Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3–5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

Bi-allelic loss-of-function variants in genes that encode subunits of the adaptor protein complex 4 (AP-4) lead to prototypical yet poorly understood forms of childhood-onset and complex hereditary spastic paraplegia: SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). Here, we report a detailed cross-sectional analysis of clinical, imaging and molecular data of 156 patients from 101 families. Enrolled patients were of diverse ethnic backgrounds and covered a wide age range (1.0–49.3 years). While the mean age at symptom onset was 0.8 ± 0.6 years [standard deviation (SD), range 0.2–5.0], the mean age at diagnosis was 10.2 ± 8.5 years (SD, range 0.1–46.3). We define a set of core features: early-onset developmental delay with delayed motor milestones and significant speech delay (50% non-verbal); intellectual disability in the moderate to severe range; mild hypotonia in infancy followed by spastic diplegia (mean age: 8.4 ± 5.1 years, SD) and later tetraplegia (mean age: 16.1 ± 9.8 years, SD); postnatal microcephaly (83%); foot deformities (69%); and epilepsy (66%) that is intractable in a subset. At last follow-up, 36% ambulated with assistance (mean age: 8.9 ± 6.4 years, SD) and 54% were wheelchair-dependent (mean age: 13.4 ± 9.8 years, SD). Episodes of stereotypic laughing, possibly consistent with a pseudobulbar affect, were found in 56% of patients. Key features on neuroimaging include a thin corpus callosum (90%), ventriculomegaly (65%) often with colpocephaly, and periventricular white-matter signal abnormalities (68%). Iron deposition and polymicrogyria were found in a subset of patients. AP4B1-associated SPG47 and AP4M1-associated SPG50 accounted for the majority of cases. About two-thirds of patients were born to consanguineous parents, and 82% carried homozygous variants. Over 70 unique variants were present, the majority of which are frameshift or nonsense mutations. To track disease progression across the age spectrum, we defined the relationship between disease severity as measured by several rating scales and disease duration. We found that the presence of epilepsy, which manifested before the age of 3 years in the majority of patients, was associated with worse motor outcomes. Exploring genotype-phenotype correlations, we found that disease severity and major phenotypes were equally distributed among the four subtypes, establishing that SPG47, SPG50, SPG51 and SPG52 share a common phenotype, an ‘AP-4 deficiency syndrome’. By delineating the core clinical, imaging, and molecular features of AP-4-associated hereditary spastic paraplegia across the age spectrum our results will facilitate early diagnosis, enable counselling and anticipatory guidance of affected families and help define endpoints for future interventional trials.

Adaptor protein complex 4 (AP-4)-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of AP-4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by AP-4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z’-factor robust > 0.3, strictly standardized mean difference > 3). The ‘ATG9A ratio’ is increased in fibroblasts of 18 well-characterized AP-4-associated hereditary spastic paraplegia patients (mean: 1.54 ± 0.13 vs. 1.21 ± 0.05 (standard deviation) in controls) and receiver-operating-characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849–0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect AP-4 function. Our findings establish the ‘ATG9A ratio’ as a diagnostic marker of AP-4-associated hereditary spastic paraplegia.

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PURPOSE: Bi-allelic TECPR2 variants have been associated with a complex syndrome with features of both a neurodevelopmental and neurodegenerative disorder. Here, we aim to provide a comprehensive clinical description and variant interpretation framework. METHODS: Through an international collaboration, we identified 13 individuals from 12 families with bi-allelic TECPR2-variants. We systemically reviewed clinical and molecular data of this cohort and 11 cases previously reported. Phenotypes were standardized using Human Phenotype Ontology terms. RESULTS: A cross-sectional analysis revealed global developmental delay/intellectual disability, muscular hypotonia, ataxia, hyporeflexia, respiratory infections and central/nocturnal hypopnea as core manifestations. A review of brain MRI scans demonstrated a thin corpus callosum in 59%. We evaluated 14 distinct variants. Missense variants in TECPR2 are predominantly located in the N- and C-terminal regions containing β-propeller repeats. Despite constituting nearly half of disease associated TECPR2 variants, classifying missense variants as (likely) pathogenic according to ACMG criteria remains difficult. We estimate a pathogenic variant carrier frequency of 1/1,221 in the general and 1/155 in the Jewish Ashkenazi population. CONCLUSION: Based on clinical, neuroimaging and genetic data, we provide recommendations for variant reporting, clinical assessment, and surveillance/treatment of individuals with TECPR2-associated disorder. This sets the stage for future prospective natural history studies.

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