RINT1 Bi-allelic Variations Cause Infantile-Onset Recurrent Acute Liver Failure and Skeletal Abnormalities

Abstract

Pediatric acute liver failure (ALF) is life threatening with genetic, immunologic, and environmental etiologies. Approximately half of all cases remain unexplained. Recurrent ALF (RALF) in infants describes repeated episodes of severe liver injury with recovery of hepatic function between crises. We describe bi-allelic RINT1 alterations as the cause of a multisystem disorder including RALF and skeletal abnormalities. Three unrelated individuals with RALF onset ≤3 years of age have splice alterations at the same position (c.1333+1G>A or G>T) in trans with a missense (p.Ala368Thr or p.Leu370Pro) or in-frame deletion (p.Val618_Lys619del) in RINT1. ALF episodes are concomitant with fever/infection and not all individuals have complete normalization of liver function testing between episodes. Liver biopsies revealed nonspecific liver damage including fibrosis, steatosis, or mild increases in Kupffer cells. Skeletal imaging revealed abnormalities affecting the vertebrae and pelvis. Dermal fibroblasts showed splice-variant mediated skipping of exon 9 leading to an out-of-frame product and nonsense-mediated transcript decay. Fibroblasts also revealed decreased RINT1 protein, abnormal Golgi morphology, and impaired autophagic flux compared to control. RINT1 interacts with NBAS, recently implicated in RALF, and UVRAG, to facilitate Golgi-to-ER retrograde vesicle transport. During nutrient depletion or infection, Golgi-to-ER transport is suppressed and autophagy is promoted through UVRAG regulation by mTOR. Aberrant autophagy has been associated with the development of similar skeletal abnormalities and also with liver disease, suggesting that disruption of these RINT1 functions may explain the liver and skeletal findings. Clarifying the pathomechanism underlying this gene-disease relationship may inform therapeutic opportunities.

Keywords: RINT1; autophagy; autosomal recessive; disorder of intracellular trafficking; recurrent acute liver failure; skeletal anomalies.

Figure 1

Liver Biopsies from Proband 1 Show Non-specific Findings Consistent with Liver Damage (A) H&E shows moderate macrovesicular steatosis (200× magnification). (B) Trichrome stain shows bridging fibrosis (100× magnification). (C) Numerous lipid droplets are present in hepatocytes (HV = 80.0 kV; 2,100× direct magnification). (D) Glycogen contents are in the normal range and there are no visible abnormalities in the endoplasmic reticulum (HV = 80.0 kV; 11,000× direct magnification). (E) The bile canaliculus (center) is normal. Electron dense material consistent with biliary material is seen in the hepatocellular cytoplasm surrounding the bile canaliculus (HV = 80.0 kV; 11,000× direct magnification). (F) There are no abnormalities of mitochondria and peroxisomes are present (HV = 80.0 kV; 21,000× direct magnification).

Figure 2

Radiographic Skeletal Findings (A) Proband 1: 15-month-old male with beaking of several lower thoracic and upper lumbar spine vertebrae (black arrows) with a hypoplastic T12 vertebra (white arrow) and platyspondyly of the thoracic vertebrae (asterisks). The acetabula are mildly shallow (white arrowheads) with an asymmetrically small right femoral head epiphysis. Dense metaphyseal bands were also noted in the long bones (black arrowheads). (B) Proband 2: 7-year-old female. Lateral radiograph of the spine shows anterior beaking of lower thoracic and lumbar vertebra (black arrows). Frontal pelvic radiograph shows mild irregularity of the acetabular rims (white arrowheads), as well as decreased spherical contour of the femoral head epiphyses (black arrowheads). (C) Proband 3: 8-year-old female. Lateral radiograph of the spine shows findings of mild anterior vertebral irregularity and beaking of lower thoracic and upper lumbar vertebrae (black arrows), with mild hypoplasia of the anterior aspects of L2 and L5 vertebrae (white arrowheads). Frontal pelvic radiograph shows the femoral head epiphyses to be flattened medially (black arrowheads) with decreased spherical contour, which may be due to an unusually large fovea capitis.

Figure 3

Bi-allelic RINT1 Variants Gene (GenBank: NM_021930) and protein schematics showing the location of the variants identified in RINT1 and the species conservation of the affected residues of the missense and in-frame deletion alterations. Introns are not to scale.

Figure 4

RNA Sequencing from Fibroblasts Reveals Skipping of Exon 9 of RINT1 Sashimi plot representing RNA sequencing reads supporting splicing junctions of exons 8, 9, and 10 of RINT1 (GenBank: NM_021930) from fibroblast cultures from proband 1 and a control (±puromycin). The junction reads from proband 1 reveal skipping of exon 9 with recovery of additional reads supporting exon 9 skipping with puromycin treatment. No exon 9 skipping was observed in the control.

Figure 5

Fibroblasts Demonstrate Decreased RINT1 Protein, but Not NBAS or p31 (A) There is a significant difference in RINT1 protein levels among the genetic variants (control [Ctrl], proband 1 [Pr 1], or proband 3 [Pr 3]) (single factor effect; F_2,12 = 98.17, p = 3.65e−8). There is no significant effect of temperature (37°C versus 40°C) (single factor effect; F_1,12 = 4.73, p = 0.05). There is no significant effect on RINT1 protein levels of the interaction between the genetic variants (Ctrl, Pr 1, or Pr 3) and temperature (37°C or 40°C) (F_2,12 = 1.30, p = 0.31). (B and C) No statistically significant reduction in NBAS protein (B) or p31 protein (C) levels compared to control were observed. 2-way analysis of variance (2-way ANOVA) from three independent experiments each. ^∗p < 0.05.

Figure 6

Fibroblasts Have Abnormal Golgi Morphology and Impaired Autophagic Flux (A and B) Fibroblasts from affected individuals and a control were cultured at 37°C or 40°C and show (A) an expanded Golgi network with proband 1 also showing fragmentation of the Golgi at 40°C, compared to control by GM130, and (B) possible expansion of the ER-Golgi intermediate compartment (ERGIC) by ERGIC53. (C) Fibroblasts cultured with and without bafilomycin A1 (BafA1) show increased LC3 by immunofluorescent imaging that was not further increased by bafilomycin A1 treatment compared to control. (D) Whole-cell lysates from each proband show an increase in LC3-II/Actin ratio compared to control. The LC3-II/Actin ratio in control cells was increased with bafilomycin A1 treatment, but remained unchanged in probands 1 and 3. Ctrl, control; Pr, proband; bars represent mean ± standard deviation for three independent experiments. Two-tailed paired t test.