Loss-of-function variants in the CAPN1 activator CD99L2 cause X-linked spastic ataxia

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: major_revision

Abstract
Most patients with a rare movement disorder (MD) do not receive a molecular diagnosis, and the underlying genetic variants and mediating genes remain elusive. Here, we evaluate the diagnostic accuracy of conventional and next-generation sequencing-based genetic testing strategies in a cohort of 2,811 individuals with ataxia, spastic paraplegia and dystonia. Exome sequencing establishes genetic diagnoses in 19.3% of cases, and specificity of phenotypic features and age at testing are positive predictors. Genome analysis ‘beyond the exome’ increases the diagnostic yield by 7.5%, mostly due to the improved detection of structural variants and repeat expansions. Unsolved cases are included in the Solve-RD cohort and subjected to gene-burden analysis, providing evidence for loss-of-function variants in X-chromosomal CD99L2 causing spastic ataxia. Cellular studies show that the transmembrane protein CD99L2 occurs mainly in a ubiquitinated form and serves as an activating interactor of the calcium-dependent protease CAPN1. Ablation of cytoplasmic or extracellular domains of CD99L2 leads to its intracellular mislocalization and abrogation of its interplay with CAPN1. Transcriptome analysis in CD99L2 patient-derived fibroblasts reveals synaptic function-specific disturbances. Impaired CAPN1 activation and dysregulation of downstream neuronal pathways constitute the likely molecular cause for neurodegeneration.

Introduction
About 70% of the ~10,000 unique rare diseases (RDs) are believed to have a genetic cause1. However, the genetic aetiologies of more than half of these diseases remain unknown1. Depending on the disease entity and testing strategy applied, the diagnostic yield varies between 20 and 70%2,3. Strategies to increase diagnostic rates for patients include streamlined genome sequencing (GS) and phenotyping, which also enables the discovery of novel gene-disease associations by cohort-based statistical approaches3–5. In Germany, a structured diagnostic process that involved multidisciplinary expertise has been established at centres for rare diseases, and was evaluated at the national level through the TRANSLATE-NAMSE study6,7. At the pan-European level, genomic data collection of individuals affected by rare diseases has been substantially advanced by the Solve-RD initiative, through European Reference Networks8,9. Associated infrastructures such as RD-Connect GPAP10, can accelerate rare disease gene discovery by matchmaking of a prioritised candidate gene in relevant disease cohorts, e.g. Rare Neurological Diseases (RND, https://www.ern-rnd.eu/). The latter include rare movement disorders (MDs), that are a prototypical example of a clinically and genetically heterogenous group of disorders with a high rate of negative testing results (38–88%)11–13, which could be partially explained by an enrichment of sequence variations that are difficult to detect with standard next-generation sequencing analysis, such as tandem repeat expansions14,15. Systematic evaluation of results from conventional fragment analysis (FA) for repeat expansion testing, exome sequencing (ES), genome sequencing (GS), and combinations thereof, can guide genetic testing in routine diagnostics. Combined with standardised clinical information, these data enable the systematic evaluation of factors impacting the diagnostic yield towards improved expectation management of patients and referring clinicians.

A better mechanistic understanding of rare disease biology by in-depth characterisation of novel disease-associated factors is key for the development of targeted therapy and bears the potential to modulate pathways for the treatment of common disease entities. Calpain biology serves as an excellent example, with components and pathology-related activation disturbances being linked to ultra-rare disorders but also Alzheimer’s and Parkinson’s disease, with the full set of mediating factors and cellular interactions awaiting full definition16–18. For instance, previous affinity capture-based mass spectrometry found the transmembrane protein CD99 antigen-like 2 (CD99L2) as an interactor of calpain-1 (CAPN1), a calcium-dependent cysteine protease involved in neuronal plasticity, and linked to forms of recessive spastic paraplegia and cerebellar ataxia19,20. Similar to CD99, CD99L2 plays a role in leucocyte transmigration through endothelial cell-cell contacts21. However, besides an association of the SNP rs11796490 with autism spectrum disorder22, and a single case observation in cerebral palsy23, it has so far not been linked to neurological disease.

In this work, we systematically assess the diagnostic yields of conventional and next-generation sequencing-based testing strategies across a large cohort of individuals with ataxia, spastic paraplegia, and dystonia, and identify factors predictive of establishing a firm molecular diagnosis. We perform a gene-burden analysis on in-house sequencing datasets from a phenotypically diverse collection of 13,132 individuals and subsequent matchmaking to provide evidence that loss-of-function variants in CD99L2 cause X-linked spastic ataxia. We use overexpression cell models and patient-derived fibroblasts to characterise the function of CD99L2 and its interplay with the calpain system and to decipher the pathomolecular consequences of CD99L2 dysfunction. Our investigations reveal a reciprocal interaction between CD99L2 and CAPN1, which involves ubiquitination as a regulatory posttranslational modification (PTM). Loss of CD99L2 in patients’ fibroblasts triggers transcriptional dysregulation of genes linked to neuronal and synaptic function. These disturbances likely represent the molecular cause of neurodegeneration triggered by the absence of CD99L2 in patients.

Results
Definition of cohort, diagnostic yield, and predictive factors
Within 6 years, 2811 individuals [of these, 2337 adults (83.1%) and 474 children (16.9%)] with a suspected rare movement disorder were referred for genetic testing from 26 sites across Germany. A total of 7241 genetic tests were performed, including 4714 FAs in 1067 cases, 2041 ES, and 486 GS (Supplementary Fig. 1a). The testing strategy initially consisted of a staged approach of FAs followed by ES, but then evolved into a prospectively evaluated first-line GS approach with subsequent FA to exclude or confirm specific repeat expansions. About 12% of cases had received previous gene panel-based testing with inconclusive findings.

The majority of cases studied by ES and/or GS were investigated under a multidisciplinary diagnostic care concept based on clinical expertise at university hospital centres for rare diseases24. Clinical information were collected using standardised human phenotype ontology (HPO) terms, and based on key clinical features, affected individuals were assigned to the disease categories ataxia, spasticity, dystonia and combination thereof (for details see Online methods). The positioning of individuals according to their original HPO annotations was visualised by projection into a clinical feature space. Most patients within the same disease group were closely located to one another. However, there was some overlap among clusters (Fig. 1a), consistent with the clinical observation that categorising patients into major MD disease groups may oversimplify their conditions.

Diagnostic yield of fragment analyses and exome sequencing ranges from 10.9 to 19.3%
At a total of 4714 FAs were performed on 1067 subjects, most of them with ataxia (n=988; 92.6%), resulting in a diagnosis in 116 cases (10.9%). The most frequently observed expansions were FXN (25%; Friedreich ataxia, MIM #229300), ATXN3 (17.2%; SCA03, MIM #109150), and CACNA1A (16.4%; SCA06, MIM #601011). The main entities investigated by ES were ataxia (n=899; 44%), spasticity (n=573; 28%), and dystonia (n=265; 13%), as well as individuals with mixed MDs (n=304; 15%). Firm diagnoses (likely pathogenic or pathogenic variants) were established in 394 (19.3%) cases, with the highest (28.1%) diagnostic yield in the spasticity group and the lowest (12.1%) in dystonia patients (Supplementary Fig. 2c). The gene-specific modes of inheritance observed in solved cases were dominant (51%), recessive (42%), X-linked (4%), and mitochondrial (3%), and the most frequently affected genes were SPAST (n=46; 16%), SPG7 (n=19; 6.6%) and CACNA1A (n=15; 5.2%). Further details of the cohort and genetic results for ES are shown in Supplementary Figs. 2 and 3.

Genome sequencing increases diagnostic yield by ~7.5%
Using a bioinformatic pipeline optimised for the detection of complex variation [repeat expansions (RE), structural variants (SV), copy-number variations (CNV)] and interpretation ‘beyond the exome’25, the added diagnostic yield and clinical feasibility of diagnostic genome sequencing were assessed in a total of 486 MD individuals [ataxia (n=219), dystonia (n=19), spasticity (n=134), and combined MDs (n=114)]. This included patients in whom a diagnosis could not be established after previous FA and/or ES (n=272; 56%), as well as n=214 (44%) MD individuals who were investigated in a GS first approach. In the latter, the diagnostic yield was higher than in the ES first cohort (25.2% versus 21.96%, respectively; Fig. 1b). In 200 patients with GS and previous ES plus optional FA, the diagnostic yield was substantially lower but still remarkable with 10.5% (21 solved cases, Fig. 1b). Of the reported clinically relevant findings in these cases, 15 (71%) had been missed in previous analyses, and an additional one (5%) has been technically detected but did not meet the criteria of being formally classified as a likely pathogenic or pathogenic variant at the time of initial testing. The majority of missed variation included SVs and REs that could only be detected by GS and which were accordingly enriched in the second-line GS cohort [16.5% (11/67) GS-first vs 33.3% (9/27) second-line GS; P=0.09475, Fig. 1c]. However, the added diagnostic sensitivity of GS of about 7.5% encompassed all types of genomic variation, including small variants. Illustrative examples can be found in the Supplementary information, together with a detailed description of the GS cohort in Supplementary Figs. 1 and 4.

Specificity of HPO terms, disease group and age at diagnosis are positive predictors for establishing a molecular diagnosis
We next aimed to assess whether factors beyond the genetic tests used could influence the diagnostic rate. A multivariate regression analysis was performed to investigate the impact of various factors, including specific subgroups of MDs, age at genetic testing, and the number of HPO terms, as well as the mean specificity of all assigned human phenotype ontology (HPO) terms per patient (derived from the relative position of HPO terms in their HPO branch). Least absolute shrinkage and selection operator (LASSO) analysis indicated a high mean specificity of HPO terms, young age at genetic testing, as well as a combined MD including spasticity as predictive parameters for establishing a firm genetic diagnosis (Fig. 1d, e). While our model was trained on the exome cohort [AUCROC on test data 0.7 (95% CI: 0.64–0.761)], it was able to discriminate between solved and unsolved cases in the genome cohort [AUCROC 0.609 (95% CI: 0.532–0.687), Supplementary Fig. 5].

Cohort analyses discover novel disease gene
Gene-burden analysis infers three known and one novel genetic association. Taking advantage of available standardised expert-curated phenotype and sequencing datasets, we next performed a gene-burden analysis to identify genetic associations between coding genes and MD phenotypes. To account for clinical heterogeneity among cases sharing the same genetic aetiology as observed with established MD entities (Fig. 1a), we merged the different MD subgroups into one with the aim of boosting power to identify statistical genetic associations. The curated dataset comprised 2287 MD cases and 10,845 controls. Using the two-sided Fisher exact test, gene-wise association analyses were conducted for high-quality protein-truncating variants (PTVs), and a combined set of high and moderate impact variants, considering only variants for which population-specific minor allele frequencies (MAFs) were likely <0.1% (Methods). Both analyses were performed for a recessive and a dominant model of inheritance. Given that copy-number variants (CNVs), at least in the homozygous state, can be detected with a high sensitivity and specificity in genomes and in uniformly processed exome datasets, we extended the recessive stringent model to include high-quality homozygous exon deletions. Of the top three associations identified, two (SPAST and SACS) were already known according to OMIM and PanelApp, and one (CD99L2) was previously unidentified. Notably, of the top 20 ranked genes, 11 (ABCD1, KIF1C, POLR3A, REEP1, SACS, SIL1, SLC44A4, SPAST, SPG11, STUB1, UCHL1) have previously been linked to neurological phenotypes, confirming the utility of the chosen approach (Fig. 1f, an extended prioritisation list is provided in Supplementary Data 1). To prioritise candidates for further investigation, we evaluated supportive evidence from co-segregation and performed a comprehensive review of the literature for each gene to assess whether an association was supported by biological function or previously known disease associations for related genes. Overall, CD99L2 remained the most compelling candidate and underwent further investigation through replication in other rare disease cohorts and in-depth functional studies.

Loss-of-function variants in CD99L2 cause X-linked spastic ataxia.
Subsequent to the seven index cases identified in the initial analysis, an additional eight index cases were detected during routine genome-based diagnostic work-up, one by an extended search of in-house research datasets, and four families via the matchmaking functionality of the RD-Connect Genome-Phenome Analysis Platform (GPAP)10 from the Solve-RD rare neurological diseases cohort26. Of the total 25 affected male individuals from 20 families (Fig. 2a–k and Supplementary Fig. 7a–i), 12 had received a primary diagnosis of hereditary spastic paraplegia (HSP), ten of spastic ataxia, and three of cerebellar ataxia. Age at onset ranged from 10 to 68 years (median: 47 years; interquartile range (IQR): 13 years), and the main presenting features were gait disturbances and spasticity (22/25; 88%), predominantly affecting the lower limbs. During the course of the disease, 41.6% (5/12) of the patients with HSP showed signs of ataxia. Dysarthria (21/25; 84%), oculomotor abnormalities (14/25; 56%), sensory deficits (13/24, 54%), and dysphagia (10/25; 40%) were additional common clinical features (Supplementary Fig. 6 and Supplementary Data 2; see Supplementary information: Case reports CD99L2 families). Nine patients were dependent on a walking aid at a median age of 59 years (range 44–62 years; IQR: 7.25 years). No specific pathologies were detected in the brain MRI examinations performed in 19 probands; only in two cases cerebellar atrophy was observed. Female carriers showed no specific phenotype (Table 1, Supplementary Data 2 and Supplementary Fig. 6).

In summary, the identification of 11 different disease alleles (Fig. 2l), most of which predict a loss of CD99L2 function, in a total of 25 affected members of 20 families with overlapping phenotypes, provides strong evidence that CD99L2 is implicated in X-linked spastic ataxia.

Consequences of identified CD99L2 variants on transcript and protein levels.
To further support the functional relevance of identified variants, we conducted transcriptome sequencing (TS) and western blot studies on available patients’ biospecimens. These included analyses of PAXgene Blood RNA Tubes, representing all identified variants, and fibroblasts from affected individuals carrying six different variants (Fig. 3 and Supplementary Fig. 7j–o). For all PTVs studied, CD99L2 mRNA levels were significantly reduced compared to controls, in line with nonsense-mediated decay (NMD) of mutant transcripts (Fig. 3b, c). When quantified as read counts, CD99L2 mRNA levels of individuals with other mutations, including predicted splice-site alterations and exon deletions, were not significantly altered. However, no wild-type transcripts were observed, only transcripts indicating exon skipping, intron retention, and/or variable use of alternative splice sites (Fig. 3a), as well as a potential upregulation of the shorter transcript ENST00000418547.2 (Ensembl ID) (Fig. 3a; transcript c) of variant c.655+1G>A). Apart from the exon 8/9 deletion, which results in an in-frame transcript missing the entire transmembrane domain, all other aberrant mRNAs predict mainly prematurely truncated proteins. The use of alternative splice sites as shown in Fig. 3a; transcript b) of variant c.535G>C and c.655+1G>A was only seen in a smaller subset of reads.

To analyse the consequences of the identified CD99L2 variants at the protein level, we performed western blotting of fibroblast-derived protein extracts using antibodies directed against either the N-terminus (targeting residues 27–117) or the C-terminus (targeting residues 209–254) of the protein (Fig. 3d–i and Supplementary Fig. 7j–o). In controls, a strong band was detected between 37 and 50 kDa using the N-terminal antibody—higher than the predicted molecular weight of 28 kDa for full-length CD99L2. However, the specificity of this signal was confirmed by parallel detection using the C-terminal antibody.

In patients carrying PTVs, CD99L2-specific bands were entirely absent (Fig. 3d–f and Supplementary Fig. 7j–l). In contrast, patients with splice-site alterations or a deletion of exons 8/9 showed faint bands of lower molecular weight when probed with the N-terminal antibody (Fig. 3g–i) and—exclusively in the case of the exon 8/9 deletion—also with the C-terminal antibody (Supplementary Fig. 7m–o). These may represent internally or C-terminally truncated CD99L2 protein forms, likely corresponding to translation products from the aberrant transcripts detected in the transcriptome analysis (Fig. 3a).

In conclusion, all CD99L2 variants investigated led to a near-complete loss of full-length CD99L2, supporting a loss-of-function as the likely pathomechanism.

Characterisation of CD99L2 function in health and disease.
Similar to CD99, CD99L2 has been postulated to play a role in leucocyte transmigration through endothelial cell-cell contacts21. However, apart from an association with autism spectrum disorder22 and cerebral palsy23, it has not yet been linked to neuronal phenotypes in humans, while a study in mice has suggested that it functions as a synaptic cell adhesion molecule important for regulating neuronal activation27.

CD99L2 primarily exists as a ubiquitinated protein and is degraded via autophagy and proteasomes. To investigate the discrepancy between the calculated (~28 kDa) and observed (37–50 kDa) molecular weight of CD99L2 in control fibroblasts (Fig. 3g–i and Supplementary Fig. 7j–o), we first replicated these findings in 293T cells and SH-SY5Y neuroblastoma cells, both at baseline and upon overexpression of a CD99L2 construct. Only upon overexpression, at levels several-fold higher than the endogenous levels, we were able to detect the apparently unmodified full-length form of CD99L2, along with a potential N-terminal fragment, while the modified form represented the predominant species (Fig. 4a and Supplementary Fig. 8a–d). We then explored possible sources of posttranslational modification (PTM) in 293T cells using pharmacological strategies. While previous studies suggested O-glycosylation as the primary PTM of CD99L2 responsible for the molecular weight shift28, we ruled out O-GlcNAcylation as the main modification (Supplementary Fig. 8e–g). On the other hand, inhibition of proteasomes and autophagy (Fig. 4b)—two protein degradation pathways that rely on substrate ubiquitination as a process-specific marker—led to a marked increase in both the modified and unmodified full-length forms of CD99L2 (Fig. 4c, d). Notably, proteasomal inhibition by MG132 was the most effective in elevating CD99L2 levels, leading to an ~12-fold increase of full-length CD99L2 compared to a five-fold increase upon autophagy inhibition using bafilomycin A1 (BafA1) (Fig. 4d). Moreover, while BafA1 treatment increased levels of the unmodified full-length CD99L2, the levels of the modified, potentially ubiquitinated form declined, likely reflecting of a compensatory upregulation of proteasomal activity in response to autophagy inhibition.

To confirm that CD99L2 undergoes ubiquitination, we performed immunoprecipitation experiments targeting ubiquitinated substrate proteins in CD99L2-expressing 293T cells, which confirmed the presence of ubiquitinated CD99L2 at the expected molecular size (Fig. 4e, f and Supplementary Fig. 8h–k). We validated these findings by mutating all 14 lysine residues in CD99L2 (Fig. 4g) to arginine (CD99L2 K0) to prevent canonical ubiquitination, which successfully resulted in a shift from the higher molecular weight form of CD99L2 to its unmodified full-length form (Fig. 4h, i). While this effect was pronounced, it did not completely eliminate the presence of modified CD99L2, which can be attributed to residual ubiquitination within the protein tag, non-canonical ubiquitination, or the contribution of previously reported glycosylation.

Protein ubiquitination is regulated by a dynamic interplay between substrate-specific E3 ubiquitin ligases (E3s) and deubiquitinases (DUBs)29. As the CD99L2-specific E3s and DUBs remain unidentified, we co-transfected the DUB ataxin-3, a polyglutamine protein associated with spinocerebellar ataxia type 330, with CD99L2 in 293T cells to analyse its effects on CD99L2 ubiquitination. As expected, ataxin-3 overexpression led to a decrease in lysine-48-linked polyubiquitin chains. Interestingly, we also observed a reduction in ubiquitinated CD99L2, accompanied by increased levels of the unmodified full-length protein (Supplementary Fig. 8l–n), further supporting our findings and suggesting a potential pathomechanistic link.

CD99L2 binds CAPN1 and triggers calpain activation.
Previous affinity capture-based mass spectrometry identified CD99L2 as an interactor of calpain-1 (CAPN1), a calcium-activated cysteine protease involved in neuronal plasticity and implicated in recessive spastic paraplegia and cerebellar ataxia19,20. To validate these findings, we performed co-immunoprecipitation (co-IP) experiments using two differently tagged CD99L2 constructs overexpressed in 293T cells. Western blot analysis confirmed that CD99L2 directly interacts with CAPN1 and apparently co-precipitates its regulatory small subunit CSS1 (Fig. 5a, b and Supplementary Fig. 9a–d). A potential weak binding to calpain-2 (CAPN2) was also observed (Extended Data Fig. 10d), whereas no interaction was detected with the non-classical calpain-10 (CAPN10), which structurally differs from CAPN1 by lacking the C-terminal calcium-binding penta-EF-hand domain—and thereby the ability to interact with CSS131—or with the endogenous proteinaceous inhibitor calpastatin (CAST).

In our co-IP experiments, we observed increased autoproteolysis of CAPN1, reduced levels of the endogenous proteinaceous inhibitor calpastatin (CAST), and a fragmentation-related decrease in full-length CSS1, suggesting activation of the calpain system upon CD99L2 transfection. To confirm this, we repeated CD99L2 overexpression in 293T and SH-SY5Y cell lines and quantitatively analysed key protein markers, including the known calpain substrate α-spectrin, whose calpain-mediated proteolysis generates a characteristic ~150 kDa fragment. All analysed proteins exhibited enhanced proteolytic processing in both cell models (Fig. 5c, d and Supplementary Fig. 9e–h), corroborating CD99L2-induced activation of calpains.

The calpain dependency of this effect was further validated through inhibition experiments. Both co-overexpression of CAST and a 24-h treatment with calpain inhibitor III (CI III) were able to counteract CD99L2-dependent calpain activation, as demonstrated by prevention of α-spectrin cleavage (Fig. 5e–h and Supplementary Fig. 10). Notably, CI III treatment showed stronger effects than CAST modulation. Interestingly, CAST overexpression and CI III administration led to increased levels of full-length CD99L2, along with a reduction of the N-terminal CD99L2 fragment, indicating that CD99L2 itself is a calpain substrate. In conclusion, we show that CD99L2 specifically interacts with CAPN1 and activates the calpain system when overexpressed.

CD99L2 is a calpain substrate and is cleaved at its cytoplasmic C-terminus.
Both genetic and pharmacological inhibition of calpains increased levels of full-length CD99L2 while reducing levels of the observed N-terminal fragment. To confirm that CD99L2 is indeed a substrate of calpain-mediated proteolysis and that the fragment is a product of this process, we first performed esiRNA-mediated knockdown of CAPN1 and CAPN2 in 293T cells. This analysis revealed that only CAPN1 depletion affected endogenously expressed CD99L2. Interestingly, CAPN1 knockdown increased the upper, high-molecular-weight fraction of ubiquitinated CD99L2, while lower bands disappeared, suggesting that both full-length and cleaved CD99L2 exist in ubiquitinated forms (Fig. 6a, b and Supplementary Fig. 11a).

To investigate this further, we analysed fibroblasts from a patient with CAPN1-related spastic paraplegia type 76 (SPG76)32, which exhibit loss of the CAPN1 protein. In comparison to a control line, the SPG76 fibroblasts showed increased levels of CAST and reduced α-spectrin cleavage (Fig. 6c, e). Notably, SPG76 fibroblasts also showed reduced fragmentation of ubiquitinated CD99L2, confirming CAPN1 as a key proteolytic regulator of CD99L2 (Fig. 6d, f).

Since effects on the unmodified full-length CD99L2 and its N-terminal fragment are difficult to assess at endogenous expression levels, we next analysed calpain-mediated cleavage in 293T cells overexpressing CD99L2. When incubating protein extracts with exogenous CAPN1, we observed rapid degradation of both full-length and ubiquitinated CD99L2, along with an accumulation of N-terminal ubiquitinated fragments (Fig. 6g, h). Using a less acute, cell-based approach, we treated transfected 293T cells with the calcium ionophore ionomycin to induce calpain activation. This resulted in a marked reduction of both ubiquitinated and unmodified full-length CD99L2, accompanied by an increase in the N-terminal fragment (Fig. 6i, j and Supplementary Fig. 11b, c).

In silico prediction tools suggested that the observed N-terminal fragment likely results from a cleavage site within the calpain-accessible cytoplasmic domain of CD99L2, located around serine 250 (Fig. 6k, l)33. To confirm the presence of this calpain cleavage site, we employed a mutagenesis strategy previously developed for the ataxin-3 protein34. Substitution of residues 247–250 with tryptophan rendered CD99L2 resistant to calpain-mediated fragmentation, effectively blocking formation of the N-terminal breakdown product (Fig. 6l, m). These results demonstrate that CD99L2 is a calpain substrate with a specific cleavage site located at serine 250.

Ubiquitination of CD99L2 modulates CAPN1 binding and calpain activation.
After demonstrating that CD99L2 directly binds to CAPN1 and induces calpain system activation, we explored whether its ubiquitination capacity contributes to this function. To investigate this, we overexpressed both myc-tagged wild-type CD99L2 and its lysine-free variant CD99L2 K0 in 293T cells. Fluorescence microscopy revealed no differences in subcellular localisation of the two CD99L2 variants, including their limited colocalization with CAPN1 at membrane-distal contact sites, indicating that lysine residues—and their ubiquitination—do not alter CD99L2 targeting to the plasma membrane (Fig. 7a).

To assess changes in protein interaction, we performed co-IP via the myc tag of the CD99L2 constructs. Western blot analysis revealed a markedly increased binding affinity of CD99L2 K0 to CAPN1 compared to wild-type CD99L2 (Fig. 7b). Notably, the absence of lysine residues in CD99L2 did not enhance its weak binding to CAPN2.

Next, we assessed the impact of lysine residue loss on CD99L2’s ability to activate calpains. Western blot analysis of calpain system markers—including CAPN1 and CAST levels, as well as α-spectrin and CSS1 cleavage—indicated a further amplification of proteolytic activation in cells expressing CD99L2 K0 (Fig. 7c, d).

Together, these findings suggest that the ubiquitination of CD99L2 modulates its interaction with CAPN1 and dampens calpain activation, potentially serving as a regulatory mechanism to prevent excessive and harmful calpain activity within cells.

CD99L2 domain truncation compromises intracellular localisation, calpain interaction and calpain activation.
We next aimed to characterise the functional role of individual CD99L2 domains by overexpressing myc-tagged CD99L2 truncation constructs containing either the extracellular region, including the N-terminal signal peptide (CD99L2 1–185), or the cytoplasmic portion (CD99L2 207–262) (Fig. 8a). Our analysis underscored the importance of CD99L2’s structural integrity: while all constructs showed limited colocalization with CAPN1, CD99L2 1–185 displayed a compartmentalised cytoplasmic pattern reminiscent of the endoplasmic reticulum (ER), which was confirmed via co-expression with a DsRed-tagged ER reporter. In contrast, CD99L2 207–262 exhibited a more diffused distribution in the cytoplasm and, to a lower extent, the nucleus. Full-length CD99L2, as expected, localised to the plasma membrane (Fig. 8b and Supplementary Fig. 12a, b).

Importantly, while not disturbing CD99L2 protein levels, the truncation of CD99L2 abolished its ubiquitination (Fig. 8c) and impaired calpain activation due to disrupted CAPN1 binding (Fig. 8d–g and Supplementary Fig. 12c–e), highlighting the necessity of membrane anchoring for proper function.

Given the previously observed bimodal degradation of full-length CD99L2, we next explored whether specific domains influence its proteostatic fate. Notably, CD99L2 1–185 accumulated upon autophagy inhibition, while CD99L2 207–262 levels increased following proteasome inhibition (Fig. 8h–j and Supplementary Fig. 12f–i). These findings suggest that CD99L2’s bimodal degradation is domain-dependent, with the N-terminal region driving its autophagosomal clearance and the C-terminal domain directing proteasomal degradation.

CD99L2 patient fibroblasts show transcriptional dysregulation of genes associated with neuronal function.
After functionally dissecting the CD99L2 protein, we next analysed the downstream consequences of CD99L2 variants to better explain the neurological phenotype observed in affected individuals. To this end, we performed transcriptomic profiling of fibroblasts from patients carrying PTVs, splice-site variants, or deletions, compared to fibroblasts from unaffected matched controls. RNA sequencing identified 515 significantly differentially expressed genes (DEGs), including 191 downregulated and 324 upregulated transcripts (Fig. 9a). Since the physical interaction of CD99L2 and the calpain system occurs at the protein level, we specifically assessed potential transcriptional dysregulations of related genes. However, no significant expression changes were detected for CAPN1, CAPN2, CAPNS1 (encoding CSS1), CAST or SPTAN1 (encoding the calpain substrate α-spectrin) (Supplementary Fig. 13a). Furthermore, we analysed protein levels of CAPN1 and CAST, as well as calpain-mediated cleavage of α-spectrin, in fibroblasts derived from CD99L2 patients and healthy controls. While no significant alterations in CAPN1 or CAST levels were detected, spectrin cleavage was reduced in patient fibroblasts, consistent with the absence of the CAPN1 activator CD99L2 (Supplementary Fig. 13b, c).

To identify functional themes among the DEGs, we performed STRING-based gene ontology (GO) enrichment analysis35. Among the top ten enriched biological processes, eight were directly linked to neuronal function. Similarly, six of the top ten enriched cellular compartments were related to neurons, particularly the synapse (Fig. 9b, c). Beyond the general GO term “cell junction”, the strongest enrichment was observed for “synapse,” which encompassed 55 DEGs showing marked dysregulation and a high degree of functional interaction (Fig. 9d, e and Supplementary Fig. 13d).

Although fibroblasts are not the model of choice for directly studying neuron-specific pathology, the consistent dysregulation of neuron- and synapse-related genes highlights the likely impact of CD99L2 variants on neuronal function and degeneration. In light of previous findings that loss of CAPN1 impairs synaptic function and triggers cerebellar ataxia36, and given that CD99L2 may regulate synapse development27, our data suggests that perturbed CD99L2 function could exert similarly detrimental effects in the human brain.

Methods
Cohorts
Patients with a clinical diagnosis of a rare MD who had received diagnostic testing between 10/2016 and 06/2022 at the Institute of Medical Genetics and Applied Genomics Tübingen were eligible for the study. Only patients without a prior genetic diagnosis were investigated by ES and/or GS, with about 12% of probands having previously received gene panel testing at the Institute. Selection and assignment to subgroups was done according to the documented HPO terms (for details see Supplementary Table 1). The majority of cases studied by exome sequencing (ES) and/or genome sequencing (GS) were investigated under a multidisciplinary diagnostic care concept based on clinical expertise at university hospital centres for rare diseases24. Visualisation of phenotype space was done using uniform manifold approximation and projection (UMAP, Fig. 1a; for methods details see Supplementary information). Following identification of the novel disease gene, additional index cases were detected during routine diagnostics, extended search of in-house research datasets, and the RD-Connect Genome-Phenome Analysis Platform (GPAP)10.

Oversight
All procedures involving human subjects were approved by the ethics committee of the Medical Faculty of the University of Tübingen, Germany (Genome+, ClinicalTrial.gov #NCT04315727; 066/2021BO2 for retrospective data analysis; 437/2018BO1 for Solve-RD and secondary use, ClinicalTrial.gov #NCT03491280; Biobank 199/2011 BO1, 423/2019BO1 for cell studies, Treat-HSP, ClinicalTrial.gov #NCT03981276). All probands or their legal representatives provided written informed consent for genetic testing. Consent for publication was obtained from all individuals whose detailed clinical data were presented. Participants did not receive compensation for their participation in the study. The authors vouch for the accuracy and completeness of the data in this report.

Visualisation of phenotypic space
As a first step, phenotypic characteristics annotated for known disease genes were downloaded from the HPO website (hp.obo data-version: hp/releases/2023-10-09 file: genes_to_phenotype.txt, downloaded on 16.11.2023). The data were merged with the annotations of individuals with ES or GS of the different subgroups (ataxia, dystonia, spasticity and the respective overlap groups) by treating each disease-ID as one individual. Subsequently, the similarities of the HPO terms between all pairs of individuals were calculated using the R package ontologySimilarity (version 2.5). The similarities were then converted into a distance matrix and projected to a four-dimensional space by uniform manifold approximation and projection (UMAP). Then, the first two dimensions of this projection were plotted using ggplot2 (https://github.com/GermanDemidov/phenotypes_plot).

Details on genetic studies
Conventional repeat expansion testing using FA. Pseudonymized metadata from FA were retrieved from the local clinical information system (GenLab, Projodis) based on the documented respective analyses. These included diagnostically validated testing for the trinucleotide repeat expansion disorders spinocerebellar ataxia 1 (SCA1, ATXN1), spinocerebellar ataxia 2 (SCA2, ATXN2), Machado-Joseph disease (MJD/SCA3, ATXN3), spinocerebellar ataxia 6 (SCA6, CACNA1A), spinocerebellar ataxia 7 (SCA7, ATXN7), spinocerebellar ataxia 8 (SCA8, ATXN8, ATXN8OS), spinocerebellar ataxia 10 (SCA10, ATXN10), spinocerebellar ataxia 12 (SCA12, PPP2R2B), spinocerebellar ataxia 17 (SCA17, TBP), dentatorubral-pallidoluysian atrophy (DRPLA, ATN1), fragile X tremor/ataxia syndrome (FXTAS, FMR1) and Friedreich ataxia (FRDA, FXN). For the determination of the respective repeat length using fragment length analyses, the section of the respective gene (ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, ATXN8T/ATX8OS, ATXN10, PPP2R2B and FXN) was amplified in a fluorescence-labelled PCR. The length of the respective fragments was determined by capillary electrophoresis (CEQ8000, Beckman Coulter) against an internal size standard (Size Standard 450 or 600, Beckman Coulter). For annotation of the respective alleles, the GenomeLab™ Genetic Analysis System software from Beckman Coulter. Singular signals are interpreted as homozygous genotypes. A heterozygous genomic deletion, which would mimic a homozygous presence, as well as an ‘allelic drop-out’ due to a very rare SNP among the primer binding sites, cannot be excluded with this method. The allele size is determined with an accuracy of ±1 repeat. For ATXN8T/ATX8OS, ATXN10 and FXN fragment length analysis included also repeat primed PCR reactions. For amplification of the CGG repeat in FMR1 the Asuragen AmplideX® PCR/CE FMR1 Kit was used, followed by capillary electrophoresis on the ABI Genetic Analyzer 3730xl with the ROX1000 Size Ladder (Asuragen) and evaluation of the data in GeneMapper Software.

Exome and genome analyses. Exome and genome sequencing, as well as subsequent bioinformatic analysis were performed as described previously25,45.
For ES, exonic and adjacent intronic regions were enriched from 3 µg genomic DNA using SureSelect XT Human All Exon v6 or v7 kits (Agilent Technologies, Santa Clara, California). Sequencing as 2×100 bp paired-end reads was performed on a HiSeq 2500 or NovaSeq 6000 system (Illumina, San Diego, California). Mean coverage of the target region was 132.81X.
For GS, 1 µg of genomic DNA was further processed using the TruSeq PCR-Free Library Prep kit (Illumina, San Diego, CA, USA) and the generated libraries were sequenced on a NovaSeq 6000 system as 2×150 bp paired-end reads to an average 44.82X coverage. Deletions detected by exome sequencing in three individuals of family 2, 9 and 10 were subsequently confirmed by shallow GS with an average coverage of 5.27, 9.58 and 6.44 reads, respectively.
Mapping, variant calling and annotation of the data were performed using the megSAP pipeline (https://github.com/imgag/megSAP) developed at the Institute of Medical Genetics and Applied Genomics, University Hospital Tübingen, Germany. Details about the used tools and databases can be found in the megSAP documentation and have been published previously25.
Clinical variant interpretation was conducted independently by two trained diagnostic molecular geneticists according to an in-house standard operating procedure. Variant classification was done according to the guidelines of the American College of Medical Genetics and Genomics (ACMG)46,47. For recessive disorders, the combination of a likely pathogenic or pathogenic variant with a ‘hot VUS’ on the other allele was considered a significant finding.

Sanger sequencing. Segregation analyses and carrier testing in CD99L2 families was by Sanger Sequencing. PCR primers were designed using the UCSC Exon Primer-Primer Design Programme. Primer sequences and PCR conditions are provided below.

PCR conditions: 12.8 µl H2O, 2.5 µl 10x PCR buffer + MgCl2, 0.5 µl dNTP mix, 2.0 µl betaine (5 M), 2.0 µl Q-Solution (Qiagen), 0.2 µl Taq Polymerase (Thermo Fisher Scientific), 150 ng DNA sample, 1.0 µl each of 10 µM forward and reverse primer. PCR cycles: one cycle of 95 °C for 5 min, 10 cycles with 94 °C for 30 s, 65 °C (-1 °C per cycle) for 30 s, and 72 °C for 30 s, followed by 30 cycles with 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, followed by 72 °C for 7 min and cooling to 10 °C.

RNA sequencing and transcriptome analyses. RNA was extracted from PAXgene tubes (14 patients and 54 male controls) and patients’ (n=8) and male control (n=6) fibroblasts using the QIAsymphony RNA kits on a QIAsymphony SP with the protocol RNA CT 400 V7. To reduce preanalytical artefacts, cell lines were cultured and harvested according to an in-house standard operating procedure (also see section cell culture below). RNA quality was assessed with the Agilent 2100 Fragment Analyzer total RNA kit (Agilent Technologies, Inc., Santa Clara, United States). From 100 ng of total RNA, mRNAs were enriched using polyA capture on a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Libraries were prepared with Next Ultra II Directional RNA Library Prep Kits for Illumina (NEB) on a Biomek i7 (Beckman Sequencing). 215 pM cDNA libraries were sequenced on an Illumina NovaSeq 6000 system as 2×100 bp paired-end reads with ~50 million clusters per sample.
Bioinformatic processing and data interpretation were essentially done as previously described25. Generated RNA sequences were analysed with respect to aberrant expression, aberrant splicing and allelic imbalance using the megSAP pipeline (version 2022_08, https://github.com/imgag/megSAP). In brief, the ngs-bits tool collection (version 2022_08-92, https://github.com/imgag/ngs-bits) was used for quality control (ReadQC) and pre-processing (SeqPurge) of fastq files. STAR (version 2.7.10a, https://www.ncbi.nlm.nih.gov/pubmed/23104886, https://github.com/alexdobin/STAR/) was used for read alignment and detection of splice junctions, which were postprocessed with SplicingToBed. After mapping, MappingQC was used for quality control and Subread (version 2.0.3, https://pubmed.ncbi.nlm.nih.gov/30783653/, https://sourceforge.net/projects/subread/) for read counting based on an Ensembl gene annotation file (GRCh38, release 107, http://www.ensembl.org/index.html). Upon normalisation (megSAP) and quality assessment (RnaQC), expression values of genes and exons were compared with an in-house cohort (same tissue and processing system) using NGSDAnnotateRNA. For differential gene expression analysis, gene names were annotated and expression values (TPM, CPM, FPKM) filtered and normalised using EdgeR. Sample correlation is computed in order to detect outliers. Then, differential gene expression and statistical significance are calculated using EdgeR (https://www.rdocumentation.org/packages/edgeR/versions/3.14.0/topics/glmQLFit, https://rdrr.io/bioc/edgeR/man/glmQLFTest.html). Gene network and functional enrichment analysis of significantly differentially expressed genes (P≤0.05; dotted horizontal line; logFC2 ≤-0.5, logFC2 ≥0.5) were performed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database35. Data obtained from STRING-based analysis was graphically visualised by bubble plots that were generated using the ggplot2 package in R 4.2.248,49. Full script can be provided upon request.

Predictors of diagnostic yield. About 1690 cases with ES and significant (n=394) or non-significant findings (n=1296) were analysed by using the least absolute shrinkage and selection operator (LASSO). Patients with uncertain candidate variants or genes were excluded from the analysis. The dataset was randomly divided into a training set incorporating 1368 cases (1053 unsolved, 315 solved) and a test set incorporating 322 cases (243 unsolved and 79 solved). The binary response of a case being solved or not solved was regressed on 17 subcategories of clinical features and controlling for gender (HPO mean, HPO count, ataxia, spasticity, dystonia, ataxia/dystonia, ataxia/spasticity, dystonia/spasticity, ataxia/dystonia/spasticity, gender, 0–5 y, 5–20 y, 20–30 y, 30–40 y, 40–50 y, 50-60 y and 60–100 y). The HPO mean was calculated as the mean value of relative heights (derived from the relative position of HPO terms in the respective branch of the HPO tree) of all HPO terms assigned to one patient.
The model was fitted on the training data, and the penalty parameter was tuned via ten-fold cross-validation. The resulting model was then applied to the independent test set and validated its predictive performance using the receiver operator characteristics (ROC) curve [AUCROC 0.7 (0.64–0.761)]. We then refitted the model on the whole cohort of 1690 cases and validated the resulting model on an independent genome cohort of 261 cases (69 solved and 192 unsolved) [AUCROC 0.609 (0.532–0.687)].

Gene-burden analyses
Data preparation. MD individuals and controls were selected based on phenotypic criteria and diagnostic-grade quality (https://github.com/imgag/ngs-bits/blob/master/doc/GSvar/sample_quality_control.md) of sequencing datasets. Datasets from relatives and duplicate experiments were excluded from both groups. Variant calling and annotation were conducted uniformly using the megSAP pipeline as described above. High-impact variants included PTVs and canonical splice-site alternations, as well as homozygous exon deletions. Predicted deleterious variants were defined as missense variants having a CADD score ≥20. As different kits were used for enrichment, we restricted the analysis to genomic regions that were well represented across assays. We did not preselect for European ancestry.

Gene-wise burden analysis. The curated dataset comprised 2287 MD cases and 10,845 controls. To account for clinical heterogeneity among cases sharing the same genetic aetiology as observed with established MD entities, we merged the different MD subgroups into one with the aim of boosting power to identify statistical genetic associations. Association analyses were carried out for each gene separately, with pre-filtering of included variants adapted to recessive or dominant models of inheritance as well as different effect sizes of detected variation. Under a dominant model, variants with a likely minor allele frequency (MAF) <0.1 were included, with high-impact (PTVs and canonical splice sites) variants in the stringent and high-impact plus predicted deleterious (CADD score >20) variants in the relaxed analysis. Under a recessive model, only homozygous or hemizygous calls were included, predicting variants with a likely MAF <1%, again with high impact of combined effects for the stringent or relaxed analyses, respectively. In a fifth approach, we added high-quality CNVs for samples with a reference cohort correlation ≥0.9 for ES and ≥0.55 for GS, that were called with a log-likelihood of 15 or more and a copy-number status=0. Alterations in CNV polymorphism regions or a putative MAF >1% as estimated from overlapping in-house deletions were excluded. To reduce the noise of false-positive prioritisation due to technical artefacts, CNVs were only considered as additional evidence for genes that were concomitantly impacted by small variants in at least two cases. Finally, case-control gene-wise burden analysis was performed on 2287 selected diagnostic cases and 10,845 controls using Fisher’s exact test. To adjust for multiple testing, the P value was adjusted according to Bonferroni (αcorr = 1–(1–α)1/n ≈ α/n where α is the desired overall alpha level and n is the number of hypotheses). When correcting for 10,165 genes affected by variants in the MD cohort, a P value <4.9×10−6 was considered as genome-wide significant. All P values were two-sided.

Functional analyses methods
Expression constructs and siRNAs. For overexpression of human CD99L2 (canonical isoform 1, 262 aa; UniProt identifier: Q8TCZ2-1), its lysine-free variant (CD99L2 K0) in which all 14 lysine residues were mutated to arginine or its truncated forms spanning from amino acids 1–185 or 207–262, pcDNA3.1 vectors carrying cDNA for C-terminally myc-His6-tagged forms of the proteins were generated (BioCat, Heidelberg, Germany). A C-terminally myc-DDK-tagged CD99L2 construct carried by a pCMV6-Entry vector was additionally purchased (RC207079, OriGene). Ataxin-3 overexpression was performed using pcDNA3.1 vectors harbouring C-terminally V5-tagged constructs with 15 or 77 glutamines50. Overexpression of calpastatin (CAST) was achieved using a pRK5 vector carrying the cDNA for full-length human CAST51. FLAG-tagged ubiquitin was overexpressed using a pcDNA3.1 vector. Empty pcDNA3.1 and pRK5 vectors were used for control transfections.
For marking the endoplasmic reticulum in fluorescence microscopy analysis, a psDsRed-ER vector (632409, Takara Bio Inc.) was used.
Gene knockdown was achieved using endoribonuclease-prepared siRNAs (esiRNAs) directed against human CAPN1 (EHU032581) and CAPN2 (EHU025391), with siRNA against Renilla luciferase (MISSION® esiRNA EHURLUC) (all MISSION® esiRNAs, Merck) as control.

Cell culture
After receiving written informed consent from the probands, fibroblast cell lines were established from skin punches according to routine procedures. HEK293T (293T) cells (ATCC: CRL-11268), SH-SY5Y cells (ATCC: CRL-2266) and primary fibroblasts derived from patients and healthy control subjects were cultured in Dulbecco’s modified Eagle’s medium (DMEM), high glucose, GlutaMAX (10566016) supplemented with 10% (v/v) foetal bovine serum (FBS), 1% (v/v) non-essential amino acids (MEM NEAA) and 1% (v/v) Antibiotic-Antimycotic (A/A) (all Gibco®, Thermo Fisher Scientific) using 75 cm2 cell culture flasks (REF 658175, Cellstar®, Greiner Bio-One) in 5% CO2 and at 37 °C.
For RNA extraction, fibroblasts were harvested from a 145/20 mm cell culture dish (REF 639160, Cellstar®, Greiner Bio-One) at 80% confluence. For protein extraction, fibroblasts were cultured in 100/20 mm tissue culture dishes (REF 353003, Falcon®, Corning Inc.). In experiments using 293T and SH-SY5Y cells, 6-well tissue culture plates (REF 353046, Falcon®, Corning Inc.) or 100/20 mm tissue culture dishes were employed.

Cell treatments. Transfections of 293T and SH-SY5Y cells were conducted for 72 h using Turbofectin 8.0 (Origene) for overexpression vectors or with Attractene transfection reagent (Qiagen) for esiRNA-based knockdown experiments according to the manufacturers’ protocols.
Cellular O-GlcNAc cycling was targeted by inhibiting the enzyme O-GlcNAc transferase (OGT) with 10 µM OSMI-1 (Sigma-Aldrich) or the O-GlcNAcase with 10 µM Thiamet-G (Sigma-Aldrich) for 24 h. Blockage of proteasomal and autophagosomal pathways was conducted using 2.5 µM proteasome inhibitor MG132 (Merck) and 0.05 µM vacuolar-type H+-ATPase inhibitor bafilomycin A1 (Enzo Life Science) for 24 h. Dimethyl sulfoxide (DMSO) served as the vehicle control. Before administration, all compounds were prediluted in Opti-MEM® I Reduced Serum Media (Gibco®, Thermo Fisher Scientific).
Cell-based calpain activation assays were performed as previously described34. Briefly, to activate endogenous calpains in transiently transfected 293T cells, the cell medium was replaced by Opti-MEM® I Reduced Serum Media containing 1.0 or 1.5 µM calcium ionophore ionomycin (in DMSO) (407950, Merck Millipore) and 5 mM CaCl2. Cells were incubated under standard culture conditions for 1 h. For inhibitor controls, cells were pre-treated with 20 µM calpain inhibitor III (CI III; carbobenzoxy-valinyl-phenylalaninal; in DMSO) (Merck) starting 1 h before ionomycin administration. DMSO served as the vehicle control.

Immunostaining and fluorescence microscopy
For immunocytochemistry staining, 293T cells were seeded on 8-well or 12-well chamber slides (80841 or 81201, Ibidi), transfected with respective constructs, and cultured for 72 h. Subsequently, cells were pre-fixed by supplementing the medium with 0.4% (w/v) paraformaldehyde (PFA) and incubating at 37 °C for 10 min. Then, medium was aspirated, cells were washed once with 1× DPBS (Gibco®, Thermo Fisher Scientific) and incubated in 4% (w/v) PFA in 1× DPBS for 15 min. Next, the fixative was removed, and cells were washed three times with 1× DPBS for 5 min. Afterwards, cells were incubated in blocking/permeabilisation buffer (10% (w/v) BSA, 0.5% (v/v) Triton X-100, 0.02% (w/v) NaN3 in 1× DPBS) at room temperature for 1 h. Cells were probed with mouse anti-myc-tag antibody (1:500; clone 9B11, #2276, Cell Signalling Technology) and rabbit anti-CAPN1 antibody (1:200; 10538-1-AP, Proteintech) in antibody diluent (1% (w/v) BSA, 0.5% (v/v) Triton X-100, 0.02% (w/v) NaN3 in 1× DPBS) at 4 °C overnight. Then the next day, cells were washed four times with 1× DPBS and incubated with goat anti-mouse Alexa Fluor 488 (1:500; A32723, Thermo Fisher Scientific), goat anti-mouse Alexa Fluor 594 (1:500, A32742, Thermo Fisher Scientific) and goat anti-rabbit Alexa Fluor 488 (1:500, A27034, Thermo Fisher Scientific) secondary antibody at room temperature for 1 h. After washing four times with 1× DPBS for 5 min, the media chamber was removed, cells mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (H-1200, Vector Laboratories) using coverslips and sealed with a transparent nail polish.
Fluorescent images were taken at 200× and 630× magnifications on an Axioplan 2 imaging microscope equipped with an ApoTome, Plan-Neofluar 20×/0.50 objective, Plan-Neofluar 40×/0.75 and Plan-Neofluar 63×/1.4 Oil objectives, and an AxioCam MRm camera, using the AxioVision 4.3 imaging software (all Zeiss).

Protein extraction
Extraction of proteins was performed as follows. 293T cells were dissociated by gentle pipetting and transferred to 2 mL tubes. Cell pellets were obtained by centrifugation at 500×g for 5 min, followed by aspiration of the supernatant. Pellets were washed once with cold 1× DPBS. SH-SY5Y cells and fibroblasts obtained from healthy control subjects and patients were harvested by scraping cells in 1× DPBS and subsequent pelleting at 500×g for 5 min, followed by aspiration of the supernatant. Cell lysis was conducted by resuspending the pellet in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% (w/v) SDS, 0.5% sodium deoxycholate and 1% (v/v) Triton X-100) containing cOmplete™ protease inhibitor cocktail and PhosSTOP™ phosphatase inhibitors (both Roche), and incubated on ice for 15 min (293T and SH-SY5Y cells) or 30 min (fibroblasts). Afterwards, samples were centrifuged for 15 min (293T and SH-SY5Y cells) or 30 min (fibroblasts) at 4 °C and 20,000×g, and supernatants were transferred to a fresh, pre-cooled reaction tube. Protein concentrations were measured spectrophotometrically in a microtiter plate using Bradford reagent (Bio-Rad Laboratories). Samples were stored at 80 °C until further analysis.

Immunoprecipitation
FLAG- and myc-tagged proteins were immunoprecipitated using DYKDDDDK Fab-Trap (ffa-20) and myc-Trap agarose (yta-20) (both ChromoTek/Proteintech Germany, Planegg-Martinsried, Germany). Ubiquitinated proteins were purified using Ubiquitin-Trap agarose (uta-20, ChromoTek/Proteintech). All immunoprecipitation experiments were performed according to the manufacturer’s protocol with the following modifications: Transfected and/or treated 293T cells were harvested by resuspension in culture medium (content of a 100/20 mm cell culture dish) and subsequent centrifugation for 5 min at 4 °C and 500×g. Cell pellets were washed once with 1× DPBS and centrifuged again. Afterwards, cell pellets were lysed in 150 µL Trap lysis buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% IGEPAL CA-630) containing EDTA-free cOmplete protease inhibitor cocktail and PhosSTOP phosphatase Inhibitor cocktail (Roche). After measuring protein concentrations using Bradford reagent, lysate concentrations were adjusted with Trap lysis buffer to 5 µg/µL and then further diluted in Trap dilution buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) containing EDTA-free cOmplete protease inhibitor cocktail and PhosSTOP phosphatase Inhibitor cocktail to a final concentration of 2 µg/µL. For immunoprecipitation, 500 µg total protein were incubated with 12.5 µL of agarose bead slurry, rotating end-over-end at 4 °C for 1 h. Subsequently, beads were washed three times with Trap wash buffer (10 mM Tris/Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA and 0.05% IGEPAL CA-630) containing EDTA-free cOmplete protease inhibitor cocktail and heat-denatured at 70 °C for 10 min in 90 µL of 4× LDS sample buffer (1 M Tris, pH 8.5, 50% (v/v) glycerol, 8% (w/v) LDS, 2 mM EDTA and 0.1% (w/v) Orange G) mixed with Trap dilution buffer in a ratio 1:1 and supplemented with 0.1 M dithiothreitol (DTT). Samples were subsequently analysed by western blotting.

Calpain cleavage assays in vitro
In vitro calpain cleavage assays were performed as previously described52. Briefly, 25 µg protein amount of 293T cell protein extracts after lysis in calpain reaction buffer (CRB) (20 mM HEPES/KOH, pH 7.6, 10 mM KCl, 1.5 mM MgCl2 and 1 mM DTT). Reactions were prepared by diluting protein extracts in CRB and incubating them with 2 mM CaCl2 and 100 ng of purified calpain-1 (208712, Merck) in a total volume of 20 µL at room temperature. Reactions were terminated by addition of 4× LDS buffer (1 M Tris, pH 8.5, 50% (v/v) glycerol, 8% (w/v) LDS, 2 mM EDTA and 0.1% (w/v) Orange G) in a ratio 3:1 with 0.1 M DTT, and heat-denaturing at 70 °C for 10 min. All samples were further analysed via western blotting.

Western blotting
For western blotting, desired amounts of diluted protein extracts were mixed with 4× LDS sample buffer in a ratio of 3:1 supplemented with 0.1 M DTT and heat-denatured for 10 min at 70 °C. Afterwards, protein samples were electrophoretically separated using Bis-Tris gels and MOPS electrophoresis buffer (50 mM MOPS, 50 mM Tris, pH 7.7, 0.1% (w/v) SDS and 1 mM EDTA). Proteins were transferred on Amersham™ Protran™ Premium 0.2 µm nitrocellulose (Cytiva) using Bicine/Bis-Tris transfer buffer (25 mM Bicine, 25 mM Bis-Tris, pH 7.2, 1 mM EDTA and 15% (v/v) methanol) and a TE22 Transfer Tank (Hoefer) at 80 V at 25 °C for 2 h.
After transfer, membranes were stained for total protein using Ponceau S staining solution (0.1% (w/v) Ponceau S, 5% (v/v) acetic acid) for 1 min, or SYPRO™ Ruby Protein Blot Stain (Thermo Fisher) according to the manufacturer’s protocols. Fluorescent SYPRO Ruby signals were detected using the LI-COR ODYSSEY® FC system (LI-COR Biosciences). If stained with Ponceau S, membranes were destained briefly with 1× TBS (10 mM Tris, pH 7.5, 150 mM NaCl). Afterwards, membranes were blocked for 45 min in 5% (w/v) skim milk powder (Sigma-Aldrich) diluted in 1× TBS at room temperature, and probed overnight at 4 °C with primary antibodies diluted in 1× TBS-T (TBS with 0.1% (v/v) Tween 20). All applied primary antibodies and their specifications are listed in Supplementary Table 2. Thereafter, membranes were washed with 1× TBS-T and incubated at room temperature for 1 h with the respective secondary IRDye® antibodies, goat anti-mouse 680LT (P/N 926−68020), goat anti-mouse 800CW (P/N 926-32210), goat anti-rabbit 680RD (P/N 926-68071) and goat anti-rabbit 800CW (P/N 926-32211) (all 1:5,000; LI-COR Biosciences). After final washing with 1× TBS-T, fluorescence signals were acquired using the LI-COR ODYSSEY® FC and quantified with Image Studio 4.0 software (both LI-COR Biosciences).

In silico calpain cleavage site prediction
Computational analyses of features within the sequence of CD99L2 were based on its canonical isoform 1 (UniProt identifier: Q8TCZ2-1). For in silico calpain cleavage site prediction, Group-based Prediction System—Calpain Cleavage Detector (GPS-CCD) (available at http://ccd.biocuckoo.org/) or Labelling calpain substrate cleavage sites from amino acid sequence using conditional random fields (LabCaS) (available at http://www.csbio.sjtu.edu.cn/bioinf/LabCaS/) tools were employed33,53. Likelihood scores for every amino acid position were graphically visualised using GraphPad Prism 9.1.2 (GraphPad Software).

Statistics and reproducibility
For qualitative analyses, all experiments were independently repeated at least three times and yielded comparable results, ensuring validity and reproducibility. Quantitative data obtained from functional analyses were statistically analysed using GraphPad Prism 9.1.2 (GraphPad Software). Results are presented as individual data points with means and s.e.m. Statistical outliers indicated by the Grubbs test (α=0.05) were excluded from the analysis. Two-tailed one-sample t-test, two-tailed Student’s t-test or one-way ANOVA with Šídák’s or Tukey’s post hoc analysis were applied. Significance was assumed with a P value ≤0.05. Exact P values are shown in graphs. For further details, see the respective figure and figure legends, and the Source Data files.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Menden et al compared different genetic testing strategies in a very large cohort of 2811 subjects affected\nby various movement disorders, exploiting the efforts of a national genome sequencing program. The main outcomes of this\nstudy are:\n- The overall diagnostic yield was nearly 20%, with the best outcome for spastic paraplegia and the worst for dystonia\n- Genome sequencing significantly increased the diagnostic rate by about 7.5%, mainly due to structural variants and\nexpansions not detectable with conventional WES\n- Gene-burden analysis of negative cases identified loss of function variants in the CD99L2 gene as causative of a novel\nform of X-linked spastic ataxia\n- Functional studies demonstrated the role of CD99L2-encoded protein as activating interactor of the protease CAPN1,\nalready linked to forms of recessive spastic paraplegia and ataxia.\nComments to the authors\n1) I find the general term “movement disorders” questionable in this study. The authors selected three major categories,\nwhich are dystonia, cerebellar ataxia and spastic paraplegia. While the inclusion of the latter is justified by the relevant\noverlap with cerebellar ataxia phenotype, spastic paraplegia cannot be strictly considered a “movement disorder”. On the\nother hand, other movement disorders were excluded from the HPO term selection, namely\nchorea, myoclonus and\nparkinsonism, just to name the most relevant ones from a genetic point of view. This should be clearly acknowledged and\ndiscussed / justified, as it should be made clear that the reported diagnostic yield cannot be generalized to rare movement\ndisorders but only to a subset of them. I would suggest the title should also be changed to reflect this.\n2) The authors mention that panel analysis, likely performed in the early days before the widespread diffusion of WES,\nwere\nnot counted. However, it is likely that a subset of patients underwent NGS-panel analysis from this cohort. Can the authors\nprovide an estimate of these figures? Did all patients who previously tested negative at panel analysis undergo WES or\nWGS? Conversely, were patients diagnosed through panel-based analysis included or excluded by the present study? As\nthis is a large nationwide-based project with likely “epidemiological” implications, it is important to have a precise knowledge\nof inclusion and exclusion criteria.\n3) Supplementary Figure 2: does 2B panel refer to the age at onset? Please check the letters in the panels, as they do not\nalways correspond to the legend.\n4) Supplementary Figure 3: does 3A panel refer to the age at onset? 3B: why patients with no significant findings were\ntested later? Is there any correlation with the phenotype (HPO categories), or the age at onset? Also in panel 3C, explain\nwhether this is age at onset or age at diagnosis. Same comments for figure 4.\n5) In Supplementary Figures 2 and 4, please explain the criteria to assign “candidate genes” (yellow colour). How many of\nthese candidate genes were validated / confirmed? They should be reported in a table.\n6) Page 7, Gene-burden analysis refers to an extended prioritization list presented in Supplementary Table 3, but such\nTable is missing. Similarly, I cannot find Supplementary Table 4, quoted on page 7.\n7) Page 7: Please check the number of index cases as they seem to be 21 (8+8+1+4), while later and in the figures the total\nnumber of families are 20.\n8) Legend to Figure 2: please correct “blue arrowheads in (r) and (d)” to “blue arrowheads in (r) and (s)”\n9) Non-truncating variants are of interest and should be characterized more in detail. Figure 2m and 2n show that RNA\nlevels from blood or fibroblasts of patients carrying splicing variants or a multiexon deletion are significantly reduced\ncompared to controls (although not abolished as shown for truncating variants). Western blotting (shown for three variants)\nlack the wild type band but show weaker, smaller bands. Only some variants were assessed at the transcriptional level\n(Supplementary Figure 8). Were the bands observed on Western blotting correspond to the transcripts detected on\nRNAseq? Supplementary Figure 8 should be expanded to comprehensively describe the impact of non-truncating variants\nat the cDNA and protein level (by the way, on figure 2, panel m referts to c.535G>C while the legend refers to c.535G>A –\nplease check)\n10) Please comment why autophagy inhibition results in a reduction of ubiquitinated CD99L2 while the unmodified protein\nincreases (Figure 3d).\n11) Legend to figure 3: please move description of grey arrowheads/white arrowheads/black arrowheads to panels a-c.\n12) Figure 4h-j: can the authors explain this divergent behavior of truncated CD99L2 in response to proteasome or\nautophagy inhibition?\n13) Figure 4k: it would be useful to have a brief explanation of this schematic cartoon to help the readers draw the right\nconclusion about the postulated mechanism\n14) Supplementary Figure 9: it seems that anti-CD99L2 antibody does not recognize any endogenous protein either in 293T\ncells or SH-SY5Y cells (mock transfected lane is perfectly clear not only in the myc-antibody panels but also in the CD99L2\npanels and overexposure shows a very faint modified band). Is the protein expected to have a very rapid turnover in cells?\nFigure 3C seems to show that even inhibition of proteasome or autophagy do not result in increased levels of endogenous\nprotein in mock-transfected cells. However, in Supplementary Figure 12 it seems that endogenous CD99L2 is detected in\ncontrol and SPG76 fibroblasts. Please provide an explanation for these observations or clarify in the text\n15) The authors discuss a cellular mislocalization of truncated forms of CD99L2 compared to wild type. By assessing\ncolocalization with an ER-marker, it seems that truncated forms of the protein have increased co-localization with ER\ncompared to wild type, however this is not explained in the text. Why the authors specifically assessed colocalization with\nER? Have they also tested colocalization with CAPN1?", "reviewer_2": "This manuscript describes the approach to increase diagnostic power for rare diseases by rationally combining the\nmeasures to detect potential cause at the level of gene sequence and their association with clinical phenotypes. In their\nexperimental settings using movement disorders as study subjects, improvements in diagnostic yields were suggested and\nthe novel disease gene, CDL99L2, was identified. Then, properties of the gene product were explored demonstrating that\nCD99L2 is constitutively ubiquitinated and its turnover is regulated by proteasome and autophagy. In addition, CD99L2\ninteracts with CAPN1, a catalytic subunit of intracellular protease calpain-1, and activates it. Further, truncated mutants of\nCD99L2, either the N- or C-terminal, were shown to be defective in localization, calpain interaction/activation as well as\nubiquitination. It is suggested that pathogenicity of the CDL99L2 variants identified in the study is manifested via\ndysregulation of calpain and/or compromise in degradative regulation of CDL99L2.\nThe first part of the study shows the impact of systematically performed evaluation of the huge sets of sequence data.\nIdentification of a novel causative gene, CDL99L2, for the movement disorder is a great outcome without question. In the\nfollowing experiment, basic properties of CDL99L2 was examined, however, the results as shown in Fig. 4 are not sufficient\nto support what the authors claim in discussion, i.e., functional investigation on CDL99L2 is missing. Therefore, it is not clear\nif there is any real association between the proteolytic systems, either proteasome, autophagy or calpain, and CD99L2.\nTo demonstrate the relevance of the present result in terms of pathogenic mechanism stemming from the CDL99L2 variants,\nfollowing points, at minimum, should be clarified; is there any functional relationship between ubiquitination and CAPN1-\ninteraction of CD99L2?, in terms of calpain-1 activation function of CD99L2, what cellular functions are compromised in\nthe cells from patients harboring CD99L2 mutations? (is activation of calpain-1 impaired ?), what is functional impact of\nubiquitination of CD99L2 function?\nComments on minor points:\n(line 176) The case number for FA should be 1,067 (as in Supl. Fig. 1).\n(line 270) The change in molecular weight by ubiquitination is ca. 22 kDa at maximum corresponding to 3 ubiquitins,\ntherefore, “highly” is unnecessary.\n(line 226 in supplement) What mutation(s) does “lysine-free” stand for?\n(line 445 in supplement) (l) should be (i).\nIn Figure legends, definitions for abbreviation and labelling (e.g., SBDP, “full”, “*” for statistical significance, etc.) should be\nprovided when they first appeared.\nMethods used for statistical significance should be consistently provided (missing in some figure legends).\nWhat does labelling “*” in pedigree schemes in Fig. 1(a-k) and Fig. S7(a-i) indicate?\nWhat is the rational in testing spectrin cleavage and including CAPN10 in the experiment shown in Fig. 3 etc.?\nWhat does “mislocalization” (as in descriptions for Fig. 4b and Fig.13a) mean exactly?\nThe title could be modified to better represent the article's content."}, "author_response": "Response: We thank the reviewer for this remark and propose to change the title of our\nmanuscript to “Loss-of-function variants in the CAPN1 activator CD99L2 cause X-linked\nspastic ataxia,” as this more precisely reflects the central clinical and functional findings of the\npresent study. Furthermore, we made changes across the text to more precisely reference the\ndisease entities under investigation and to avoid premature conclusions regarding possible\nadditional diagnostic benefits of genome sequencing in clinical conditions not covered by this\nstudy.\n2) The authors mention that panel analysis, likely performed in the early days before the\nwidespread diffusion of WES, were not counted. However, it is likely that a subset of patients\nunderwent NGS-panel analysis from this cohort. Can the authors provide an estimate of these\nfigures? Did all patients who previously tested negative at panel analysis undergo WES or\nWGS? Conversely, were patients diagnosed through panel-based analysis included or\nexcluded by the present study? As this is a large nationwide-based project with likely\n“epidemiological” implications, it is important to have a precise knowledge of inclusion and\nexclusion criteria.\nResponse: We thank the reviewer for these important comments and agree on a putative\nascertainment bias introduced by previous targeted single gene or panel testing. In general,\nexome sequencing (ES) or genome sequencing (GS) was performed only in affected\nindividuals without a likely diagnosis. We reviewed the available documentation indicating a\nprevious panel test in 262 out of 2,041 (12.8%) patients investigated by ES between 10/2016-\n12/2020, and in 59 out of 486 (12.1%) patients prior to GS. However, due to the sometimes\nlong diagnostic odyssey and diversity of health care / genetic testing providers, we assume\nthat this is a likely underestimate, especially in the early days of ES & GS. We included this\ninformation and caveat of our study in the manuscript.\n3) Supplementary Figure 2: does 2B panel refer to the age at onset? Please check the letters\nin the panels, as they do not always correspond to the legend.\nResponse: We have corrected the legend, and thank the reviewer for the thorough\nproofreading. Age always refers to age at diagnostic testing. We have added this information.\n4) Supplementary Figure 3: does 3A panel refer to the age at onset? 3B: why patients with no\nsignificant findings were tested later? Is there any correlation with the phenotype (HPO\ncategories), or the age at onset? Also in panel 3C, explain whether this is age at onset or age\nat diagnosis. Same comments for figure 4.\nResponse: Age always refers to age at diagnostic testing. We assume, that patients with a\nlater onset are tested later in life and in these cases, a genetic diagnosis is made less often.\nThis is also shown in Suppl. Fig. 2b by a declining diagnostic yield with increasing age. The\noutcome in the different subgroups is shown in Suppl. Fig. 2c with the highest diagnostic yield\nfor spasticity (28.1%) and the lowest for dystonia (12.1%). The outcome by age in the different\nsubgroups is shown in Suppl. Fig. 3c. Corresponding data for GS are shown in Suppl. Fig. 4d.\nHere the overlapping movement disorders show the highest diagnostic yield.\n5) In Supplementary Figures 2 and 4, please explain the criteria to assign “candidate genes”\n(yellow colour). How many of these candidate genes were validated / confirmed? They should\nbe reported in a table.\nResponse: The designation ‘candidate gene’ refers to changes in genes that lack substantial\npublicly available evidence of a confident gene-phenotype association (e.g. report of several\ndisease alleles in more than three unrelated families and supportive functional studies). Criteria\nof an assignment as ‘candidate gene’ are weighted at an individual’s level and include but are\nnot limited to i) conservation of the gene in control cohorts (e.g. loss-of-function de novo\nchange in a gene with expected loss-of-function intolerance in gnomAD: o/e score = 0), ii) bi-\nallelic/hemizygous predicted loss-of-function variant in linkage region / ROH in several family\nmembers or families without such variants in in-house or public controls, iii) conserved\nalterations in a factor with a putative mechanistic implication based on known protein function\nand/or specific phenotype (e.g. variant in gene coding for mitochondrial respiratory chain\ncomplex I (RCCI) subunit in a patient with mitochondrial encephalopathy and biochemically\nconfirmed RCC1 deficiency), iv) clear variant but not matching the currently associated\nphenotype (alternative pathomechanism, e.g. truncated protein vs nonse-mediated decay, and\nv) calls for patients from collaborators. Although dependent on the specific context of the family,\nsuch changes are disclosed to the referring clinicians in a scientific report accompanying the\ndiagnostic report, elaborating on the available literature/evidence and perspective functional\nstudies.\nOf the 26 cases with a candidate gene in ES samples, as of today, in 21 cases the candidate\ngenes could be subsequently established as likely causal.\nCandidate gene Reporting publication Number of cases\nC12orf66 PMID: 39824192 2 cases\nHPDL PMID: 32707086 4 cases\nCD99L2 this paper 7 cases\nAOPEP PMID: 35587627 2 cases\nNUDT2 PMID: 38141063 2 cases\nVWA1 PMID: 33459760 1 case\nPRDX3 PMID: 31782998 1 case\nSPATA5L1 PMID: 34626583 1 case\nKLC4 PMID: 26423925 1 case\nOf the 4 cases with a candidate gene in GS samples, CD99L2 could be further acknowledged\nas causal through the functional studies done for this paper.\n6) Page 7, Gene-burden analysis refers to an extended prioritization list presented in\nSupplementary Table 3, but such Table is missing. Similarly, I cannot find Supplementary Table\n4, quoted on page 7.\nResponse: We thank the reviewer for this remark and have attached the tables.\n7) Page 7: Please check the number of index cases as they seem to be 21 (8+8+1+4), while\nlater and in the figures the total number of families are 20.\nResponse: We thank the reviewer for the thorough proofreading. The index case counted\nhere, had died at 9 months of age of probable unrelated disease and was not able for follow-\nup. For the clarity of presentation, we have excluded this case and corrected the numbers\naccordingly.\n8) Legend to Figure 2: please correct “blue arrowheads in (r) and (d)” to “blue arrowheads in\n(r) and (s)”\nResponse: We have corrected this mistake, and thank the reviewer for the thorough\nproofreading.\n9) Non-truncating variants are of interest and should be characterized more in detail. Figure\n2m and 2n show that RNA levels from blood or fibroblasts of patients carrying splicing variants\nor a multiexon deletion are significantly reduced compared to controls (although not abolished\nas shown for truncating variants). Western blotting (shown for three variants) lack the wild type\nband but show weaker, smaller bands. Only some variants were assessed at the transcriptional\nlevel (Supplementary Figure 8). Were the bands observed on Western blotting correspond to\nthe transcripts detected on RNAseq? Supplementary Figure 8 should be expanded to\ncomprehensively describe the impact of non-truncating variants at the cDNA and protein level\n(by the way, on figure 2, panel m refers to c.535G>C while the legend refers to c.535G>A –\nplease check)\nResponse: We thank the reviewer for the additional comments and the requests to clarify the\nlink between the transcripts detected in RNA sequencing and the protein products analysed\nvia western blotting. In response, we have expanded and revised the representation of all\nviable transcripts resulting from splice-site alterations of exon deletions. This updated overview\nis now incorporated as an extension of Fig. 2l, and Supplementary Fig. 8 has been removed\naccordingly. All relevant transcripts are shown, while transcript resulting from PTVs were\nsubstantially reduced and thus assumed to undergo nonsense-mediated decay.\nBy comparing the immunogenic regions of the N-terminal and C-terminal CD99L2 antibodies\nused in our western blot analyses, we can reasonably infer – though not conclusively prove –\nthat the transcripts lacking exons 10 and 11, such as those arising from the c.535G>C\n(p.Gly179Arg) variant (see transcript a)) and c.655+1G>A (see transcript a) and c)), may be\nthe templates for the CD99L2 protein forms detected in Fig. 2r and 2s.\nFurthermore, the detection of a protein band in fibroblasts from a patient carrying the c.497-\n612_655+2267delinsC variant using both N-terminal and C-terminal antibodies supports the\ninterpretation that the template transcript encodes an internally truncated CD99L2 protein\nlacking exons 8 and 9. This is consistent with the revised schematic in Fig. 2l (transcript a) for\nvariant c.497-612_655+2267delinsC), the protein signal in Fig.2t, and Supplementary Fig. 7o.\nIn addition to modifying the figure and legend accordingly, we have revised the manuscript text\nto include a new paragraph titled “Consequences of identified CD99L2 variants on transcript\nand protein levels” in the Result section, which now reads as follows:\nTo further support the functional relevance of identified variants, we conducted\ntranscriptome sequencing (TS) and western blot studies on available patients’\nbiospecimens. These included analyses of PAXgene Blood RNA Tubes, representing\nall identified variants, and fibroblasts from affected individuals carrying six different\nvariants (Fig. 2o-t; Supplementary Fig. 7j-o). For all PTVs studied, CD99L2 mRNA\nlevels were significantly reduced compared to controls, in line with nonsense-mediated\ndecay (NMD) of mutant transcripts. When quantified as read counts, CD99L2 mRNA\nlevels of individuals with predicted splice site alterations and exon deletions were not\nsignificantly altered. However, no wild-type transcripts were observed, only transcripts\nindicating exon skipping, intron retention, and/or variable use of alternative splice sites,\nas well as a potential upregulation of the shorter transcript ENST00000418547.2 (Fig.\n2l; transcript c) of variant c.655+1G>A). Apart from the exon 8/9 deletion, which results\nin an in-frame transcript missing the entire transmembrane domain, all other aberrant\nmRNAs predict mainly prematurely truncated proteins. The use of alternative splice\nsites as shown in Fig. 2l; transcript b) of variant c.535G>C and c.655+1G>A was only\nseen in a smaller subset of reads.\nTo analyse the consequences of the identified CD99L2 variants at the protein level, we\nperformed western blotting of fibroblast-derived protein extracts using antibodies\ndirected against either the N-terminus (targeting residues 27-117) or the C-terminus\n(targeting residues 209-254) of the protein (Fig. 2o-t; Supplementary Fig. 7j-o). In\ncontrols, a strong band was detected between 37 kDa and 50 kDa using the N-terminal\nantibody – higher than the predicted molecular weight of 28 kDa for full-length CD99L2.\nHowever, the specificity of this signal was confirmed by parallel detection using the C-\nterminal antibody.\nIn patients carrying PTVs, CD99L2-specific bands were entirely absent (Fig. 2o-q;\nSupplementary Fig. 7j-l). In contrast, patients with splice-site alterations or a deletion\nof exons 8/9 showed faint bands of lower molecular weight when probed the N-terminal\nantibody (Fig. 2r-t) and – exclusively in the case of the exon 8/9 deletion – also with the\nC-terminal antibody (Suppl. Fig. 7m-o). These may represent internally or C-terminally\ntruncated CD99L2 protein forms, likely corresponding to translation products from the\naberrant transcripts detected in the transcriptome analysis (Fig. 2l).\nIn conclusion, all CD99L2 variants investigated led to a near-complete loss of full-length\nCD99L2, supporting a loss-of-function as the likely pathomechanism.”\nThe error in legend to Fig. 2 has been corrected to c.535G>C.\n10) Please comment why autophagy inhibition results in a reduction of ubiquitinated CD99L2\nwhile the unmodified protein increases (Figure 3d).\nResponse: We thank the reviewer for this request and appreciate their careful observation.\nOur data, derived from analyses of both full-length and truncated CD99L2 constructs, clearly\ndemonstrate that CD99L2 undergoes bimodal degradation via both the proteasomal and\nautophagosomal pathways. The C-terminus appears to mediate proteasomal degradation,\nwhile the N-terminus is associated with autophagosomal degradation, although the\nproteasomal route remains the predominant pathway. As demonstrated in many studies,\nincluding ours, there is a strong interplay between autophagy and the proteasomal system\n(10.1016/j.tplants.2023.01.013). Inhibition of one pathway often triggers compensatory\nactivation of the other. This is reflected in our data, where we observe decreased K48-linked\npolyubiquitin levels upon BafA1 treatment and increased LC3B-II levels following MG132\nadministration (see Fig. 3b; Reviewer Fig. 1). We believe the modified (i.e., ubiquitinated) forms\nof CD99L2 shown in Fig. 3d are primarily targeted by the UPS. Accordingly, these forms\naccumulate strongly upon proteasome inhibition but are reduced upon autophagy inhibition -\nlikely due to enhanced UPS-mediated turnover as a compensatory response. Conversely, the\nfull-length, unmodified CD99L2 accumulates under autophagy inhibition, suggesting that this\nfraction may be preferentially degraded via autophagy, potentially through interactions specific\nto membrane proteins or organelles, rather than via a ubiquitination-dependent mechanism.\nWhile this explanation is plausible, it requires further investigation, which we hope to address\nin future studies focused on the precise modulatory roles of post-translational modifications\nand catabolic pathways in regulating CD99L2 turnover.\nTo additionally address this observation, we have included the following sentences to the\nrespective result section:\n“Notably, proteasomal inhibition by MG132 was the most effective in elevating CD99L2\nlevels, leading to an approximately twelve-fold increase of fl-CD99L2 compared to a\nfive-fold increase upon autophagy inhibition using bafilomycin A1 (BafA1) (Fig. 3d).\nMoreover, while BafA1 treatment increased levels of the unmodified full-length\nCD99L2, the levels of the modified, potentially ubiquitinated form declined, likely\nreflecting of a compensatory upregulation of proteasomal activity in response to\nautophagy inhibition.”\n11) Legend to figure 3: please move description of grey arrowheads/white arrowheads/black\narrowheads to panels a-c.\nResponse: We have added a description of the grey, white, and black arrowheads after the\ntext describing panel a.\n12) Figure 4h-j: can the authors explain this divergent behavior of truncated CD99L2 in\nresponse to proteasome or autophagy inhibition?\nResponse: We thank the reviewer for this insightful question. We believe that the N- and C-\nterminal domains, as well as the subcellular localization of the protein or its domains, play a\ncrucial role in mediating CD99L2 degradation, which we have characterized as bimodal in our\nmanuscript. This is likely facilitated by the presence of specific sites for post-translational\nmodifications – such as distinct lysine residues for ubiquitination – and the presence or\nabsence of defined recognition motifs for the two degradation pathways. Further research on\nCD99L2 is needed to identify which specific sites and motifs are key determinants of its\nproteostatic fate.\nTo address this point more clearly in the manuscript, we have expanded the description of this\nexperiment, which now reads as follows:\n“Given the previously observed bimodal degradation of full-length CD99L2, we next\nexplored whether specific domains influence its proteostatic fate. Notably, CD99L2\n1-185\naccumulated upon autophagy inhibition, while CD99L2 levels increased following\n207-262\nproteasome inhibition (Fig. 6h-j; Supplementary Fig. 12f-i). These findings suggest that\nCD99L2’s bimodal degradation is domain-dependent, with the N-terminal region driving\nits autophagosomal clearance and the C-terminal domain directing proteasomal\ndegradation.\n13) Figure 4k: it would be useful to have a brief explanation of this schematic cartoon to help\nthe readers draw the right conclusion about the postulated mechanism.\nResponse: We are grateful to the reviewer for pointing this out. We have amended the legend\nto Fig. 4k (now Fig. 6k) by including a brief description of the schematic, which outlines the\npostulated mechanism. The respective section reads as follows:\n“Model depicting the functional interaction between CD99L2 and CAPN1, and the\nproteostatic regulation of CD99L2. Inactive CAPN1 binds to unmodified full-length\nCD99L2 and becomes activated. Active CAPN1 then cleaves CD99L2, removing its C-\nterminus, which in turn inhibits the further activation of additional CAPN1 molecules.\nFurthermore, an interplay between E3 ubiquitin ligases (E3) and deubiquitinases (DUB)\nmaintains a balance between ubiquitinated and unmodified full-length CD99L2. This\nensures continuous degradation of the protein via the ubiquitin proteasome system\n(UPS), or to a lesser extent, autophagy, thereby preventing excessive activation of the\ncalpain system. CP, cytoplasm; EC, extracellular space.”\n14) Supplementary Figure 9: it seems that anti-CD99L2 antibody does not recognize any\nendogenous protein either in 293T cells or SH-SY5Y cells (mock transfected lane is perfectly\nclear not only in the myc-antibody panels but also in the CD99L2 panels and overexposure\nshows a very faint modified band). Is the protein expected to have a very rapid turnover in\ncells? Figure 3C seems to show that even inhibition of proteasome or autophagy do not result\nin increased levels of endogenous protein in mock-transfected cells. However, in\nSupplementary Figure 12 it seems that endogenous CD99L2 is detected in control and SPG76\nfibroblasts. Please provide an explanation for these observations or clarify in the text.\nResponse: We thank the reviewer for this insightful comment. Indeed, the difference in\nexpression levels between endogenous and overexpressed CD99L2 is substantial, making it\nextremely challenging – if not impossible – to track the fate of the endogenous protein when\nsamples are loaded side by side on the same Western blot. The strong signal from the\noverexpressed CD99L2 even caused bleed-over into adjacent lanes. However, only with such\nhigh expression levels were we able to clearly visualize the unmodified full-length form of\nCD99L2, as well as its potential fragments.\nNevertheless, our data in Fig. 4a and 4b, as well as Supplementary Fig. 8c and 8d,\ndemonstrate that CD99L2 is detectable in its ubiquitinated/modified form in both 293T and SH-\nSY5Y cells. Moreover, we show that this modified form can be modulated, for example, via\nCAPN1 knockdown (Fig. 4a). As the reviewer suggests, low baseline expression, rapid\nturnover, and cell- or tissue-specific differences in CD99L2 expression and degradation may\nall contribute to the challenges with analyzing this protein. We propose that CD99L2’s high\nturnover rate may serve as an important regulatory mechanism, potentially preventing\nexcessive calpain activation, as hypothesized in our schematic model. To address this further,\nwe repeated the analysis in mock-transfected cells treated with MG132 and BafA1. These\nexperiments confirm that CD99L2 is detectable in its modified form and that this form is indeed\nmodulated by treatment, specifically upon proteasomal inhibition (see Reviewer Fig. 2).\nIn line with this, we conducted a pilot experiment to assess the half-life of CD99L2 in 293T\ncells using a cycloheximide (CHX) chase assay in the presence or absence of the\ndeubiquitinase ataxin-3, which plays a role in the UPS. While replication and validation of this\nassay is warranted and will be included in our follow-up studies, our initial data indicate that\nCD99L2 has a mean half-life of approximately 18 hours in 293T WT cells, which is substantially\nshorter than the 30-hour half-life predicted by the Expasy ProtParam tool\n(web.expasy.org/protparam) (see Reviewer Fig. 3).\nFinally, we have revised the corresponding section in the Results to include a more detailed\nexplanation of our findings, which now reads as follows:\n“To investigate the discrepancy between the calculated (~28 kDa) and observed (37–\n50 kDa) molecular weight of CD99L2 in control fibroblasts (Fig. 2o-t; Supplementary\nFig. 7j-o), we first replicated these findings in 293T cells and SH-SY5Y neuroblastoma\ncells, both at baseline and upon overexpression of a CD99L2 construct. Only upon\noverexpression, at levels several-fold higher than the endogenous levels, we were able\nto detect the apparently unmodified full-length form of CD99L2, along with potential\nfragments, while the modified form represented the predominant species (Fig. 3a;\nSupplementary Fig. 8a-d).”\n15) The authors discuss a cellular mislocalization of truncated forms of CD99L2 compared to\nwild type. By assessing colocalization with an ER-marker, it seems that truncated forms of the\nprotein have increased co-localization with ER compared to wild type, however this is not\nexplained in the text. Why the authors specifically assessed colocalization with ER?\nHave they also tested colocalization with CAPN1?\nResponse: We thank the reviewer for this request and their insightful comments. The initial\nobservation of mislocalization was made without the use of additional counterstaining or cell\ncompartment-specific reporters. However, we observed a marked contrast between the\nexpected plasma membrane localization of full-length CD99L2 and the granular, cytoplasmic\n(non-nuclear) distribution of the N-terminal CD99L2 fragment, while the smaller C-terminal\n1-185\nCD99L2 fragment displayed a diffuse distribution in both the cytoplasm and nucleus.\n207-262\nGiven that the pattern of CD99L2 resembled the endoplasmic reticulum (ER), we\n1-185\nperformed co-transfection with a dsRed-tagged ER reporter. This confirmed colocalization,\nindicating that the N-terminal fragment is mislocalized to the ER (now Supplementary Fig. 12b).\nTo better illustrate this observational sequence, we have now included a panel with the first\nmicroscopy-based analysis of the CD99L2 truncation constructs, performed without co-\nexpression of the ER reporter (new Supplementary Fig. 12a) and updated the referencing to\nthe figure accordingly.\nThe observed mislocalization of CD99L2 may result from the retention of the plasma\n1-185\nmembrane-targeting signal peptide at the N-terminus (residues 1 – 25), coupled with the\nabsence of the transmembrane domain in this fragment. Since we had not previously\nmentioned the N-terminal signal peptide in our manuscript or schematics, we have now revised\nthe manuscript accordingly to include this detail.\nWe had not initially tested co-localization of CD99L2 and CAPN1 via microscopy. However,\nfollowing the reviewer’s suggestion, we have now performed the relevant co-immunostainings.\nThese new results show that all CD99L2 constructs partially share the subcellular space with\nCAPN1, offering potential contact sites for an interaction. Nevertheless, as demonstrated by\nour co-immunoprecipitation experiments (Fig. 6f, g), this partial spatial overlap is insufficient to\ninduce calpain activation or binding. The new co-staining results have been included in our\nmanuscript as the new Fig. 6b, and we have updated the result section accordingly.\nIn addition, we have expanded the corresponding section in the results to provide a more\ndetailed explanation, as suggested by the reviewer. The revised section now reads as follows:\n“Our analysis underscored the importance of CD99L2’s structural integrity: while all\nconstructs showed limited colocalization with CAPN1, CD99L2 displayed a\n1-185\ncompartmentalized cytoplasmic pattern reminiscent of the endoplasmic reticulum (ER),\nwhich was confirmed via co-expression with a DsRed-tagged ER reporter. In contrast,\nCD99L2 exhibited a more diffuse distribution in the cytoplasm and, to a lower\n207-262\nextent, the nucleus. Full-length CD99L2, as expected, localized to the plasma\nmembrane (Fig. 6b; Supplementary Fig. 12a, b).”\nResponse: We would like to express our sincere gratitude to the reviewer for their thoughtful\nassessment of our manuscript, as well as for the valuable comments and recommendations\naimed at improving the significance and conclusiveness of our study, both in major and minor\naspects. While we firmly believe that our molecular analysis, which validated and elucidated\nkey aspects of CD99L2 function and the CD99L2–calpain axis – including structural features,\npost-translational modifications, protein interactions, degradation pathways, and downstream\nproteolytic activation of the calpain system – convincingly demonstrates the relevance of the\nproposed pathway, we acknowledge that there is still room for refinement and improvement.\nAccordingly, we have made every effort to thoroughly address all points raised by the reviewer\nto the best of our knowledge and within the limits of our currently available resources.\nWe have addressed the major points raised by the reviewer as follows:\n1. Is there any functional relationship between ubiquitination and CAPN1-interaction of\nCD99L2?\nResponse: The reviewer raised an insightful and valid question regarding the relationship\nbetween the ubiquitinated form of CD99L2 and its interaction with CAPN1, which we had\npreviously addressed only hypothetically in our schematic summary (now Figure 6k). To\nexplore this experimentally, we utilized our lysine-free CD99L2 construct (CD99L2 ) and\nK0\nconducted new co-immunoprecipitation (co-IP) experiments and fluorescence microscopy\nanalysis to assess the effect of lysine depletion – and thus loss of ubiquitination – on the\ninteraction between CD99L2 and CAPN1.\nInterestingly, our western blot analysis revealed that the absence of lysine residues (via lysine-\nto-arginine mutagenesis), and therefore the lack of CD99L2 ubiquitination, resulted in a\nmarkedly increased binding to CAPN1. In contrast, the negligible interaction with CAPN2\nremained unchanged (see new Fig. 5b).\nTo ensure that this increased binding was not due to altered subcellular localization, we\nexamined the localization of CD99L2 via fluorescence microscopy. The lysine-free construct\nK0\nmaintained the same plasma membrane-specific localization as the wild-type protein (see new\nFig. 5a), indicating that ubiquitination does not influence CD99L2 targeting. Consequently, the\nlimited, membrane-proximal interaction site between CD99L2 and CAPN1 was not altered, as\ndemonstrated by our co-staining with CAPN1. Therefore, the observed increase in CAPN1\nbinding is not attributable to changes in protein localization.\nThese new findings are presented in the new Fig. 5 and in the updated Results section of the\nmanuscript, which reads as follows:\n“After demonstrating that CD99L2 directly binds to CAPN1 and induces calpain system\nactivation, we explored whether its ubiquitination capacity contributes to this function.\nTo investigate this, we overexpressed both myc-tagged wild-type CD99L2 and its\nlysine-free variant CD99L2 in 293T cells. Fluorescence microscopy revealed no\nK0\ndifferences in subcellular localization of the two CD99L2 variants, including their limited\ncolocalization with CAPN1 at membrane-distal contact sites, indicating that lysine\nresidues – and their ubiquitination – do not alter CD99L2 targeting to the plasma\nmembrane (Fig. 5a).\nTo assess changes in protein interaction, we performed co-IP via the myc tag of the\nCD99L2 constructs. Western blot analysis revealed a markedly increased binding\naffinity of CD99L2 to CAPN1 compared to wild-type CD99L2 (Fig. 5b). Notably, the\nK0\nabsence of lysine residues in CD99L2 did not enhance its weak binding to CAPN2.”\n2. In terms of calpain-1 activation function of CD99L2, what cellular functions are\ncompromised in the cells from patients harboring CD99L2 mutations?\nResponse: Although our functional analysis using CD99L2 truncation constructs provides\nsome indications of the molecular consequences expected in the patient context, we agree\nwith the reviewer that downstream pathway analyses in patient-derived models would offer\nmore substantial insight into the cellular functions compromised by CD99L2 variants and the\nmechanisms underlying the observed phenotype. While a neuronal system would be optimal\nto assess these potential dysregulations in a more physiological context—a consideration for\nfuture studies—we have now performed RNA sequencing–based transcriptome analysis of\nprimary fibroblasts derived from CD99L2 patients and healthy controls, followed by pathway\nand enrichment analyses.\nWe observed significant dysregulation of more than 500 genes in patient fibroblasts. Notably,\nbiological process and cellular compartment enrichment analyses revealed a characteristic\ndysregulation of genes involved in neuronal and synaptic function. Among the top 10\nenriched biological processes and cellular compartments, the majority were related to neuronal and\nsynaptic functions. These findings are consistent with previous studies investigating the effects\nof CAPN1 loss (Wang et al., 2016; PMID: 27320912) and with recent work on the neuronal\nrole of CD99L2 (PMID: 39808524), in which synaptic function was primarily affected and linked\nto cerebellar ataxia or hippocampal function.\nTogether, these results strongly support our hypothesis that CD99L2 influences neuronal\nfunction and may play a role in the CAPN1-related regulatory axis.\nWe have incorporated these data as new Fig. 7 and Supplementary Fig. 13, including their\nrespective legends, along with the following textual additions in the manuscript:\nResults:\n“CD99L2 patient fibroblasts show transcriptional dysregulation of genes\nassociated with neuronal function\nAfter functionally dissecting the CD99L2 protein, we next analysed the downstream\nconsequences of CD99L2 variants to better explain the neurological phenotype\nobserved in affected patients. To this end, we performed transcriptomic profiling of\nfibroblasts from patients carrying PTVs, splice-site variants, or deletions, compared to\nfibroblasts from unaffected matched controls. RNA sequencing identified 515 significant\ndifferentially expressed genes (DEGs), including 191 downregulated and 324\nupregulated transcripts (Fig. 7a). Since the physical interaction of CD99L2 and the\ncalpain system occurs at the protein level, we specifically assessed potential\ntranscriptional dysregulations of related genes. However, no significant expression\nchanges were detected for CAPN1, CAPN2, CAPNS1 (encoding CSS1), CAST, or\nSPTAN1 (encoding the calpain substrate α-spectrin) (Supplementary Fig. 13a).\nTo identify functional themes among the DEGs, we performed STRING-based gene\nontology (GO) enrichment analysis (Szklarczyk et al., 2023). Among the top 10\nenriched biological processes, 8 were directly linked to neuronal function. Similarly, 6\nof the top 10 enriched cellular compartments were related neurons, particularly the\nsynapse (Fig. 7b, c). Beyond the general GO term “cell junction”, the strongest\nenrichment was observed for “synapse,” which encompassed 55 DEGs showing\nmarked dysregulation and a high degree of functional interaction (Fig. 7d, e;\nSupplementary Fig. 13d).\nAlthough fibroblasts are not the model of choice for directly studying neuron-specific\npathology, the consistent dysregulation of neuron- and synapse-related genes\nhighlights the likely impact of CD99L2 variants on neuronal function and degeneration.\nIn light of previous findings that loss of CAPN1 impairs synaptic function and triggers\ncerebellar ataxia (Wang et al., 2016), and given that CD99L2 may regulate synapse\ndevelopment (Kang et al., 2025), our data suggests that perturbed CD99L2 function\ncould exert similarly detrimental effects in the patient brain.”\nDiscussion:\n“Our transcriptome analysis of patient-derived fibroblasts revealed a pronounced and\nspecific dysregulation of genes, with strong enrichment for biological processes linked\nto neuronal function, particularly the synapse. This transcriptional signature overlaps\nwith synaptic pathways known to be affected by the loss of CAPN1, whose LOF variants\ncause SPG76 and forms of cerebellar ataxia (Gan-Or et al., 2016; Méreaux et al., 2021;\nWang et al., 2016).”\n3. Is activation of calpain-1 impaired [in the cells from patients harboring CD99L2 mutations]?\nResponse: We thank the reviewer for this relevant question. As CD99L2-mediated CAPN1\nactivation appears to be a conserved mechanism across our cell model systems, the\nabsence of the protein is expected to reduce calpain activation to a certain extent. To\naddress this, we followed the reviewer’s suggestion and investigated calpain-mediated α-\nspectrin fragmentation, as well as CAPN1 and CAST levels, in the fibroblast lines available\nin our laboratory. Western blot analysis did not reveal significant changes in CAPN1 or\nCAST levels, although some variability was observed. However, cleavage of α-spectrin\nwas significantly reduced in patient-derived fibroblasts. This finding is consistent with the\nexpected effect of CD99L2 absence or loss, when considered as a calpain activator.\nWe have included this analysis in the new Supplementary Fig. 13b, c and added a\ncorresponding description of our findings in the Results section, which now reads as\nfollows:\nFurthermore, we analysed protein levels of CAPN1 and CAST, as well as calpain-mediated\ncleavage of α-spectrin, in fibroblasts derived from CD99L2 patients and healthy controls.\nWhile no significant alterations in CAPN1 or CAST levels were detected, spectrin cleavage\nwas reduced in patient fibroblasts, consistent with the absence of the CAPN1 activator\nCD99L2 (Supplementary Fig. 13b, c).\n4. What is functional impact of ubiquitination of CD99L2 function?\nResponse: Following our results indicating the increased CAPN1 binding of CD99L2 ,\nK0\nsubsequently investigated whether this enhanced interaction leads to stronger activation of the\ncalpain system, in order to highlight the functional relevance of CD99L2 ubiquitination, as\nrequested by the reviewer. By analysing the protein levels and calpain-mediated proteolysis of\nCAPN1, CAST, α-spectrin, and CSS1, we were able to demonstrate a clear increase in calpain\nactivation (see new Fig. 5c, d). These findings suggest that the ubiquitination may not only\nregulate CD99L2 degradation via the proteasomal pathway, but also modulate its function as\na calpain activator.\nWe have included this data in the new Fig. 5 and in the respective paragraphs of the Results\nand Discussion section, which read as follows:\nResults:\n“Next, we assessed the impact of lysine residue loss on CD99L2’s ability to activate\ncalpains. Western blot analysis of calpain system markers – including CAPN1 and\nCAST levels, as well as α-spectrin and CSS1 cleavage – indicated a further\namplification of proteolytic activation in cells expressing CD99L2 (Fig. 5c, d).\nK0\nTogether, these findings suggest that the ubiquitination of CD99L2 modulates its\ninteraction with CAPN1 and dampens calpain activation, potentially serving as a\nregulatory mechanism to prevent excessive and harmful calpain activity within cells.”\nDiscussion:\n“Specifically, ubiquitination of CD99L2 appears to reduce its binding to CAPN1 and\nattenuate calpain system activation, consistent with established roles of this PTM in\nmodulating protein-protein interactions (Tracz & Bialek, 2021). This regulatory\nmechanism modulation may help prevent excessive proteolytic activity, which has been\nimplicated in neurodegenerative diseases such as polyglutamine disorders.”\nWe have addressed the reviewer’s comments on minor points as follows:\n1. (line 176) The case number for FA should be 1,067 (as in Supl. Fig. 1).\nResponse: We thank the reviewer for this remark. We have corrected the number respectively.\n2. (line 270) The change in molecular weight by ubiquitination is ca. 22 kDa at maximum\ncorresponding to 3 ubiquitins, therefore, “highly” is unnecessary.\nResponse: We agree with the reviewer’s assessment and apologize for the inadequate\nwording regarding the ubiquitination of CD99L2. Our intention was to emphasize that the\nCD99L2 protein appears in its ubiquitinated form, rather than in its unmodified state. The\nsection containing the term “highly” has been removed, and we have avoided using it\nelsewhere.\n3. (line 226 in supplement) What mutation(s) does “lysine-free” stand for?\nResponse: We thank the reviewer for the request and clarified the characteristic of our\nconstruct respectively. The reader will find the following description in the main text:\n“We validated these findings by mutating all 14 lysine residues in CD99L2 (Fig. 3g) to\narginine (CD99L2 ) to prevent canonical ubiquitination, which successfully resulted in a\nK0\nshift from the higher molecular weight form of CD99L2 to its unmodified full-length form\n(Fig. 4h, i).”\nWe have also amended the respective figure legend:\n“h, Western blot analysis of 293T cells overexpressing wild-type (WT) myc-His -CD99L2\n6\nand a lysine-free (K0) variant in which all 14 lysine residues were mutated to arginine, using\na myc tag-specific antibody. GAPDH was used as a loading control.”\n4. (line 445 in supplement) (l) should be (i).\nResponse: Thank you. We have corrected this mistake in the figure legend to Supplementary\nFig. 9 (now Supplementary Fig. 8).\n5. In Figure legends, definitions for abbreviation and labelling (e.g., SBDP, “full”, “*” for\nstatistical significance, etc.) should be provided when they first appeared.\nResponse: We thank the reviewer for noticing these inconsistencies. We have edited our\nfigure legends and included the missing explanation of abbreviations and labelling according\nto their first appearance in the respective figure.\n6. Methods used for statistical significance should be consistently provided (missing in some\nfigure legends).\nResponse: We have checked and updated the figure legends in this regard and included the\nmissing information where necessary.\n7. What does labelling “*” in pedigree schemes in Fig. 1(a-k) and Fig. S7(a-i) indicate?\nResponse: We thank the reviewer for pointing out this missing information. In both figure\nlegend, we added the following explanatory sentence:\n“Asterisks (*) indicate fully characterised patients.”\n8. What is the rational in testing spectrin cleavage and including CAPN10 in the experiment\nshown in Fig. 3 etc.?\nResponse: In the previous version of the manuscript, the conciseness of our descriptions may\nhave left some of the rationale behind our analysis insufficiently clear. We have addressed this\nin the revised manuscript by providing more detailed explanations throughout. Specifically,\nregarding the reviewer’s request for clarification of the marker proteins α-spectrin and\nCAPN10, we have integrated the following sections into the corresponding Result paragraph:\nRegarding spectrin cleavage:\n“In our co-IP experiments, we observed increased autoproteolysis of CAPN1, reduced\nlevels of the endogenous proteinaceous inhibitor calpastatin (CAST), and a fragmentation-\nrelated decrease in full-length CSS1, suggesting activation of the calpain system upon\nCD99L2 transfection. To confirm this, we repeated CD99L2 overexpression in 293T and\nSH-SY5Y cell lines and quantitatively analysed key protein markers, including the known\ncalpain substrate α-spectrin, whose calpain-mediated proteolysis generates a\ncharacteristic ~150 kDa fragment. All analysed proteins exhibited enhanced proteolytic\nprocessing in both cell models (Fig. 3l, m; Supplementary Fig. 9e-h), corroborating\nCD99L2-induced activation of calpains.”\nRegarding CAPN10:\n“Previous affinity capture–based mass spectrometry identified CD99L2 as an interactor of\ncalpain-1 (CAPN1), a calcium-activated cysteine protease involved in neuronal plasticity\nand implicated in recessive spastic paraplegia and cerebellar ataxia (Huttlin et al., 2021;\nBaudry et al., 2016). To validate these findings, we performed co-immunoprecipitation (co-\nIP) experiments using two differently tagged CD99L2 constructs overexpressed in 293T\ncells. Western blot analysis confirmed that CD99L2 directly interacts with CAPN1 and\napparently co-precipitates its regulatory small subunit CSS1 (Fig. 3j, k; Supplementary Fig.\n9a–d). A potential weak binding to calpain-2 (CAPN2) was also observed (Extended Data\nFig. 10d), whereas no interaction was detected with the non-classical calpain-10\n(CAPN10), which structurally differs from CAPN1 by lacking the C-terminal calcium-binding\npenta-EF-hand domain—and thereby the ability to interact with CSS1 (Ono et al., 2016)—\nor with the endogenous proteinaceous inhibitor calpastatin (CAST).”\n9. What does “mislocalization” (as in descriptions for Fig. 4b and Fig.13a) mean exactly?\nResponse: As mentioned above, the previous version of our manuscript was highly concise\nand thus did not allow for a detailed explanation of our observations. Pertaining to the findings\npreviously shown in Fig. 4b and Fig. 13a (now revised, extended, and re-labeled as Fig. 6b\nand Suppl. Fig. 12a, b), we have incorporated the following explanatory passage into the\ncorresponding Results section:\n“Our analysis underscored the importance of CD99L2’s structural integrity: while all\nconstructs showed limited colocalization with CAPN1, CD99L2 displayed a\n1-185\ncompartmentalized cytoplasmic pattern reminiscent of the endoplasmic reticulum (ER),\nwhich was confirmed via co-expression with a DsRed-tagged ER reporter. In contrast,\nCD99L2 exhibited a more diffuse distribution in the cytoplasm and, to a lower extent,\n207-262\nthe nucleus. Full-length CD99L2, as expected, localized to the plasma membrane (Fig. 6b;\nSupplementary Fig. 12a, b).”\n10. The title could be modified to better represent the article's content.\nResponse: We concur with the reviewer that modifying of the manuscript title is beneficial.\nAccordingly, we have changed the title to “Loss-of-function variants in the CAPN1 activator\nCD99L2 cause X-linked spastic ataxia”, as it more precisely reflects the central clinical and\nfunctional findings of the present study."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I appreciate the work made by the authors to improve the manuscript and address the criticisms raised by the reviewers. A lot of additional experimental work has been added and several issues have been clarified. I am overall satisfied of the data presented here.\nEnza Maria Valente, MD, PhD", "reviewer_2": "The revised manuscript shows great improvement in the overall construction as well as data presentation. It is greatly appreciated that the authors have added much work and I believe that an important lead for the future studies is established.\nA few minor corrections:\nPage 6, line 165; the number “1,076” should be “1,067” (as in line 181).\nPage 7, line 224; considering the expression “Of the 3 associations identified, 2… were already known…and 1… was … unidentified (line 236-238), “three known” should be “two known”?\nPage 9, line 331; should be “Fig. 3h, i”?\nChanging the order of Supplementary Tables could better suit the flow of the manuscript; the present Table S2 showing a list of used antibodies could be Table S4 since the content is not referred to in the main text and stands as a component in supplementary methods."}, "author_response": "Response: We thank the reviewer for the positive feedback and appreciate the recognition of\nour efforts to strengthen the manuscript in response to the reviewers’ comments.\nResponse: We thank the reviewer for the positive assessment and greatly appreciate the\nrecognition of the additional work incorporated during revision. We are pleased that the\nreviewer views the study as establishing an important lead for future research.\nA few minor corrections:\nPage 6, line 165; the number “1,076” should be “1,067” (as in line 181).\nResponse: We thank the reviewer for noticing this mistake. We have corrected the number\naccordingly.\nPage 7, line 224; considering the expression “Of the 3 associations identified, 2… were already\nknown…and 1… was … unidentified (line 236-238), “three known” should be “two known”?\nResponse: We appreciate the reviewer’s careful reading. We have revised the wording\naccordingly.\nPage 9, line 331; should be “Fig. 3h, i”?\nResponse: We thank the reviewer for pointing this out. We had originally updated the figure\nreference following the reviewer’s correct remark. However, because we subsequently\nrearranged and split figures to meet the journal’s formatting requirements, the correct\nreference in the final layout is now again “Fig. 4h, i.”\nChanging the order of Supplementary Tables could better suit the flow of the manuscript; the\npresent Table S2 showing a list of used antibodies could be Table S4 since the content is not\nreferred to in the main text and stands as a component in supplementary methods.\nResponse: We thank the reviewer for this helpful suggestion. Because the former\nSupplementary Tables S3 and S4 were redesignated as Supplementary Data 1 and\nSupplementary Data 2 to comply with the journal’s formatting guidelines, only two\nSupplementary Tables remain. For this reason, the antibody table continues to be numbered\n“Supplementary Table 2.”"}]

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