Nolan Kamitaki

Schizophrenia risk from complex variation of complement component 4

Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia’s strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

Lymphatic and Other Vascular Malformative/Overgrowth Disorders Are Caused by Somatic Mutations in PIK3CA

OBJECTIVES To test the hypothesis that somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) mutations would be found in patients with more common disorders including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS). STUDY DESIGN We used next generation sequencing, droplet digital polymerase chain reaction, and single molecule molecular inversion probes to search for somatic PIK3CA mutations in affected tissue from patients seen at Boston Childrens Hospital who had an isolated LM (n = 17), KTS (n = 21), fibro-adipose vascular anomaly (n = 8), or congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (n = 33), the disorder for which we first identified somatic PIK3CA mutations. We also screened 5 of the more common PIK3CA mutations in a second cohort of patients with LM (n = 31) from Seattle Childrens Hospital. RESULTS Most individuals from Boston Childrens Hospital who had isolated LM (16/17) or LM as part of a syndrome, such as KTS (19/21), fibro-adipose vascular anomaly (5/8), and congenital lipomatous overgrowth with vascular, epidermal, and skeletal anomalies syndrome (31/33) were somatic mosaic for PIK3CA mutations, with 5 specific PIK3CA mutations accounting for ∼ 80% of cases. Seventy-four percent of patients with LM from Seattle Childrens Hospital also were somatic mosaic for 1 of 5 specific PIK3CA mutations. Many affected tissue specimens from both cohorts contained fewer than 10% mutant cells. CONCLUSIONS Somatic PIK3CA mutations are the most common cause of isolated LMs and disorders in which LM is a component feature. Five PIK3CA mutations account for most cases. The search for causal mutations requires sampling of affected tissues and techniques that are capable of detecting low-level somatic mosaicism because the abundance of mutant cells in a malformed tissue can be low.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.

Endothelial Cells from Capillary Malformations Are Enriched for Somatic GNAQ Mutations.

Background: A somatic mutation in GNAQ (c.548G>A; p.R183Q), encoding G&agr;q, has been found in syndromic and sporadic capillary malformation tissue. However, the specific cell type containing the mutation is unknown. The purpose of this study was to determine which cells in capillary malformations have the GNAQ mutation. Methods: Human capillary malformation tissue was obtained from 13 patients during a clinically indicated procedure. Droplet digital polymerase chain reaction, capable of detecting mutant allelic frequencies as low as 0.1 percent, was used to quantify the abundance of GNAQ mutant cells in capillary malformation tissue. Six specimens were fractionated by fluorescence-activated cell sorting into hematopoietic, endothelial, perivascular, and stromal cells. The frequency of GNAQ mutant cells in these populations was quantified by droplet digital polymerase chain reaction. Results: Eight capillary malformations contained GNAQ p.R183Q mutant cells, two lesions had novel GNAQ mutations (p.R183L and p.R183G), and three capillary malformations did not have a detectable GNAQ p.R183 mutation. Mutant allelic frequencies ranged from 2 to 11 percent. Following fluorescence-activated cell sorting, the GNAQ mutation was found in the endothelial but not the platelet-derived growth factor receptor-&bgr;–positive cell population; mutant allelic frequencies were 3 to 43 percent. Conclusion: Endothelial cells in capillary malformations are enriched for GNAQ mutations and are likely responsible for the pathophysiology underlying capillary malformation.

A Rapid Molecular Approach for Chromosomal Phasing

Determining the chromosomal phase of pairs of sequence variants – the arrangement of specific alleles as haplotypes – is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11–116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain

The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in 690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.

A Single-Cell Atlas of Cell Types, States, and Other Transcriptional Patterns from Nine Regions of the Adult Mouse Brain

The mammalian brain is composed of diverse, specialized cell populations, few of which we fully understand. To more systematically ascertain and learn from cellular specializations in the brain, we used Drop-seq to perform single-cell RNA sequencing of 690,000 cells sampled from nine regions of the adult mouse brain: frontal and posterior cortex (156,000 and 99,000 cells, respectively), hippocampus (113,000), thalamus (89,000), cerebellum (26,000), and all of the basal ganglia – the striatum (77,000), globus pallidus externus/nucleus basalis (66,000), entopeduncular/subthalamic nuclei (19,000), and the substantia nigra/ventral tegmental area (44,000). We developed computational approaches to distinguish biological from technical signals in single-cell data, then identified 565 transcriptionally distinct groups of cells, which we annotate and present through interactive online software we developed for visualizing and re-analyzing these data (DropViz). Comparison of cell classes and types across regions revealed features of brain organization. These included a neuronal gene-expression module for synthesizing axonal and presynaptic components; widely shared patterns in the combinatorial co-deployment of voltage-gated ion channels by diverse neuronal populations; functional distinctions among cells of the brain vasculature; and specialization of glutamatergic neurons across cortical regions to a degree not observed in other neuronal or non-neuronal populations. We describe systematic neuronal classifications for two complex, understudied regions of the basal ganglia, the globus pallidus externus and substantia nigra reticulata. In the striatum, where neuron types have been intensely researched, our data reveal a previously undescribed population of striatal spiny projection neurons (SPNs) comprising 4% of SPNs. The adult mouse brain cell atlas can serve as a reference for analyses of development, disease, and evolution.