Loss-of-Function Mutations in UNC45A Cause a Syndrome Associating Cholestasis, Diarrhea, Impaired Hearing, and Bone Fragility

Abstract

Despite the rapid discovery of genes for rare genetic disorders, we continue to encounter individuals presenting with syndromic manifestations. Here, we have studied four affected people in three families presenting with cholestasis, congenital diarrhea, impaired hearing, and bone fragility. Whole-exome sequencing of all affected individuals and their parents identified biallelic mutations in Unc-45 Myosin Chaperone A (UNC45A) as a likely driver for this disorder. Subsequent in vitro and in vivo functional studies of the candidate gene indicated a loss-of-function paradigm, wherein mutations attenuated or abolished protein activity with concomitant defects in gut development and function.

Keywords: GCUNC-45.

Figure 1

UNC45A Variants and Gene Structure (A) Pedigrees of the three families with mutations in UNC45A. (B) Structural organization of UNC45A transcript (Ensembl: ENST00000394275) and protein with known conserved protein domains and localization of amino acid residues affected by mutations identified in the three families. Exonic (in purple) regions are not drawn to scale. (C) Venn diagram showing the overlap between clinical signs of the four individuals.

Figure 2

Western Blot Detection and Quantification of UNC45A Protein in Lymphoblastoid Cells and Fibroblasts (A) Detection of UNC45A by western blot analysis in lymphoblastoid cells and fibroblasts using a monoclonal antibody. GAPDH was used as a loading control. (B and C) Quantification of UNC45A protein levels with Fiji software shows significantly lowered levels of the expression of UNC45A in all individuals’ lymphoblastoid cells (B) and fibroblasts of individual A.II.3 (C). Values are mean ratio ± SD, n = 3; Welch’s two sample t test (affected individual versus control) (^∗p < 0.04).

Figure 3

Microgavage Assay to Assess Intestinal Motility Fluorescent microspheres were gavaged and their transit was observed at 0, 3, 6, 9, and 24 hr in unc45 morphants and mutants. (A) Numbers of embryos with observed fluorescence in the four zones of the alimentary canal at four different time points. Compared to controls (left set), in which fluorescent beads peak in zone 4 at time 1 and the progressively expelled from the gut (times 2–4). Both morphants (middle set) and mutants (right set) show presence of fluorescent beads in zones 1 and 2 of the gut. (B) Representative images of the gut from controls, morphants, and mutants, demarcating the four zones and showing characteristic evidence of GFP in zones 2 and 3.

Figure 4

Histological Study of Transverse Sections of Zones 2 and 3 of 5 dpf Zebrafish Markers for F-actin (Phalloidin) and intestinal brush borders (4e8) were used and revealed defects (lack of epithelial folds in the intestinal tube) in the structure of kus^tr12 larvae compared to controls.

Figure 5

Assessment of Human UNC45A c.1268T>A, Resulting in a p.Val423Asp Change (V423N), on Zones 2 and 3 of Mutant unc45a Zebrafishes Mutant embryos were injected with either WT or V423N-encoding mRNA and intestinal folds were observed using brightfield microscopy. Mutants injected with WT encoding mRNA were able to restore folds in zones 2 and 3, but those injected with V423N-encoding mRNA were not restored, illustrating that V423N is pathogenic.