Abstract Biallelic pathogenic repeat expansions in RFC1 were recently identified as molecular origin of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) as well as of one of the ...most common causes of adult-onset ataxia. In the meantime, the phenotypic spectrum has expanded massively and now includes mimics of multiple system atrophy or parkinsonism. After identifying a patient with a clinical diagnosis of amyotrophic lateral sclerosis (ALS) as a carrier of biallelic pathogenic repeat expansions in RFC1 , we studied a cohort of 106 additional patients with a clinical main phenotype of motor neuron disease (MND) to analyze whether such repeat expansions are more common in MND patients. Indeed, two additional MND patients (one also with ALS and one with primary lateral sclerosis/PLS) have been identified as carrier of biallelic pathogenic repeat expansions in RFC1 in the absence of another genetic alteration explaining the phenotype, suggesting motor neuron disease as another extreme phenotype of RFC1 spectrum disorder. Therefore, MND might belong to the expanding phenotypic spectrum of pathogenic RFC1 repeat expansions, particularly in those MND patients with additional features such as sensory and/or autonomic neuropathy, vestibular deficits, or cerebellar signs. By systematically analyzing the RFC1 repeat array using Oxford nanopore technology long-read sequencing, our study highlights the high intra- and interallelic heterogeneity of this locus and allows the identification of the novel repeat motif ‘ACAAG’.
Abstract
Instability of simple DNA repeats has been known as a common cause of hereditary ataxias for over 20 years. Routine genetic diagnostics of these phenotypically similar diseases still rely on ...an iterative workflow for quantification of repeat units by PCR-based methods of limited precision.
We established and validated clinical nanopore Cas9-targeted sequencing, an amplification-free method for simultaneous analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. The method combines target enrichment by CRISPR–Cas9, Oxford Nanopore long-read sequencing and a bioinformatics pipeline using the tools STRique and Megalodon for parallel detection of length, sequence, methylation and composition of the repeat loci.
Clinical nanopore Cas9-targeted sequencing allowed for the precise and parallel analysis of 10 repeat loci associated with adult-onset ataxia and revealed additional parameter such as FMR1 promotor methylation and repeat sequence required for diagnosis at the same time. Using clinical nanopore Cas9-targeted sequencing we analysed 100 clinical samples of undiagnosed ataxia patients and identified causative repeat expansions in 28 patients. Parallel repeat analysis enabled a molecular diagnosis of ataxias independent of preconceptions on the basis of clinical presentation. Biallelic expansions within RFC1 were identified as the most frequent cause of ataxia. We characterized the RFC1 repeat composition of all patients and identified a novel repeat motif, AGGGG.
Our results highlight the power of clinical nanopore Cas9-targeted sequencing as a readily expandable workflow for the in-depth analysis and diagnosis of phenotypically overlapping repeat expansion disorders.
Erdmann et al. establish and validate Clin-CATS, an amplification-free method for parallel analysis of 10 repeat loci associated with clinically overlapping hereditary ataxias. They apply the method to 100 undiagnosed patients and identify causative repeat expansions in 28 individuals, including biallelic expansions in RFC1.
Abstract
Genetic diagnosis of facioscapulohumeral muscular dystrophy (FSHD) remains a challenge in clinical practice as it cannot be detected by standard sequencing methods despite being the third ...most common muscular dystrophy. The conventional diagnostic strategy addresses the known genetic parameters of FSHD: the required presence of a permissive haplotype, a size reduction of the D4Z4 repeat of chromosome 4q35 (defining FSHD1) or a pathogenic variant in an epigenetic suppressor gene (consistent with FSHD2). Incomplete penetrance and epistatic effects of the underlying genetic parameters as well as epigenetic parameters (D4Z4 methylation) pose challenges to diagnostic accuracy and hinder prediction of clinical severity.
In order to circumvent the known limitations of conventional diagnostics and to complement genetic parameters with epigenetic ones, we developed and validated a multistage diagnostic workflow that consists of a haplotype analysis and a high-throughput methylation profile analysis (FSHD-MPA). FSHD-MPA determines the average global methylation level of the D4Z4 repeat array as well as the regional methylation of the most distal repeat unit by combining bisulphite conversion with next-generation sequencing and a bioinformatics pipeline and uses these as diagnostic parameters. We applied the diagnostic workflow to a cohort of 148 patients and compared the epigenetic parameters based on FSHD-MPA to genetic parameters of conventional genetic testing. In addition, we studied the correlation of repeat length and methylation level within the most distal repeat unit with age-corrected clinical severity and age at disease onset in FSHD patients. The results of our study show that FSHD-MPA is a powerful tool to accurately determine the epigenetic parameters of FSHD, allowing discrimination between FSHD patients and healthy individuals, while simultaneously distinguishing FSHD1 and FSHD2. The strong correlation between methylation level and clinical severity indicates that the methylation level determined by FSHD-MPA accounts for differences in disease severity among individuals with similar genetic parameters. Thus, our findings further confirm that epigenetic parameters rather than genetic parameters represent FSHD disease status and may serve as a valuable biomarker for disease status.
Erdmann et al. analyse the correlation between methylation of the D4Z4 locus and clinical status in FSHD, and show that epigenetic rather than genetic parameters determine disease manifestation and severity. Distal methylation can thus serve as a powerful diagnostic but also prognostic marker.
X-ray structures of enamines and iminium ions derived from 2-tritylpyrrolidine (Maruoka catalyst) and 2-(triphenylsilyl)pyrrolidine (Bolm–Christmann–Strohmann catalyst) have been determined. Kinetic ...investigations showed that enamines derived from phenylacetaldehyde and pyrrolidine (R = H) or 2-(triphenylsilyl)pyrrolidine (R = SiPh3) have similar reactivities toward benzhydryl cations Ar2CH+ (reference electrophiles), while the corresponding enamine derived from 2-tritylpyrrolidine (R = CPh3) is 26 times less reactive. The rationalization of this phenomenon by negative hyperconjugative interaction of the trityl group with the lone pair of the enamine nitrogen is supported by the finding that the trityl group in the 2-position of the pyrrolidine increases the electrophilic reactivity of iminium ions derived from cinnamaldehyde by a factor of 14. The consequences of these observations for the rationalization of the reactivity of the Jørgensen–Hayashi catalyst (diphenylprolinol trimethylsilyl ether) are discussed.
We synthesised and characterised four copper complexes (with copper in the oxidation states I and II) with the bis(pyrazolyl)methane ligands HC(3‐tBuPz)2(Py) and HC(3‐tBuPz)2(Qu). With the quinolinyl ...ligand (2‐quinolinyl)bis(3‐tert‐butylpyrazolyl)methane HC(3‐tBuPz)2(Qu) we obtained the tetrahedral monofacial complex CuCl{HC(3‐tBuPz)2(Qu)} (C1) and with the pyridinyl ligand (2‐pyridinyl)bis(3‐tert‐butylpyrazolyl)methane HC(3‐tBuPz)2(Py) we obtained the three complexes CuCl{HC(3‐tBuPz)2(Py)} (C2), CuBr2{HC(3‐tBuPz)2(Py)} (C3) and CuCl2{HC(3‐tBuPz)2(Py)} (C4), which are also monofacially coordinated. The molecular structures were analysed and compared with density functional theory calculations, which included natural bond orbital (NBO) analysis. C1 can, when generated in situ, serve as part of a precursor, used for the activation of oxygen as tyrosinase model. We observe the self‐assembly of a peroxo–dicopper complex P with the HC(3‐tBuPz)2(Qu) ligand, which is able to perform catalytic hydroxylation catalysis with phenols. DFT calculations were also carried out to understand the electronic transitions responsible for the UV/Vis bands in the corresponding spectra of the peroxo species.
Herein, we present four new bis(pyrazolyl)methane–copper complexes. Furthermore, we studied the self‐assembly of a peroxo–dicopper species with catalytic hydroxylation activity of phenols by UV/Vis spectroscopy. The donor competition between pyrazolyl and pyridinyl units as well as the UV transitions of the tyrosinase model have been investigated by density functional theory.
Bis(pyrazolyl)methane ligands are excellent components of model complexes used to investigate the activity of the enzyme tyrosinase. Combining the N donors 3‐tert‐butylpyrazole and 1‐methylimidazole ...results in a ligand that is capable of stabilising a (μ‐η2:η2)‐dicopper(II) core that resembles the active centre of tyrosinase. UV/Vis spectroscopy shows blueshifted UV bands in comparison to other known peroxo complexes, due to donor competition from different ligand substituents. This effect was investigated with the help of theoretical calculations, including DFT and natural transition orbital analysis. The peroxo complex acts as a catalyst capable of hydroxylating a variety of phenols by using oxygen. Catalytic conversion with the non‐biological phenolic substrate 8‐hydroxyquinoline resulted in remarkable turnover numbers. In stoichiometric reactions, substrate‐binding kinetics was observed and the intrinsic hydroxylation constant, kox, was determined for five phenolates. It was found to be the fastest hydroxylation model system determined so far, reaching almost biological activity. Furthermore, Hammett analysis proved the electrophilic character of the reaction. This sheds light on the subtle role of donor strength and its influence on hydroxylation activity.
Enhancing reactivity: Efficient catalysis is shown by a copper peroxide tyrosinase model complex consisting of pyrazolyl and imidazolyl moieties. The important role of the third N donor function (turquoise in figure) is highlighted in a reactivity study including a Hammett analysis.
•LRSAM1 mutations define the subgroup of axonal neuropathy CMT2P.•Clinical and electrophysiological data of 14 patients out of 12 families confirm a late onset axonal neuropathy with predominance of ...sensorimotor impairment.•Due to variable inheritance patterns and clustering of pathogenic variants in 3´-prime exons, interpretation of genetic variants in LRSAM1 is challenging.•The majority follows dominant inheritance, variants at the 3`end may or may not escape from nonsense-mediated decay (NMD), thereby defining the pattern of inheritance.
More than 80 genes are known to be associated with Charcot-Marie-Tooth disease (CMT). Mutations of LRSAM1 were identified as a rare cause and define the subgroup of axonal neuropathy CMT2P. We identified additional 14 patients out of 12 families. Clinical and electrophysiological data confirm a late-onset axonal neuropathy with a predominance of sensorimotor impairment. The patients harbored ten different variants in LRSAM1, seven of which were novel. Due to variable inheritance patterns and clustering of pathogenic variants in 3´-prime exons, interpretation of genetic variants in LRSAM1 is challenging. The majority follows dominant inheritance, whereas recessive inheritance has been described for one variant. Variants at the 3`end may or may not escape from nonsense-mediated decay, thereby defining the pattern of inheritance. Our data emphasize the importance of the C-terminal RING domain, which exerts a dominant-negative effect on protein function, whenever affected by an altered or truncated protein. In conclusion, CMT2P is a rare, but nevertheless relevant cause of adult-onset axonal and painful neuropathy. ACMG (American College of Medical Genetics and genomics) criteria should be carefully applied in variant interpretation, with special attention to premature termination codon-introducing variants and their location within the gene.
Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease, with no effective pharmacological treatment. Its pathogenesis is unknown, although a subset of the cases is linked to ...genetic mutations. A significant fraction of the mutations occur in one protein, copper, zinc superoxide dismutase (SOD1). The toxic function of mutant SOD1 has not been elucidated, but damage to the metal site of the protein is believed to play a major role. In this work, we study the electrostatic loop of SOD1, which we had previously proposed to work as a "solvent seal" isolating the metal site from water molecules. Out of the five contact points identified between the electrostatic loop and its dock in the rest of the protein, three points were found to be affected by ALS-linked mutations, with a total of five mutations identified. The effect of the five mutations was studied using methods of computational chemistry. We found that four of the mutations destabilize the proposed solvent seal, while the fifth mutation directly affects the metal-site stability. In the two contact points unaffected by ALS-linked mutations, the side chains of the residues were not found to play a stabilizing role. Our results show that the docking of the electrostatic loop to the rest of SOD1 plays a role in ALS pathogenesis, in support of that structure acting as a solvent barrier for the metal site. The results provide a unified pathogenic mechanism for five different ALS-linked mutations of SOD1.
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