Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

BiP inactivation due to loss of the deAMPylation function of FICD causes a motor neuron disease.

PURPOSE: The chaperone protein BiP is the master regulator of the unfolded protein response in the endoplasmic reticulum. BiP chaperone activity is regulated by the post-translational modification AMPylation, exclusively provided by FICD. We investigated whether FICD variants identified in patients with motor neuron disease could interfere with BiP activity regulation. METHODS: Exome sequencing was performed to identify causative pathogenic variants associated with motor neuron diseases. Functional studies were conducted on fibroblasts from patients to explore the molecular mechanism of the disease. RESULTS: We identified biallelic variants in FICD causing a neurodegenerative disease of upper and lower motor neurons. Affected individuals harbor a specific missense variant, Arg374His, positioned in the catalytic motif of the enzyme and important for adenosine triphosphate binding. The mutated residue abolishes intramolecular interaction with the regulatory residue Glu234, essential to inhibit AMPylation and to promote de-AMPylation by FICD. Consequently, fibroblasts from patients with FICD variants have abnormally increased levels of AMPylated and thus inactivated BiP. CONCLUSION: Loss of BiP chaperone activity in patients likely results in a chronic impairment of the protein quality control system in the endoplasmic reticulum. These findings will guide the development of therapeutic strategies for motoneuron and related diseases linked to proteotoxic stress.

De Novo and Dominantly Inherited SPTAN1 Mutations Cause Spastic Paraplegia and Cerebellar Ataxia.

BACKGROUND: Pathogenic variants in SPTAN1 have been linked to a remarkably broad phenotypical spectrum. Clinical presentations include epileptic syndromes, intellectual disability, and hereditary motor neuropathy. OBJECTIVES: We investigated the role of SPTAN1 variants in rare neurological disorders such as ataxia and spastic paraplegia. METHODS: We screened 10,000 NGS datasets across two international consortia and one local database, indicative of the level of international collaboration currently required to identify genes causative for rare disease. We performed in silico modeling of the identified SPTAN1 variants. RESULTS: We describe 22 patients from 14 families with five novel SPTAN1 variants. Of six patients with cerebellar ataxia, four carry a de novo SPTAN1 variant and two show a sporadic inheritance. In this group, one variant (p.Lys2083del) is recurrent in four patients. Two patients have novel de novo missense mutations (p.Arg1098Cys, p.Arg1624Cys) associated with cerebellar ataxia, in one patient accompanied by intellectual disability and epilepsy. We furthermore report a recurrent missense mutation (p.Arg19Trp) in 15 patients with spastic paraplegia from seven families with a dominant inheritance pattern in four and a de novo origin in one case. One further patient carrying a de novo missense mutation (p.Gln2205Pro) has a complex spastic ataxic phenotype. Through protein modeling we show that mutated amino acids are located at crucial interlinking positions, interconnecting the three-helix bundle of a spectrin repeat. CONCLUSIONS: We show that SPTAN1 is a relevant candidate gene for ataxia and spastic paraplegia. We suggest that for the mutations identified in this study, disruption of the interlinking of spectrin helices could be a key feature of the pathomechanism. © 2022 International Parkinson and Movement Disorder Society.

Overarching pathomechanisms in inherited peripheral neuropathies, spastic paraplegias, and cerebellar ataxias.

International consortia collaborating on the genetics of rare diseases have significantly boosted our understanding of inherited neurological disorders. Historical clinical classification boundaries were drawn between disorders with seemingly different etiologies, such as inherited peripheral neuropathies (IPNs), spastic paraplegias, and cerebellar ataxias. These clinically defined borders are being challenged by the identification of mutations in genes displaying wide phenotypic spectra and by shared pathomechanistic themes, which are valuable indications for therapy development. We highlight common cellular alterations that underlie this genetic landscape, including alteration of cytoskeleton, axonal transport, mitochondrial function, and DNA repair response. Finally, we discuss venues for future research using the long axonopathies of the PNS as a model to explore other neurogenetic disorders.

Biallelic variants in COX18 cause a mitochondrial disorder primarily manifesting as peripheral neuropathy.

Defects in mitochondrial dynamics are a common cause of Charcot-Marie-Tooth disease (CMT), while primary deficiencies in the mitochondrial respiratory chain (MRC) are rare and atypical for this etiology. This study aims to report COX18 as a novel CMT-causing gene. This gene encodes an assembly factor of mitochondrial Complex IV (CIV) that translocates the C-terminal tail of MTCO2 across the mitochondrial inner membrane. Exome sequencing was performed in four affected individuals. The patients and available family members underwent thorough neurological and electrophysiological assessment. The impact of one of the identified variants on splicing, protein levels, and mitochondrial bioenergetics was investigated in patient-derived lymphoblasts. The functionality of the mutant protein was assessed using a Proteinase K protection assay and immunoblotting. Neuronal relevance of COX18 was assessed in a Drosophila melanogaster knockdown model. Exome sequencing coupled with homozygosity mapping revealed a homozygous splice variant c.435-6A>G in COX18 in two siblings with early-onset progressive axonal sensory-motor peripheral neuropathy. By querying external databases, we identified two additional families with rare deleterious biallelic variants in COX18 . All affected individuals presented with axonal CMT and some patients also exhibited central nervous system symptoms, such as dystonia and spasticity. Functional characterization of the c.435-6A>G variant demonstrated that it leads to the expression of an alternative transcript that lacks exon 2, resulting in a stable but defective COX18 isoform. The mutant protein impairs CIV assembly and activity, leading to a reduction in mitochondrial membrane potential. Downregulation of the COX18 homolog in Drosophila melanogaster displayed signs of neurodegeneration, including locomotor deficit and progressive axonal degeneration of sensory neurons. Our study presents genetic and functional evidence that supports COX18 as a newly identified gene candidate for autosomal recessive axonal CMT with or without central nervous system involvement. These findings emphasize the significance of peripheral neuropathy within the spectrum of primary mitochondrial disorders and the role of mitochondrial CIV in the development of CMT. Our research has important implications for the diagnostic workup of CMT patients.

Comprehensive reanalysis for CNVs in ES data from unsolved rare disease cases results in new diagnoses.

We report the results of a comprehensive copy number variant (CNV) reanalysis of 9171 exome sequencing datasets from 5757 families affected by a rare disease (RD). The data reanalysed was extremely heterogeneous, having been generated using 28 different enrichment kits by 42 different research groups across Europe partnering in the Solve-RD project. Each research group had previously undertaken their own analysis of the data but failed to identify disease-causing variants. We applied three CNV calling algorithms to maximise sensitivity, and rare CNVs overlapping genes of interest, provided by four partner European Reference Networks, were taken forward for interpretation by clinical experts. This reanalysis has resulted in a molecular diagnosis being provided to 51 families in this sample, with ClinCNV performing the best of the three algorithms. We also identified partially explanatory pathogenic CNVs in a further 34 individuals. This work illustrates the value of reanalysing ES cold cases for CNVs.

Heterozygous loss-of-function variants in SPTAN1 cause a novel early childhood onset distal myopathy with chronic neurogenic features.

Background: Neurogenetic disorders caused by pathogenic variants in four genes encoding non-erythrocytic spectrins ( SPTAN1, SPTBN1, SPTBN2, SPTBN4) range from peripheral and central nervous system involvement to complex syndromic presentations. Heterozygous pathogenic variants in SPTAN1 are exemplary for this diversity with phenotypes spanning almost the entire spectrum. Methods: Through international collaboration we identified 14 families with genetically unsolved distal weakness and unreported heterozygous SPTAN1 loss-of-function variants including frameshift, nonsense and splice-acceptor variants. Clinical data, electrophysiology, muscle CT or MRI and muscle biopsy findings were collected and standardized. SPTAN1 protein, mRNA expression analysis and cDNA sequencing was performed on muscle tissue from two patients. Results: All 20 patients presented with early childhood onset distal weakness. The severity varied both within families and between different families. Foot abnormalities ranged from hammer toes and pes cavus to distal arthrogryposis. Electrophysiology showed mixed myogenic and neurogenic features. Muscle MRI or CT in 10 patients showed fatty infiltration of the distal lower limb anterior compartment and/or selective involvement of the extensor hallucis longus muscle. Muscle biopsy revealed myopathic changes with mild dystrophic and chronic neurogenic changes in 7 patients. Finally, we provide proof for nonsense mediated decay in tissues derived from two patients. Conclusions: We provide evidence for the association of SPTAN1 loss-of-function variants with childhood onset distal myopathy in 14 families. This finding extends the phenotypic spectrum of SPTAN1 loss-of-function variants ranging from intellectual disability to distal weakness with a predominant myogenic cause. KEY MESSAGES: SPTAN1 loss-of-function variants, including frameshift, nonsense and splice site variants cause a novel childhood onset distal weakness syndrome with primarily skeletal muscle involvement. Hereditary motor neuropathies and distal myopathic disorders present a well-known diagnostic challenge as they demonstrate substantial clinical and genetic overlap. The emergence of SPTAN1 loss-of-function variants serves as a noteworthy example, highlighting a growing convergence in the spectrum of genotypes linked to both hereditary motor neuropathies and distal myopathies.

Unravelling undiagnosed rare disease cases by HiFi long-read genome sequencing.

Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

Translational Diagnostics: An In-House Pipeline to Validate Genetic Variants in Children with Undiagnosed and Rare Diseases.

Diagnosis is essential for the management and treatment of patients with rare diseases. In a group of patients, the genetic study identifies variants of uncertain significance or inconsistent with the phenotype; therefore, it is urgent to develop novel strategies to reach the definitive diagnosis. Herein, we develop the in-house Translational Diagnostics Program (TDP) to validate genetic variants as part of the diagnostic process with the close collaboration of physicians, clinical scientists, and research scientists. The first 7 of 33 consecutive patients for whom exome-based tests were not diagnostic were investigated. The TDP pipeline includes four steps: (i) phenotype assessment, (ii) literature review and prediction of in silico pathogenicity, (iii) experimental functional studies, and (iv) diagnostic decision-making. Re-evaluation of the phenotype and re-analysis of the exome allowed the diagnosis in one patient. In the remaining patients, the studies included either cDNA cloning or PCR-amplified genomic DNA, or the use of patients' fibroblasts. A comparative computational analysis of confocal microscopy images and studies related to the protein function was performed. In five of these six patients, evidence of pathogenicity of the genetic variant was found, which was validated by physicians. The current research demonstrates the feasibility of the TDP to support and resolve intramural medical problems when the clinical significance of the patient variant is unknown or inconsistent with the phenotype.