Phenotypic and genome-wide studies on dicarbonyls: major associations to glomerular filtration rate and gamma-glutamyltransferase activity.

BACKGROUND: The dicarbonyl compounds methylglyoxal (MG), glyoxal (GO) and 3-deoxyglucosone (3-DG) have been linked to various diseases. However, disease-independent phenotypic and genotypic association studies with phenome-wide and genome-wide reach, respectively, have not been provided. METHODS: MG, GO and 3-DG were measured by LC-MS in 1304 serum samples of two populations (KORA, n = 482; BiDirect, n = 822) and assessed for associations with genome-wide SNPs (GWAS) and with phenome-wide traits. Redundancy analysis (RDA) was used to identify major independent trait associations. FINDINGS: Mutual correlations of dicarbonyls were highly significant, being stronger between MG and GO (ρ = 0.6) than between 3-DG and MG or GO (ρ = 0.4). Significant phenotypic results included associations of all dicarbonyls with sex, waist-to-hip ratio, glomerular filtration rate (GFR), gamma-glutamyltransferase (GGT), and hypertension, of MG and GO with age and C-reactive protein, of GO and 3-DG with glucose and antidiabetics, of MG with contraceptives, of GO with ferritin, and of 3-DG with smoking. RDA revealed GFR, GGT and, in case of 3-DG, glucose as major contributors to dicarbonyl variance. GWAS did not identify genome-wide significant loci. SNPs previously associated with glyoxalase activity did not reach nominal significance. When multiple testing was restricted to the lead SNPs of GWASs on the traits selected by RDA, 3-DG was found to be associated (p = 2.3 × 10-5) with rs1741177, an eQTL of NF-κB inhibitor NFKBIA. INTERPRETATION: This large-scale, population-based study has identified numerous associations, with GFR and GGT being of pivotal importance, providing unbiased perspectives on dicarbonyls beyond the current state. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Munich, German Centre for Cardiovascular Research (DZHK), German Federal Ministry of Research and Education (BMBF).

Episignature analysis of moderate effects and mosaics.

DNA methylation classifiers ("episignatures") help to determine the pathogenicity of variants of uncertain significance (VUS). However, their sensitivity is limited due to their training on unambiguous cases with strong-effect variants so that the classification of variants with reduced effect size or in mosaic state may fail. Moreover, episignature evaluation of mosaics as a function of their degree of mosaicism has not been developed so far. We improved episignatures with respect to three categories. Applying (i) minimum-redundancy-maximum-relevance feature selection we reduced their length by up to one order of magnitude without loss of accuracy. Performing (ii) repeated re-training of a support vector machine classifier by step-wise inclusion of cases in the training set that reached probability scores larger than 0.5, we increased the sensitivity of the episignature-classifiers by 30%. In the newly diagnosed patients we confirmed the association between DNA methylation aberration and age at onset of KMT2B-deficient dystonia. Moreover, we found evidence for allelic series, including KMT2B-variants with moderate effects and comparatively mild phenotypes such as late-onset focal dystonia. Retrained classifiers also can detect mosaics that previously remained below the 0.5-threshold, as we showed for KMT2D-associated Kabuki syndrome. Conversely, episignature-classifiers are able to revoke erroneous exome calls of mosaicism, as we demonstrated by (iii) comparing presumed mosaic cases with a distribution of artificial in silico-mosaics that represented all the possible variation in degree of mosaicism, variant read sampling and methylation analysis.

Epigenetic Association Analyses and Risk Prediction of RLS.

BACKGROUND: As opposed to other neurobehavioral disorders, epigenetic analyses and biomarkers are largely missing in the case of idiopathic restless legs syndrome (RLS). OBJECTIVES: Our aims were to develop a biomarker for RLS based on DNA methylation in blood and to examine DNA methylation in brain tissues for dissecting RLS pathophysiology. METHODS: Methylation of blood DNA from three independent cohorts (n = 2283) and post-mortem brain DNA from two cohorts (n = 61) was assessed by Infinium EPIC 850 K BeadChip. Epigenome-wide association study (EWAS) results of individual cohorts were combined by random-effect meta-analysis. A three-stage selection procedure (discovery, n = 884; testing, n = 520; validation, n = 879) established an epigenetic risk score including 30 CpG sites. Epigenetic age was assessed by Horvath's multi-tissue clock and Shireby's cortical clock. RESULTS: EWAS meta-analysis revealed 149 CpG sites linked to 136 genes (P < 0.05 after Bonferroni correction) in blood and 23 CpG linked to 18 genes in brain (false discovery rate [FDR] < 5%). Gene-set analyses of blood EWAS results suggested enrichments in brain tissue types and in subunits of the kainate-selective glutamate receptor complex. Individual candidate genes of the brain EWAS could be assigned to neurodevelopmental or metabolic traits. The blood epigenetic risk score achieved an area under the curve (AUC) of 0.70 (0.67-0.73) in the validation set, comparable to analogous scores in other neurobehavioral disorders. A significant difference in biological age in blood or brain of RLS patients was not detectable. CONCLUSIONS: DNA methylation supports the notion of altered neurodevelopment in RLS. Epigenetic risk scores are reliably associated with RLS but require even higher accuracy to be useful as biomarkers. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Recessive NUP54 Variants Underlie Early-Onset Dystonia with Striatal Lesions.

Infantile striatonigral degeneration is caused by a homozygous variant of the nuclear-pore complex (NPC) gene NUP62, involved in nucleo-cytoplasmic trafficking. By querying sequencing-datasets of patients with dystonia and/or Leigh(-like) syndromes, we identified 3 unrelated individuals with biallelic variants in NUP54. All variants clustered in the C-terminal protein region that interacts with NUP62. Associated phenotypes were similar to those of NUP62-related disease, including early-onset dystonia with dysphagia, choreoathetosis, and T2-hyperintense lesions in striatum. In silico and protein-biochemical studies gave further evidence for the argument that the variants were pathogenic. We expand the spectrum of NPC component-associated dystonic conditions with localized basal-ganglia abnormalities. ANN NEUROL 2023;93:330-335.

Fast versus slow disease progression in amyotrophic lateral sclerosis-clinical and genetic factors at the edges of the survival spectrum.

Patients with amyotrophic lateral sclerosis (ALS) show substantial differences in disease progression and survival. However, the genetic contribution to the extremes of this spectrum remains poorly characterized. We unbiasedly selected and genotyped 102 ALS patients with very short (<15 months) and 90 with very long survival (>100 months) from the ALS registry of Ulm University using whole-exome sequencing and C9orf72 repeat expansion testing followed by a clinicogenetic correlation analysis. Clinically, groups significantly differed regarding site of disease onset, age at onset, BMI at diagnosis, disease progression rates, and diagnostic latency. We found a monogenic disease cause in 31 patients (16%) without significant differences in patients with short and long survival (19% vs. 13%; p = 0.41), but differences in the genotypic architecture. C9orf72 expansions and FUS mutations were only found in fast progressors, whereas SOD1 variants were frequent in both groups contributing 52% of all monogenic cases-33% among fast and 75% among slow variants. Our genotype-phenotype correlation may be relevant for genetic counseling, estimation of prognosis, and therapeutic decisions.

Reassessment of candidate gene studies for idiopathic restless legs syndrome in a large genome-wide association study dataset of European ancestry.

STUDY OBJECTIVES: Several candidate gene studies have been published for idiopathic restless legs syndrome (RLS) in populations of European ancestry, but the reported associations have not been confirmed in independent samples. Our aim was to reassess these findings in a large case-control dataset in order to evaluate their validity. METHODS: We screened PubMed for RLS candidate gene studies. We used the genome-wide association study (GWAS) dataset of the International EU-RLS-GENE Consortium as our replication sample, which provided genome-wide single-variant association data based on at most 17 220 individuals of European ancestry. We performed additional gene-based tests using the software MAGMA and assessed the power of our study using the genpwr R package. RESULTS: We identified 14 studies conducted in European samples which assessed 45 variants in 27 genes of which 5 variants had been reported as significantly associated. None of these individual variants were replicated in our GWAS-based reassessment (nominal p > 0.05) and gene-based tests for the respective five genes ADH1B, GABRR3, HMOX1, MAOA, and VDR, were also nonsignificant (nominal p > 0.05). Our replication dataset was well powered to detect the reported effects, even when adjusting for effect size overestimation due to winner's curse. Power estimates were close to 100% for all variants. CONCLUSION: In summary, none of the significant single-variant associations from candidate gene studies were confirmed in our GWAS dataset. Therefore, these associations were likely false positive. Our observations emphasize the need for large sample sizes and stringent significance thresholds in future association studies for RLS.

ExomeChip-based rare variant association study in restless legs syndrome.

Restless legs syndrome (RLS) is a common sleep-related movement disorder in populations of European descent and disease risk is strongly influenced by genetic factors. Common variants have been assessed extensively in several genome-wide association studies, but the contribution of rarer genetic variation has not been investigated at this scale. We therefore genotyped a case-control set of 9246 individuals for mainly rare and low frequency exonic variants using the Illumina ExomeChip. However, standard single variant and gene-level association tests were negative. This does not preclude a role of rare variants in RLS, but is likely due to the small sample size and the limited selection of rare genetic variation captured on the array. Therefore, exome or whole genome sequencing should be performed rather than increasing the sample size of ExomeChip studies in order to identify rare risk variants for RLS.

Blood DNA methylation provides an accurate biomarker of KMT2B-related dystonia and predicts onset.

Dystonia is a prevalent, heterogeneous movement disorder characterized by involuntarily abnormal postures. Biomarkers of dystonia are notoriously lacking. Here, a biomarker is reported for histone lysine methyltransferase (KMT2B)-deficient dystonia, a leading subtype among the individually rare monogenic dystonias. It was derived by applying a support vector machine to an episignature of 113 DNA CpG sites, which, in blood cells, showed significant epigenome-wide association with KMT2B deficiency and at least 1× log-fold change of methylation. This classifier was accurate both when tested on the general population and on samples with various other deficiencies of the epigenetic machinery, thus allowing for definitive evaluation of variants of uncertain significance and identifying patients who may profit from deep brain stimulation, a highly successful treatment in KMT2B-deficient dystonia. Methylation was increased in KMT2B deficiency at all 113 CpG sites. The coefficients of variation of the normalized methylation levels at these sites also perfectly classified the samples with KMT2B-deficient dystonia. Moreover, the mean of the normalized methylation levels correlated well with the age at onset of dystonia (P = 0.003)-being lower in samples with late or incomplete penetrance-thus serving as a predictor of disease onset and severity. Similarly, it may also function in monitoring the recently envisioned treatment of KMT2B deficiency by inhibition of DNA methylation.

Intronic elements associated with insomnia and restless legs syndrome exhibit cell-type-specific epigenetic features contributing to MEIS1 regulation.

A highly evolutionarily conserved myeloid ecotropic viral integration site 1 (MEIS1) intronic region is strongly associated with restless legs syndrome (RLS) and insomnia. To understand its regulatory function, we dissected the region by analyzing chromatin accessibility, enhancer-promoter contacts, DNA methylation and expression quantitative trait locus (eQTLs) in different human neural cell types and tissues. We observed specific activity with respect to cell type and developmental maturation, indicating a prominent role for distinct highly conserved intronic elements in forebrain inhibitory neuron differentiation. Two elements were hypomethylated in neural cells with higher MEIS1 expression, suggesting a role of enhancer demethylation in gene regulation. MEIS1 eQTLs showed a striking modular chromosomal distribution, with forebrain eQTLs clustering in intron 8/9. Clustered regularly interspersed short palindromic repeats interference targeting of individual elements in this region attenuated MEIS1 expression, revealing a complex regulatory interplay of distinct elements. In summary, we found that MEIS1 regulation is organized in a modular pattern. Disease-associated intronic regulatory elements control MEIS1 expression with cell type and maturation stage specificity, particularly in the inhibitory neuron lineage. The precise spatiotemporal activity of these elements likely contributes to the pathogenesis of insomnia and RLS.

Clinico-genetic findings in 509 frontotemporal dementia patients.

Frontotemporal dementia (FTD) is a clinically and genetically heterogeneous disorder. To which extent genetic aberrations dictate clinical presentation remains elusive. We investigated the spectrum of genetic causes and assessed the genotype-driven differences in biomarker profiles, disease severity and clinical manifestation by recruiting 509 FTD patients from different centers of the German FTLD consortium where individuals were clinically assessed including biomarker analysis. Exome sequencing as well as C9orf72 repeat analysis were performed in all patients. These genetic analyses resulted in a diagnostic yield of 18.1%. Pathogenic variants in C9orf72 (n = 47), GRN (n = 26), MAPT (n = 11), TBK1 (n = 5), FUS (n = 1), TARDBP (n = 1), and CTSF (n = 1) were identified across all clinical subtypes of FTD. TBK1-associated FTD was frequent accounting for 5.4% of solved cases. Detection of a homozygous missense variant verified CTSF as an FTD gene. ABCA7 was identified as a candidate gene for monogenic FTD. The distribution of APOE alleles did not differ significantly between FTD patients and the average population. Male sex was weakly associated with clinical manifestation of the behavioral variant of FTD. Age of onset was lowest in MAPT patients. Further, high CSF neurofilament light chain levels were found to be related to GRN-associated FTD. Our study provides large-scale retrospective clinico-genetic data such as on disease manifestation and progression of FTD. These data will be relevant for counseling patients and their families.

Scoring Algorithm-Based Genomic Testing in Dystonia: A Prospective Validation Study.

BACKGROUND: Despite the established value of genomic testing strategies, practice guidelines for their use do not exist in many indications. OBJECTIVES: We sought to validate a recently introduced scoring algorithm for dystonia, predicting the diagnostic utility of whole-exome sequencing (WES) based on individual phenotypic aspects (age-at-onset, body distribution, presenting comorbidity). METHODS: We prospectively enrolled a set of 209 dystonia-affected families and obtained summary scores (0-5 points) according to the algorithm. Singleton (N = 146), duo (N = 11), and trio (N = 52) WES data were generated to identify genetic diagnoses. RESULTS: Diagnostic yield was highest (51%) among individuals with a summary score of 5, corresponding to a manifestation of early-onset segmental or generalized dystonia with coexisting non-movement disorder-related neurological symptoms. Sensitivity and specificity at the previously suggested threshold for implementation of WES (3 points) was 96% and 52%, with area under the curve of 0.81. CONCLUSIONS: The algorithm is a useful predictive tool and could be integrated into dystonia routine diagnostic protocols. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Therapeutic effectiveness of thalidomide in a patient with treatment-resistant restless legs syndrome.

None: Recent developments in the genetics of restless legs syndrome (RLS) revealed associations of disease risk with genetic loci containing the genes coding cereblon, the protein bound by thalidomide, and its endogenous substrate MEIS2, whose degradation is inhibited by the thalidomide-cereblon interaction. Therefore it was hypothesized that thalidomide may be a potential treatment option for RLS. Here we report on the therapeutic effect of thalidomide in a patient with otherwise treatment-resistant RLS who received 100 mg thalidomide off-label for 3 weeks. The female patient, severely affected by RLS before treatment, experienced significant amelioration of the symptoms, increased self-reported sleep quality, and better daytime functioning during thalidomide treatment. This therapeutic success warrants larger studies investigating the efficacy of drugs of the thalidomide class in RLS.

Lifetime risk of autosomal recessive mitochondrial disorders calculated from genetic databases.

BACKGROUND: Mitochondrial disorders are a group of rare diseases, caused by nuclear or mitochondrial DNA mutations. Their marked clinical and genetic heterogeneity as well as referral and ascertainment biases render phenotype-based prevalence estimations difficult. Here we calculated the lifetime risk of all known autosomal recessive mitochondrial disorders on basis of genetic data. METHODS: We queried the publicly available Genome Aggregation Database (gnomAD) and our in-house exome database to assess the allele frequency of disease-causing variants in genes associated with autosomal recessive mitochondrial disorders. Based on this, we estimated the lifetime risk of 249 autosomal recessive mitochondrial disorders. Three of these disorders and phenylketonuria (PKU) served as a proof of concept since calculations could be aligned with known birth prevalence data from newborn screening reports. FINDINGS: The estimated lifetime risks are very close to newborn screening data (where available), supporting the validity of the approach. For example, calculated lifetime risk of PKU (16·0/100,000) correlates well with known birth prevalence data (18·7/100,000). The combined estimated lifetime risk of 249 investigated mitochondrial disorders is 31·8 (20·9-50·6)/100,000 in our in-house database, 48·4 (40·3-58·5)/100,000 in the European gnomAD dataset, and 31·1 (26·7-36·3)/100,000 in the global gnomAD dataset. The disorders with the highest lifetime risk (> 3 per 100,000) were, in all datasets, those caused by mutations in the SPG7, ACADM, POLG and SLC22A5 genes. INTERPRETATION: We provide a population-genetic estimation on the lifetime risk of an entire class of monogenic disorders. Our findings reveal the substantial cumulative prevalence of autosomal recessive mitochondrial disorders, far above previous estimates. These data will be very important for assigning diagnostic a priori probabilities, and for resource allocation in therapy development, public health management and biomedical research. FUNDING: German Federal Ministry of Education and Research.

Identification of Restless Legs Syndrome Genes by Mutational Load Analysis.

OBJECTIVE: Restless legs syndrome is a frequent neurological disorder with substantial burden on individual well-being and public health. Genetic risk loci have been identified, but the causatives genes at these loci are largely unknown, so that functional investigation and clinical translation of molecular research data are still inhibited. To identify putatively causative genes, we searched for highly significant mutational burden in candidate genes. METHODS: We analyzed 84 candidate genes in 4,649 patients and 4,982 controls by next generation sequencing using molecular inversion probes that targeted mainly coding regions. The burden of low-frequency and rare variants was assessed, and in addition, an algorithm (binomial performance deviation analysis) was established to estimate independently the sequence variation in the probe binding regions from the variation in sequencing depth. RESULTS: Highly significant results (considering the number of genes in the genome) of the conventional burden test and the binomial performance deviation analysis overlapped significantly. Fourteen genes were highly significant by one method and confirmed with Bonferroni-corrected significance by the other to show a differential burden of low-frequency and rare variants in restless legs syndrome. Nine of them (AAGAB, ATP2C1, CNTN4, COL6A6, CRBN, GLO1, NTNG1, STEAP4, VAV3) resided in the vicinity of known restless legs syndrome loci, whereas 5 (BBS7, CADM1, CREB5, NRG3, SUN1) have not previously been associated with restless legs syndrome. Burden test and binomial performance deviation analysis also converged significantly in fine-mapping potentially causative domains within these genes. INTERPRETATION: Differential burden with intragenic low-frequency variants reveals putatively causative genes in restless legs syndrome. ANN NEUROL 2020;87:184-193.

[Exome diagnostics in neurology]. / Exomdiagnostik in der Neurologie.

After an impressively successful application as a research instrument, whole-exome sequencing (WES) now enters the clinical practice due to its high diagnostic, time, and economic efficiency. WES is the diagnostic method of choice for symptoms that may be due to many different monogenic causes. Neurological indications include movement disorders, especially in cases of early symptom onset, familial clustering and complex manifestation. Starting from a blood sample, enrichment and sequencing of the exome enable the examination of all coding DNA regions for point mutations and small insertions/deletions. The identification of variants as the cause of a disease requires a professional evaluation pipeline, variant prioritization schemes and variant classification databases. Whereas many variants can be reliably classified as pathogenic or benign, variants of unclear significance (VUS) remain a challenge for the clinical evaluation and necessitate a periodic reanalysis of WES data. As a genetic examination WES requires adequate patient informed consent which in particular should address possible secondary findings as well as data security. A positive molecular result ends diagnostic odysseys, enables accurate genetic counseling and can point to targeted preventive measures and treatment. A WES significantly contributes to the understanding of the genetic architecture and pathophysiology of neurological diseases, enriching and enabling precision medicine.

Common Grounds for Family Maladies.

Rare variants cause Mendelian family aggregation in subsets of common diseases, and common variants may contribute to rare diseases. In this issue of Neuron, Gormley et al. (2018) report that the common variant burden in familial migraine is larger than in migraine of the general population.

Identification of novel risk loci for restless legs syndrome in genome-wide association studies in individuals of European ancestry: a meta-analysis.

BACKGROUND: Restless legs syndrome is a prevalent chronic neurological disorder with potentially severe mental and physical health consequences. Clearer understanding of the underlying pathophysiology is needed to improve treatment options. We did a meta-analysis of genome-wide association studies (GWASs) to identify potential molecular targets. METHODS: In the discovery stage, we combined three GWAS datasets (EU-RLS GENE, INTERVAL, and 23andMe) with diagnosis data collected from 2003 to 2017, in face-to-face interviews or via questionnaires, and involving 15 126 cases and 95 725 controls of European ancestry. We identified common variants by fixed-effect inverse-variance meta-analysis. Significant genome-wide signals (p≤5 × 10-8) were tested for replication in an independent GWAS of 30 770 cases and 286 913 controls, followed by a joint analysis of the discovery and replication stages. We did gene annotation, pathway, and gene-set-enrichment analyses and studied the genetic correlations between restless legs syndrome and traits of interest. FINDINGS: We identified and replicated 13 new risk loci for restless legs syndrome and confirmed the previously identified six risk loci. MEIS1 was confirmed as the strongest genetic risk factor for restless legs syndrome (odds ratio 1·92, 95% CI 1·85-1·99). Gene prioritisation, enrichment, and genetic correlation analyses showed that identified pathways were related to neurodevelopment and highlighted genes linked to axon guidance (associated with SEMA6D), synapse formation (NTNG1), and neuronal specification (HOXB cluster family and MYT1). INTERPRETATION: Identification of new candidate genes and associated pathways will inform future functional research. Advances in understanding of the molecular mechanisms that underlie restless legs syndrome could lead to new treatment options. We focused on common variants; thus, additional studies are needed to dissect the roles of rare and structural variations. FUNDING: Deutsche Forschungsgemeinschaft, Helmholtz Zentrum München-Deutsches Forschungszentrum für Gesundheit und Umwelt, National Research Institutions, NHS Blood and Transplant, National Institute for Health Research, British Heart Foundation, European Commission, European Research Council, National Institutes of Health, National Institute of Neurological Disorders and Stroke, NIH Research Cambridge Biomedical Research Centre, and UK Medical Research Council.

Analysis of Factor D Isoforms in Malpuech-Michels-Mingarelli-Carnevale Patients Highlights the Role of MASP-3 as a Maturase in the Alternative Pathway of Complement.

Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation. However, the necessity of MASP-3 for pro-FD cleavage has been questioned, because AP activity is still observed in sera from MASP-1/3-deficient Malpuech-Michels-Mingarelli-Carnevale (3MC) patients. The identification of a novel 3MC patient carrying a previously unidentified MASP-3 G665S mutation prompted us to develop an analytical isoelectric focusing technique that resolves endogenous FD variants in complex samples. This enabled us to show that although 3MC patients predominantly contain pro-FD, they also contain detectable levels of mature FD. Moreover, using isoelectric focusing analysis, we show that both pro-FD and FD are present in the circulation of healthy donors. We characterized the naturally occurring 3MC-associated MASP-3 mutants and found that they all yielded enzymatically inactive proteins. Using MASP-3-depleted human serum, serum from 3MC patients, and Masp1/3-/- mice, we found that lack of enzymatically active MASP-3, or complete MASP-3 deficiency, compromises the conversion of pro-FD to FD. In summary, our observations emphasize that MASP-3 acts as an important maturase in the AP of complement, while also highlighting that there exists MASP-3-independent pro-FD maturation in 3MC patients.