Dominantly acting variants in ATP6V1C1 and ATP6V1B2 cause a multisystem phenotypic spectrum by altering lysosomal and/or autophagosome function.

The vacuolar H+-ATPase (V-ATPase) is a functionally conserved multimeric complex localized at the membranes of many organelles where its proton-pumping action is required for proper lumen acidification. The V-ATPase complex is composed of several subunits, some of which have been linked to human disease. We and others previously reported pathogenic dominantly acting variants in ATP6V1B2, the gene encoding the V1B2 subunit, as underlying a clinically variable phenotypic spectrum including dominant deafness-onychodystrophy (DDOD) syndrome, Zimmermann-Laband syndrome (ZLS), and deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures (DOORS) syndrome. Here, we report on an individual with features fitting DOORS syndrome caused by dysregulated ATP6V1C1 function, expand the clinical features associated with ATP6V1B2 pathogenic variants, and provide evidence that these ATP6V1C1/ATP6V1B2 amino acid substitutions result in a gain-of-function mechanism upregulating V-ATPase function that drives increased lysosomal acidification. We demonstrate a disruptive effect of these ATP6V1B2/ATP6V1C1 variants on lysosomal morphology, localization, and function, resulting in a defective autophagic flux and accumulation of lysosomal substrates. We also show that the upregulated V-ATPase function affects cilium biogenesis, further documenting pleiotropy. This work identifies ATP6V1C1 as a new gene associated with a neurodevelopmental phenotype resembling DOORS syndrome, documents the occurrence of a phenotypic continuum between ZLS, and DDOD and DOORS syndromes, and classify these conditions as lysosomal disorders.

Epilepsy due to potential loss of ATP6V1B2 function with mechanistic insight by a Drosophila Vha55 model.

ATP6V1B2 encodes the subunit of the vacuolar H+-ATPase, which is an enzyme responsible for the acidification of intracellular organelles and essential for cell signaling and neurotransmitter release. The aim of the study is to identify the correlation between ATP6V1B2 and epilepsy. Trio-exome sequencing was performed. Reverse Transcription-PCR and Quantitative real-time PCR analyses were carried out to determine whether this variant leads to nonsense-mediated mRNA decay (NMD). Drosophila models with knocked-down homologous genes of ATP6V1B2 were generated to study the causal relationship between the ATP6V1B2 and the phenotype of epilepsy. We described a 5-year-old male with a novel variant c.1163delT(p.Tyr389IlefsTer13) in ATP6V1B2, who presented with epilepsy. The expression level of the premature termination codon (PTC) transcript was normal in the patient, which indicated that NMD evasion existed in the PTC transcript. We generated an animal model using Drosophila to study the knock down effects of Vha55, which is the ATP6V1B2 ortholog in fly. The Vha55 knockdown flies show seizure-like behaviors and climbing defects. This study expands the variation spectrum of the ATP6V1B2 gene. Cross-species animal model demonstrates the causal relationship between ATP6V1B2 defect and epilepsy, and shed new insights into the disease mechanism caused by ATP6V1B2 LOF variants.

The ATP6V1B2 DDOD/DOORS-Associated p.Arg506* Variant Causes Hyperactivity and Seizures in Mice.

The vacuolar H+-ATPase is a multisubunit enzyme which plays an essential role in the acidification and functions of lysosomes, endosomes, and synaptic vesicles. Many genes encoding subunits of V-ATPases, namely ATP6V0C, ATP6V1A, ATP6V0A1, and ATP6V1B2, have been associated with neurodevelopmental disorders and epilepsy. The autosomal dominant ATP6V1B2 p.Arg506* variant can cause both congenital deafness with onychodystrophy, autosomal dominant (DDOD) and deafness, onychodystrophy, osteodystrophy, mental retardation, and seizures syndromes (DOORS). Some but not all individuals with this truncating variant have intellectual disability and/or epilepsy, suggesting incomplete penetrance and/or variable expressivity. To further explore the impact of the p.Arg506* variant in neurodevelopment and epilepsy, we generated Atp6v1b2emR506* mutant mice and performed standardized phenotyping using the International Mouse Phenotyping Consortium (IMPC) pipeline. In addition, we assessed the EEG profile and seizure susceptibility of Atp6v1b2emR506* mice. Behavioral tests revealed that the mice present locomotor hyperactivity and show less anxiety-associated behaviors. Moreover, EEG analyses indicate that Atp6v1b2emR506* mutant mice have interictal epileptic activity and that both heterozygous (like patients) and homozygous mice have reduced seizure thresholds to pentylenetetrazol. Our results confirm that variants in ATP6V1B2 can cause seizures and that the Atp6v1b2emR506* heterozygous mouse model is a valuable tool to further explore the pathophysiology and potential treatments for vacuolar ATPases-associated epilepsy and disorders.

ATP6V1B2-related disorders featuring Lennox-Gastaut-syndrome: A case-based overview.

BACKGROUND: ATP6V1B2 (ATPase, H+ transporting, lysosomal VI subunit B, isoform 2) encodes for a subunit of a ubiquitous transmembrane lysosomal proton pump, implicated in the acidification of intracellular organelles and in several additional cellular functions. Variants in ATP6V1B2 have been related to a heterogeneous group of multisystemic disorders sometimes associated with variable neurological involvement. However, our knowledge of genotype-phenotype correlations and the neurological spectrum of ATP6V1B2-related disorders remain limited due to the few numbers of reported cases. CASE STUDY: We hereby report the case of an 18-year-old male Sicilian patient affected by a global developmental delay, skeletal abnormalities, and epileptic encephalopathy featuring Lennox-Gastaut syndrome (LGS), in which exome sequencing led to the identification of a novel de novo variant in ATP6V1B2 (NM_001693.4: c.973G > C, p.Gly325Arg). CONCLUSIONS: Our report provides new insights on the inclusion of developmental epileptic encephalopathies (DEEs) within the continuum group of ATP6V1B2-related disorders, expanding the phenotypic and molecular spectrum associated with these conditions.

A novel pathogenic ATP6V1B2 variant: Widening the genotypic spectrum of the epileptic neurodevelopmental phenotype.

ATP6V1B2 pathogenic variants are linked with variable phenotypes, such as dominant deafness-onychodystrophy syndrome (DDOD), autosomal dominant Zimmermann-Laband syndrome type 2 (ZLS2), and some cases of DOORS (deafness, onychodystrophy, osteodystrophy, intellectual disability [ID], and seizures). Epilepsy was first linked to ATP6V1B2, when the p.(Glu374Gln) missense variant was detected in a patient with ID and seizures, but without characteristic features of DDOD or ZLS2 syndromes. We herein report a novel pathogenic ATP6V1B2:p.Glu374Gly variant detected in an adult patient with ID and myoclonic-atonic seizures. The (re)occurrence of different variants affecting the same highly conserved hydrophilic glutamic acid on position 374 of the V-proton ATPase subunit B, indicates a potential novel pathogenic hotspot and a critical role for the specific residue in the development of epilepsy. ATP6V1B2 gene defects should be considered when analyzing patients with epilepsy, even in the absence of most cardinal features of DDOD, DOORS, or ZLS such as deafness, onychodystrophy, and osteodystrophy.

Case Report: Exome Sequencing Identified Variants in Three Candidate Genes From Two Families With Hearing Loss, Onychodystrophy, and Epilepsy.

A cohort of 542 individuals in 166 families with congenital hearing loss was recruited for whole-exome sequencing analysis. Here, we report the identification of three variants in five affected individuals in two unrelated families. In family 1, a nonsense mutation (c.1516C>T, p.R506*) in the ATP6V1B2 gene, a known causal allele for dominant deafness-onychodystrophy (DDOD), was identified in the mother and son with DDOD. However, a novel heterozygous variant (c.1590T>G, p.D530E) in TJP2, a known causal gene for hearing-loss, was also detected in the patients. In family 2, the same mutation (c.1516C>T, p.R506*) of ATP6V1B2 was detected from the father and daughter with DDOD. Furthermore, a novel heterozygous variant (c.733A>G, p.M245V) in the KIF11 gene was identified from the spouse with sensorineural hearing-loss and epilepsy. Notably, genotype-phenotype analysis of KIF11-associated disorders revealed that the p.M245V and two reported hearing-loss-associated variants (p.S235C and p.H244Y) are all mapped to a single ß-sheet (Ser235∼M245) in the kinesin motor domain. Together, this is the first demonstration that ATP6V1B2-caused DDOD is an autosomal dominant genetic disease, compared to previous cases with de novo mutation. Our findings expand the variant spectrum of hearing-loss-associated genes and provide new insights on understanding of hearing-loss candidate genes ATP6V1B2, TJP2, and KIF11.

Syndromic Deafness Gene ATP6V1B2 Controls Degeneration of Spiral Ganglion Neurons Through Modulating Proton Flux.

ATP6V1B2 encodes the V1B2 subunit in V-ATPase, a proton pump responsible for the acidification of lysosomes. Mutations in this gene cause DDOD syndrome, DOORS syndrome, and Zimmermann-Laband syndrome, which share overlapping feature of congenital sensorineural deafness, onychodystrophy, and different extents of intellectual disability without or with epilepsy. However, the underlying mechanisms remain unclear. To investigate the pathological role of mutant ATP6V1B2 in the auditory system, we evaluated auditory brainstem response, distortion product otoacoustic emissions, in a transgenic line of mice carrying c.1516 C > T (p.Arg506∗) in Atp6v1b2, Atp6v1b2 Arg506*/Arg506* . To explore the pathogenic mechanism of neurodegeneration in the auditory pathway, immunostaining, western blotting, and RNAscope analyses were performed in Atp6v1b2Arg506*/Arg506* mice. The Atp6v1b2Arg506*/Arg506* mice showed hidden hearing loss (HHL) at early stages and developed late-onset hearing loss. We observed increased transcription of Atp6v1b1 in hair cells of Atp6v1b2Arg506*/Arg506* mice and inferred that Atp6v1b1 compensated for the Atp6v1b2 dysfunction by increasing its own transcription level. Genetic compensation in hair cells explains the milder hearing impairment in Atp6v1b2Arg506*/Arg506* mice. Apoptosis activated by lysosomal dysfunction and the subsequent blockade of autophagic flux induced the degeneration of spiral ganglion neurons and further impaired the hearing. Intraperitoneal administration of the apoptosis inhibitor, BIP-V5, improved both phenotypical and pathological outcomes in two live mutant mice. Based on the pathogenesis underlying hearing loss in Atp6v1b2-related syndromes, systemic drug administration to inhibit apoptosis might be an option for restoring the function of spiral ganglion neurons and promoting hearing, which provides a direction for future treatment.

DOORS syndrome and a recurrent truncating ATP6V1B2 variant.

PURPOSE: Biallelic variants in TBC1D24, which encodes a protein that regulates vesicular transport, are frequently identified in patients with DOORS (deafness, onychodystrophy, osteodystrophy, intellectual disability [previously referred to as mental retardation], and seizures) syndrome. The aim of the study was to identify a genetic cause in families with DOORS syndrome and without a TBC1D24 variant. METHODS: Exome or Sanger sequencing was performed in individuals with a clinical diagnosis of DOORS syndrome without TBC1D24 variants. RESULTS: We identified the same truncating variant in ATP6V1B2 (NM_001693.4:c.1516C>T; p.Arg506*) in nine individuals from eight unrelated families with DOORS syndrome. This variant was already reported in individuals with dominant deafness onychodystrophy (DDOD) syndrome. Deafness was present in all individuals, along with onychodystrophy and abnormal fingers and/or toes. All families but one had developmental delay or intellectual disability and five individuals had epilepsy. We also describe two additional families with DDOD syndrome in whom the same variant was found. CONCLUSION: We expand the phenotype associated with ATP6V1B2 and propose another causal gene for DOORS syndrome. This finding suggests that DDOD and DOORS syndromes might lie on a spectrum of clinically and molecularly related conditions.

Clinicopathological Relationships in an Aged Case of DOORS Syndrome With a p.Arg506X Mutation in the ATP6V1B2 Gene.

DOORS [deafness, onychodystrophy, osteodystrophy, intellectual disability (mental retardation), and seizures] syndrome can be caused by mutations in the TBC1D24 and ATP6V1B2 genes, both of which are involved in endolysosomal function. Because of its extreme rarity, to date, no detailed neuropathological assessment has been performed to establish clinicopathological relationships and, thereby, understand better the neurobiology of this disease in aged cases. Accordingly, the aim of the current study was to highlight the clinicopathological characteristics of a novel case with a presumable de novo mutation in the ATP6V1B2 gene from a neuropathological point of view. This Caucasian male patient, who died at the age of 72 years, presented all the typical cardinal signs of DOORS syndrome. In addition, behavioral alterations, pyramidal signs, and Parkinsonism were observed. The p.R506X pathogenic mutation identified in the ATP6V1B2 gene was responsible for the clinical phenotype. The detailed neuropathological assessment revealed a limbic-predominant tauopathy in the forms of argyrophilic grain disease, primary age-related tauopathy, and age-related tau-astrogliopathy. In summary, we present the first detailed clinicopathological report of a patient with DOORS syndrome harboring a pathogenic mutation in the ATP6V1B2 gene. The demonstrated tauopathy may be considered as a consequence of lysosomal and/or mitochondrial dysfunction, similar to that found in Niemann-Pick type C disease, which is another lysosomal disorder characterized by premature neurodegenerative disorder.

ATP6V1B2-related epileptic encephalopathy.

ATP6V1B2 encodes a subunit of the lysosomal transmembrane proton pump necessary for adequate functioning of several acid hydrolases. De novo monoallelic variants of this gene have been associated with two distinct phenotypes: Zimmermann-Laband syndrome 2 (ZLS2), an intellectual deficiency/multiple malformation syndrome, and dominant deafness onychodystrophy (DDOD), a multiple malformation syndrome without cognitive involvement. Epilepsy is not observed in DDOD, is variably present in ZLS2, but is a common feature in Zimmermann-Laband syndrome 1 (ZLS1) (caused by monoallelic pathogenic variants in KCNH1) and Zimmermann-Laband syndrome-like (ZLSL) (associated with KCNK4 variants). Herein, we report a case of an infant with severe epileptic encephalopathy with microcephaly and profound developmental delay, associated with a novel de novo loss-of-function variant in ATP6V1B2, diagnosed by whole-exome sequencing. This finding expands the spectrum of ATP6V1B2-associated disorders and adds ATP6V1B2 as a new member for the growing list of early-onset epileptic encephalopathy genes. [Published with video sequence].

EXOME REPORT: Novel mutation in ATP6V1B2 segregating with autosomal dominant epilepsy, intellectual disability and mild gingival and nail abnormalities.

Mutations in ATP6V1B2, which encodes the B2 subunit of the vacuolar H + ATPase have previously been associated with Zimmermann-Laband syndrome 2 (ZLS2) and deafness-onychodystrophy (DDOD) syndrome. Recently epilepsy has also been described as a potentially associated phenotype. Here we further uncover the role of ATP61VB2 in epilepsy and report autosomal dominant inheritance of a novel missense variant in ATP6V1B2 in a large Polish family with relatively mild gingival and nail problems, no phalangeal hypoplasia and with generalized epilepsy. In light of our findings and review of the literature, we propose that the ATP6V1B2 gene should be considered in families with autosomal dominant epilepsy both with or without intellectual disability, and that presence of subtle gingival and nail problems may be another characteristic calling card of affected individuals with ATP6V1B2 mutations.

A subunit of V-ATPases, ATP6V1B2, underlies the pathology of intellectual disability.

BACKGROUND: Dominant deafness-onychodystrophy (DDOD) syndrome is a rare disorder mainly characterized by severe deafness, onychodystrophy and brachydactyly. We previously identified c.1516C > T (p.Arg506X) in ATP6V1B2 as cause of DDOD syndrome, accounting for all cases of this genetic disorder. Clinical follow-up of DDOD syndrome patients with cochlear implantation revealed the language rehabilitation was unsatisfactory although the implanted cochlea worked well, which indicates there might be learning and memory problems in DDOD syndrome patients. However, the underlying mechanisms were unknown. METHODS: atp6v1b2 knockdown zebrafish and Atp6v1b2 c.1516C > T knockin mice were constructed to explore the phenotypes and related mechanism. In mutant mice, auditory brainstem response test and cochlear morphology analysis were performed to evaluate the auditory function. Behavioral tests were used to investigate various behavioral and cognitive domains. Resting-state functional magnetic resonance imaging was used to evaluate functional connectivity in the mouse brain. Immunofluorescence, Western blot, and co-immunoprecipitation were performed to examine the expression and interactions between the subunits of V-ATPases. FINDINGS: atp6v1b2 knockdown zebrafish showed developmental defects in multiple organs and systems. However, Atp6v1b2 c.1516C > T knockin mice displayed obvious cognitive defects but normal hearing and cochlear morphology. Impaired hippocampal CA1 region and weaker interaction between the V1E and B2 subunits in Atp6v1b2Arg506X//Arg506X mice were observed. INTERPRETATION: Our study extends the phenotypic range of DDOD syndrome. The impaired hippocampal CA1 region may be the pathological basis of the behavioral defects in mutant mice. The molecular mechanism underlying V-ATPases dysfunction involves a weak interaction between subunits, although the assembly of V-ATPases can still take place.

Dominant deafness-onychodystrophy syndrome caused by an ATP6V1B2 mutation.

Our report clarifies the role of ATP6V1B2 in patients with deafness and onycho-osteodystrophy and confirms that a recurring ATP6V1B2 c.1516C>T [p.(Arg506*)], variant causes dominant deafness-onychodystrophy (DDOD) syndrome.