Monogenic variants in dystonia: an exome-wide sequencing study

Abstract

Background: Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia.

Methods: For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow.

Findings: We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism.

Interpretation: In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations.

Funding: Else Kröner-Fresenius-Stiftung, Technische Universität München, Helmholtz Zentrum München, Medizinische Universität Innsbruck, Charles University in Prague, Czech Ministry of Education, the Slovak Grant and Development Agency, the Slovak Research and Grant Agency.

Figure 1. The landscape of genetic etiologies in the dystonia cohort

(A) Spectrum of genes containing diagnostic variants. By using WES, a total of 135 presumptive diagnoses were established, representing 78 distinct disease entities. The counts of individuals with presumptive diagnosis per gene are indicated for different dystonia clinical categories. VUS refers to genes in which variants were formally classified as variants of uncertain significance. Non-MD symptoms, non-movement disorder-related neurological symptoms.

Figure 2. Pie charts summarizing the molecular diagnostic results for the dystonia cohort

(A) Overall presumptive diagnostic rates for different dystonia clinical categories, represented by 388 (isolated dystonia), 98 (combined dystonia), and 222 (isolated or combined dystonia with coexisting non-movement disorder-related neurological symptoms) individuals. “Known gene/novel variant” includes genes with compound heterozygous variants if at least one identified variant was novel. Non-MD symptoms, non-movement disorder-related neurological symptoms. (B) - (D) Characteristics of diagnostic variants. Distribution of variants by inheritance pattern (B) and type (C). Percentages shown for “variant inheritance” and “variant type” do not total 100 because of rounding. Of the 160 variants identified, 91 were not found in ClinVar or the literature (D). Additional variant characteristics can be found in the appendix (p-16) (E) Proposed novel associations between known disease genes and dystonia. There were 13 individuals carrying diagnostic variants in 11 genes (green box) whose dystonia manifestations were interpreted as expansions of the previously appreciated gene-specific phenotypes. For the remaining 66 variant-harboring genes, dystonia and/or (dystonic) tremor has already been documented to be part of the associated disease spectra. VUS refers to a gene in which variants were formally classified as variants of uncertain significance. (F) Fraction of 135 WES-based diagnoses that pointed towards a clinical management and/or treatment implication. Breakdown by practical importance is shown (for details, see appendix pp-50-51).

Figure 3. Clinical score predicting the diagnostic success rate of WES in individuals with dystonia

(A) Schematic overview of the proposed scoring system. We selected as scoring parameters clinical predictors of a diagnostic WES finding, as determined by multiple logistic regression analysis (appendix pp-52-53). The assigned scoring points add up to yield a summary score, ranging from 0–5. Non-MD symptoms, non-movement disorder-related neurological symptoms. (B) Receiver operating characteristic (ROC) curve plot for the proposed score with indication of the specificities and sensitivities (specificity, sensitivity) at the thresholds postulated in (C). A summary score threshold of 3 points implies a low rate (4%) of individuals that are erroneously excluded from WES and an acceptable rate (38%) of erroneously included subjects. (C) Summary scores (0–5), diagnostic yields, and recommendations for the clinical application of WES in dystonia. On the basis of our diagnostic WES results and multivariable logistic model, we suggest to routinely implement WES in dystonia diagnostics if the derived summary score is 3 or higher.

Figure 4. The landscape of genetic etiologies associated with dystonic cerebral palsy

(A) Double ring diagram correlating dystonic cerebral palsy (DCP) subtypes (outer ring) and the number of individuals who received a presumptive diagnosis via WES (inner ring). “Known gene/novel variant” includes genes with compound heterozygous variants if at least one identified variant was novel. Of the individuals with idiopathic DCP, 66% had a diagnostic variant. Overall, we were able to define the (likely) etiology of disease for three quarters of DCP-affected cases (variant-positive individuals plus individuals who showed evidence of perinatal brain injury). (B) Spectrum of genes containing diagnostic variants in the DCP cohort. By using WES, a total of 37 presumptive diagnoses were established, representing 27 distinct disease entities. The counts of variant-harboring individuals per gene are shown. SPAST carried diagnostic variants in 9% of individuals with idiopathic DCP. VUS refers to a gene in which variants were formally classified as variants of uncertain significance.

Figure 5. Spatially clustering de novo variants in IMPDH2 are associated with a neurodevelopmental disorder with or without dystonia

(A) Pedigrees with de novo IMPDH2 variants. Symbols are defined as follows: square, male; circle, female; filled, neurodevelopmental disorder (NDD)-affected (with or without dystonia); empty, unaffected; wt/wt denotes homozygous wild-type sequence, and wt/m1–6 denotes a heterozygous IMPDH2 variant; m1, c.338G>A (p.Gly113Glu); m2, c.337G>A (p.Gly113Arg); m3, c.478_480delTCC (p.Ser160del); m4, c.729G>C (p.Gln243His); m5, c.619G>C (p.Gly207Arg); m6, c.619G>A (p.Gly207Arg); NA, no DNA available. All families were analyzed by trio WES (indicated with asterisks). (B) Linear view of inosine-5`-monophosphate dehydrogenase 2 (IMPDH2). The herein described variants are situated in and around the cystathionine-β-synthase (CBS) domain, a regulatory element in which clustering of pathogenic missense variants has been already demonstrated for IMPDH2`s homolog, IMPDH1^,. Note variant recurrences at positions gly113 and gly207.