Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing

Acknowledgments Some of the figures (Figures 4–6 and Supplementary Material S1) were generated using BioRender and draw.io, respectively. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Wellcome Foundation (grant #222941/Z/21/Z), the South African Medical Research Council, American Association of Zoo Veterinarians Wild Animal Health Fund [S005651 and S007355], the National Research Foundation South African Research Chair Initiative [grant #86949], and MHM was supported by Wellcome Trust (grant #216634/Z/19/Z). AGL is supported by the EDCTP TESA III network (CSA2020NoE-3104).Peer reviewedPublisher PD

Investigation of the role of vitamin D metabolism in South African breast cancer patients using a pathology-supported genetic testing platform

Thesis (PhD)--Stellenbosch University, 2020.ENGLISH ABSTRACT: The high global breast cancer incidence drives the development of novel genomic approaches for disease prevention and targeted treatment. Towards this goal, a pathology-supported genetic testing (PSGT) platform was established to facilitate risk management of non-communicable diseases across the continuum of care, ranging from early-stage to metastatic disease and cancer survivorship. The causes and consequences of low vitamin D levels recently reported in the majority of postmenopausal breast cancer patients treated with aromatase inhibitors at the Tygerberg Academic Hospital, in the Western Cape Province of South Africa, were addressed in this study using PSGT to translate genomic findings into clinical practice. The aim was to determine the relationship between clinical characteristics, tumour histopathology and genetic variation underlying vitamin D metabolism in postmenopausal breast cancer patients at increased risk of osteoporosis, identified as a significant co-morbidity in the study population. Clinical and lifestyle information of 116 postmenopausal women with known vitamin D status diagnosed with breast carcinoma between 2014 and 2017 was extracted from a central genomics database linked to a biobank of DNA samples extracted from blood. Whole exome sequencing (WES) was performed on the Ion Torrent platform, followed by variant calling of vitamin D-related genes, while simultaneously assessing BRCA1/2 mutation status. Allele-specific real-time polymerase chain reaction (PCR), Sanger sequencing and long-range nanopore sequencing using the pocket-size MinION device were used to verify the WES results and to screen for variants in the vitamin D receptor (VDR) and E-cadherin (CDH1) genes beyond the coding regions covered by WES. Seasonal variation (p = 0.009) and high body mass index (BMI) (p = 0.032) contributed significantly to vitamin D levels, with the lowest values recorded during winter. WES initially performed in 10 breast cancer patients selected based on vitamin D levels at extreme upper and lower ranges, identified GC rs4588 (c.1364C>A, T455K) as a potential contributing factor vitamin D deficiency in the five patients with ultra-low vitamin D levels (≤12 ng/mL). However, 2/5 patients with levels in the upper extreme of vitamin D (>30 ng/mL), also tested positive for this variant and no significant association was detected after extended genotyping in 100 South African patients using real-time PCR. Sanger sequencing subsequently performed in 14 breast cancer patients diagnosed with osteoporosis prior to initiation of aromatase inhibitor therapy, highlighted the potential significance of genetic variation in the VDR gene. WES analysis of VDR in an extended sample of 55 breast cancer patients furthermore confirmed the significant effect of genetic variation in this gene on bone health (p < 0.001). The CDH1 gene known to be activated by VDR was furthermore analysed in patients stratified by tumour type. CDH1 c.G671A (p.R224H) detected in a breast cancer patient with invasive carcinoma of no special type (ICNST) was classified as benign, since pathogenic germline CDH1 variants are associated with invasive lobular carcinoma and diffuse gastric cancer, but not ICNST. CDH1 c.A1298G (p.D433G) was detected in a patient with invasive lobular carcinoma together with a pathogenic BRCA1 variant detected by WES. Although this finding supports a likely benign classification for CDH1 p.D433G as reported in the international ClinVar database, a family history of stomach cancer raised the possibility of a CDH1 modifier gene effect on BRCA1 gene expression. New insights gained through integration of pathology and genomic findings were incorporated into a pharmaco-diagnostic algorithm applicable to hormone receptor-positive postmenopausal breast cancer patients. The PSGT platform facilitated interpretation of research results of study participants through use of WES and recommendation of genetic counselling where appropriate.AFRIKAANSE OPSOMMING:Die hoë globale voorkoms van borskanker dryf die ontwikkeling van nuwe genomiese benaderings tot siekte voorkoming and geteikende behandeling. Om hierdie doel te verwesenlik is ‘n patologie-ondersteunde genetiese toets platform (POGT) daargestel om risikohantering oor die kontinuum van sorg te fasiliteer, wat strek van vroeë tot metastatiese siekte en oorlewing van kanker. Die oorsaak en gevolge van lae vitamien D vlakke wat onlangs gerapporteer is in die meerderheid van postmenopousale borskanker pasiënte wat met aromatase inhibitore behandel word by die Tygerberg Akademiese Hospitaal, in die Wes Kaap provinsie van Suid-Afrika, is aangespreek in hierdie studie deur POGT te gebruik om genomiese bevindings om te skakel in kliniese praktyk. Die doel was om die verband te bepaal tussen kliniese karaktertrekke, tumor histopatologie en genetiese variasie onderliggend aan vitamien D tekort in postmenopousale borskanker pasiënte met verhoogde risiko vir osteoporose, wat as ‘n belangrike ko-morbiditeit geïdentifiseer is in die studiepopulasie. Kliniese en leefstyl inligting van 116 postmenopousale vroue met bekende vitamien D status, nuut gediagnoseer met borskanker tussen 2014 and 2017, is selekteer uit ‘n sentrale genomiese databasis wat gekoppel is aan ‘n biobank van DNA monsters wat geëkstraheer is uit bloed. Heel eksoom volgordebepaling (HEV) is uitgevoer op die Ion Torrent platform, gevolg deur variant selektering van vitamien D-verwante gene, met gelyktydige bepaling van BRCA1/2 mutasie status. Alleel-spesifieke reël-tyd polymerase ketting reaksie (PKR), Sanger DNA volgordebepaling, en lang-fragment nanopore volgordebepaling is uitgevoer met gebruik van ‘n sak-grootte MinION apparaat om die HEV uitslae te bevestig en te sif vir variante in die vitamien D reseptor (VDR) en E-cadherin (CDH1) gene buite die kodering areas wat deur HEV gedek word. Seisoenale variasie (p = 0.009) en hoë liggaam gewig indeks (p = 0.032) het statisties betekenisvol bygedra tot vitamien D vlakke, met die laagste waardes tydens winter gemeet. HEV wat aanvanklik gedoen is in 10 geselekteerde borskanker pasiënte gebaseer op vitamien D wat in the uiterste van die hoogste en laagste vlakke gemeet het, het GC rs4588 (c.1364C>A, T455K) geïdentifiseer as ‘n potensieël bydraende faktor tot vitamien D tekort in vyf pasiënte met ultra-laag vitamien D vlakke (≤12 ng/mL). Twee uit vyf pasiënte met die hoogste vlakke van vitamin D (>30 ng/mL) het ook postief getoets vir hierdie GC variant, en ‘n uitgebreide genotipering deur gebruik van reël-tyd PKR in 100 Suid-Afrikaanse pasiënte nie ‘n statisties betekenisvolle assosiasie met vitamien D vlakke getoon het nie. Sanger volgordebepaling wat is daarna uitgevoer in 14 borskanker pasiënte gediagnoseer met osteoporose voor die aanvang van aromatase inhibitor behandeling, het die kliniese relevansie van genetiese variasie in die VDR geen beklemtoon. HEV analise van VDR in ‘n uitgebreide aantal van 55 borskanker pasiënte het die betekenisvolle effek van VDR genetiese variasie op beengesondheid verder bevestig (p < 0.001). Die CDH1 geen wat deur VDR geaktiveer word is verder ondersoek in pasiënte wat gestratisifeer is volgens tumor tipe. Die CDH1 c.G671A (p.R224H) variant wat waargeneem is in ‘n borskanker pasiënt met infiltrerende karsinoom van geen spesiale tipe is geklassifiseer as benigne, aangesien patogeniese kiemlyn CDH1 variante geassosieer word met infiltrerende lobulêre karsinoom (ILK) en diffuse maagkanker, en nie infiltrerende karsinoom van geen spesiale tipe nie. CDH1 c.A1298G (p.D433G) is in ‘n pasiënt met ILK tesame met ‘n patogeniese BRCA1 variant waargeneem met gebruik van HEV. Alhoewel hierdie bevinding ‘n waarskynlik benigne klassifikasie ondersteun soos gerappporteer in die internasionale ClinVar databasis, het die familiegeskiedenis van maagkanker die moontlikheid van ‘n CDH1 modifiseerder geen effek op BRCA1 geen uitdrukking uitgelig. Nuwe insigte soos verkry deur die integrasie van patologie en genomiese bevindinge in hierdie studie, is geïnkorporeer in n farmako-diagnostiese algoritme, toepaslik tot hormoon-positiewe postmenopousale borskanker pasiënte. Die POGT platform fasiliteer interpretasie van navorsingresultate van studie deelnemers deur die gebruik van HEV asook aanbeveling van genetiese raadgewing waar nodig.Doctora

Pathology-supported genetic testing for the application of breast cancer pharmacodiagnostics: family counselling, lifestyle adjustments and change of medication

Pathology-supported genetic testing (PSGT) enables transitioning of risk stratification from the study population to the individual. We provide an overview of the translational research performed in postmenopausal breast cancer patients at increased risk of osteoporosis due to aromatase inhibitor therapy, as the indication for referral. Both tumor histopathology and blood biochemistry levels were assessed to identify actionable disease pathways using whole exome sequencing (WES). The causes and consequences of inadequate vitamin D levels as a modifiable risk factor for bone loss were highlighted in 116 patients with hormone receptor-positive breast cancer. Comparison of lifestyle factors and WES data between cases with vitamin D levels at extreme upper and lower ranges identified obesity as a major discriminating factor, with the lowest levels recorded during winter. Functional polymorphisms in the vitamin D receptor gene contributed independently to therapy-related osteoporosis risk. In a patient with invasive lobular carcinoma, genetic counseling facilitated investigation of the potential modifying effect of a rare CDH1 variant co-occurring with BRCA1 c.66dup (p.Glu23ArgfsTer18). Validation of PSGT as a three-pronged pharmacodiagnostics tool for generation of adaptive reports and data reinterpretation during follow-up represents a new paradigm in personalized medicine, exposing significant limitations to overcome.</p