Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA

American Journal of Human Genetics · 14 authors, 10 centres

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This study presents GROFFFY, a novel bioinformatics tool designed to prioritize functional non-coding variants in whole-genome sequencing (WGS) data by integrating experimentally validated regulatory regions. Applied to HHT patient data from the 100,000 Genomes Project, GROFFFY reduced the mean variant count per individual from 4,867,167 to 21,486 without removing known pathogenic variants. Functional validation via RNA-seq in patient-derived peripheral blood mononuclear cells showed altered SMAD4 expression compared to controls, particularly under stress conditions. A limitation is the focus on a single gene for discovery, justified by immediate clinical implications for HHT diagnosis.

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internal medicine

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ORIGINAL PAPER

PMC10645545

Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA

American Journal of Human Genetics

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ABSTRACT

Summary Despite 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.

CITATION EXPORT

Sihao Xiao, Zhentian Kai, Daniel Murphy et al.. Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA. American Journal of Human Genetics. (2023). https://doi.org/10.1016/j.ajhg.2023.09.005

AUTHORS

14 · 10 CENTRES

Sihao Xiao, Zhentian Kai, Daniel Murphy, Dongyang Li, Dilip Patel, Adrianna M. Bielowka, Maria E. Bernabeu-Herrero, Awatif Abdulmogith, Andrew D. Mumford, Sarah K. Westbury, Micheala A. Aldred, Neil Vargesson, Mark J. Caulfield, Claire L. Shovlin

1National Heart and Lung Institute, Imperial College London, W12 ONN London, UK
2National Institute for Health Research (NIHR) Imperial Biomedical Research Centre, W2 1NY London, UK
3Topgen Biopharm Technology Co. Ltd., Shanghai 201203, China
4Women’s, Children’s & Clinical Support (Pharmacy), Imperial College Healthcare NHS Trust, W2 1NY London, UK
5School of Cellular and Molecular Medicine, University of Bristol, BS8 1QU Bristol, UK
6Division of Pulmonary, Critical Care, Sleep & Occupational Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
7School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, AB25 2ZD Aberdeen, UK
8William Harvey Research Institute, Queen Mary University of London, E1 4NS London, UK
9Genomics England, EC1M 6BQ London, UK
10Specialist Medicine, Imperial College Healthcare NHS Trust, W12 OHS London, UK