Utilizing RNA and outlier analysis to identify an intronic splice-altering variant in AP4S1 in a sibling pair with progressive spastic paraplegia.

We report a likely pathogenic splice-altering AP4S1 intronic variant in two sisters with progressive spastic paraplegia, global developmental delay, shy character, and foot deformities. Sequencing was completed on whole-blood messenger RNA (mRNA) and analyzed for gene expression outliers after exome sequencing analysis failed to identify a causative variant. AP4S1 was identified as an outlier and contained a rare homozygous variant located three bases upstream of exon 5 (NC_000014.8(NM_007077.4):c.295-3C>A). Confirmed by additional RNA-seq, reverse-transcription polymerase chain reaction, and Sanger sequencing, this variant corresponded with exon 5, including skipping, altered isoform usage, and loss of expression from the canonical isoform 2 (NM_001128126.3). Previously, loss-of-function variants within AP4S1 were associated with a quadriplegic cerebral palsy-6 phenotype, AP-4 Deficiency Syndrome. In this study, the inclusion of mRNA-seq allowed for the identification of a previously missed splice-altering variant, and thereby expands the mutational spectrum of AP-4 Deficiency Syndrome to include impacts to some tissue-dependent isoforms.

Identification of mutations in AP4S1/SPG52 through next generation sequencing in three families.

BACKGROUND AND PURPOSE: The term hereditary spastic paraplegia (HSP) covers a spectrum of genetically heterogeneous disorders in which lower limb spasticity is the common clinical feature. Many patients with childhood-onset HSP are mistakenly diagnosed with cerebral palsy (CP). METHODS: A group of as yet molecularly undiagnosed HSP patients were analyzed using SpastoPlex, a customized target re-sequencing panel able to investigate the coding regions of 72 genes linked to HSP, spastic ataxias or related motor diseases. RESULTS: Our investigations identified loss-of-function mutations in AP4S1/SPG52 in four children (three families) who had previously received a diagnosis of diplegic/quadriplegic CP. The patients presented spastic paraparesis, mild facial dysmorphisms, moderate-to-severe intellectual disability and severe speech delay. Two patients manifested febrile seizures and childhood-onset focal seizures. In all the patients, brain magnetic resonance imaging (MRI) showed a peculiar hypoplastic posterior corpus callosum, often associated with ventriculomegaly, white matter loss and cerebral atrophy. CONCLUSION: Adaptor protein 4 (AP-4) deficiency disorders should be suspected in children with spastic paraparesis, cognitive deficit and absent speech accompanied by suggestive MRI features. Seizures might be amongst the clinical manifestations of the syndrome.

Ap4s1 truncation leads to axonal defects in a zebrafish model of spastic paraplegia 52.

Biallelic mutations in AP4S1, the σ4 subunit of the adaptor protein complex 4 (AP-4), lead to autosomal recessive spastic paraplegia 52 (SPG52). It is a subtype of AP-4-associated hereditary spastic paraplegia (AP-4-HSP), a complex childhood-onset neurogenetic disease characterized by progressive spastic paraplegia of the lower limbs. This disease has so far lacked effective treatment, in part due to a lack of suitable animal models. Here, we used CRISPR/Cas9 technology to generate a truncation mutation in the ap4s1 gene in zebrafish. The ap4s1 truncation led to motor impairment, delayed neurodevelopment, and distal axonal degeneration. This animal model is useful for further research into AP-4 and AP-4-HSP.

Axonal autophagosome maturation defect through failure of ATG9A sorting underpins pathology in AP-4 deficiency syndrome.

Adaptor protein (AP) complexes mediate key sorting decisions in the cell through selective incorporation of transmembrane proteins into vesicles. Little is known of the roles of AP-4, despite its loss of function leading to a severe early onset neurological disorder, AP-4 deficiency syndrome. Here we demonstrate an AP-4 epsilon subunit knockout mouse model that recapitulates characteristic neuroanatomical phenotypes of AP-4 deficiency patients. We show that ATG9A, critical for autophagosome biogenesis, is an AP-4 cargo, which is retained within the trans-Golgi network (TGN) in vivo and in culture when AP-4 function is lost. TGN retention results in depletion of axonal ATG9A, leading to defective autophagosome generation and aberrant expansions of the distal axon. The reduction in the capacity to generate axonal autophagosomes leads to defective axonal extension and de novo generation of distal axonal swellings containing accumulated ER, underlying the impaired axonal integrity in AP-4 deficiency syndrome.Abbreviations: AP: adaptor protein; AP4B1: adaptor-related protein complex AP-4, beta 1; AP4E1: adaptor-related protein complex AP-4, epsilon 1; ATG: autophagy-related; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GFAP: glial fibrillary acidic protein; GOLGA1/Golgin-97/GOLG97: golgi autoantigen, golgin subfamily a, 1; GOLGA2/GM130: golgi autoantigen, golgin subfamily a, 2; HSP: hereditary spastic paraplegia; LC3/MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK8IP1/JIP1: mitogen-acitvated protein kinase 8 interacting protein 1; NEFH/NF200: neurofilament, heavy polypeptide; RBFOX3/NeuN (RNA binding protein, fox-1 homolog [C. elegans] 3); SQSTM1/p62: sequestosome 1; TGN: trans-Golgi network; WIPI2: WD repeat domain, phosphoinositide interacting protein 2.