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Cellular Assay Fluorescence Microscopyįor immunohistochemistry, perfused brain samples were cryoprotected in 30% sucrose, embedded in OCT, and stored at −80☌. Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) and a ChemiDoc XRS+ imaging system (Bio-Rad). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 45 min at room temperature. Membranes were blocked in phosphate-buffered saline containing 5% non-fat milk powder and 0.1% Tween-20 and then incubated overnight at 4☌ with primary antibody. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes using a TransBlot Turbo blotting apparatus (Bio-Rad). Cell lysates were cleared by centrifugation at 10,000 × g for 3 min at 4☌. Cells were lysed for 10 min at 4☌ with 100 mM Tris (pH 7.5), 150 mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1% PMSF, and protease inhibitor cocktail. Assessment of Clustering of De Novo MutationsĬells were transfected in 6-well plates and cultured for 48 hr. The resulting p value was adjusted for the use of two models by Bonferroni correction. The loss-of-function enrichment and the combined functional statistic were compared and the better performing model was selected. A combined functional de novo statistic was calculated by using Fisher’s method to combine p values from enrichment of functional mutations with clustering of missense mutations. The expected number of mutations in each class was assumed to be the mean of a Poisson distribution, and the probability of drawing from that distribution a number of mutations equal or greater than the observed number of mutations was calculated. The loss-of-function and functional mutation rates were multiplied by the number of gene transmissions (twice the number of probands) to give the total expected number of mutations given the number of probands sequenced. The functional mutation rate was estimated by summing the loss-of-function mutation rate with the rate of missense variants and in-frame indels. The loss-of-function mutation rate was estimated by summing the mutation rates of nonsense, canonical splice sites, and frameshift variants. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype.

The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Intellectual disability (ID) is a common condition with considerable genetic heterogeneity.