time in Asia; Hb J-Broussais, Hb G-Honolulu and J-Wenchang-Wuming, they were first reported in Guangxi; 1 case of Hb Anti-Lepore Liuzhou, which was a newly discovered hemoglobin variant; hemoglobin variants Hb G-Siriraj, Hb Handsworth, Hb Q-Thailand, Hb Ube-2, Hb NewYork were also detected.ConclusionThere are a few studies on rare hemoglobin variants in Z region in Southern China. Ten rare hemoglobin variants were found in this study. The hematological phenotype and component content of hemoglobin variants are related to the occurrence of thalassemia. This study enriched the data of rare hemoglobin variants in Southern China and provided a comprehensive data basis for prenatal diagnosis of hemoglobin variants in this area.

CDNA sequencing increases the molecular diagnostic yield in Chediak-Higashi syndrome.

Introduction: Chediak-Higashi syndrome (CHS) is rare autosomal recessive disorder caused by bi-allelic variants in the Lysosomal Trafficking Regulator (LYST) gene. Diagnosis is established by the detection of pathogenic variants in LYST in combination with clinical evidence of disease. Conventional molecular genetic testing of LYST by genomic DNA (gDNA) Sanger sequencing detects the majority of pathogenic variants, but some remain undetected for several individuals clinically diagnosed with CHS. In this study, cDNA Sanger sequencing was pursued as a complementary method to identify variant alleles that are undetected by gDNA Sanger sequencing and to increase molecular diagnostic yield. Methods: Six unrelated individuals with CHS were clinically evaluated and included in this study. gDNA Sanger sequencing and cDNA Sanger sequencing were performed to identify pathogenic LYST variants. Results: Ten novel LYST alleles were identified, including eight nonsense or frameshift variants and two in-frame deletions. Six of these were identified by conventional gDNA Sanger sequencing; cDNA Sanger sequencing was required to identify the remaining variant alleles. Conclusion: By utilizing cDNA sequencing as a complementary technique to identify LYST variants, a complete molecular diagnosis was obtained for all six CHS patients. In this small CHS cohort, the molecular diagnostic yield was increased, and canonical splice site variants identified from gDNA Sanger sequencing were validated by cDNA sequencing. The identification of novel LYST alleles will aid in diagnosing patients and these molecular diagnoses will also lead to genetic counseling, access to services and treatments and clinical trials in the future.

Abstract P5-03-07: Prevalence of Pathogenic Variants in Cancer Predisposition Genes in Women with Young Onset Breast Cancer

Abstract Introduction: Approximately 5% of breast cancers are diagnosed in women 40 years of age or younger. Known risk factors for young-onset breast cancer are few and can only account for a very small proportion of cases. In this study, we evaluated the contribution of mutations in 24 breast cancer predisposition genes in unselected Canadian women diagnosed with breast cancer at age 40 or younger. Methods: This study is a sub-study of the larger Reducing the bUrden of Breast cancer in Young women (RUBY) Study. In the RUBY study, women diagnosed with breast cancer at the age of 40 years or younger are recruited at the time of diagnosis from 33 centres across Canada. Participants in RUBY provided detailed demographic and clinical data, in addition to provision of serial biospecimens. Participants could elect to consent into the genetics substudy, and have genetic testing performed for pathogenic variants in 24 breast cancer predisposition genes, including ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11, MSH2, MSH6, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, RECQL, STK11, TP53 and XRCC2. Sequencing was performed and all potentially pathogenic variants were confirmed with conventional Sanger sequencing. Pathogenic and likely pathogenic mutations were reported for all 24 genes. CanRisk scores for likelihood of having a pathogenic variant in 8 cancer predisposition genes (BRCA1 BRCA2, PALB2, CHEK2, ATM, RAD51C, RAD51D, and BRIP1) were generated for each participant. Results: 714 women consented and genetic testing was performed on the blood samples provided as a component of the RUBY study. The mean age of the participants was 35.8 years (range 23-40 years), and the mean CanRisk score was 13.7 (range 2.3-98.0). Overall, 150 pathogenic mutations (21.0%) were detected in 147 women (three participants had mutations in two genes). The most common pathogenic variants detected were in BRCA1 (48), BRCA2 (40), CHEK2 (24), ATM (10), and PALB2 (9), representing 87.3% of all pathogenic variants identified. The mean CanRisk score was 28.8% (range 3.2-98.0%) for those identified with a pathogenic variant compared to 9.6% (range 1.0-88.9%) for those with a negative result (p &amp;lt; 0.0001). The prevalence of pathogenic variants was 32.9% for women age 20-30 years, 27.5% for 31-35 years, and 16.7% for 36-40 years. Conclusions: Twenty-one percent of women with breast cancer at age 40 or younger had a pathogenic variant in a breast-cancer predisposition gene. The great majority of these pathogenic variants were found in genes (BRCA1, BRCA2, CHEK2, PALB2) for which there are validated breast cancer treatment recommendations. All women with young-onset breast cancer should be offered germline genetic testing at the time of breast cancer diagnosis to make informed surgical and medical treatment decisions. Citation Format: Kelly Metcalfe, May Lynn Quan, Steven Narod, Ellen Warner, Christine Friedenreich, Nancy Baxter, Aletta J. Poll, Mohammad Akbari. Prevalence of Pathogenic Variants in Cancer Predisposition Genes in Women with Young Onset Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-07.

Potential outbreak by herpesvirus in equines: detection, clinical, and genetic analysis of equid gammaherpesvirus 2 (EHV-2).

Equid herpesvirus (EHV) commonly affects horses causing neurologic and respiratory symptoms beside spontaneous abortions, meaning huge economic losses for equine industry worldwide. In foals, the virus can facilitate secondary infections by Rhodococcus equi, important in morbidity and mortality in equines. A total of five genotypes of EHV were previously described in Brazil including EHV-1, EHV-2, EHV-3, EHV-4, and EHV-5. EHV-2 genotype had only been previously described in Brazil in asymptomatic animals. We report the investigation of the dead of 11 foals in Middle-west region of Brazil showing respiratory and neurological symptoms, as well as several abortions in mares from the same farm. Clinical and laboratory exams were performed in this case study. Lung, whole blood, serum, and plasma samples were analyzed by necroscopic and histopathologic techniques followed by molecular assays (conventional and qPCR and Sanger sequencing). Laboratory exams revealed neutrophilia leukocytosis. Necroscopic and histopathologic findings were suppurative bronchopneumonia and ulcerative enteritis. Molecular assays point to the absence of the bacteria Rhodococcus equi and other viruses (including other EHV). The presence of EHV-2 DNA was confirmed by sequencing in serum sample from one foal. This is the first confirmed outbreak of EHV-2 causing disease in Brazilian horses with confirmed presence of the virus, and which highlight the important role of EHV-2 in equine respiratory disease and spontaneous abortions in equid in Brazil.

First molecular identification of multiple tick-borne pathogens in livestock within Kassena-Nankana, Ghana

The risk of pathogen transmission continues to increase significantly in the presence of tick vectors due to the trade of livestock across countries. In Ghana, there is a lack of data on the incidence of tick-borne pathogens that are of zoonotic and veterinary importance. This study, therefore, aimed to determine the prevalence of such pathogens in livestock using molecular approaches. A total of 276 dry blood spots were collected from cattle (100), sheep (95) and goats (81) in the Kassena-Nankana Districts. The samples were analyzed using Polymerase Chain Reaction (qPCR) and conventional assays and Sanger sequencing that targeted pathogens including Rickettsia, Coxiella, Babesia, Theileria, Ehrlichia and Anaplasma. An overall prevalence of 36.96% was recorded from the livestock DBS, with mixed infections seen in 7.97% samples. Furthermore, the prevalence of infections in livestock was recorded to be 19.21% in sheep, 14.13% in cattle, and 3.62% in goats. The pathogens identified were Rickettsia spp. (3.26%), Babesia sp. Lintan (8.70%), Theileria orientalis (2.17%), Theileria parva (0.36%), Anaplasma capra (18.48%), Anaplasma phagocytophilum (1.81%), Anaplasma marginale (3.26%) and Anaplasma ovis (7.25%). This study reports the first molecular identification of the above-mentioned pathogens in livestock in Ghana and highlights the use of dry blood spots in resource-limited settings. In addition, this research provides an update on tick-borne pathogens in Ghana, suggesting risks to livestock production and human health. Further studies will be essential to establish the distribution and epidemiology of these pathogens in Ghana.

Validation of multi-gene panel next-generation sequencing for the detection of BRCA mutation in formalin-fixed, paraffin-embedded epithelial ovarian cancer tissues

ObjectiveThe therapeutic effect of poly (ADP-ribose) polymerase (PARP) inhibitors in patients with epithelial ovarian cancer (EOC) with somatic BRCA mutations is consistent with that observed in patients with germline BRCA mutations, indicating the importance of detecting both germline and somatic BRCA mutations concurrently. We compared the efficacy of multi-gene panel next generation sequencing (NGS) in EOC patients’ formalin-fixed, paraffin-embedded (FFPE) tissue to that of conventional Sanger sequencing in blood samples. Materials and methodsThis study included 48 patients with EOC, and both blood Sanger sequencing and FFPE tissue NGS were conducted in all of them. Clinical and pathological data were reviewed, including age at diagnosis, histology, and stage. Blood Sanger sequencing was performed using peripheral blood leukocytes. The target regions of 90 cancer-related genes were identified using FFPE tissue. ResultsThe median age of patients was 56.1 years, with serous carcinoma (n = 40, 83.3%) and stage III (n = 37, 77.1%) being the most common histology and International Federation of Gynecology and Obstetrics (FIGO) stage, respectively. FFPE tissue NGS identified ten pathogenic variants, including all eight pathogenic variants identified by blood Sanger sequencing and two additional pathogenic variants. Furthermore, FFPE tissue NGS identified 19 variants of uncertain significance (VUS), including all ten VUS identified by blood Sanger sequencing and nine additional VUS. ConclusionThe FFPE tissue multi-gene panel NGS had 100% sensitivity for detecting BRCA germline mutations and could detect additional somatic mutations. Furthermore, performing FFPE tissue multi-gene panel NGS followed by blood Sanger sequencing sequentially may help differentiate germline from somatic BRCA mutations for genetic counseling.