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Browsing by Author "Bernabeu-Herrero, Maria E."
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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 Mutational and phenotypic characterization of hereditary hemorrhagic telangiectasia(American Society of Hematology, 2020-10-22) Shovlin, Claire L.; Simeoni, Ilenia; Downes, Kate; Frazer, Zoe C.; Megy, Karyn; Bernabeu-Herrero, Maria E.; Shurr, Abigail; Brimley, Jennifer; Patel, Dilipkumar; Kell, Loren; Stephens, Jonathan; Turbin, Isobel G.; Aldred, Micheala A.; Penkett, Christopher J.; Ouwehand, Willem H.; Jovine, Luca; Turro, Ernest; Medicine, School of MedicineHereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular dysplasia. Care delivery for HHT patients is impeded by the need for laborious, repeated phenotyping and gaps in knowledge regarding the relationships between causal DNA variants in ENG, ACVRL1, SMAD4 and GDF2, and clinical manifestations. To address this, we analyzed DNA samples from 183 previously uncharacterized, unrelated HHT and suspected HHT cases using the ThromboGenomics high-throughput sequencing platform. We identified 127 rare variants across 168 heterozygous genotypes. Applying modified American College of Medical Genetics and Genomics Guidelines, 106 variants were classified as pathogenic/likely pathogenic and 21 as nonpathogenic (variant of uncertain significance/benign). Unlike the protein products of ACVRL1 and SMAD4, the extracellular ENG amino acids are not strongly conserved. Our inferences of the functional consequences of causal variants in ENG were therefore informed by the crystal structure of endoglin. We then compared the accuracy of predictions of the causal gene blinded to the genetic data using 2 approaches: subjective clinical predictions and statistical predictions based on 8 Human Phenotype Ontology terms. Both approaches had some predictive power, but they were insufficiently accurate to be used clinically, without genetic testing. The distributions of red cell indices differed by causal gene but not sufficiently for clinical use in isolation from genetic data. We conclude that parallel sequencing of the 4 known HHT genes, multidisciplinary team review of variant calls in the context of detailed clinical information, and statistical and structural modeling improve the prognostication and treatment of HHT.