Major goals of the parent application (R01 MH109648-04, entitled “Neurobiology of Autism with Macrocephaly) are to investigate developmental alterations in ASD with large brain size (macrocephaly) and normal brain size (normocephaly) in order to understand whether these represent biologically distinct subtypes of ASD. To this end, we are collecting and analyzing neurobiological measures, transcriptome and gene regulatory element (enhancers) activities in induced pluripotent stem cell (iPSC)- derived organoids obtained from ASD patients with macrocephaly or normocephaly. Differential gene expression and network analyses in an initial set of macrocephalic and normocephalic proband-control pairs suggest that more than 75% of differentially expressed genes between probands and their unaffected fathers were specific to the macrocephalic or normocephalic groups, confirming our hypothesis that these represent different subtypes of ASD, and validating our approach to separately analyze these sets of families and compare their neurobiology. However, power analysis indicate that, in order to discover 90% of the predicted total number of enhancer elements active in our model, we need to analyze primary datasets from 10 additional macrocephalic and 7 additional normocephalic proband-control pairs. For macrocephalic individuals, 8 families are already available, requiring the recruitment of only 2 additional families. For normocephalic families, all 7 need to be newly enrolled. All together, under the auspices of this supplement we will recruit 9 families (18 subjects), generate iPSC lines, and produce transcriptome and enhancer datasets. Thus, this supplement application requests funds to complete recruitment, produce iPSC lines, and generate primary data which has been halted by the COVID pandemic. Over the years, the Vaccarino lab has a demonstrated ability to perform this research, as shown by deposition in the NIMH Stem Cell Resource at Infinity BiologiX LLC of a total of 82 primary cell lines and 121 iPSC lines from families with ASD (NDA collections C1201 and C2424).