Molecular Analysis, Pathogenic Mechanisms, and Readthrough Therapy on a Large Cohort ofKabuki Syndrome Patients

Abstract

Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients’ lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.

RAP1-mediated MEK/ERK pathway defects in Kabuki syndrome

“…We first purified GTP-bound RAP1A (mutant or WT) with RalGDS-RBD beads (23); although the overall protein amounts of the mutant protein were not altered appreciably, we observed a reduction of active, GTP-bound RAP1A after stimulation of cells with EGF ( Figure 2B), suggesting a loss-of-function mechanism. Since expression of target genes has been shown (13), the downstream pathomechanism of KS has not yet been elucidated.…”

Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons

“…In our study, we focused on nonsense or frameshift mutations leading to PTC resulting in an almost complete absence of particular key proteins involved in lysosomal function and cell homeostasis maintenance. Preclinical studies with gentamicin or PTC124 had succeeded by enhancing stop codon readthrough and were able to be used as therapeutic agents for genetic diseases [9][10][11][12][13][14][15][16][17][18][36][37][38][39][40][41][42]. PTC124 has been successfully extended into clinical trials [18,43], although preliminary results are not clear for Duchenne muscular dystrophy [44].…”

“…Although major progress has been made in gene therapy, it is still far from achieving real clinical application. In the last few years, other potential strategies such as pharmacological chaperones [8], or the suppression of pathogenic nonsense mutations through the induction of translational readthrough have emerged [2,[9][10][11][12][13][14][15][16][17][18]. To this effect, aminoglycoside antibiotics such as gentamicin [16,17], and small molecules such as PTC124 [18] and NB84 [19], have been described to induce PTC readthrough, eluding the NMD mechanism and allowing the formation of stable mRNAs encoding for full-length mutant, but probably still quite active, proteins [20,21].…”

Epigenetic control of the immune system: a lesson from Kabuki syndrome

“…It has been postulated, and recently demonstrated, that in most cases KS1 results from KMT2D haploinsufficiency, as transcripts carrying nonsense and frameshift mutations, are destructed by nonsense-mediated decay, resulting in decreased levels and reduced activity of the KMT2D protein, as assessed by the expression of transcription factor HOXC6 target gene in KS1 cell lines [27]. Alternatively, KMT2D missense mutations may disrupt the protein conformation and cause altered protein-protein interaction or affect posttranslational modifications.…”