Functional analysis of novel and recurrent RINT1 variants in patients with infantile liver dysfunction.

Rad50-interacting protein (RINT1) interacts with the endoplasmic reticulum (ER) tethering and SNARE complex, playing a central role in membrane trafficking and lipid metabolism. Loss-of-function variants of RINT1 have been related to episodic severe transaminitis with skeletal dysplasia or spastic paraplegia. We report two unrelated patients with recurrent markedly elevated aminotransferase triggered by fever, accompanied by coagulopathy and hyperammonemia. Liver biopsy revealed liver steatosis and bridging fibrosis in one patient, while the other displayed mild hepatocyte enlargement. Trio-whole-exome sequencing identified biallelic pathogenic RINT1 variants in the two patients. A novel missense variant [c.662 A > C, p.(His221Pro)] and a recurrent splice-site variant (c.1333+1 G > A) were identified in the first case. In the second case, a recurrent pathogenic RINT1 homozygous missense variant [c.1102 G > A, p.(Ala368Thr)] was identified. We investigated the pathogenicity of these variants through immunoprecipitation. Recombinant proteins produced from the mutant RINT1 transcript (p.His221Pro or p.Ala368Thr) displayed disrupted ER tether and SNARE interactions. Since the inhibition of ER-Golgi transport is associated with ER-stress activation, unfolded protein response (UPR)-related gene expression was investigated by qPCR. TIP20, a RINT1 homolog in Saccharomyces cerevisiae, is needed for autophagosome formation; therefore, an LC3-II turnover assay was performed and revealed disrupted autophagic flux. In addition, we created a fat-body-specific Rint1 knockdown in Drosophila. In the mutant larva, tissue atrophy and decreased lipid droplets in the fat body were observed. These results indicated that a loss of RINT1 function activated the UPR, impairs autophagy, and led to lipid storage abnormalities, contributing to the pathogenesis of liver disease.