Recent progress in the cultivation of induced pluripotent cell (iPSC) lines from individual patients—using technical methods pioneered in our research center—allow deep structural and molecular exploration of the impact of any given patient’s individual mutation on the function of individual neurons, a capability that was heretofore available only from tissues obtained in rare surgical procedures, brain biopsies, or autopsy. Use of iPSC disease models has major advantages over other disease models, including: (1) an iPSC model incorporates the exact genotype of a specific patient (because it is derived from the patient), thus the genotype is specified not only for the mutation in question but with respect to the entire genome (see below); (2) it examines effects of the condition that are appreciable in individual cells, and therefore identifies pathological mechanisms can be therapeutically targeted by treatments that are designed to reach cells or tissues affected by the disease process.

Experimental modeling of human genetic disorders provides insight into the cellular and molecular mechanisms involved, and the underlying genetic components influencing the disease characteristics of a particular patient. IPSC's provide a self-renewing and, thus, unlimited source of pluripotent cells derived from a single patient that can be developed into any cell type in the human body. Therefore, iPSC technology, and the increasingly refined abilities to differentiate iPSCs into disease-relevant target cells, has far-reaching implications for understanding how different cell types in the body are affected by the disease process (eg. why brain or muscle cells are more affected than skin or blood cells), and developing precise therapeutic approaches to modifying the disease processes that impair the function of cells.

For the frontier of M-CM research, It is particularly important to consider the diversity of genetic causes of M-CM and the gaps in understanding how a particular mutation initiates the cascade of changes that result in increased signalling activity in the molecular pathway that has been implicated as the mechanism of disease, and therefore how the cellular effects of specific mutations could be intercepted by biological therapies. The use of cellular models will allow comparisons to be made across different mutations that result in the same condition (M-CM), and therefore identify what is common to disease processes that result in M-CM, and therefore what might help many patients irrespective of their specific genetic abnormality or whether a specific genetic cause has been identified. Moreover, there are a number of new drug and gene-editing therapies that are theoretically extremely promising, but there do not exist cellular assays that would allow prediction of whether a specific therapy might be expected to help (or harm) a given patient. Use of iPSC's to test the effects of these new therapies (i.e. how the therapies affect established disease mechanisms and cellular signatures) will lend major insights into which patients are most likely to respond and least likely to be harmed by a novel therapy.

Here, we propose the development of an initial library of iPS lines derived from non-invasively acquired epithelial tissue from 4 individual patients. PIK3CA-Related Overgrowth Spectrum (PROS) syndromes feature mosaicism, in which neuronal / epithelial cells may be affected by a mutation but other cells and tissues such as blood unaffected. The induced pluripotent cell lines will be made available to all qualified researchers studying these conditions and their effects on any tissue. At Washington University, the iPSCs will be differentiated into cortical neurons, and their development and function will be compared a) with one another; b) to those of normal iPSCs/neurons of unaffected individuals; c) to iPSCs/neurons of patients with autism spectrum disorders established in our laboratory; and d) to a companion library of 50 iPSC lines generated in our laboratory from a single clonal progenitor cell line, in which loss or gain of function of 50 autism-associated genes (one per cell line) have been experimentally induced, and the cellular effects examined. Once M-CM-specific abnormalities in the neuronal cell lines have been identified, the effect of correction of the deleterious mutation in restoring normative cell structure and function will be determined.

To conduct M-CM research in this context has major advantages. First, the use of common methodologies implemented across neurodevelopmental disorder models will help ensure the reliability and interpretability of cellular signatures of disease. Second, the ability to characterize what DISTINGUISHES M-CM related mechanisms of disease from those of other neurodevelopmental disorders will accelerate understanding of what can uniquely be done to target the pathogenic process in these conditions. Third, there may be shared mechanisms across specific subsets of neurodevelopmental disorders (for example those associated with macrocephaly, or those associated with specific neurodevelopmental delays) even when they are caused by abnormalities in different genes or gene networks. Some of the analyses of such commonalities may reveal reasons why different mutations giving rise to M-CM result in contrasting outcomes.

(1) All of the disorders in the PIK3CA-Related Overgrowth Spectrum (PROS)
(2) Other neurodevelopmental disorders represented in our iPSC library whose downstream consequences overlap with those of M-CM and related overgrowth syndromes, including the RASopathies which feature abnormalities in an overlapping signaling pathway

Research on disorders of human brain and behavioral development is at a disadvantage in comparison to other branches of biomedical research in which it is feasible and ethical to harvest diseased tissue of an actual patient for direct examination. This is not possible for brain disorders of childhood. iPSC's of a given patient carry the exact genotype of that patient, not only respect to the disease gene in question but with respect to all background genetic factors across the genome which may have participated in the causation of the disease phenotype of that particular patient. As such, iPSCs represent the next best option to a biopsy and therefore represent a revolutionary new opportunity to understand disease mechanisms affecting specific patients. As clinician-scientists who have devoted our career efforts to the field of developmental disorders for decades, we view this opportunity as the most exciting path forward in the development of higher-impact therapies for our patients.