BACKGROUND: Next-generation sequencing-based molecular assessment has benefited the diagnosis of hereditary spastic paraplegia (HSP) subtypes. However, the clinical and genetic spectrum of HSP due to large fragment deletions/duplications has yet to be fully defined.
OBJECTIVE: We aim to better characterize the clinical phenotypes and genetic features of HSP and to provide new thoughts on diagnosis.
METHODS: Whole-exome sequencing (WES) was performed in patients with clinically suspected HSP, followed by multiple ligation-dependent probe amplification (MLPA) sequentially carried out for those with negative findings in known causative genes. Genotype-phenotype correlation analyses were conducted under specific genotypes.
RESULTS: We made a genetic diagnosis in 60% (162/270) of patients, of whom 48.9% (132/270) had 24 various subtypes due to point mutations (SPG4/SPG11/SPG35/SPG7/SPG10/SPG5/SPG3A/SPG2/SPG76/SPG30/SPG6/SPG9A/SPG12/SPG15/SPG17/SPG18/SPG26/SPG49/SPG55/SPG56/SPG57/SPG62/SPG78/SPG80). Thirty patients were found to have causative rearrangements by MLPA (11.1%), among which SPG4 was the most prevalent (73.3%), followed by SPG3A (16.7%), SPG6 (3.3%), SPG7 (3.3%), and SPG11 (3.3%). Clinical analysis showed that some symptoms were often related to specific subtypes, and rearrangement-related SPG3A patients seemingly had later onset. We observed a presumptive anticipation among SPG4 and SPG3A families due to rearrangement.
CONCLUSIONS: Based on the largest known Asian HSP cohort, including the largest subgroup of rearrangement-related pedigrees, we gain a comprehensive understanding of the clinical and genetic spectrum of HSP. We propose a diagnostic flowchart to sequentially detect the causative genes in practice. Large fragment mutations account for a considerable proportion of HSP, and thus, MLPA screening acts as a beneficial supplement to routine WES. © 2024 International Parkinson and Movement Disorder Society.

Haploinsufficiency is widely accepted as the pathogenic mechanism of hereditary spastic paraplegias type 4 (SPG4). However, there are some cases that cannot be explained by reduced function of the spastin protein encoded by SPAST. The aim of this study was to identify the causative variant of SPG4 in a large Chinese family and explore its pathological mechanism.
A five-generation family with 49 members including nine affected (4 males and 5 females) and 40 unaffected individuals in Mongolian nationality was recruited. Whole exome sequencing was employed to investigate the genetic etiology. Western blotting and immunofluorescence were used to analyze the effects of the mutant proteins in vitro.
A novel frameshift variant NM_014946.4: c.483_484delinsC (p.Val162Leufs*2) was identified in SPAST from a pedigree with SPG4. The variant segregated with the disease in the family and thus determined as the disease-causing variant. The c.483_484delinsC variant produced two truncated mutants (mutant M1 and M87 isoforms). They accumulated to a higher level and presented increased stability than their wild-type counterparts and may lost the microtubule severing activity.
SPAST mutations leading to premature stop codons do not always act through haploinsufficiency. The potential toxicity to the corticospinal tract caused by the intracellular accumulation of truncated spastin should be considered as the pathological mechanism of SPG4.

The transmembrane 63 (TMEM63) family of proteins are originally identified as homologs of the osmosensitive calcium-permeable (OSCA) channels in plants. Mechanosensitivity of OSCA and TMEM63 proteins are recently demonstrated in addition to their proposed activation mechanism by hyper/hypo-osmolarity. TMEM63 proteins exist in all animals, with a single member in Drosophila (TMEM63) and three members in mammals (TMEM63 A/B/C). In humans, monoallelic variants of TMEM63A have been reported to cause transient hypomyelination during infancy, or severe hypomyelination and global developmental delay. Heterozygous variants of TMEM63B are found in patients with intellectual disability and abnormal motor function and brain morphology. Biallelic variants of TMEM63C are associated with hereditary spastic paraplegias accompanied by mild or no intellectual disability. Physiological functions of TMEM63 proteins clearly recognized so far include detecting food grittiness and environmental humidity in Drosophila, and supporting hearing in mice by regulating survival of cochlear hair cells. In this review, we summarize current knowledge about the activation mechanisms and biological functions of TMEM63 channels, and provide a concise reference for researchers interested in investigating more physiological and pathogenic roles of this family of proteins with ubiquitous expression in the body.

UNASSIGNED: Hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative diseases in which little is known about the most appropriate clinical outcome assessments (COAs) to capture disease progression. The objective of this study was to prospectively determine disease progression after 4.5 years of follow-up with different clinician-reported (ClinRO) and gait performance outcomes (PerFOs).
UNASSIGNED: Twenty-six HSP patients (15 SPG4, 5 SPG7, 4 SPG5, 2 SPG3A) participated in this single-center cohort study in which the ClinRO: Spastic Paraplegia Rating Scale; and the PerFOs: 10-meters walking test and timed-up and go (TUG), at self-selected and maximal walking speeds; Locomotor Rehabilitation Index; and 6-min walking test were performed at baseline and after 1.5 (18 patients) and 4.5 (13 patients) years.
UNASSIGNED: In the 3-year interval between the second and third assessments, significant progressions were only found in PerFOs, while in the overall 4.5 years of follow-up, both PerFOs and ClinROs presented significant progressions. The progression slopes of COAs modeled according to the disease duration allowed the estimation of the annual progression of the outcomes and sample size estimations for future clinical trials of interventions with different effect sizes. TUG at maximal walking speed was the only COA capable of differentiating subjects with a worse compared to a stable/better impression of change and would require the smallest sample size if chosen as the primary endpoint of a clinical trial.
UNASSIGNED: These findings indicate that both performance and clinician-reported outcomes can capture long-term progression of HSPs, with some PerFOs presenting greater sensitivity to change. The presented data are paramount for planning future disease-modifying and symptomatic therapy trials for this currently untreatable group of diseases.

Spastic paraplegia type 11 (SPG11) is a common autosomal recessive form of hereditary spastic paraplegia (HSP) characterized by the degeneration of cortical motor neuron axons, leading to muscle spasticity and weakness. Impaired lipid trafficking is an emerging pathology in neurodegenerative diseases including SPG11, though its role in axonal degeneration of human SPG11 neurons remains unknown. Here, we established a pluripotent stem cell-based SPG11 model by knocking down the SPG11 gene in human embryonic stem cells (hESCs). These stem cells were then differentiated into cortical projection neurons (PNs), the cell types affected in HSP patients, to examine axonal defects and cholesterol distributions. Our data revealed that SPG11 deficiency led to reduced axonal outgrowth, impaired axonal transport, and accumulated swellings, recapitulating disease-specific phenotypes. In SPG11-knockdown neurons, cholesterol was accumulated in lysosome and reduced in plasma membrane, revealing impairments in cholesterol trafficking. Strikingly, the liver-X-receptor (LXR) agonists restored cholesterol homeostasis, leading to the rescue of subsequent axonal defects in SPG11-deficient cortical PNs. To further determine the implication of impaired cholesterol homeostasis in SPG11, we examined the cholesterol distribution in cortical PNs generated from SPG11 disease-mutation knock-in hESCs, and observed a similar cholesterol trafficking impairment. Moreover, LXR agonists rescued the aberrant cholesterol distribution and mitigated the degeneration of SPG11 disease-mutated neurons. Taken together, our data demonstrate impaired cholesterol trafficking underlying axonal degeneration of SPG11 human neurons, and highlight the therapeutic potential of LXR agonists for SPG11 through restoring cholesterol homeostasis.

UNASSIGNED: Hereditary spastic paraplegias (HSPs) are genetic neurodegenerative diseases. The most common form of pure HSP that is inherited in an autosomal dominant manner is spastic paraplegia type 4 (SPG4), which is caused by mutations in the SPAST gene. Different theories have been proposed as the mechanism underlying SPAST-HSP for different types of genetic mutations, including gain- and loss-of-function mechanisms. To better understand the mutation mechanisms, we performed genetic analysis and investigated a truncating SPAST variant that segregated with disease in one family.
UNASSIGNED: We described a pure HSP pedigree with family members across four generations. We performed genetic analysis and investigated a novel frameshift pathogenic variant (c.862_863dupAC, p. H289Lfs*27) in this family. We performed reverse transcription-polymerase chain reaction (RT-PCR), Sanger sequencing, and quantitative RT-PCR using total RNA from an Epstein-Barr virus-induced lymphoblastoid cell line produced from the proband. We also performed Western blotting on cell lysates to investigate if the protein expression of spastin is affected by this variant.
UNASSIGNED: This variant (c.862_863dupAC, p. H289Lfs*27) co-segregated with pure HSP in this family and is not registered in any public database. Measurement of SPAST transcripts in lymphoblasts from the proband demonstrated a reduction of SPAST transcript levels through likely nonsense-mediated mRNA decay. Immunoblot analyses demonstrated a reduction of spastin protein expression levels in lymphoblasts.
UNASSIGNED: We report an SPG4 family with a novel heterozygous frameshift variant p.H289Lfs*27 in SPAST. Our study implies haploinsufficiency as the pathogenic mechanism for this variant and expands the known mutation spectrum of SPAST.

Chromosome 3-linked frontotemporal dementia (FTD3) is caused by a gain-of-function mutation in CHMP2B, resulting in the production of a truncated toxic protein, CHMP2BIntron5. Loss-of-function mutations in spastin are the most common genetic cause of hereditary spastic paraplegias (HSP). How these proteins might interact with each other to drive pathology remains to be explored. Here we found that spastin binds with greater affinity to CHMP2BIntron5 than to CHMP2BWT and colocalizes with CHMP2BIntron5 in p62-positive aggregates. In cultured cells expressing CHMP2BIntron5, spastin level in the cytoplasmic soluble fraction is decreased while insoluble spastin level is increased. These pathological features of spastin are validated in brain neurons of a mouse model of FTD3. Moreover, genetic knockdown of spastin enhances CHMP2BIntron5 toxicity in a Drosophila model of FTD3, indicating the functional significance of their association. Thus, our study reveals that the enhanced association between mutant CHMP2B and spastin represents a novel potential pathological link between FTD3 and HSP.

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).

Hereditary ataxias (HA) are a rare group of heterogeneous disorders. Here, we present the results of molecular testing of a group of ataxia patients using a custom-designed next-generation sequencing (NGS) panel. Due to the genetic and clinical overlapping of hereditary ataxias and spastic paraplegias (HSP), the panel encompasses together HA and HSP genes. The NGS libraries, comprising coding sequences for 152 genes, were performed using KAPA HyperPlus and HyperCap Target Enrichment Kit, sequenced on the MiSeq instrument. The results were analyzed using the BaseSpace Variant Interpreter and Integrative Genomics Viewer. All pathogenic and likely pathogenic variants were confirmed using Sanger sequencing. A total of 29 patients with hereditary ataxias were enrolled in the NGS testing, and 16 patients had a confirmed molecular diagnosis with diagnostic accuracy rate of 55.2%. Pathogenic or likely pathogenic mutations were identified in 10 different genes: POLG (PEOA1, n = 3; SCAE, n = 2), CACNA1A (EA2, n = 2), SACS (ARSACS, n = 2), SLC33A1 (SPG42, n = 2), STUB1 (SCA48, n = 1), SPTBN2 (SCA5, n = 1), TGM6 (SCA35, n = 1), SETX (AOA2, n = 1), ANO10 (SCAR10, n = 1), and SPAST (SPG4, n = 1). We demonstrated that an approach based on the targeted use of the NGS panel can be highly effective and a useful tool in the molecular diagnosis of ataxia patients. Furthermore, we highlight the fact that a sequencing panel targeting both ataxias and HSP genes increases the diagnostic success level.

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