Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases
Authors: Pagnamenta Alistair T. , Camps Carme , Giacopuzzi Edoardo , Taylor John M. , Hashim Mona , Calpena Eduardo , Kaisaki Pamela J. , Hashimoto Akiko , Yu Jing , Sanders Edward , Schwessinger Ron , Hughes Jim R. , Lunter Gerton , Dreau Helene , Ferla Matteo , Lange Lukas , Kesim Yesim , Ragoussis Vassilis , Vavoulis Dimitrios V. , Allroggen Holger , Ansorge Olaf , Babbs Christian , Banka Siddharth , Baños-Piñero Benito , Beeson David , Ben-Ami Tal , Bennett David L. , Bento Celeste , Blair Edward , Brasch-Andersen Charlotte , Bull Katherine R. , Cario Holger , Cilliers Deirdre , Conti Valerio , Davies E. Graham , Dhalla Fatima , Dacal Beatriz Diez , Dong Yin , Dunford James E. , Guerrini Renzo , Harris Adrian L. , Hartley Jane , Hollander Georg , Javaid Kassim , Kane Maureen , Kelly Deirdre , Kelly Dominic , Knight Samantha J. L. , Kreins Alexandra Y. , Kvikstad Erika M. , Langman Craig B. , Lester Tracy , Lines Kate E. , Lord Simon R. , Lu Xin , Mansour Sahar , Manzur Adnan , Maroofian Reza , Marsden Brian , Mason Joanne , McGowan Simon J. , Mei Davide , Mlcochova Hana , Murakami Yoshiko , Németh Andrea H. , Okoli Steven , Ormondroyd Elizabeth , Ousager Lilian Bomme , Palace Jacqueline , Patel Smita Y. , Pentony Melissa M. , Pugh Chris , Rad Aboulfazl , Ramesh Archana , Riva Simone G. , Roberts Irene , Roy Noémi , Salminen Outi , Schilling Kyleen D. , Scott Caroline , Sen Arjune , Smith Conrad , Stevenson Mark , Thakker Rajesh V. , Twigg Stephen R. F. , Uhlig Holm H. , van Wijk Richard , Vona Barbara , Wall Steven , Wang Jing , Watkins Hugh , Zak Jaroslav , Schuh Anna H. , Kini Usha , Wilkie Andrew O. M. , Popitsch Niko , Taylor Jenny C. 
Published: November 2023

Abstract
Background
Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome.

Methods
We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants.

Results
Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving.

Conclusions
Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.