#4688 ATP6V0A4 GENE MUTATION-ASSOCIATED NEPHROPATHY

Abstract

Abstract Background and Aims The ATP6V0A4 gene is localized to chromosome 7q33∼34 and encodes for vacuolar H+-ATPase (V-ATPase) α4 subunit. Decreased V-ATPase function due to mutations in the ATP6V0A4 genes could cause H+ excretion defect of α-intercalated cells in the renal collecting duct, resulting in hereditary distal renal tubular acidosis (dRTA). However, crucially, no gain-of-function mutations in the ATP6V0A4 gene have been reported previously. This study reports a gain-of-function mutation of ATP6V0A4 gene occurred in a 32-year-old male with metabolic alkalosis, hypokalemia and hearing loss, and further explore potential pathogenic mechanisms of this mutation. Method Clinical information was collected from the proband and his family members. Kidney tissue samples were collected for morphological observation and immunohistochemical staining. High-throughput sequencing analyses were done for the proband and his parents. ATP6V0A4 wild-type and mutant plasmids were constructed and used to transfect the 293T cells. After 48 hours of transfection, the expression of ATP6V0A4 was verified using WB analyses and V-ATPase activity was measured by ultraviolet spectrophotometry. Results The proband and his father both suffered from severe hypertension and hearing loss. Blood tests showed hypokalemia, metabolic alkalosis and renal insufficiency. Urinalysis indicated acidic urine. Laboratory tests on admission are shown in Table 1. Renal biopsy suggested malignant hypertensive kidney injury. Whole-exome sequencing demonstrated that the proband carried the heterozygous mutation c.1534G>T; p.V512L in exon 15 of the ATP6V0A4 gene. Sanger sequencing confirmed that the variant was inherited from his father. The immunohistochemical results implied the expression of V-ATPase α4 was significantly higher in renal tissues from proband than that of the control who was diagnosed with minimal change disease (Figure 1B).WB analysis showed 293T cells transfected with ATP6V0A4 mutant plasmids with darker protein bands, suggesting higher expression of V-ATPase α4 (Figure 1D). The cells transfected with mutant ATP6V0A4 plasmids showed significantly higher V-ATPase activity compared with wild-type ATP6V0A4 plasmids transfected cells (Figure 1E). Conclusion The ATP6V0A4 c.1534G>T; p.V512L mutation is a gain-of-function mutation and this result indicates the possibility that this mutation might enhance the normal physiological function of the V-ATPase, and likely contributes to the increased hydrogen ions excretion and thereby to the development of metabolic alkalosis (Figure 1F).