Novel c.G630A TCIRG1 mutation causes aberrant splicing resulting in an unusually mild form of autosomal recessive osteopetrosis

• Published in: Journal of cellular biochemistry 2019-10, Vol.120 (10), p.17180-17193
• Main Authors: Zirngibl, Ralph A., Wang, Andrew, Yao, Yeqi, Manolson, Morris F., Krueger, Joerg, Dupuis, Lucie, Mendoza‐Londono, Roberto, Voronov, Irina
• Format: Article
• Language: English
• Subjects: Acidification; Adenosine triphosphatase; Biomedical materials; Bone density; Bone diseases; Bone growth; Exons; Gene expression; Monocytes; Mutation; Nucleotides; osteoclast; Osteoclastogenesis; Osteoclasts; Osteopetrosis; Peripheral blood; Phenotypes; Plasmids; Proteins; Splicing; Translocation; T‐cell immune regulator 1; V‐ATPase; Yeast; Yeasts; Zinc
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.28979

Autosomal recessive osteopetrosis (ARO) is a severe genetic bone disease characterized by high bone density due to mutations that affect formation or function of osteoclasts. Mutations in the a3 subunit of the vacuolar‐type H+‐ATPase (encoded by T‐cell immune regulator 1 [TCIRG1]) are responsible for ~50% of all ARO cases. We identified a novel TCIRG1 (c.G630A) mutation responsible for an unusually mild form of the disease. To characterize this mutation, osteoclasts were differentiated using peripheral blood monocytes from the patient (c.G630A/c.G630A), male sibling (+/+), unaffected female sibling (+/c.G630A), and unaffected parent (+/c.G630A). Osteoclast formation, bone‐resorbing function, TCIRG1 protein, and mRNA expression levels were assessed. The c.G630A mutation did not affect osteoclast differentiation; however, bone‐resorbing function was decreased. Both TCIRG1 protein and full‐length TCIRG1 mRNA expression levels were also diminished in the affected patient's sample. The c.G630A mutation replaces the last nucleotide of exon 6 and may cause splicing defects. We analyzed the TCIRG1 splicing pattern between exons 4 to 8 and detected deletions of exons 5, 6, 7, and 5‐6 (ΔE56). These deletions were only observed in c.G630A/c.G630A and +/c.G630A samples, but not in +/+ controls. Among these deletions, only ΔE56 maintained the reading frame and was predicted to generate an 85 kDa protein. Exons 5‐6 encode an uncharacterized portion of the cytoplasmic N‐terminal domain of a3, a domain not involved in proton translocation. To investigate the effect of ΔE56 on V‐ATPase function, we transformed yeast with plasmids carrying full‐length or truncated Vph1p, the yeast ortholog of a3. Both proteins were expressed; however, ΔE56‐Vph1p transformed yeast failed to grow on Zn2+‐containing plates, a growth assay dependent on V‐ATPase‐mediated vacuolar acidification. In conclusion, our results show that the ΔE56 truncated protein is not functional, suggesting that the mild ARO phenotype observed in the patient is likely due to the residual full‐length protein expression. Here we characterize a novel unusually mild osteopetrotic T‐cell immune regulator 1 (TCIRG1) c.G630A mutation on cellular, gene, and protein levels. We propose a possible molecular mechanism of how this mutation affects V‐ATPase function using yeast as a model system. In addition, this novel TCIRG1 c.G630A mutation provides us with an unexpected insight into structure and function of a3, the V‐ATPase