Zimmermann-Laband syndrome (ZLS; MIM135500) is a rare developmental disorder characterized by facial dysmorphism, including coarsening of the face, bulbous soft nose and gingival enlargement, and nail aplasia or hypoplasia, hypertrichosis, and intellectual disability, with or without seizures. Recently, our group reported that heterozygous missense mutations in KCNH1 and ATP6V1B2 genes account for a significant proportion of ZLS cases (Kortüm & Caputo et al., 2015). KCNH1 encodes the voltage-gated K+ channel Eag1 (Kv10.1) (Garg et al., 2012), that is highly expressed in brain. ATP6V1B2 encodes the B2 subunit of the multimeric vacuolar H+ ATPase. Notably, while biochemical and functional studies shed light on the molecular mechanisms underlying KCNH1 mutations, no data has been collected on the consequences of the ATP6V1B2 mutation on the assembly/function of the ATPase complex. Based on these considerations, major goals of my PhD project are to identify the “missing” disease-gene(s) implicated in ZLS and to explore the functional impact of the ATP6V1B2 mutations by using cellular and yeast models. With the aim of identifying novel ZLS causative gene(s), a selected panel of clinically well-characterized ZLS patients was enrolled to be analyzed by WES. WES analysis was performed in five subjects negative for mutations in KCNH1 and ATP6V1B2. This approach allowed us to identify a de novo missense mutation in ATP6V1C1 (c.865G>A; p.Glu289Lys) in a patient showing coarse face, gingival hyperplasia and aplasia/hypoplasia of nails and terminal phalanges. Interestingly, these clinical features resembles the cardinal signs observed in the ATP6V1B2 mutation-positive subjects that has been described by our group (Kortüm & Caputo et al., 2015). Of note, mosaicism for the ATP6V1C1 mutant allele was recognized in the father’s proband, consistent with the relatively mild clinical ZLS features observed in this subject. ATP6V1C1 encodes the C1 subunit of the V1 subcomplex of the V-ATPase (the same complex involving ATP6V1B2). 3D modeling indicated a probably deleterious effect of the Glu289Lys amino acid substitution on proper assembly and activity of the ATPase complex. To test this hypothesis and explore the impact of the ATP6V1B2 and ATP6V1C1 mutations on subcellular localization, lysosomal function, and autophagy, we performed functional studies. These assays allowed to demonstrate that in fibroblasts derived from the patient carrying the Arg485Pro change, the ATP6V1B2 subunit co-localize predominantly with perinuclear lysosomes, suggesting that the V-ATPase containing the mutant subunit does not work properly and might prevent the lysosomes to reach the right pH required for proper localization within the cell. Confocal microscopy analysis revealed defects in the de-quenching process of DQ-BSA in fibroblasts from ZLS patients, indicating impaired lysosomal function. Furthermore, ceramide and cholesterol were strongly enhanced in patient’s fibroblasts compared to control cells, indicating a disruptive effect of the disease-causing mutations on ceramide and cholesterol catabolism. Finally, fibroblasts from ZLS patients displayed high levels of LC3-II in basal condition, and higher levels of this protein compared to control cells at any times after starvation. In the presence of the vacuolar V-ATPase inhibitor bafilomycin A1, we did not observe a significant LC3 increase in ZLS starved cells. These data indicate impaired autophagic flux in ZLS. Yeast data confirmed the deleteriousness of both the ATP6V1B2 mutations, with a stronger disrupting effect of the Arg485 change, while no effect of the mutant ATP6V1C1 subunit was observed in this system. Overall, these findings provide first evidence that ZLS caused by dysregulated V-ATPase function underlies a previously unrecognized metabolic disorder characterized by aberrant storage of undigested material inside the cell.

Zimmerman Laband syndrome: identification of novel disease causative genes and characterization of the underlying molecular mechanisms / Carpentieri, Giovanna. - (2018 Feb 12).

Zimmerman Laband syndrome: identification of novel disease causative genes and characterization of the underlying molecular mechanisms

CARPENTIERI, GIOVANNA
12/02/2018

Abstract

Zimmermann-Laband syndrome (ZLS; MIM135500) is a rare developmental disorder characterized by facial dysmorphism, including coarsening of the face, bulbous soft nose and gingival enlargement, and nail aplasia or hypoplasia, hypertrichosis, and intellectual disability, with or without seizures. Recently, our group reported that heterozygous missense mutations in KCNH1 and ATP6V1B2 genes account for a significant proportion of ZLS cases (Kortüm & Caputo et al., 2015). KCNH1 encodes the voltage-gated K+ channel Eag1 (Kv10.1) (Garg et al., 2012), that is highly expressed in brain. ATP6V1B2 encodes the B2 subunit of the multimeric vacuolar H+ ATPase. Notably, while biochemical and functional studies shed light on the molecular mechanisms underlying KCNH1 mutations, no data has been collected on the consequences of the ATP6V1B2 mutation on the assembly/function of the ATPase complex. Based on these considerations, major goals of my PhD project are to identify the “missing” disease-gene(s) implicated in ZLS and to explore the functional impact of the ATP6V1B2 mutations by using cellular and yeast models. With the aim of identifying novel ZLS causative gene(s), a selected panel of clinically well-characterized ZLS patients was enrolled to be analyzed by WES. WES analysis was performed in five subjects negative for mutations in KCNH1 and ATP6V1B2. This approach allowed us to identify a de novo missense mutation in ATP6V1C1 (c.865G>A; p.Glu289Lys) in a patient showing coarse face, gingival hyperplasia and aplasia/hypoplasia of nails and terminal phalanges. Interestingly, these clinical features resembles the cardinal signs observed in the ATP6V1B2 mutation-positive subjects that has been described by our group (Kortüm & Caputo et al., 2015). Of note, mosaicism for the ATP6V1C1 mutant allele was recognized in the father’s proband, consistent with the relatively mild clinical ZLS features observed in this subject. ATP6V1C1 encodes the C1 subunit of the V1 subcomplex of the V-ATPase (the same complex involving ATP6V1B2). 3D modeling indicated a probably deleterious effect of the Glu289Lys amino acid substitution on proper assembly and activity of the ATPase complex. To test this hypothesis and explore the impact of the ATP6V1B2 and ATP6V1C1 mutations on subcellular localization, lysosomal function, and autophagy, we performed functional studies. These assays allowed to demonstrate that in fibroblasts derived from the patient carrying the Arg485Pro change, the ATP6V1B2 subunit co-localize predominantly with perinuclear lysosomes, suggesting that the V-ATPase containing the mutant subunit does not work properly and might prevent the lysosomes to reach the right pH required for proper localization within the cell. Confocal microscopy analysis revealed defects in the de-quenching process of DQ-BSA in fibroblasts from ZLS patients, indicating impaired lysosomal function. Furthermore, ceramide and cholesterol were strongly enhanced in patient’s fibroblasts compared to control cells, indicating a disruptive effect of the disease-causing mutations on ceramide and cholesterol catabolism. Finally, fibroblasts from ZLS patients displayed high levels of LC3-II in basal condition, and higher levels of this protein compared to control cells at any times after starvation. In the presence of the vacuolar V-ATPase inhibitor bafilomycin A1, we did not observe a significant LC3 increase in ZLS starved cells. These data indicate impaired autophagic flux in ZLS. Yeast data confirmed the deleteriousness of both the ATP6V1B2 mutations, with a stronger disrupting effect of the Arg485 change, while no effect of the mutant ATP6V1C1 subunit was observed in this system. Overall, these findings provide first evidence that ZLS caused by dysregulated V-ATPase function underlies a previously unrecognized metabolic disorder characterized by aberrant storage of undigested material inside the cell.
12-feb-2018
File allegati a questo prodotto
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1126441
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact