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Leap forward for LEOPARD syndrome patients

By Maria H. Ledran

Researchers, overseen by Bruce Gelb and Ihor Lemischka at the Mount Sinai School of Medicine (New York), have described the derivation of two separate induced pluripotent stem cell (iPSC) lines from LEOPARD syndrome patients.  LEOPARD syndrome is a multifaceted autosomal-dominant developmental disease (closely related to Noonan syndrome), which can result in facial dysmorphia, growth retardation, cardiac anomalies and deafness, among a plethora of other potential clinical features.  The break-though, reported in the June 9th issue of Nature, describes how, for the first time, stem cells have been differentiated in vitro into cells with a cardiomyopathay – that is, with a cardiac disease phenotype.  The vast majority of LEOPARD syndrome cases, and about half of Noonan syndrome cases result from missense mutations in the ubiquitously expressed PTPN11 gene, which encodes the protein tyrosine phosphatise SHP2 – a crucial regulator of normal development.  These mutations have been shown to result in a gain of function phenotype, by destabilising the catalytically inactive protein conformation, and thus inhibiting growth factor induced ERK1/2 signalling.  Additionally a distinct class of somatic PTPN11 mutations contribute to juvenile leukaemogenesis.  Despite the description of animal models of LEOPARD syndrome in both Drosophila and zebrafish, a detailed molecular basis of the disease phenotypes has remained obscure, but the group hopes that these disease specific cells might be able to contribute fresh insights.

Approximately 80% of LEOPARD syndrome patients exhibit hypertrophic cardiomyopathy: thickening of heart cardiomyocytes resulting from increased cell size with an enlarged sarcomeric (contractile) protein component.  Infact this aspect of the disease is the most life threatening, because of the association with cardiac arrhythmia and sudden death.  The researchers revealed that the cardiomyocytes differentiated from LEOPARD iPSCs showed a perturbed phenotype – with a significantly increased average cell size and increased sarcomere assembly – essentially recapitulating the disease state in vitro.  Upon further investigation it also became apparent that the transcription factor NFAT4 was nearly three times as likely to be localised in the nucleus of LEOPARD syndrome iPSCs compared to wild type iPSCs – highlighting the importance of the calcineurin-NFAT pathway in the regulation of cardiac hypertrophy.  Moreover, phosphorylation of EGFR and MEK1 proteins was significantly increased in LEOPARD syndrome iPSCs, making a promising future line of investigation, given that MEK1 is the upstream kinase of ERK1/2 (the protein known to be perturbed in this disease), and since RAS-mitogen-activated kinase (MAPK) is the major deregulated signalling pathway in SHP2 mutants.  Basal levels of phosphorylated ERK were increased in LEOPARD iPSCs, and giving further confidence to these disease specific stem cells as an in vitro disease model, receptor tyrosine kinase stimulation failed to increase activation of ERK, mimicking results previously obtained in an alternative SHP2 mutated model.  Taken together these findings offer insight into a previously inpenetrateable problem underlying the molecular basis of the disease phenotype and potentially implicate the perturbation of RAS-MAPK signalling from the earliest stem cell origins.


Of principal importance to this study and the majority of stem cell based research, is ease of access to theoretically unlimited numbers of disease affected stem cells and their derivatives: in this case of particular value when studying cardiac related phenotypes since access to diseased human heart is very limited, and cardiac cell types do not readily proliferate in vitro.  Indeed amongst the most eagerly anticipated applications of iPSC technologies include the prospects of using these types of diseased and normal cells in the large scale screening for novel drugs, for therapeutic treatment strategies and for illuminating underlying disease pathogenesis.  For instance in the case of LEOPARD syndrome this could include the identification of molecules that could block the overgrowth of cardiomyocytes, an application with a wider potential benefits in the treatment of all cardiomyopic disease.

The description of LEOPARD patient specific iPSCs also contributes to a growing body of work initiated by the first description of cellular reprogramming by Shinya Yamanaka in 2007.  Indeed, in the week that Yamanaka has  been honoured with yet another prize in recognition of his achievements (the 26th annual Kyoto Prize in Advanced Technology), it is undoubted that the insights that iPSCs already gained and likely ahead are phenomenal.  However, as the researchers behind the generation of LEOPARD iPSCs themselves note, there are still many important hurdles to overcome with this and other pluripotent cell technologies.  For instance in the case of LEOPARD iPSCs, differentiation efficiency was variable and the group was unable to generate a sufficiently pure population of cardiac cells to fully asses essential disease characteristics such as the reactivation of fetal gene programs and the assessment of cardiac protein synthesis rates.  Nonetheless, without a doubt the continued generation of patient specific iPSCs will be of chief concern for many labs around the world and is likely to give fresh insight into many disease types that would otherwise be much more difficult to study.