Functional Modeling

Functional modeling of genomic variation

ASD Genes

Gene discovery studies in autism spectrum disorder (ASD) have identified mutations in regulatory genes and genes involved in synaptic processes. The challenge lies in translating this data to biologically relevant molecular pathways to understand the underlying disease mechanisms. We CRISPR-engineered human iPSC against two different backgrounds to disrupt synaptic and chromatin-associated genes commonly hit by rare de novo mutations in ASD, e.g., CHD8, SCN2A, AUTS2, FOXP1, POGZ. The derived neuronal models enable us to measure and follow the effects of early neurodevelopmental events on different types of functional neural cells in time. Transcriptional and epigenetic profiling, as well as synaptic phenotyping of ASD neuronal models, will be used to identify points of molecular and functional convergence associated with these perturbations. We hypothesize that there is a convergent transcriptional signature in neuronal models of perturbations of functionally different ASD genes.

Prader-Willi Syndrome

Paternally inherited deletions of chromosome 15q11.2-q13, Type I (6 Mb) and Type II (5.3 Mb, are the most common cause of Prader-Willi syndrome (PWS). PWS is highly penetrant and characterized by hyperphagia (life-threatening childhood obesity), muscular hypotonia, and intellectual impairment and is associated with psychiatric disorders, including bipolar disorder and autism spectrum disorder. A genetically distinct disorder, yet the pathological mechanisms remain unclear. Most of the genes within this region are not expressed from the maternal allele due to being imprinted. To explore the causative genetic driver(s) of PWS, we CRISPR engineered an allelic series of isogenic human iPSC with PWS region mutations and reprogrammed them into relevant cell lineages (e.g., hypothalamic neurons). Our analyses have begun to characterize the transcriptional and functional deficits associated with these models and individual genetic targets for precision therapeutic rescue.

Genomic Disorders

Reciprocal copy number variant (rCNV) of a segment of chromosome 16p11.2 (OMIM #611913) is one of the most significant recurrent genomic disorders. It has been associated with neurodevelopmental disorders (NDDs), including intellectual disability, autism spectrum disorder (ASD), schizophrenia, and obesity. The mechanism of NAHR-mediated CNV formation involves the mispairing of the flanking segmental duplications (SDs), resulting in either the loss or gain of the unique 593 kb genic segment, containing 25 protein-coding genes. However, the pathogenic mechanism, the functional relevance of individual genes within 16p11.2 RGD, and the combined contributions of multiple genes are unknown. 16p11.2 RGD is highly penetrant for NDDs, delineating the critical drivers of 16p11.2 RGD and the highly interconnected hub genes will shed light on disease pathogenesis and therapeutics.

X-linked Dystonia Parkinsonism (XDP)

X-linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disorder indigenous to the Philippines that occurs in males around the age 40 years and symptoms are temporally separated by dystonic and Parkinsonian phenotypes. We developed novel assembly-based functional genomics methods in iPSC derived neuronal models to explore the genomes and transcriptomes of XDP patients. These studies discovered the causal variant in XDP to be a novel noncoding sine-VNTR-alu (SVA) retrotransposition insertion into intron 32 of TAF1, a critical gene involved in the TIID transcriptional complex. Transcriptome assembly revealed that this SVA resulted in aberrant splicing and anomalous intron retention, with concomitant reduction of TAF1 expression. Remarkably, we were able to ameliorate this deficit by excising the SVA using CRISPR/Cas9. Currently, we are exploring the critical questions of functional mechanism, in vivo alterations in the XDP brain, and the potential for precision therapeutics using an anti-sense oligonucleotide (ASO) library against XDP transcriptomic signatures.