Many patients with severe, chronic diseases remain without a diagnosis despite extensive medical evaluation, including some cases with clinical exome sequencing. The goal of the NIH-funded Undiagnosed Diseases Network (UDN) is to provide a diagnosis for these challenging cases and to identify biological characteristics of newly discovered disease genes. The UDN uses a collaborative multidisciplinary approach that combines comprehensive medical workup, exome/genome sequencing, bioinformatic analysis, with functional studies in model organisms including zebrafish, Drosophila, and C. elegans. Through the UDN, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant, p.Asp136His, in the Ras/Rab GTPases family nucleotide binding domain in RAB5B was identified. Functional studies were performed in the C. elegans Model Organism Screening Center at Washington University in St. Louis. We used CRISPR/Cas9 to knock the proband variant into the conserved position (Asp135) of the ortholog,
rab-5. Analyses of organismal phenotypes such as locomotion and size demonstrated that
rab-5[Asp135His] is damaging. We also show data indicating that
rab-5[Asp135His] heterozygotes were defective in endocytosis and early endosome (EE) fusion. Dosage analysis by adding extra copies of wild type
rab-5 transgene revealed that
rab-5[D135H] has a strong dominant negative effect, requiring three wild type copies to suppress the variant's poisonous effect. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in type II pneumocytes, and mature SP-B and SP-C were significantly reduced, while ProSP-B and ProSP-C were normal. Furthermore, staining of normal lung showed co-localization of RAB5B and EEA1 with ProSP-B and ProSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, inducing interstitial lung disease, and that RAB5B and EEs normally function in the regulated surfactant secretion pathway. Together, the data suggest a non-canonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for surfactant dysfunction disorder.