Comparison of the ABC and ACMG systems for variant classification.

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as "maybe report" after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

Polygenic adaptation from standing genetic variation allows rapid ecotype formation.

Adaptive ecotype formation can be the first step to speciation, but the genetic underpinnings of this process are poorly understood. Marine midges of the genus Clunio (Diptera) have recolonized Northern European shore areas after the last glaciation. In response to local tide conditions they have formed different ecotypes with respect to timing of adult emergence, oviposition behavior and larval habitat. Genomic analysis confirms the recent establishment of these ecotypes, reflected in massive haplotype sharing between ecotypes, irrespective of whether there is ongoing gene flow or geographic isolation. QTL mapping and genome screens reveal patterns of polygenic adaptation from standing genetic variation. Ecotype-associated loci prominently include circadian clock genes, as well as genes affecting sensory perception and nervous system development, hinting to a central role of these processes in ecotype formation. Our data show that adaptive ecotype formation can occur rapidly, with ongoing gene flow and largely based on a re-assortment of existing alleles.

Genomic variants causing mitochondrial dysfunction are common in hereditary lower motor neuron disease.

Hereditary lower motor neuron diseases (LMND) other than 5q-spinal muscular atrophy (5q-SMA) can be classified according to affected muscle groups. Proximal and distal forms of non-5q-SMA represent a clinically and genetically heterogeneous spectrum characterized by significant overlaps with axonal forms of Charcot-Marie-Tooth (CMT) disease. A consensus for the best approach to molecular diagnosis needs to be reached, especially in light of continuous novel gene discovery and falling costs of next-generation sequencing (NGS). We performed exome sequencing (ES) in 41 families presenting with non-5q-SMA or axonal CMT, 25 of which had undergone a previous negative neuromuscular disease (NMD) gene panel analysis. The total diagnostic yield of ES was 41%. Diagnostic success in the cohort with a previous NMD-panel analysis was significantly extended by ES, primarily due to novel gene associated-phenotypes and uncharacteristic phenotypic presentations. We recommend early ES for individuals with hereditary LMND presenting uncharacteristic or significantly overlapping features. As mitochondrial dysfunction was the underlying pathomechanism in 47% of the solved individuals, we highlight the sensitivity of the anterior horn cell and peripheral nerve to mitochondrial imbalance as well as the necessity to screen for mitochondrial disorders in individuals presenting predominant lower motor neuron symptoms.

The importance of DNA barcode choice in biogeographic analyses - a case study on marine midges of the genus Clunio.

DNA barcodes are widely used for species identification and biogeographic studies. Here, we compare the use of full mitochondrial genomes versus DNA barcodes and other mitochondrial DNA fragments for biogeographic and ecological analyses. Our dataset comprised 120 mitochondrial genomes from the genus Clunio (Diptera: Chironomidae), comprising five populations from two closely related species (Clunio marinus and Clunio balticus) and three ecotypes. We extracted cytochrome oxidase c subunit I (COI) barcodes and partitioned the mitochondrial genomes into non-overlapping windows of 750 or 1500 bp. Haplotype networks and diversity indices were compared for these windows and full mitochondrial genomes (15.4 kb). Full mitochondrial genomes indicate complete geographic isolation between populations, but do not allow for conclusions on the separation of ecotypes or species. COI barcodes have comparatively few polymorphisms, ideal for species identification, but do not resolve geographic isolation. Many of the similarly sized 750 bp windows have higher nucleotide and haplotype diversity than COI barcodes, but still do not resolve biogeography. Only when increasing the window size to 1500 bp, two windows resolve biogeography reasonably well. Our results suggest that the design and use of DNA barcodes in biogeographic studies must be carefully evaluated for each investigated species.

De Novo and Inherited Variants in GBF1 are Associated with Axonal Neuropathy Caused by Golgi Fragmentation.

Distal hereditary motor neuropathies (HMNs) and axonal Charcot-Marie-Tooth neuropathy (CMT2) are clinically and genetically heterogeneous diseases characterized primarily by motor neuron degeneration and distal weakness. The genetic cause for about half of the individuals affected by HMN/CMT2 remains unknown. Here, we report the identification of pathogenic variants in GBF1 (Golgi brefeldin A-resistant guanine nucleotide exchange factor 1) in four unrelated families with individuals affected by sporadic or dominant HMN/CMT2. Genomic sequencing analyses in seven affected individuals uncovered four distinct heterozygous GBF1 variants, two of which occurred de novo. Other known HMN/CMT2-implicated genes were excluded. Affected individuals show HMN/CMT2 with slowly progressive distal muscle weakness and musculoskeletal deformities. Electrophysiological studies confirmed axonal damage with chronic neurogenic changes. Three individuals had additional distal sensory loss. GBF1 encodes a guanine-nucleotide exchange factor that facilitates the activation of members of the ARF (ADP-ribosylation factor) family of small GTPases. GBF1 is mainly involved in the formation of coatomer protein complex (COPI) vesicles, maintenance and function of the Golgi apparatus, and mitochondria migration and positioning. We demonstrate that GBF1 is present in mouse spinal cord and muscle tissues and is particularly abundant in neuropathologically relevant sites, such as the motor neuron and the growth cone. Consistent with the described role of GBF1 in Golgi function and maintenance, we observed marked increase in Golgi fragmentation in primary fibroblasts derived from all affected individuals in this study. Our results not only reinforce the existing link between Golgi fragmentation and neurodegeneration but also demonstrate that pathogenic variants in GBF1 are associated with HMN/CMT2.

A Dipteran's Novel Sucker Punch: Evolution of Arthropod Atypical Venom with a Neurotoxic Component in Robber Flies (Asilidae, Diptera).

Predatory robber flies (Diptera, Asilidae) have been suspected to be venomous due to their ability to overpower well-defended prey. However, details of their venom composition and toxin arsenal remained unknown. Here, we provide a detailed characterization of the venom system of robber flies through the application of comparative transcriptomics, proteomics and functional morphology. Our results reveal asilid venoms to be dominated by peptides and non-enzymatic proteins, and that the majority of components in the crude venom is represented by just ten toxin families, which we have named Asilidin1-10. Contrary to what might be expected for a liquid-feeding predator, the venoms of robber flies appear to be rich in novel peptides, rather than enzymes with a putative pre-digestive role. The novelty of these peptides suggests that the robber fly venom system evolved independently from hematophagous dipterans and other pancrustaceans. Indeed, six Asilidins match no other venom proteins, while three represent known examples of peptide scaffolds convergently recruited to a toxic function. Of these, members of Asilidin1 closely resemble cysteine inhibitor knot peptides (ICK), of which neurotoxic variants occur in cone snails, assassin bugs, scorpions and spiders. Synthesis of one of these putative ICKs, U-Asilidin1-Mar1a, followed by toxicity assays against an ecologically relevant prey model revealed that one of these likely plays a role as a neurotoxin involved in the immobilization of prey. Our results are fundamental to address these insights further and to understand processes that drive venom evolution in dipterans as well as other arthropods.

Dominant optic atrophy, OPA1, and mitochondrial quality control: understanding mitochondrial network dynamics.

Mitochondrial quality control is fundamental to all neurodegenerative diseases, including the most prominent ones, Alzheimer's Disease and Parkinsonism. It is accomplished by mitochondrial network dynamics - continuous fission and fusion of mitochondria. Mitochondrial fission is facilitated by DRP1, while MFN1 and MFN2 on the mitochondrial outer membrane and OPA1 on the mitochondrial inner membrane are essential for mitochondrial fusion. Mitochondrial network dynamics are regulated in highly sophisticated ways by various different posttranslational modifications, such as phosphorylation, ubiquitination, and proteolytic processing of their key-proteins. By this, mitochondria process a wide range of different intracellular and extracellular parameters in order to adapt mitochondrial function to actual energetic and metabolic demands of the host cell, attenuate mitochondrial damage, recycle dysfunctional mitochondria via the mitochondrial autophagy pathway, or arrange for the recycling of the complete host cell by apoptosis. Most of the genes coding for proteins involved in this process have been associated with neurodegenerative diseases. Mutations in one of these genes are associated with a neurodegenerative disease that originally was described to affect retinal ganglion cells only. Since more and more evidence shows that other cell types are affected as well, we would like to discuss the pathology of dominant optic atrophy, which is caused by heterozygous sequence variants in OPA1, in the light of the current view on OPA1 protein function in mitochondrial quality control, in particular on its function in mitochondrial fusion and cytochrome C release. We think OPA1 is a good example to understand the molecular basis for mitochondrial network dynamics.

A clinically complex form of dominant optic atrophy (OPA8) maps on chromosome 16.

Dominant optic atrophy (DOA) is genetically heterogeneous and pathogenic mutations have been identified in the OPA1 and OPA3 genes, both encoding for mitochondrial proteins. We characterized clinical and laboratory features in a large OPA1-negative family with complicated DOA. Search for mitochondrial dysfunction was performed by studying muscle biopsies, fibroblasts, platelets and magnetic resonance (MR) spectroscopy. Genetic investigations included mitochondrial DNA (mtDNA) analysis, linkage analysis, copy number variation (CNV) analysis and candidate gene screening. Optic neuropathy was undistinguishable from that in OPA1-DOA and frequently associated with late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials and various cardiac abnormalities. Serum lactic acid after exercise, platelet respiratory complex activities, adenosine triphosphate (ATP) content in fibroblasts and muscle phosphorus MR spectroscopy all failed to reveal a mitochondrial dysfunction. However, muscle biopsies and their mtDNA analysis showed increased mitochondrial biogenesis. Furthermore, patient's fibroblasts grown in the galactose medium were unable to increase ATP content compared with controls, and exhibited abnormally high rate of fusion activity. Genome-wide linkage revealed a locus on chromosome 16q21-q22 with a maximum two-point LOD score of 8.84 for the marker D16S752 and a non-recombinant interval of ∼ 6.96 cM. Genomic screening of 45 genes in this interval including several likely candidate genes (CALB2, CYB5B, TK2, DHODH, PLEKHG4) revealed no mutation. Moreover, we excluded the presence of CNVs using array-based comparative genome hybridization. The identification of a new OPA locus (OPA8) in this pedigree demonstrates further genetic heterogeneity in DOA, and our results indicate that the pathogenesis may still involve mitochondria.

Solving a 50 year mystery of a missing OPA1 mutation: more insights from the first family diagnosed with autosomal dominant optic atrophy.

BACKGROUND: Up to the 1950s, there was an ongoing debate about the diversity of hereditary optic neuropathies, in particular as to whether all inherited optic atrophies can be ascribed to Leber's hereditary optic neuropathy (LHON) or represent different disease entities. In 1954 W. Jaeger published a detailed clinical and genealogical investigation of a large family with explicit autosomal dominant segregation of optic atrophy thus proving the existence of a discrete disease different from LHON, which is nowadays known as autosomal dominant optic atrophy (ADOA). Since the year 2000 ADOA is associated with genomic mutations in the OPA1 gene, which codes for a protein that is imported into mitochondria where it is required for mitochondrial fusion. Interestingly enough, the underlying mutation in this family has not been identified since then. RESULTS: We have reinvestigated this family with the aim to identify the mutation and to further clarify the underlying pathomechanism. Patients showed a classical non-syndromic ADOA. The long term deterioration in vision in the two teenagers examined 50 years later is of particular note 5/20 to 6/120. Multiplex ligation probe amplification revealed a duplication of the OPA1 exons 7-9 which was confirmed by long distance PCR and cDNA analysis, resulting in an in-frame duplication of 102 amino acids. Segregation was verified in 53 available members of the updated pedigree and a penetrance of 88% was calculated. Fibroblast cultures from skin biopsies were established to assess the mitochondrial network integrity and to qualitatively and quantitatively study the consequences of the mutation on transcript and protein level. Fibroblast cultures demonstrated a fragmented mitochondrial network. Processing of the OPA1 protein was altered. There was no correlation of the OPA1 transcript levels and the OPA1 protein levels in the fibroblasts. Intriguingly an overall decrease of mitochondrial proteins was observed in patients' fibroblasts, while the OPA1 transcript levels were elevated. CONCLUSIONS: The thorough study of this family provides a detailed clinical picture accompanied by a molecular investigation of patients' fibroblasts. Our data show a classic OPA1-associated non-syndromic ADOA segregating in this family. Cell biological findings suggest that OPA1 is regulated by post-translational mechanisms and we would like to hypothesize that loss of OPA1 function might lead to impaired mitochondrial quality control. With the clinical, genetic and cell biological characterisation of a family described already more than 50 years ago, we span more than half a century of research in optic neuropathies.

Electrophysiological and histologic assessment of retinal ganglion cell fate in a mouse model for OPA1-associated autosomal dominant optic atrophy.

PURPOSE: The main disease features of autosomal dominant optic atrophy (ADOA) are a bilateral reduction of visual acuity, cecocentral scotoma, and frequently tritanopia, which have been ascribed to a progressive loss of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve. The main disease-causing gene is OPA1. Here, we examine a mouse carrying a pathogenic mutation in Opa1 by electrophysiological measurements and assess the fate of RGCs. METHODS: Two-year-old animals underwent a full examination by electroretinography (ERG) and visually evoked potential (VEP) measurements to assess the function of the outer and inner retina and the optic nerve. Retrograde Fluorogold labeling was performed to determine the number of surviving RGCs and to assess axonal transport by neurofilament counterstaining. Phagocytosis-dependent labeled microglial cells were identified by an Iba-1 staining. RESULTS: ERG responses were normal in aged Opa1 mice. VEP measurements revealed significantly reduced amplitudes but no change in the latencies in contrast to extended latencies found in glaucoma. Retrograde labeling of RGCs showed a significant reduction in the number of RGCs in Opa1 mice. Long-term experiments revealed the presence of microglial cells with ingested fluorescent dye. CONCLUSIONS: This is the first electrophysiological demonstration of a visual function deficit in aged Opa1 mice. VEP measurements and retrograde labeling experiments show that the number of RGCs is reduced whereas the remaining RGCs and axons function normally. Taken together, these findings support an ascending progress of degeneration from the soma toward the axon.

Subtle neurological and metabolic abnormalities in an Opa1 mouse model of autosomal dominant optic atrophy.

The ubiquitously expressed gene OPA1 is the main disease causing gene for autosomal dominant optic atrophy (ADOA). These patients present with bilateral reduction in visual acuity, central visual field defects and impaired color vision, secondary to the progressive loss of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve. Up to now, it is not clear why a mutation in a ubiquitously expressed gene affects only RGCs and the optic nerve. Twenty-two-month-old Opa1 animals underwent a full examination following the Shirpa protocol. Weight, food intake and life span were monitored. Rotarod treadmill experiments were performed to assess neuromuscular function. Limb skeletal muscle was evaluated morphologically, mitochondrial cytochrome c oxidase (COX) activity was studied histochemically and mtDNA integrity was determined by long-range PCR. The Shirpa test showed that 33% of the Opa1 mice suffered from tremor and 52% of the Opa1 animals showed an abnormal clutching reflex. Control animals performed well in the accelerating Rotarod treadmill experiment whereas the Opa1 mice performed significantly worse. Skeletal muscle fibers were morphologically normal, had normal COX activity and showed no evidence of secondary mtDNA damage in contrast to patients with syndromic ADOA. We also found a highly significant difference in body weight. Our results demonstrate that OPA1 mutations affect not only RGCs but also other tissues and cell types, though to a lesser extent. In particular we found deficits in both neuromuscular and metabolic function. We therefore want to encourage clinicians to be vigilant about to extra-ocular manifestations in ADOA patients.

Comprehensive cDNA study and quantitative transcript analysis of mutant OPA1 transcripts containing premature termination codons.

Autosomal dominant optic atrophy (adOA) is most commonly caused by mutations in the OPA1 gene. There is a considerable allelic heterogeneity among adOA-associated OPA1 mutations, however these mutations have mostly been identified and studied only at the genomic DNA level. Here we report the identification of 22 novel OPA1 mutations and their analysis at the cDNA level along with 15 already known OPA1 mutations. We found that 18 of these mutations cause splice defects that involve either skipping of the adjacent exon or the activation of a cryptic splice site. We also observed a reduced level of the mutant transcript in several adOA subjects. Allele-specific quantification of the transcript steady-state level was performed for 13 different OPA1 mutations applying pyrosequencing to a RT-PCR amplified cSNP (c.2109C>T) in OPA1. Using this new assay we could demonstrate that the majority of OPA1 mutations that lead to a premature termination codon (PTC) undergo nonsense-mediated mRNA decay (NMD). Mutant transcript levels were reduced between 1.25- and 2.5-fold and varied between PTC containing mutations, and between subjects. Our results emphasize the value of cDNA analysis in the characterization of OPA1 mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in OPA1-associated adOA.