Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. Here, we identify a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. Our study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, our in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules.

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ObjectiveHuman genomics established that pathogenic variation in diverse genes can underlie a single disorder. For example, hereditary spastic paraplegia is associated with >80 genes, with frequently only few affected individuals described for each gene. Herein, we characterize a large cohort of individuals with biallelic variation in ENTPD1, a gene previously linked to spastic paraplegia 64 (Mendelian Inheritance in Man # 615683).MethodsIndividuals with biallelic ENTPD1 variants were recruited worldwide. Deep phenotyping and molecular characterization were performed.ResultsA total of 27 individuals from 17 unrelated families were studied; additional phenotypic information was collected from published cases. Twelve novel pathogenic ENTPD1 variants are described (NM 001776.6): c.398_399delinsAA; p.(Gly133Glu), c.540del; p.(Thr181Leufs*18), c.640del; p.(Gly216Glufs*75), c.185 T > G; p.(Leu62*), c.1531 T > C; p.(*511Glnext*100), c.967C > T; p.(Gln323*), c.414‐2_414‐1del, and c.146 A > G; p.(Tyr49Cys) including 4 recurrent variants c.1109 T > A; p.(Leu370*), c.574‐6_574‐3del, c.770_771del; p.(Gly257Glufs*18), and c.1041del; p.(Ile348Phefs*19). Shared disease traits include childhood onset, progressive spastic paraplegia, intellectual disability (ID), dysarthria, and white matter abnormalities. In vitro assays demonstrate that ENTPD1 expression and function are impaired and that c.574‐6_574‐3del causes exon skipping. Global metabolomics demonstrate ENTPD1 deficiency leads to impaired nucleotide, lipid, and energy metabolism.InterpretationThe ENTPD1 locus trait consists of childhood disease onset, ID, progressive spastic paraparesis, dysarthria, dysmorphisms, and white matter abnormalities, with some individuals showing neurocognitive regression. Investigation of an allelic series of ENTPD1 (1) expands previously described features of ENTPD1‐related neurological disease, (2) highlights the importance of genotype‐driven deep phenotyping, (3) documents the need for global collaborative efforts to characterize rare autosomal recessive disease traits, and (4) provides insights into disease trait neurobiology. ANN NEUROL 2022;92:304–321

Undiagnosed genetic disease imposes significant burden on families and healthcare resources, especially in cases with a complex phenotype. Here we present a child with suspected leukodystrophy in the context of additional features, including hearing loss, clinodactyly, rotated thumbs, tapered fingers, and simplified palmar crease. Trio genome sequencing (GS) identified three molecular diagnoses in this individual: compound heterozygous missense variants associated with Pol III-related leukodystrophy, a 4 Mb de novo copy number loss including the MYCN gene associated with Feingold syndrome, and a mosaic single nucleotide variant (SNV) associated with COL2A1-related disorders. These variants fully account for the individual's features, but also illustrate the potential for superimposed and unclear contributions of multiple diagnoses to a individual's overall presentation. This report demonstrates the advantage of GS in detection of multiple variant types, including low-level mosaic variants, and emphasizes the need for comprehensive genetic analysis and detailed clinical phenotyping to provide individuals and their families with the maximum benefit for clinical care and genetic counseling.