Zimmermann-Laband syndrome (ZLS) is a rare developmental disorder characterized by facial dysmorphism, gingival enlargement, nail aplasia or hypoplasia, hypertrichosis, and intellectual disability, with or without epilepsy. The genetic basis of ZLS has been recently reported by the identification of two disease genes by using a whole exome sequencing approach, KCNH1 and ATP6V1B2. ZLS has been proven as a genetic and phenotypic heterogeneous disease. Approximately 40% of ZLS cases do not carry mutations in the identified disease genes, KCNH1 and ATP6V1B2, indicating the existence of additional genes implicated in the pathogenesis. Preliminary functional studies on KCNH1 mutants demonstrated that mutations in KCNH1 perturb channel activity. However, to date, the functional role of KCNH1 in cells and in the pathogenesis of ZLS, is still incomplete. To gain insights into the molecular bases of ZLS, the applicant proposes studies with the aim of identify novel disease genes by using an exome sequencing strategy on a cohort of selected subjects with clinical features fitting ZLS, and of the characterization of their functional role of KCNH1 and molecular mechanisms of pathogenesis. Functional validation using in vitro approaches will be performed to study KCNH1 wild type and mutant protein subcellular localization, splicing variants, and the role in ciliogenesis.
Efforts aimed at disclosing molecular functions of KCNH1 and its dysfunctions in ZLS pathogenesis are a fundamental prerequisite that will provide molecular information with direct impact on diagnosis, prognosis, counselling and patient management.
ZLS is a rare disorder characterized by gingival fibromatosis, dysplastic/absent nails and distal phalanges, hirsutism, epilepsy, and recognized as a genetic disease only recently (Korthum, Caputo et al. 2015).
The mutation screening of ZLS known genes KCNH1 and ATP6V1B2 on newly recruited patients will allow the identification of new and/or novel mutations, allowing a better genotype-phenotype correlation.
A significant proportion of patients with features within the ZLS clinical spectrum does not harbor mutation in the recently identified disease genes. Since the causative disease genes belong to apparently distant biological processes, the identification of novel disease gene/s could shed light on molecular network involved in the pathogenesis of this disorder characterized by multisystem involvement.
Following the identification of 6 patients with de novo missense KCNH1 mutations causing ZLS, several papers reported further patients with KCNH1 mutations associated to Temple-Baraitser syndrome and a variable phenotype characterized by intellectual disability, facial dysmorphism, developmental delay, hypotonia and seizures. These results suggest that KCNH1 exerts a role in human cognitive development and that, when mutated, may cause a phenotypic continuum of neurodevelopmental disorders with some distinctive phenotypic features (e.g. epilepsy).
Recently, KCNH1 has been implicated in ciliogenesis (Sànchez et al. 2016), highlighting the relevance of non-canonical functions of the ion channel, whose perturbation could underlie developmental processes. Cilia transduce signals from extracellular stimuli to a cellular response that regulates proliferation, and differentiation. Dysfunctions of this organelle impair the cellular responses to differentiation signals, leading to a group of disease collectively termed as ¿ciliopathies¿, characterized by visceral cysts, defective digits, oral anomalies, cognitive impairment, and tumors. KCNH1 mutated patients show orofacial, digial anomalies, and mental retardation, and it has been suggested that their morphological features are highly reminiscent of ciliopathies (Pardo et al. 2016). Preliminary results reported that at least one KCNH1 mutation causing ZLS exerts a functional effect on ciliary resoption (Pardo et al. 2016). Therefore, we suggest that efforts aimed at disclosing molecular functions of KCNH1 and its dysfunctions in ZLS pathogenesis are a fundamental prerequisite that will provide molecular information with direct impact on diagnosis, prognosis, counselling and patient management.
To date, 12 mutations have been identified in KCNH1 gene, all affecting highly conserved residues located in the voltage-sensing transmembrane helix and other transmembrane stretches. First comparative phenotype analysis indicated that there is no correlation between the location of the affected residues and the clinical diagnosis. A plausible explanation could be the presence of still unrecognized transcript variants that, at least in some tissue, could exert specific cell functions that could be specifically altered by the disease causing mutations. Therefore, the study of splicing variability of the KCNH1 could aim the study of cell mechanisms involving KCNH1 and aid in the genotype-phenotype correlation studies.
KCNH1 is a channel protein mainly expressed in the mammalian brain and its overexpression contributes to tumour development and malignancy. It is considered a clinically relevant marker and therapeutic target for several tumours. In cancer cells, KCNH1 expression is coupled to cell cycle and its expression promotes both the progression through G1 and G2/M phases, facilitating proliferation (Urrego et al. 2016). Several reports showed KCNH1 channels localization at the plasma membrane, but it was observed also in intracellular sites, e.g. the inner nuclear membrane. Additional studies aimed at localize KCNH1 isoforms in the cell and during cell cycle progression could be useful to better understand oncogenic proprieties of the channel.
To date, only preliminary studies have been accomplished to study the functional role of KCNH1 mutants on the pathogenesis of human disease, therefore functional assays to explore the mechanism involving this potassium channel could fill this gap, providing clues on the differential effect that exerts, when mutated, on ZLS related developmental processes.