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Solving unsolved rare neurological diseases—a Solve-RD viewpoint

A Correction to this article was published on 25 August 2021

This article has been updated

Introduction

Rare genetic neurological disorders (RND; ORPHA:71859) are a heterogeneous group of disorders comprising >1700 distinct genetic disease entities. However, genetic discoveries have not yet translated into dramatic increases of diagnostic yield and indeed rates of molecular genetic diagnoses have been stuck at about 30–50% across NGS modalities and RND phenotypes [1, 2]. Existence of yet unknown disease genes as well as shortcomings of commonly employed NGS technologies and analysis pipelines in detecting certain variant types are typically cited to explain the low diagnosis rates.

To increase the diagnostic yield in RNDs - one of the four focus disease groups in Solve-RD - we follow two major approaches, that we will here present and exemplify: (i) systematic state-of the art re-analysis of large cohorts of unsolved whole-exome/genome sequencing (WES/WGS) RND datasets; and (ii) novel-omics approaches. Based on the way Solve-RD systematically organizes researchers’ expertise to channel this approach [3], the European Reference Network for Rare Neurological Diseases (ERN-RND) has established its own Data Interpretation Task Force (DITF) within SOLVE-RD, which is currently composed of clinical and genetic experts from 29 sites in 15 European countries.

Systematic re-analysis of coding variation

Unsolved WES datasets (fastq) from 2048 families with RNDs were submitted by clinical sites of ERN-RND [4] to the RD-Connect Genome-Phenome Analysis Platform. Genomic data were processed and filtered as detailed [5]. The Solve-RD SNV/Indel working group reported back 74,456 variants in 2246 individuals, which were ranked according to their likelihood of being causative. One thousand nine hundred and forty-three variants in 1155 individuals (average 1.68 variants/individuum) were classified as rank 1 (genotype matches OMIM and variant (likely) pathogenic according to ACMG).

Based on these results and the work of the RND DITF 44 cases could be solved by this systematic re-analysis approach, which equals 29% of the re-analysed cases for which feedback was available. Reasons for solving cases were firstly updates of the respective ClinVar entry of identified variants between the time of the initial genetic workup and the Solve-RD re-analysis due to now additional available evidence. One example is the re-classification of variants in highly variably genes like ITPR1 between 2016 and 2020 [6] (Fig. 1A).

Fig. 1: Clinical information and functional variant validation for families 1–3.
figure 1

A Pedigrees and cranial MRI of patient 1 (NM_001168272.1(ITPR1):c.805C>T, p.(Arg269Trp)). Mid-sagital MRI (T2) shows marked cerebellar atrophy at age 7. B Pedigree and longitudinal MRIs taken from patient 2 (pontocerebellar atrophy—NM_016042.3(EXOSC3):c.395A>C, p.(Asp132Ala)). MRIs demonstrate marked cerebellar atrophy while brainstem volume is not affected. C Pedigree, segregation analysis and functional analysis in family 3. The index cases carries two intronic POLR3A variants. Variant c.1048+5G>T is located in intron 7; RT-PCR with primers binding to sequences in exon (forward) and exon 9 (reverse) demonstrate presence of an aberrant transcript that is absent in controls. Specific amplification of this additional band and sequencing revealed that all 177 bp of intron 7 are included in the transcript. A nonsense codon in intron 7 presumably leads to termination of translation (p.Phe352_Arg353ins(23)Ter). The variant c.1909+22C>T has previously been demonstrated to lead to inclusion of the first 19 nucleotides from intron 14 into the final transcript und consequently to shift of the reading frame [8].

Second, use of human phenotype ontology-based phenotypes [7] rather than diagnostic categories as well as consideration of variant-specific rather than gene-specific phenotypes enabled detection of functionally relevant variants because initial analysis focused on disease-specific panels. Mis-classification of phenotypes in RNDs is a common problem due to the considerable overlap between diagnostic categories especially in phenotypes affecting more than one neurological system. This approach i.e. allowed identification of a causative variant in EXOSC3 (c.395A>C) that is typically associated with a ‘milder’ clinical disease course and lacking the hallmark pontine atrophy characteristic for EXOSC3-associated disease (Fig. 1B).

Analysis of non-coding variation

The relative contribution of non-coding variation to RNDs has not been established yet and will be systematically explored by Solve-RD by combining WGS and RNA Seq. We will evaluate the added value of RNA Seq in early onset sporadic cases (Trio-WGS), multiplex recessive and dominant families.

In the meantime, the exon–intron boundaries commonly covered by WES already allow at least a glimpse into the realm of non-coding variants. Indeed, the systematic Solve-RD re-analysis top-listed a single heterozygous intronic variant in the POLR3A gene (NM_007055.3(POLR3A):c.1909T>A: c.1909+22G>A, p.Tyr637Cysfs*14) that had recently been shown to be a frequent cause of spastic ataxia [8] in trans with a second loss-of-function POLR3A variant in an unsolved adult patient with a spastic ataxia phenotype. No second coding POLR3A variant was identified. However, a variant in intron 7 of the POLR3A gene was discovered in the WES data (NM_007055.3(POLR3A): c.1048+5G>T). RT-PCR from whole blood revealed an aberrant transcript that was absent in controls. Specific amplification and sequencing demonstrated the inclusion of all 177 bp of intron 7 into the final mRNA transcript. On protein level, this change is predicted to insert 23 amino acids coded by intron 7, followed by a stop codon (p.Phe352_Arg353ins(23)Ter) (Fig. 1C).

Finding novel variations through novel omics

Scientific rationale drives application of novel-omics technologies in Solve-RD. From the large variety of different omics technologies that will be used by SOLVE-RD, we here present the example of long-range WGS for ataxias, which has just been initiated. For ataxias >25% of all autosomal-dominant and >50% of all autosomal-recessive ataxia patients remain unsolved despite advanced WES analysis [9]. Ataxias are unique in so far as repeat expansions represent the most frequent disease cause. Seventy-five percent of all known autosomal-dominant ataxia cases and 50% of all known autosomal-recessive ataxia cases are caused by repeat expansions [10]. We thus hypothesize that a substantial share of repeat-expansion disorders is still to be found in the large share of still unsolved WES-negative ataxia cases. Therefore, in Solve-RD we will be using long-range WGS in family ‘triplets’ from autosomal-dominant ataxia families, which will be stringently enriched for novel repeat-expansion disorders: namely only families negative not only on WES and frequent SCA repeats, but also on short-read WGS and for which DNA from >2 affected and >2 non-affected family members are available. In a first round of submission, 20 families with 44 ‘slots’ have been submitted and we are awaiting data in 2021.

Conclusion

This viewpoint presents and exemplifies the approach being taken by Solve-RD to diagnostically solve unsolved RND. While re-analysis so far succeeded in 29% of cases, scientifically rational ‘beyond the exome’ approaches are being implemented to further unravel new RND causing genes.

Change history

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Acknowledgements

We thank the patients and their families for supporting this study.

Solve-RD-DITF-RND

Jonathan Baets13,14,15, Peter Balicza16, Patrick Chinnery17, Alexandra Dürr18,19,20, Tobias Haack12, Holger Hengel2,21, Rita Horvath22, Henry Houlden23, Erik-Jan Kamsteeg24, Christoph Kamsteeg24, Katja Lohmann25, Alfons Macaya26, Anna Marcé-Grau26, Ales Maver27, Judit Molnar16, Alexander Münchau25, Borut Peterlin27, Olaf Riess12,28, Ludger Schöls2,21, Rebecca Schüle2,21, Giovanni Stevanin18,19,20,29,30, Matthis Synofzik2,21, Vincent Timmerman31,32, Bart van de Warrenburg33, Nienke van Os33,34, Jana Vandrovcova23, Melanie Wayand2,21, Carlo Wilke2,21

The Solve-RD Consortium

Olaf Riess12,28, Tobias B. Haack12, Holm Graessner12,28, Birte Zurek12,28, Kornelia Ellwanger12,28, Stephan Ossowski12, German Demidov12, Marc Sturm12, Julia M. Schulze-Hentrich12, Rebecca Schüle2,21, Christoph Kessler2,21, Melanie Wayand2,21, Matthis Synofzik2,21, Carlo Wilke2,21, Andreas Traschütz2,21, Ludger Schöls2,21, Holger Hengel2,21, Peter Heutink2,21, Han Brunner24,33,35, Hans Scheffer24,35, Nicoline Hoogerbrugge24,36, Alexander Hoischen24,36,37, Peter A. C. ’t Hoen36,38, Lisenka E. L. M. Vissers24,33, Christian Gilissen24,36, Wouter Steyaert24,36, Karolis Sablauskas24, Richarda M. de Voer24,36, Erik-Jan Kamsteeg24, Bart van de Warrenburg33,34, Nienke van Os33,34, Iris te Paske24,36, Erik Janssen24,36, Elke de Boer24,33, Marloes Steehouwer24, Burcu Yaldiz24, Tjitske Kleefstra24,33, Anthony J. Brookes39, Colin Veal39, Spencer Gibson39, Marc Wadsley39, Mehdi Mehtarizadeh39, Umar Riaz39, Greg Warren39, Farid Yavari Dizjikan39, Thomas Shorter39, Ana Töpf40, Volker Straub40, Chiara Marini Bettolo40, Sabine Specht40, Jill Clayton-Smith41, Siddharth Banka41,42, Elizabeth Alexander41, Adam Jackson41, Laurence Faivre43,44,45,46,47, Christel Thauvin44,45,46,47, Antonio Vitobello45, Anne-Sophie Denommé-Pichon45, Yannis Duffourd45,46, Emilie Tisserant45, Ange-Line Bruel45, Christine Peyron48,49, Aurore Pélissier49, Sergi Beltran9,10, Ivo Glynne Gut10, Steven Laurie10, Davide Piscia10, Leslie Matalonga10, Anastasios Papakonstantinou10, Gemma Bullich10, Alberto Corvo10, Carles Garcia10, Marcos Fernandez-Callejo10, Carles Hernández10, Daniel Picó10, Ida Paramonov10, Hanns Lochmüller10, Gulcin Gumus50, Virginie Bros-Facer51, Ana Rath52, Marc Hanauer52, Annie Olry52, David Lagorce52, Svitlana Havrylenko52, Katia Izem52, Fanny Rigour52, Giovanni Stevanin18,19,20,29,30, Alexandra Durr19,20,29,53, Claire-Sophie Davoine19,20,29,30, Léna Guillot-Noel19,20,29,30, Anna Heinzmann19,20,29,54, Giulia Coarelli19,20,29,54, Gisèle Bonne55, Teresinha Evangelista55, Valérie Allamand55, Isabelle Nelson55, Rabah Ben Yaou55,56,57, Corinne Metay55,58, Bruno Eymard55,56, Enzo Cohen55, Antonio Atalaia55, Tanya Stojkovic55,56, Milan Macek Jr.59, Marek Turnovec59, Dana Thomasová59, Radka Pourová Kremliková59, Vera Franková59, Markéta Havlovicová59, Vlastimil Kremlik59, Helen Parkinson60, Thomas Keane60, Dylan Spalding60, Alexander Senf60, Peter Robinson61, Daniel Danis61, Glenn Robert62, Alessia Costa62, Christine Patch62,63, Mike Hanna64, Henry Houlden65, Mary Reilly64, Jana Vandrovcova65, Francesco Muntoni66,67, Irina Zaharieva66, Anna Sarkozy66, Vincent Timmerman31,32, Jonathan Baets13,14,15, Liedewei Van de Vondel13,32, Danique Beijer13,32, Peter de Jonghe14,32, Vincenzo Nigro68,69, Sandro Banfi68,69, Annalaura Torella68, Francesco Musacchia68,69, Giulio Piluso68, Alessandra Ferlini70, Rita Selvatici70, Rachele Rossi70, Marcella Neri70, Stefan Aretz17,71, Isabel Spier17,71, Anna Katharina Sommer71, Sophia Peters71, Carla Oliveira72,73,74, Jose Garcia Pelaez72,73, Ana Rita Matos72,73, Celina São José72,73, Marta Ferreira72,73, Irene Gullo72,73,74, Susana Fernandes72,75, Luzia Garrido76, Pedro Ferreira72,73,77, Fátima Carneiro72,73,74, Morris A. Swertz78, Lennart Johansson78, Joeri K. van der Velde78, Gerben van der Vries78, Pieter B. Neerincx78, Dieuwke Roelofs-Prins78, Sebastian Köhler79, Alison Metcalfe62,80, Alain Verloes81,82, Séverine Drunat81,82, Caroline Rooryck83, Aurelien Trimouille84, Raffaele Castello69, Manuela Morleo69, Michele Pinelli69, Alessandra Varavallo69, Manuel Posada De la Paz85, Eva Bermejo Sánchez85, Estrella López Martín85, Beatriz Martínez Delgado85, F. Javier Alonso García de la Rosa85, Andrea Ciolfi86, Bruno Dallapiccola86, Simone Pizzi86, Francesca Clementina Radio86, Marco Tartaglia86, Alessandra Renieri87,88,89, Elisa Benetti87, Peter Balicza90, Maria Judit Molnar90, Ales Maver91, Borut Peterlin91, Alexander Münchau92, Katja Lohmann92, Rebecca Herzog92, Martje Pauly92, Alfons Macaya93, Anna Marcé-Grau93, Andres Nascimiento Osorio94, Daniel Natera de Benito94, Hanns Lochmüller95,96,97, Rachel Thompson95,97, Kiran Polavarapu95, David Beeson98, Judith Cossins98, Pedro M. Rodriguez Cruz98, Peter Hackman99, Mridul Johari99, Marco Savarese99, Bjarne Udd99,100,101, Rita Horvath102, Gabriel Capella103, Laura Valle103, Elke Holinski-Feder104, Andreas Laner104, Verena Steinke-Lange104, Evelin Schröck105, Andreas Rump105,106

Funding

The Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 779257. Data were analysed using the RD‐Connect Genome‐Phenome Analysis Platform, which received funding from EU projects RD‐Connect, Solve-RD and EJP-RD (Grant Numbers FP7 305444, H2020 779257, H2020 825575), Instituto de Salud Carlos III (Grant Numbers PT13/0001/0044, PT17/0009/0019; Instituto Nacional de Bioinformática, INB) and ELIXIR Implementation Studies. The study was further funded by the Federal Ministry of Education and Research, Germany, through the TreatHSP network (01GM1905 to RS and LS), the National Institute of Neurological Diseases and Stroke (R01NS072248 to SZ and RS), the European Joint Program on Rare Diseases-EJP-RD COFUND-EJP N° 825575 through funding for the PROSPAX consortium (441409627 to MS, RS and BvW). CW was supported by the PATE program of the Medical Faculty, University of Tübingen. CEE received support from the Dutch Princess Beatrix Muscle Fund and the Dutch Spieren voor Spieren Muscle fund. Authors on this paper are members of the European Reference Network for Rare Neurological Diseases (ERN-RND, Project ID 739510). Open Access funding enabled and organized by Projekt DEAL.

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Correspondence to Rebecca Schüle.

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HG receives/has received research support from the Deutsche Forschungsgemeinschaft (DFG), the Bundesministerium für Bildung und Forschung (BMBF), the Bundesministerium für Gesundheit (BMG) and the European Union (EU). He has received consulting fees from Roche. He has received a speaker honorarium from Takeda. The authors declare no competing interests.

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Members of the Solve-RD-DITF-RND and The Solve-RD Consortium are listed in below Acknowledgements.

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Schüle, R., Timmann, D., Erasmus, C.E. et al. Solving unsolved rare neurological diseases—a Solve-RD viewpoint. Eur J Hum Genet 29, 1332–1336 (2021). https://doi.org/10.1038/s41431-021-00901-1

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