Functional filter for whole genome sequence data identifies stress impact, non-coding, alternate polyadenylation site variants >5kb from coding DNA

Despite whole genome sequencing (WGS), why do many single gene disorder cases remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection? 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 which 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 per 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 prime untranslated region (UTR) of the proto-oncogene SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis. Sited >5.4kb 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. Monocyte SMAD4 RNA expression differed between patients and controls in resting and cycloheximide-stressed states. Patterns predicted the mutational site for an unrelated case, where a complex insertion was subsequently identified. In conclusion, a new type of functional rare variant is described, exposing novel regulatory systems based on polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement National Institute for Health Research Imperial Biomedical Research Centre; D Almeida Charitable Trust; Imperial College Healthcare NHS Foundation Trust. AA was supported by Prince Sultan Military Medical City, Saudi Arabia. MAA was supported by the National Institutes of Health (grant R35HL140019). This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research (NIHR) and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Health Research Authority Committee East England-Cambridge South (REC Ref 14/EE/1112) and East of Scotland Research Ethics Service (EoSRES 16/ES/0095) gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The publicly available file accession numbers used to generate the code are provided in full within the Data Supplement and have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJNA596860, referencing the WGS data source under accession number SAMN13640532. Primary data from the 100,000 Genomes Project, which are held in a secure Research Environment, are available to registered users. Please see https://www.genomicsengland.co.uk/about-gecip/for-gecip-members/data-and-data-access for further information.