Patients with genetic disease of unknown causes can be rapidly diagnosed by bioinformatic analysis of disease-associated DNA sequences and phenotype.

Autosomal recessive cutis laxa type 2 (ARCL2), a syndrome of growth and developmental delay and redundant, inelastic skin, is caused by mutations in the a2 subunit of the vesicular ATPase H⁺-pump (ATP6V0A2). The goal of this study was to define the disease mechanisms that lead to connective tissue lesions in ARCL2. In a new cohort of 17 patients, DNA sequencing of ATP6V0A2 detected either homozygous or compound heterozygous mutations. Considerable allelic and phenotypic heterogeneity was observed, with a missense mutation of a moderately conserved residue p.P87L leading to unusually mild disease. Abnormal N- and/or mucin type O-glycosylation was observed in all patients tested. Premature stop codon mutations led to decreased ATP6V0A2 mRNA levels by destabilizing the mutant mRNA via the nonsense-mediated decay pathway. Loss of ATP6V0A2 either by siRNA knockdown or in ARCL2 cells resulted in distended Golgi cisternae, accumulation of abnormal lysosomes and multivesicular bodies. Immunostaining of ARCL2 cells showed the accumulation of tropoelastin (TE) in the Golgi and in large, abnormal intracellular and extracellular aggregates. Pulse–chase studies confirmed impaired secretion and increased intracellular retention of TE, and insoluble elastin assays showed significantly reduced extracellular deposition of mature elastin. Fibrillin-1 microfibril assembly and secreted lysyl oxidase activity were normal in ARCL2 cells. TUNEL staining demonstrated increased rates of apoptosis in ARCL2 cell cultures. We conclude that loss-of-function mutations in ATP6V0A2 lead to TE aggregation in the Golgi, impaired clearance of TE aggregates and increased apoptosis of elastogenic cells.