ATP6V1B1 encodes a subunit of the vacuolar H+-ATPase and pathogenic variants are associated with autosomal recessive distal renal tubular acidosis (dRTA) with deafness. Heterozygous variants predicted to affect a specific amino acid, Arg394, have been recurrently reported in dRTA but their significance has been unclear. We hypothesized that these variants are associated with a dominant disease mechanism. We conducted a retrospective analysis of cases identified in our genetic laboratories and through European nephrology organizations. Data regarding demographics, clinical presentation, laboratory findings, hearing and imaging studies of kidneys were collected from the index patient and, if available, from other family members. The potential disease mechanism was investigated through structural modelling in silico. Twenty index patients in total were included, of which 19 carried the variant c.1181G>A; p.(Arg394Gln) and one c.1180C>G; p.(Arg394Gly). In seven families, more than one member was affected and the variant segregated with the disease in those with available information (15 affected, 6 unaffected), except for the unaffected mother of 2 affected children, who was mosaic. In no patient was a second causative variant in trans identified. In eight sporadic patients and one affected parent, the variant was confirmed to be de novo. Both variants are absent in gnomAD. Sensorineural hearing loss was reported in 8 of the 22 patients with available information. Structural modelling supports a crucial role for Arg394 in nucleotide binding. We provide strong evidence for the pathogenicity of heterozygous variants affecting Arg394 and thus a novel inheritance modus for ATP6V1B1-associated dRTA. Clinically, this form differs from the recessive one by the lower prevalence of hearing loss. The prominent position of Arg394 in the nucleotide binding fold of the H+-ATPase structure is consistent with a dominant negative mechanism. Our findings inform the diagnosis and management of patients with dRTA and variants of Arg394.

V-ATPases are a class of multi-subunit protein complexes that utilize energy derived from ATP hydrolysis for mediating H+ transport across cell membranes, which plays an important role in a range of life activities by acidifying the intracellular and extracellular environment. Variants of V-ATPase genes may lead to complete or partial loss of V-ATPase activity, which in turn may impair the ability of type A intercalated cells in renal tubules to pump H+ into the tubular lumen, ultimately resulting in the onset of autosomal recessive distal renal tubular acidosis (dRTA). With the rapid development of molecular techniques, ATP6V0A4 and ATP6V1B1 have now been identified as the pathogenic genes for dRTA. Moreover, animal and cell experiments have substantiated the implication of V-ATPase subunit genes including ATP6V1C2 and ATP6V1G3 in the development of dRTA, though clinical evidence is still limited. This article has reviewed recent progress on the genetic and molecular mechanisms of V-ATPase subunit gene variants which can lead to dRTA, which may shed light on the diagnosis and treatment of this disease.

Mutations in solute carrier family 4 member 1 (SLC4A1) encoding anion exchanger 1 (AE1) are the most common cause of autosomal recessive distal renal tubular acidosis (AR dRTA) in Southeast Asians. To explain the molecular mechanism of this disease with hematological abnormalities in an affected family, we conducted a genetic analysis of SLC4A1 and studied wild-type and mutant AE1 proteins expressed in human embryonic kidney 293T (HEK293T) cells. SLC4A1 mutations in the patient and family members were analyzed by molecular genetic techniques. Protein structure modeling was initially conducted to evaluate the effects of mutations on the three-dimensional structure of the AE1 protein. The mutant kidney anion exchanger 1 (kAE1) plasmid construct was created to study protein expression, localization, and stability in HEK293T cells. We discovered that the patient who had AR dRTA coexisting with mild hemolytic anemia carried a novel compound heterozygous SLC4A1 mutations containing c.1199_1225del (p.Ala400_Ala408del), resulting in Southeast Asian ovalocytosis (SAO), and c.1331C > A (p.Thr444Asn). Homologous modeling and in silico mutagenesis indicated that these two mutations affected the protein structure in the transmembrane regions of kAE1. We found the wild-type and mutant kAE1 T444N to be localized at the cell surface, whereas the mutants kAE1 SAO and SAO/T444N were intracellularly retained. The half-life of the kAE1 SAO, T444N, and SAO/T444N mutants was shorter than that of the wild-type protein. These results suggest impaired trafficking and instability of kAE1 SAO/T444N as the likely underlying molecular mechanism explaining the pathogenesis of the novel SLC4A1 compound heterozygous mutation identified in this patient.

Homozygous autosomal recessive distal renal tubular acidosis (dRTA) is a rare entity. The intercalated cells in the collecting ducts are defective in apical proton secretion or basolateral bicarbonate reabsorption, due to mutations in genes encoding for proteins in a4 and B1 subunits of the V-ATPase and the anion exchanger Cl-/HCO- (kAE1). This results in decreased ammonium (NH4+) excretion and defective urine acidification. dRTA is characterized by hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitranuria, and nephrocalcinosis. Autosomal recessive dRTA is associated with mutation in ATP6V1B1 (2p13) or ATP6V0A4 (7q34) genes. ATP6V1B1 mutation is associated with early - onset sensory neural hearing loss (SNHL), whereas ATP6V0A4 gene mutation may be associated with early-to late-onset SNHL. We report the case of a 30-year-old married woman diagnosed with dRTA at three months of age with mild SNHL, showing homogygous nonsense mutation in exon 3 of the ATP6V0A4 gene that resulted in a stop codon and premature truncation of the protein at codon 6.

Autosomal recessive distal renal tubular acidosis (dRTA) is a rare condition, most cases of which are caused by genetic mutations. Several loss-of-function mutations in the ATP6V0A4 gene have been recently reported. A 2-month, 24-day-old Chinese girl presenting with vomiting and diarrhea. dRTA was established by metabolic acidosis and hypokalemia. Mutational analysis of the ATP6V0A4 gene revealed a homozygous deletion of exons 13 and 14. The father was found to have a heterozygous loss of both exons, whereas the mother was normal. Patient was treated with potassium citrate. The patient has shown normal pH and potassium levels. This is the first case of a homozygous deletion in ATP6V0A4 reported in the literature. Although the initial auditory screening was normal in this case, this patient will nevertheless undergo long-term auditory testing.