Patients with complex and very-early-onset ATL1-related spastic paraplegia offer insights on genotype/phenotype correlations and support for autosomal recessive forms of SPG3A.

Spastic paraplegia type 3A (SPG3A) is the second most common form of hereditary spastic paraplegia (HSP). This autosomal-dominant-inherited motor disorder is caused by heterozygous variants in the ATL1 gene which usually presents as a pure childhood-onset spastic paraplegia. Affected individuals present muscle weakness and spasticity in the lower limbs, with symptom onset in the first decade of life. Individuals with SPG3A typically present a slow progression and remain ambulatory throughout their life. Here we report three unrelated individuals presenting with very-early-onset (before 7 months) complex, and severe HSP phenotypes (axial hypotonia, spastic quadriplegia, dystonia, seizures and intellectual disability). For 2 of the 3 patients, these phenotypes led to the initial diagnosis of cerebral palsy (CP). These individuals carried novel ATL1 pathogenic variants (a de novo ATL1 missense p.(Lys406Glu), a homozygous frameshift p.(Arg403Glufs*3) and a homozygous missense variant (p.Tyr367His)). The parents carrying the heterozygous frameshift and missense variants were asymptomatic. Through these observations, we increase the knowledge on genotype-phenotype correlations in SPG3A and offer additional proof for possible autosomal recessive forms of SPG3A, while raising awareness on these exceptional phenotypes. Their ability to mimic CP also implies that genetic testing should be considered for patients with atypical forms of CP, given the implications for genetic counseling.

The mitochondrial seryl-tRNA synthetase SARS2 modifies onset in spastic paraplegia type 4.

PURPOSE: Hereditary spastic paraplegia type 4 is extremely variable in age at onset; the same variant can cause onset at birth or in the eighth decade. We recently discovered that missense variants in SPAST, which influences microtubule dynamics, are associated with earlier onset and more severe disease than truncating variants, but even within the early and late-onset groups there remained significant differences in onset. Given the rarity of the condition, we adapted an extreme phenotype approach to identify genetic modifiers of onset. METHODS: We performed a genome-wide association study on 134 patients bearing truncating pathogenic variants in SPAST, divided into early- and late-onset groups (aged ≤15 and ≥45 years, respectively). A replication cohort of 419 included patients carrying either truncating or missense variants. Finally, age at onset was analyzed in the merged cohort (N = 553). RESULTS: We found 1 signal associated with earlier age at onset (rs10775533, P = 8.73E-6) in 2 independent cohorts and in the merged cohort (N = 553, Mantel-Cox test, P < .0001). Western blotting in lymphocytes of 20 patients showed that this locus tends to upregulate SARS2 expression in earlier-onset patients. CONCLUSION: SARS2 overexpression lowers the age of onset in hereditary spastic paraplegia type 4. Lowering SARS2 or improving mitochondrial function could thus present viable approaches to therapy.

Reduced penetrance of an eastern French mutation in ATL1 autosomal-dominant inheritance (SPG3A): extended phenotypic spectrum coupled with brain 18F-FDG PET.

ATL1-related spastic paraplegia SPG3A is a pure form of hereditary spastic paraplegia. Rare complex phenotypes have been described, but few data concerning cognitive evaluation or molecular imaging of these patients are available. We relate a retrospective collection of patients with SPG3A from the Neurology Department of Nancy University Hospital, France. For each patient were carried out a 18F-FDG PET (positron emission tomography), a electromyography (EMG), a sudoscan®, a cerebral and spinal cord MRI (magnetic resonance imaging) with measurement of cervical and thoracic surfaces, a neuropsychological assessment. The present report outlines standardised clinical and paraclinical data of five patients from two east-France families carrying the same missense pathogenic variation, NM_015915.4(ATL1): c.1483C > T p.(Arg495Trp) in ATL1. Mean age at onset was 14 ± 15.01 years. Semi-quantitatively and in comparison to healthy age-matched subjects, PET scans showed a significant cerebellar and upper or mild temporal hypometabolism in all four adult patients and hypometabolism of the prefrontal cortex or precuneus in three of them. Sudoscan® showed signs of small fibre neuropathy in three patients. Cervical and thoracic patients' spinal cords were significantly thinner than matched-control, respectively 71 ± 6.59mm2 (p = 0.01) and 35.64 ± 4.35mm2 (p = 0.015). Two patients presented with a dysexecutive syndrome. While adding new clinical and paraclinical signs associated with ATL1 pathogenic variations, we insist here on the variable penetrance and expressivity. We report small fibre neuropathy, cerebellar hypometabolism and dysexecutive syndromes associated with SPG3A. These cognitive impairments and PET findings may be related to a cortico-cerebellar bundle axonopathy described in the cerebellar cognitive affective syndrome (CCAS).

Clinical and genetic spectra of 1550 index patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia refers to rare genetic neurodevelopmental and/or neurodegenerative disorders in which spasticity due to length-dependent damage to the upper motor neuron is a core sign. Their high clinical and genetic heterogeneity makes their diagnosis challenging. Multigene panels allow a high-throughput targeted analysis of the increasing number of genes involved using next-generation sequencing. We report here the clinical and genetic results of 1550 index cases tested for variants in a panel of hereditary spastic paraplegia related genes analysed in routine diagnosis. A causative variant was found in 475 patients (30.7%) in 35/65 screened genes. SPAST and SPG7 were the most frequently mutated genes, representing 142 (9.2%) and 75 (4.8%) index cases of the whole series, respectively. KIF1A, ATL1, SPG11, KIF5A and REEP1 represented more than 1% (>17 cases) each. There were 661 causative variants (382 different ones) and 30 of them were structural variants. This large cohort allowed us to obtain an overview of the clinical and genetic spectrum of hereditary spastic paraplegia in clinical practice. Because of the wide phenotypic variability, there was no very specific sign that could predict the causative gene, but there were some constellations of symptoms that were found often related to specific subtypes. Finally, we confirmed the diagnostic effectiveness of a targeted sequencing panel as a first-line genetic test in hereditary spastic paraplegia. This is a pertinent strategy because of the relative frequency of several known genes (i.e. SPAST, KIF1A) and it allows identification of variants in the rarest involved genes and detection of structural rearrangements via coverage analysis, which is less efficient in exome datasets. It is crucial because these structural variants represent a significant proportion of the pathogenic hereditary spastic paraplegia variants (∼6% of patients), notably for SPAST and REEP1. In a subset of 42 index cases negative for the targeted multigene panel, subsequent whole-exome sequencing allowed a theoretical diagnosis yield of ∼50% to be reached. We then propose a two-step strategy combining the use of a panel of genes followed by whole-exome sequencing in negative cases.

Phenoconversion from Spastic Paraplegia to ALS/FTD Associated with CYP7B1 Compound Heterozygous Mutations.

Biallelic mutations in the CYP7B1 gene lead to spastic paraplegia-5 (SPG5). We report herein the case of a patient whose clinical symptoms began with progressive lower limb spasticity during childhood, and who secondly developed amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) at the age of 67 years. Hereditary spastic paraplegia (HSP) gene analysis identified the compound heterozygous mutations c.825T>A (pTyr275*) and c.1193C>T (pPro398Leu) in CYP7B1 gene. No other pathogenic variant in frequent ALS/FTD causative genes was found. The CYP7B1 gene seems, therefore, to be the third gene associated with the phenoconversion from HSP to ALS, after the recently described UBQLN2 and ERLIN2 genes. We therefore expand the phenotype associated with CYP7B1 biallelic mutations and make an assumption about a link between cholesterol dyshomeostasis and ALS/FTD.

Evidence of mosaicism in SPAST variant carriers in four French families.

Hereditary spastic paraplegias (HSP) are heterogeneous disorders, with more than 70 causative genes. Variants in SPAST are the most frequent genetic etiology and are responsible for spastic paraplegia type 4 (SPG4). Age at onset can vary, even between patients from the same family, and incomplete penetrance is described. Somatic mosaicism is extremely rare with only three patients reported in the literature. We report here SPAST mosaic variants in four unrelated patients. We confirm that mosaicism in SPAST is a very rare event with only four identified cases on more than 300 patients with a SPAST variant previously described by our clinical diagnostic laboratory.

Increasing involvement of CAPN1 variants in spastic ataxias and phenotype-genotype correlations.

Spastic ataxias are rare neurogenetic disorders involving spinocerebellar and pyramidal tracts. Many genes are involved. Among them, CAPN1, when mutated, is responsible for a complex inherited form of spastic paraplegia (SPG76). We report the largest published series of 21 novel patients with nine new CAPN1 disease-causing variants and their clinical characteristics from two European university hospitals (Paris and Stockholm). After a formal clinical examination, causative variants were identified by next-generation sequencing and confirmed by Sanger sequencing. CAPN1 variants are a rare cause (~ 1.4%) of young-adult-onset spastic ataxia; however, together with all published cases, they allowed us to better describe the clinical and genetic spectra of this form. Truncating variants are the most frequent, and missense variants lead to earlier age at onset in favor of an additional deleterious effect. Cerebellar ataxia with cerebellar atrophy, dysarthria and lower limb weakness are often associated with spasticity. We also suggest that cognitive impairment and depression should be assessed specifically in the follow-up of SPG76 cases.

RNF170-Related Hereditary Spastic Paraplegia: Confirmation by a Novel Mutation.

BACKGROUND: Spastic paraparesis and biallelic variants functionally characterized as deleterious in the RNF170 gene have recently been reported by Wagner et al. 2019, strongly supporting the involvement of this gene in hereditary spastic paraplegia. METHODS: Exome sequencing was performed on 6 hereditary spastic paraplegia families previously tested on an hereditary spastic paraplegia-specific panel. RESULTS: We describe here a novel hereditary spastic paraplegia family with 4 affected members carrying a homozygous p.(Tyr114*) stop gain variant in RNF170. CONCLUSIONS: We confirm the involvement of biallelic truncating variants in RNF170 in a novel form of hereditary spastic paraplegia. © 2020 International Parkinson and Movement Disorder Society.

Integrated molecular characterization of chondrosarcoma reveals critical determinants of disease progression.

Chondrosarcomas are primary cancers of cartilaginous tissue with highly contrasting prognoses. These tumors are defined by recurrent mutations in the IDH genes and other genetic alterations including inactivation of CDKN2A and COL2A1; however, these have no clinical value. Here we use multi-omics molecular profiles from a series of cartilage tumors and find an mRNA classification that identifies two subtypes of chondrosarcomas defined by a balance in tumor differentiation and cell cycle activation. The microRNA classification reveals the importance of the loss of expression of the 14q32 locus in defining the level of malignancy. Finally, DNA methylation is associated with IDH mutations. We can use the multi-omics classifications to predict outcome. We propose an mRNA-only classifier to reproduce the integrated multi-omics classification, and its application to relapsed tumor samples shows the progressive nature of the classification. Thus, it may be possible to use mRNA-based signatures to detect patients with high-risk chondrosarcomas.

Loss of paraplegin drives spasticity rather than ataxia in a cohort of 241 patients with SPG7.

OBJECTIVE: We took advantage of a large multinational recruitment to delineate genotype-phenotype correlations in a large, trans-European multicenter cohort of patients with spastic paraplegia gene 7 (SPG7). METHODS: We analyzed clinical and genetic data from 241 patients with SPG7, integrating neurologic follow-up data. One case was examined neuropathologically. RESULTS: Patients with SPG7 had a mean age of 35.5 ± 14.3 years (n = 233) at onset and presented with spasticity (n = 89), ataxia (n = 74), or both (n = 45). At the first visit, patients with a longer disease duration (>20 years, n = 62) showed more cerebellar dysarthria (p < 0.05), deep sensory loss (p < 0.01), muscle wasting (p < 0.01), ophthalmoplegia (p < 0.05), and sphincter dysfunction (p < 0.05) than those with a shorter duration (<10 years, n = 93). Progression, measured by Scale for the Assessment and Rating of Ataxia evaluations, showed a mean annual increase of 1.0 ± 1.4 points in a subgroup of 30 patients. Patients homozygous for loss of function (LOF) variants (n = 65) presented significantly more often with pyramidal signs (p < 0.05), diminished visual acuity due to optic atrophy (p < 0.0001), and deep sensory loss (p < 0.0001) than those with at least 1 missense variant (n = 176). Patients with at least 1 Ala510Val variant (58%) were older (age 37.6 ± 13.7 vs 32.8 ± 14.6 years, p < 0.05) and showed ataxia at onset (p < 0.05). Neuropathologic examination revealed reduction of the pyramidal tract in the medulla oblongata and moderate loss of Purkinje cells and substantia nigra neurons. CONCLUSIONS: This is the largest SPG7 cohort study to date and shows a spasticity-predominant phenotype of LOF variants and more frequent cerebellar ataxia and later onset in patients carrying at least 1 Ala510Val variant.

Spastic paraplegia due to recessive or dominant mutations in ERLIN2 can convert to ALS.

OBJECTIVE: The aim of this study was to evaluate whether mutations in ERLIN2, known to cause SPG18, a recessive hereditary spastic paraplegia (SP) responsible for the degeneration of the upper motor neurons leading to weakness and spasticity restricted to the lower limbs, could contribute to amyotrophic lateral sclerosis (ALS), a distinct and more severe motor neuron disease (MND), in which the lower motor neurons also profusely degenerates, leading to tetraplegia, bulbar palsy, respiratory insufficiency, and ultimately the death of the patients. METHODS: Whole-exome sequencing was performed in a large cohort of 200 familial ALS and 60 sporadic ALS after a systematic screening for C9orf72 hexanucleotide repeat expansion. ERLIN2 variants identified by exome analysis were validated using Sanger analysis. Segregation of the identified variant with the disease was checked for all family members with available DNA. RESULTS: Here, we report the identification of ERLIN2 mutations in patients with a primarily SP evolving to rapid progressive ALS, leading to the death of the patients. These mutations segregated with the disease in a dominant (V168M) or recessive (D300V) manner in these families or were found in apparently sporadic cases (N125S). CONCLUSIONS: Inheritance of ERLIN2 mutations appears to be, within the MND spectrum, more complex that previously reported. These results expand the clinical phenotype of ERLIN2 mutations to a severe outcome of MND and should be considered before delivering a genetic counseling to ERLIN2-linked families.

Spastic paraplegia due to SPAST mutations is modified by the underlying mutation and sex.

Hereditary spastic paraplegias (HSPs) are rare neurological disorders caused by progressive distal degeneration of the corticospinal tracts. Among the 79 loci and 65 spastic paraplegia genes (SPGs) involved in HSPs, mutations in SPAST, which encodes spastin, responsible for SPG4, are the most frequent cause of both familial and sporadic HSP. SPG4 is characterized by a clinically pure phenotype associated with restricted involvement of the corticospinal tracts and posterior columns of the spinal cord. It is rarely associated with additional neurological signs. However, both age of onset and severity of the disorder are extremely variable. Such variability is both intra- and inter-familial and may suggest incomplete penetrance, with some patients carrying mutations remaining asymptomatic for their entire life. We analysed a cohort of 842 patients with SPG4-HSP to assess genotype-phenotype correlations. Most patients were French (89%) and had a family history of SPG4-HSP (75%). Age at onset was characterized by a bimodal distribution, with high inter-familial and intra-familial variability, especially concerning first-degree relatives. Penetrance of the disorder was 0.9, complete after 70 years of age. Penetrance was lower in females (0.88 versus 0.94 in males, P = 0.01), despite a more diffuse phenotype with more frequent upper limb involvement. Seventy-seven per cent of pathogenic mutations (missense, frameshift, splice site, nonsense, and deletions) were located in the AAA cassette of spastin, impairing its microtubule-severing activity. A comparison of the missense and truncating mutations revealed a significantly lower age at onset for patients carrying missense mutations than those carrying truncating mutations, explaining the bimodal distribution of the age at onset. The age at onset for patients carrying missense mutations was often before 10 years, sometimes associated with intellectual deficiency. Neuropathological examination of a single case showed degeneration of the spinocerebellar and spinocortical tracts, as well as the posterior columns. However, there were numerous small-diameter processes among unusually large myelinated fibres in the corticospinal tract, suggesting marked regeneration. In conclusion, this large cohort of 842 individuals allowed us to identify a significantly younger age at onset in missense mutation carriers and lower penetrance in females, despite a more severe disorder. Neuropathology in one case showed numerous small fibres suggesting regeneration.

Plasma oxysterols: biomarkers for diagnosis and treatment in spastic paraplegia type 5.

The hereditary spastic paraplegias are an expanding and heterogeneous group of disorders characterized by spasticity in the lower limbs. Plasma biomarkers are needed to guide the genetic testing of spastic paraplegia. Spastic paraplegia type 5 (SPG5) is an autosomal recessive spastic paraplegia due to mutations in CYP7B1, which encodes a cytochrome P450 7α-hydroxylase implicated in cholesterol and bile acids metabolism. We developed a method based on ultra-performance liquid chromatography electrospray tandem mass spectrometry to validate two plasma 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC) as diagnostic biomarkers in a cohort of 21 patients with SPG5. For 14 patients, SPG5 was initially suspected on the basis of genetic analysis, and then confirmed by increased plasma 25-OHC, 27-OHC and their ratio to total cholesterol. For seven patients, the diagnosis was initially based on elevated plasma oxysterol levels and confirmed by the identification of two causal CYP7B1 mutations. The receiver operating characteristic curves analysis showed that 25-OHC, 27-OHC and their ratio to total cholesterol discriminated between SPG5 patients and healthy controls with 100% sensitivity and specificity. Taking advantage of the robustness of these plasma oxysterols, we then conducted a phase II therapeutic trial in 12 patients and tested whether candidate molecules (atorvastatin, chenodeoxycholic acid and resveratrol) can lower plasma oxysterols and improve bile acids profile. The trial consisted of a three-period, three-treatment crossover study and the six different sequences of three treatments were randomized. Using a linear mixed effect regression model with a random intercept, we observed that atorvastatin decreased moderately plasma 27-OHC (∼30%, P < 0.001) but did not change 27-OHC to total cholesterol ratio or 25-OHC levels. We also found an abnormal bile acids profile in SPG5 patients, with significantly decreased total serum bile acids associated with a relative decrease of ursodeoxycholic and lithocholic acids compared to deoxycholic acid. Treatment with chenodeoxycholic acid restored bile acids profile in SPG5 patients. Therefore, the combination of atorvastatin and chenodeoxycholic acid may be worth considering for the treatment of SPG5 patients but the neurological benefit of these metabolic interventions remains to be evaluated in phase III therapeutic trials using clinical, imaging and/or electrophysiological outcome measures with sufficient effect sizes. Overall, our study indicates that plasma 25-OHC and 27-OHC are robust diagnostic biomarkers of SPG5 and shall be used as first-line investigations in any patient with unexplained spastic paraplegia.

CYP2U1 activity is altered by missense mutations in hereditary spastic paraplegia 56.

Hereditary spastic paraplegia (HSP) is an inherited disorder of the central nervous system mainly characterized by gradual spasticity and weakness of the lower limbs. SPG56 is a rare autosomal recessive early onset complicated form of HSP caused by mutations in CYP2U1. The CYP2U1 enzyme was shown to catalyze the hydroxylation of arachidonic acid. Here, we report two further SPG56 families carrying three novel CYP2U1 missense variants and the development of an in vitro biochemical assay to determine the pathogenicity of missense variants of uncertain clinical significance. We compared spectroscopic, enzymatic, and structural (from a 3D model) characteristics of the over expressed wild-type or mutated CYP2U1 in HEK293T cells. Our findings demonstrated that most of the tested missense variants in CYP2U1 were functionally inactive because of a loss of proper heme binding or destabilization of the protein structure. We also showed that functional data do not necessarily correlate with in silico predictions of variants pathogenicity, using different bioinformatic phenotype prediction tools. Our results therefore highlight the importance to use biological tools, such as the enzymatic test set up in this study, to evaluate the effects of newly identified variants in clinical settings.

Hereditary spastic paraplegia due to a novel mutation of the REEP1 gene: Case report and literature review.

RATIONALE: Hereditary spastic paraplegia (HSP) is a heterogeneous group of diseases little known in clinical practice due to its low prevalence, slow progression, and difficult diagnosis. This results in an underestimation of HSP leading to belated diagnosis and management. In depth diagnosis is based on clinical presentation and identification of genomic mutations. We describe the clinical presentation and pathogeny of HSP through a report of a case due to a novel mutation of the REEP1 gene (SPG31). PATIENT CONCERNS: A 64-year-old woman presented gait disturbances due to spasticity of the lower limbs progressing since her third decade. Previous investigations failed to find any cause. INTERVENTIONS: DNA analysis was performed to search for HSP causing mutations. DIAGNOSES: A novel heterozygote mutation (c.595 + 1G>A) of the REEP1 gene, within the splice site of intron 6, was discovered. This nucleotide change causes exon 6 skipping leading to frame shift and a truncated transcript identified by complementary DNA sequencing of reverse transcription polymerase chain reaction products. OUTCOMES: REEP1 is a known protein predominantly located in the upper motor neurons. Mutation of REEP1 primary affects the longest axons explaining predominance of pyramidal syndrome on lower limbs. LESSONS: Slow progressive pyramidal syndrome of the lower limbs should elicit a diagnosis of HSP. We describe a novel mutation of the REEP1 gene causing HSP. Pathogeny is based on resulting abnormal REEP1 protein which is involved in the development of longest axons constituting the corticospinal tracts.

New candidate loci identified by array-CGH in a cohort of 100 children presenting with syndromic obesity.

Syndromic obesity is defined by the association of obesity with one or more feature(s) including developmental delay, dysmorphic traits, and/or congenital malformations. Over 25 syndromic forms of obesity have been identified. However, most cases remain of unknown etiology. The aim of this study was to identify new candidate loci associated with syndromic obesity to find new candidate genes and to better understand molecular mechanisms involved in this pathology. We performed oligonucleotide microarray-based comparative genomic hybridization in a cohort of 100 children presenting with syndromic obesity of unknown etiology, after exhaustive clinical, biological, and molecular studies. Chromosomal copy number variations were detected in 42% of the children in our cohort, with 23% of patients with potentially pathogenic copy number variants. Our results support that chromosomal rearrangements are frequently associated with syndromic obesity with a variety of contributory genes having relevance to either obesity or developmental delay. A list of inherited or apparently de novo duplications and deletions including their enclosed genes and not previously linked to syndromic obesity was established. Proteins encoded by several of these genes are involved in lipid metabolism (ACOXL, MSMO1, MVD, and PDZK1) linked with nervous system function (BDH1 and LINGO2), neutral lipid storage (PLIN2), energy homeostasis and metabolic processes (CDH13, CNTNAP2, CPPED1, NDUFA4, PTGS2, and SOCS6).

Comprehensive analysis of PTEN status in breast carcinomas.

PTEN plays a well-established role in the negative regulation of the PI3K pathway, which is frequently activated in several cancer types, including breast cancer. A nuclear function in the maintenance of chromosomal stability has been proposed for PTEN but is yet to be clearly defined. In order to improve understanding of the role of PTEN in mammary tumorigenesis in terms of a possible gene dosage effect, its PI3K pathway function and its association with p53, we undertook comprehensive analysis of PTEN status in 135 sporadic invasive ductal carcinomas. Four PTEN status groups were defined; complete loss (19/135, 14%), reduced copy number (19/135, 14%), normal (86/135, 64%) and complex (11/135, 8%). Whereas the PTEN complete loss status was significantly associated with estrogen receptor (ER) negativity (p=0.006) and in particular the basal-like phenotype (p<0.0001), a reduced PTEN copy number was not associated with hormone receptor status or a particular breast cancer subtype. Overall, PI3K pathway alteration was suggested to be involved in 59% (79/134) of tumors as assessed by human epidermal growth factor receptor 2 overexpression, PIK3CA mutation or a complete loss of PTEN. A complex PTEN status was identified in a tumor subgroup which displayed a specific, complex DNA profile at the PTEN locus with a strikingly similar highly rearranged pan-genomic profile. All of these tumors had relapsed and were associated with a poorer prognosis in the context of node negative disease (p=1.4 × 10(-13) ) thus may represent a tumor subgroup with a common molecular alteration which could be targeted to improve clinical outcome.