- Browse by Subject
Browsing by Subject "Peripheral blood mononuclear cells"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA(Elsevier, 2023) Xiao, Sihao; Kai, Zhentian; Murphy, Daniel; Li, Dongyang; Patel, Dilip; Bielowka, Adrianna M.; Bernabeu-Herrero, Maria E.; Abdulmogith, Awatif; Mumford, Andrew D.; Westbury, Sarah K.; Aldred, Micheala A.; Vargesson, Neil; Caulfield, Mark J.; Genomics England Research Consortium; Shovlin, Claire L.; Medicine, School of MedicineDespite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.Item Stimulated peripheral blood mononuclear cells from chlamydia-infected women release predominantly Th1-polarizing cytokines(Elsevier, 2019-01) Jordan, Stephen J.; Bakshi, Rakesh K.; Brown, LaDraka’ T.; Chi, Xiaofei; Geisler, William M.; Medicine, School of MedicineChlamydia trachomatis infection (chlamydia) is the most prevalent sexually transmitted bacterial infection and causes significant reproductive morbidity in women. Little is known about how immunity to chlamydia develops in women, though animal models of chlamydia indicate that T-helper type 1 (Th1) responses are important for chlamydia clearance and protective immunity, whereas T-helper type 2 (Th2) responses are associated with persisting infection. In chlamydia-infected women, whether the predominant immune response is Th1- or Th2-polarizing remains controversial. To determine the cytokine profiles elicited by peripheral blood mononuclear cells (PBMCs) from chlamydia-infected women, we stimulated PBMCs with C. trachomatis elementary bodies and recombinant C. trachomatis Pgp3 and measured supernatant levels of select cytokines spanning Th1- and Th2-polarizing responses. We found that stimulated PBMCs from chlamydia-infected women secreted cytokines that indicate strong Th1-polarizing responses, especially interferon-gamma, whereas Th2-polarizing cytokines were expressed at significantly lower levels. In chlamydia-infected women, the predominant cytokine responses elicited on stimulation of PBMCs with C. trachomatis antigens were Th1-polarizing, with interferon-gamma as the predominant cytokine.