A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

A Dysferlin Exon 32 Nonsense Mutant Mouse Model Shows Pathological Signs of Dysferlinopathy.

Dysferlinopathies are a group of autosomal recessive muscular dystrophies caused by pathogenic variants in the DYSF gene. While several animal models of dysferlinopathy have been developed, most of them involve major disruptions of the Dysf gene locus that are not optimal for studying human dysferlinopathy, which is often caused by single nucleotide substitutions. In this study, the authors describe a new murine model of dysferlinopathy that carries a nonsense mutation in Dysf exon 32, which has been identified in several patients with dysferlinopathy. This mouse model, called Dysf p.Y1159X/p.Y1159X, displays several molecular, histological, and functional defects observed in dysferlinopathy patients and other published mouse models. This mutant mouse model is expected to be useful for testing various therapeutic approaches such as termination codon readthrough, pharmacological approaches, and exon skipping. Therefore, the data presented in this study strongly support the use of this animal model for the development of preclinical strategies for the treatment of dysferlinopathies.

Imbalance of NRG1-ERBB2/3 signalling underlies altered myelination in Charcot-Marie-Tooth disease 4H.

Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neurological disorders, affecting either axons from the motor and/or sensory neurons or Schwann cells of the peripheral nervous system (PNS) and caused by more than 100 genes. We previously identified mutations in FGD4 as responsible for CMT4H, an autosomal recessive demyelinating form of CMT disease. FGD4 encodes FRABIN, a GDP/GTP nucleotide exchange factor, particularly for the small GTPase Cdc42. Remarkably, nerves from patients with CMT4H display excessive redundant myelin figures called outfoldings that arise from focal hypermyelination, suggesting that FRABIN could play a role in the control of PNS myelination. To gain insights into the role of FGD4/FRABIN in Schwann cell myelination, we generated a knockout mouse model (Fgd4SC-/-), with conditional ablation of Fgd4 in Schwann cells. We show that the specific deletion of FRABIN in Schwann cells leads to aberrant myelination in vitro, in dorsal root ganglia neuron/Schwann cell co-cultures, as well as in vivo, in distal sciatic nerves from Fgd4SC-/- mice. We observed that those myelination defects are related to an upregulation of some interactors of the NRG1 type III/ERBB2/3 signalling pathway, which is known to ensure a proper level of myelination in the PNS. Based on a yeast two-hybrid screen, we identified SNX3 as a new partner of FRABIN, which is involved in the regulation of endocytic trafficking. Interestingly, we showed that the loss of FRABIN impairs endocytic trafficking, which may contribute to the defective NRG1 type III/ERBB2/3 signalling and myelination. Using RNA-Seq, in vitro, we identified new potential effectors of the deregulated pathways, such as ERBIN, RAB11FIP2 and MAF, thereby providing cues to understand how FRABIN contributes to proper ERBB2 trafficking or even myelin membrane addition through cholesterol synthesis. Finally, we showed that the re-establishment of proper levels of the NRG1 type III/ERBB2/3 pathway using niacin treatment reduces myelin outfoldings in nerves of CMT4H mice. Overall, our work reveals a new role of FRABIN in the regulation of NRG1 type III/ERBB2/3 NRG1signalling and myelination and opens future therapeutic strategies based on the modulation of the NRG1 type III/ERBB2/3 pathway to reduce CMT4H pathology and more generally other demyelinating types of CMT disease.

Mesenchymal stem cells derived from patients with premature aging syndromes display hallmarks of physiological aging.

Progeroid syndromes are rare genetic diseases with most of autosomal dominant transmission, the prevalence of which is less than 1/10,000,000. These syndromes caused by mutations in the <i>LMNA</i> gene encoding A-type lamins belong to a group of disorders called laminopathies. Lamins are implicated in the architecture and function of the nucleus and chromatin. Patients affected with progeroid laminopathies display accelerated aging of mesenchymal stem cells (MSCs)-derived tissues associated with nuclear morphological abnormalities. To identify pathways altered in progeroid patients' MSCs, we used induced pluripotent stem cells (hiPSCs) from patients affected with classical Hutchinson-Gilford progeria syndrome (HGPS, c.1824C&gt;T-p.G608G), HGPS-like syndrome (HGPS-L; c.1868C&gt;G-p.T623S) associated with farnesylated prelamin A accumulation, or atypical progeroid syndromes (APS; homozygous c.1583C&gt; T-p.T528M; heterozygous c.1762T&gt;C-p.C588R; compound heterozygous c.1583C&gt;T and c.1619T&gt;C-p.T528M and p.M540T) without progerin accumulation. By comparative analysis of the transcriptome and methylome of hiPSC-derived MSCs, we found that patient's MSCs display specific DNA methylation patterns and modulated transcription at early stages of differentiation. We further explored selected biological processes deregulated in the presence of <i>LMNA</i> variants and confirmed alterations of age-related pathways during MSC differentiation. In particular, we report the presence of an altered mitochondrial pattern; an increased response to double-strand DNA damage; and telomere erosion in HGPS, HGPS-L, and APS MSCs, suggesting converging pathways, independent of progerin accumulation, but a distinct DNA methylation profile in HGPS and HGPS-L compared with APS cells.

Objective Evaluation of Clinical Actionability for Genes Involved in Myopathies: 63 Genes with a Medical Value for Patient Care.

The implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of "actionable" genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date. Using the ClinGen Actionability criteria, we scored the clinical actionability of all 199 genes implicated in myopathies published by FILNEMUS for the "National French consensus on gene Lists for the diagnosis of myopathies using next generation sequencing". We objectified that 63 myopathy genes were actionable with the currently available data. Among the 36 myopathy genes with the highest actionability scores, only 8 had been scored to date by ClinGen. The data obtained through these methodological tools are an important resource for strategic choices in diagnostic approaches and the management of genetic myopathies. The clinical actionability of genes has to be considered as an evolving concept, in relation to progresses in disease knowledge and therapeutic approaches.

Novel Exon-Skipping Therapeutic Approach for the DMD Gene Based on Asymptomatic Deletions of Exon 49.

Exon skipping is a promising therapeutic approach. One important condition for this approach is that the exon-skipped form of the gene can at least partially perform the required function and lead to improvement of the phenotype. It is therefore critical to identify the exons that can be skipped without a significant deleterious effect on the protein function. Pathogenic variants in the DMD gene are responsible for Duchenne muscular dystrophy (DMD). We report for the first time a deletion of the in-frame exon 49 associated with a strikingly normal muscular phenotype. Based on this observation, and on previously known therapeutic approaches using exon skipping in DMD for other single exons, we aimed to extend the clinical use of exon skipping for patients carrying truncating mutations in exon 49. We first determined the precise genomic position of the exon 49 deletion in our patients. We then demonstrated the feasibility of skipping exon 49 using an in vitro AON (antisense oligonucleotide) approach in human myotubes carrying a truncating pathogenic variant as well as in healthy ones. This work is a proof of concept aiming to expand exon-skipping approaches for DMD exon 49.

HINT1 neuropathy: Expanding the genotype and phenotype spectrum.

Inherited peripheral neuropathy (IPN) is a heterogeneous group of disorders due to pathogenic variation in more than 100 genes. In 2012, the first cases of IPN associated with HINT1 pathogenic variations were described in 33 families sharing the same phenotype characterized by an axonal neuropathy with neuromyotonia and autosomal recessive inheritance (NMAN: OMIM #137200). Histidine Triad Nucleotide Binding Protein 1 regulates transcription, cell-cycle control, and is possibly involved in neuropsychiatric pathophysiology. Herein, we report seven French patients with NMAN identified by Next Generation Sequencing. We conducted a literature review and compared phenotypic and genotypic features with our cohort. We identified a new HINT1 pathogenic variation involved in NMAN: c.310G>C p.(Gly104Arg). This cohort is comparable with literature data regarding age of onset (7,4yo), neuronal involvement (sensorimotor 3/7 and motor pure 4/7), and skeletal abnormalities (scoliosis 3/7, feet anomalies 6/7). We expand the phenotypic spectrum of HINT1-related neuropathy by describing neurodevelopmental or psychiatric features in six out of seven individuals such as generalized anxiety disorder (GAD), obsessive-compulsive disorder (OCD), mood disorder and attention deficit hyperactivity disorder (ADHD). However, only 3/128 previously described patients had neuropsychiatric symptomatology or neurodevelopmental disorder. These features could be part of HINT1-related disease, and we should further study the clinical phenotype of the patients.

Genetic Profile of Patients with Limb-Girdle Muscle Weakness in the Chilean Population.

Hereditary myopathies are a group of genetically determined muscle disorders comprising more than 300 entities. In Chile, there are no specific registries of the distinct forms of these myopathies. We now report the genetic findings of a series of Chilean patients presenting with limb-girdle muscle weakness of unknown etiology. Eighty-two patients were explored using high-throughput sequencing approaches with neuromuscular gene panels, establishing a definite genetic diagnosis in 49 patients (59.8%) and a highly probable genetic diagnosis in eight additional cases (9.8%). The most frequent causative genes identified were DYSF and CAPN3, accounting for 22% and 8.5% of the cases, respectively, followed by DMD (4.9%) and RYR1 (4.9%). The remaining 17 causative genes were present in one or two cases only. Twelve novel variants were identified. Five patients (6.1%) carried a variant of uncertain significance in genes partially matching the clinical phenotype. Twenty patients (24.4%) did not carry a pathogenic or likely pathogenic variant in the phenotypically related genes, including five patients (6.1%) presenting an autoimmune neuromuscular disorder. The relative frequency of the different forms of myopathy in Chile is like that of other series reported from different regions of the world with perhaps a relatively higher incidence of dysferlinopathy.

miR-376a-3p and miR-376b-3p overexpression in Hutchinson-Gilford progeria fibroblasts inhibits cell proliferation and induces premature senescence.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder, in which an abnormal and toxic protein called progerin, accumulates in cell nuclei, leading to major cellular defects. Among them, chromatin remodeling drives gene expression changes, including miRNA dysregulation. In our study, we evaluated miRNA expression profiles in HGPS and control fibroblasts. We identified an enrichment of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster. Using 3D FISH, we demonstrated that overexpression of these miRNAs is associated with chromatin remodeling at this specific locus in HGPS fibroblasts. We then focused on miR-376b-3p and miR-376a-3p, both overexpressed in HGPS fibroblasts. We demonstrated that their induced overexpression in control fibroblasts decreases cell proliferation and increases senescence, whereas their inhibition in HGPS fibroblasts rescues proliferation defects and senescence and decreases progerin accumulation. By targeting these major processes linked to premature aging, these two miRNAs may play a pivotal role in the pathophysiology of HGPS.

MG132 Induces Progerin Clearance and Improves Disease Phenotypes in HGPS-like Patients' Cells.

Progeroid syndromes (PS), including Hutchinson-Gilford Progeria Syndrome (HGPS), are premature and accelerated aging diseases, characterized by clinical features mimicking physiological aging. Most classical HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type lamins. This mutation activates a cryptic splice site, leading to the production of a truncated prelamin A, called prelamin A ∆50 or progerin, that accumulates in HGPS cell nuclei and is a hallmark of the disease. Some patients with PS carry other LMNA mutations and are named "HGPS-like" patients. They produce progerin and/or other truncated prelamin A isoforms (∆35 and ∆90). We previously found that MG132, a proteasome inhibitor, induced progerin clearance in classical HGPS through autophagy activation and splicing regulation. Here, we show that MG132 induces aberrant prelamin A clearance and improves cellular phenotypes in HGPS-like patients' cells other than those previously described in classical HGPS. These results provide preclinical proof of principle for the use of a promising class of molecules toward a potential therapy for children with HGPS-like or classical HGPS.

A National French Consensus on Gene List for the Diagnosis of Charcot-Marie-Tooth Disease and Related Disorders Using Next-Generation Sequencing.

Next generation sequencing (NGS) is strategically used for genetic diagnosis in patients with Charcot-Marie-Tooth disease (CMT) and related disorders called non-syndromic inherited peripheral neuropathies (NSIPN) in this paper. With over 100 different CMT-associated genes involved and ongoing discoveries, an important interlaboratory diversity of gene panels exists at national and international levels. Here, we present the work of the French National Network for Rare Neuromuscular Diseases (FILNEMUS) genetic diagnosis section which coordinates the seven French diagnosis laboratories using NGS for peripheral neuropathies. This work aimed to establish a unique, simple and accurate gene classification based on literature evidence. In NSIPN, three subgroups were usually distinguished: (1) HMSN, Hereditary Motor Sensory Neuropathy, (2) dHMN, distal Hereditary Motor Neuropathy, and (3) HSAN, Hereditary Sensory Autonomic Neuropathy. First, we reported ClinGen evaluation, and second, for the genes not evaluated yet by ClinGen, we classified them as "definitive" if reported in at least two clinical publications and associated with one report of functional evidence, or "limited" otherwise. In total, we report a unique consensus gene list for NSIPN including the three subgroups with 93 genes definitive and 34 limited, which is a good rate for our gene's panel for molecular diagnostic use.

Compound impacts from droughts and structural vulnerability on human mobility.

Extreme dry events already disrupt populations' ability to migrate. In a warming climate, compound drought events could amplify vulnerability and drive forced migration. Here, we contribute the first multi-method research design on societal impacts from compound drought events. We show how mobility patterns are shaped by the intersection of drought and social vulnerability factors in three drought-prone countries - Madagascar, Nepal, and Mexico. We find that internal migration in agricultural communities in Mexico increased by 14 to 24 basis points from 1991 to 2018 and will prospectively increase by 2 to 15 basis points in Nepal in case of a compound drought event in 2025. We show that consecutive drought events exacerbate structural vulnerabilities, limiting migrants' adaptation options, including long-range migration. We conclude that the additional social pre-conditions, e.g., social isolation and lack of accurate information, ultimately limit migration as an adaptation option for households vulnerable to compound drought events.

A 20-year Clinical and Genetic Neuromuscular Cohort Analysis in Lebanon: An International Effort.

BACKGROUND: Clinical and molecular data on the occurrence and frequency of inherited neuromuscular disorders (NMD) in the Lebanese population is scarce. OBJECTIVE: This study aims to provide a retrospective overview of hereditary NMDs based on our clinical consultations in Lebanon. METHODS: Clinical and molecular data of patients referred to a multi-disciplinary consultation for neuromuscular disorders over a 20-year period (1999-2019) was reviewed. RESULTS: A total of 506 patients were diagnosed with 62 different disorders encompassing 10 classes of NMDs. 103 variants in 49 genes were identified. In this cohort, 81.4% of patients were diagnosed with motor neuron diseases and muscular dystrophies, with almost half of these described with spinal muscular atrophy (SMA) (40.3% of patients). We estimate a high SMA incidence of 1 in 7,500 births in Lebanon. Duchenne and Becker muscular dystrophy were the second most frequently diagnosed NMDs (17% of patients). These disorders were associated with the highest number of variants (39) identified in this study. A highly heterogeneous presentation of Limb Girdle Muscular Dystrophy and Charcot-Marie-Tooth disease was notably identified. The least common disorders (5.5% of patients) involved congenital, metabolic, and mitochondrial myopathies, congenital myasthenic syndromes, and myotonic dystrophies. A review of the literature for selected NMDs in Lebanon is provided. CONCLUSIONS: Our study indicates a high prevalence and underreporting of heterogeneous forms of NMDs in Lebanon- a major challenge with many novel NMD treatments in the pipeline. This report calls for a regional NMD patient registry.

Titration of Apparent In-Cellula Affinities of Protein-Protein Interactions.

A genetic assay permits simultaneous quantification of two interacting proteins and their bound fraction at the single-cell level using flow cytometry. Apparent in-cellula affinities of protein-protein interactions can be extracted from the acquired data through a titration-like analysis. The applicability of this approach is demonstrated on a diverse set of interactions with proteins from different families and organisms and with in-vitro dissociation constants ranging from picomolar to micromolar.

The Dysferlin Transcript Containing the Alternative Exon 40a is Essential for Myocyte Functions.

Dysferlinopathies are a group of muscular dystrophies caused by recessive mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is a transmembrane protein involved in several muscle functions like T-tubule maintenance and membrane repair. In 2009, a study showed the existence of fourteen dysferlin transcripts generated from alternative splicing. We were interested in dysferlin transcripts containing the exon 40a, and among them the transcript 11 which contains all the canonical exons and exon 40a. This alternative exon encodes a protein region that is cleaved by calpains during the muscle membrane repair mechanism. Firstly, we tested the impact of mutations in exon 40a on its cleavability by calpains. We showed that the peptide encoded by the exon 40a domain is resistant to mutations and that calpains cleaved dysferlin in the first part of DYSF exon 40a. To further explore the implication of this transcript in cell functions, we performed membrane repair, osmotic shock, and transferrin assay. Our results indicated that dysferlin transcript 11 is a key factor in the membrane repair process. Moreover, dysferlin transcript 11 participates in other cell functions such as membrane protection and vesicle trafficking. These results support the need to restore the dysferlin transcript containing the alternative exon 40a in patients affected with dysferlinopathy.

Novel partial loss-of-function variants in the tyrosyl-tRNA synthetase 1 (YARS1) gene involved in multisystem disease.

Cytoplasmic aminoacyl-tRNA synthetases (ARSs) are emerging as a cause of numerous rare inherited diseases. Recently, biallelic variants in tyrosyl-tRNA synthetase 1 (YARS1) have been described in ten patients of three families with multi-systemic disease (failure to thrive, developmental delay, liver dysfunction, and lung cysts). Here, we report an additional subject with overlapping clinical findings, heterozygous for two novel variants in tyrosyl-tRNA synthetase 1 (NM_003680.3(YARS1):c.176T>C; p.(Ile59Thr) and NM_003680.3(YARS1):c.237C>G; p.(Tyr79*) identified by whole exome sequencing. The p.Ile59Thr variant is located in the highly conserved aminoacylation domain of the protein. Compared to subjects previously described, this patient presents a much more severe condition. Our findings support implication of two novel YARS1 variants in these disorders. Furthermore, we provide evidence for a reduced protein abundance in cells of the patient, in favor of a partial loss-of-function mechanism.

Inhibition of P53-mediated cell cycle control as the determinant in dedifferentiated liposarcomas development.

Liposarcomas are a heterogeneous group of sarcomas, including well-differentiated and dedifferentiated liposarcoma, myxoid/round cell liposarcoma, and pleomorphic liposarcoma. Complete surgical resection is the key of treatment. Radiotherapy, based on the tumor grade and the vicinity of critical structures with the tumor, can be used to prevent local recurrence. The group of dedifferentiated liposarcomas (DDLS) is poorly sensitive to adjuvant chemotherapy. Improved understanding of the genetic aberrations that lead to liposarcoma initiation is necessary for the development of targeted therapies to improve tumor control and survival. DDLS share genetic abnormalities with other groups, exhibiting high-level amplifications of chromosome 12, including the MDM2 and CDK4 genes, and harbor additional amplifications of chromosomes 6 and 1. Novel therapies targeted at the gene products of chromosome 12 are currently considered in clinical trials. Our work consisted in a genomic characterization of DDLS to draw up a complete picture of alterations, including genomic signatures, tumor mutation burden, gene mutations, copy number variations, translocations, gene fusions and methylation modifications. Analysis of translocations helped to understand the mechanisms underlying the amplification processes. Combination of mutations and loss of heterozygosity or homozygous deletions were detected and led to inactivate tumor suppressor genes (TSG). In contrast, methylation anomalies seemed not linked to any particular genomic profile. All identified anomalies, whether amplifications and/or TSG inactivation, involve genes playing a role in p53 regulation, that appears to be the epicenter of the initiation process in DDLS tumorigenesis, as is also known to be responsible for Li-Fraumeni syndrome, a family cancer syndrome highly predisposing to sarcomas.

Case Report: Identification of Novel Variants in ERCC4 and DDB2 Genes in Two Tunisian Patients With Atypical Xeroderma Pigmentosum Phenotype.

Xeroderma Pigmentosum (XP) is a rare genetic disorder affecting the nucleotide excision repair system (NER). It is characterized by an extreme sensitivity to sunlight that induces cutaneous disorders such as severe sunburn, freckling and cancers. In Tunisia, six complementation groups have been already identified. However, the genetic etiology remains unknown for several patients. In this study, we investigated clinical characteristics and genetic defects in two families with atypical phenotypes originating from the central region in Tunisia. Clinical investigation revealed mild cutaneous features in two patients who develop multiple skin cancers at later ages, with no neurological disorders. Targeted gene sequencing revealed that they carried novel variants. A homozygous variation in the ERCC4 gene c.1762G>T, p.V588F, detected in patient XP21. As for patient XP134, he carried two homozygous mutations in the DDB2 gene c.613T>C, p.C205R and c.618C>A, p.S206R. Structural modeling of the protein predicted the identified ERCC4 variant to mildly affect protein stability without affecting its functional domains. As for the case of DDB2 double mutant, the second variation seems to cause a mild effect on the protein structure unlike the first variation which does not seem to have an effect on it. This study contributes to further characterize the mutation spectrum of XP in Tunisian families. Targeted gene sequencing accelerated the identification of rare unexpected genetic defects for diagnostic testing and genetic counseling.

Genotype-phenotype correlation in French patients with myelin protein zero gene-related inherited neuropathy.

BACKGROUND AND PURPOSE: Preparations for clinical trials of unfolded protein response (UPR) inhibitors (such as Sephin1) that target the upregulated UPR in patients with Charcot-Marie-Tooth disease (CMT) carrying MPZ mutations are currently underway. The inclusion criteria for these trials are still being formulated. Our objective was to characterize the relation between genotypes and phenotypes in patients with CMT caused by MPZ mutations, and to refine the inclusion criteria for future trials. METHODS: Clinical and neurophysiological data of CMT patients with MPZ mutations were retrospectively collected at 11 French reference centers. RESULTS: Forty-four mutations in MPZ were identified in 91 patients from 61 families. There was considerable heterogeneity. The same mutation was found to cause either axonal or demyelinating neuropathy. Three groups were identified according to the age at disease onset. CMT Examination Score (CMTES) tended to be higher in the early (≤22 years) and adult (23-47 years) onset groups (mean CMTESv2 = 10.4 and 10.0, respectively) than in the late onset group (>47 years, mean CMTESv2 = 8.6, p = 0.47). There was a significant positive correlation between CMTESv2 and the age of patients in Groups I (p = 0.027) and II (p = 0.023), indicating that clinical severity progressed with age in these patients. CONCLUSIONS: To optimize the selection of CMT patients carrying MPZ mutations for the upcoming trials, inclusion criteria should take into account the pathophysiology of the disease (upregulated UPR). Recruited patients should have a mild to moderate disease severity and a disease onset at between 18 and 50 years, as these patients exhibit significant disease progression over time.