Andrew J. Schork

Dynamic Changes in the Copy Number of Pluripotency and Cell Proliferation Genes in Human ESCs and iPSCs during Reprogramming and Time in Culture

Genomic stability is critical for the clinical use of human embryonic and induced pluripotent stem cells. We performed high-resolution SNP (single-nucleotide polymorphism) analysis on 186 pluripotent and 119 nonpluripotent samples. We report a higher frequency of subchromosomal copy number variations in pluripotent samples compared to nonpluripotent samples, with variations enriched in specific genomic regions. The distribution of these variations differed between hESCs and hiPSCs, characterized by large numbers of duplications found in a few hESC samples and moderate numbers of deletions distributed across many hiPSC samples. For hiPSCs, the reprogramming process was associated with deletions of tumor-suppressor genes, whereas time in culture was associated with duplications of oncogenic genes. We also observed duplications that arose during a differentiation protocol. Our results illustrate the dynamic nature of genomic abnormalities in pluripotent stem cells and the need for frequent genomic monitoring to assure phenotypic stability and clinical safety.

All SNPs are not created equal: genome-wide association studies reveal a consistent pattern of enrichment among functionally annotated SNPs.

Recent results indicate that genome-wide association studies (GWAS) have the potential to explain much of the heritability of common complex phenotypes, but methods are lacking to reliably identify the remaining associated single nucleotide polymorphisms (SNPs). We applied stratified False Discovery Rate (sFDR) methods to leverage genic enrichment in GWAS summary statistics data to uncover new loci likely to replicate in independent samples. Specifically, we use linkage disequilibrium-weighted annotations for each SNP in combination with nominal p-values to estimate the True Discovery Rate (TDR = 1−FDR) for strata determined by different genic categories. We show a consistent pattern of enrichment of polygenic effects in specific annotation categories across diverse phenotypes, with the greatest enrichment for SNPs tagging regulatory and coding genic elements, little enrichment in introns, and negative enrichment for intergenic SNPs. Stratified enrichment directly leads to increased TDR for a given p-value, mirrored by increased replication rates in independent samples. We show this in independent Crohns disease GWAS, where we find a hundredfold variation in replication rate across genic categories. Applying a well-established sFDR methodology we demonstrate the utility of stratification for improving power of GWAS in complex phenotypes, with increased rejection rates from 20% in height to 300% in schizophrenia with traditional FDR and sFDR both fixed at 0.05. Our analyses demonstrate an inherent stratification among GWAS SNPs with important conceptual implications that can be leveraged by statistical methods to improve the discovery of loci.

Subcortical brain volume differences in participants with attention deficit hyperactivity disorder in children and adults: a cross-sectional mega-analysis

BACKGROUND Neuroimaging studies have shown structural alterations in several brain regions in children and adults with attention deficit hyperactivity disorder (ADHD). Through the formation of the international ENIGMA ADHD Working Group, we aimed to address weaknesses of previous imaging studies and meta-analyses, namely inadequate sample size and methodological heterogeneity. We aimed to investigate whether there are structural differences in children and adults with ADHD compared with those without this diagnosis. METHODS In this cross-sectional mega-analysis, we used the data from the international ENIGMA Working Group collaboration, which in the present analysis was frozen at Feb 8, 2015. Individual sites analysed structural T1-weighted MRI brain scans with harmonised protocols of individuals with ADHD compared with those who do not have this diagnosis. Our primary outcome was to assess case-control differences in subcortical structures and intracranial volume through pooling of all individual data from all cohorts in this collaboration. For this analysis, p values were significant at the false discovery rate corrected threshold of p=0·0156. FINDINGS Our sample comprised 1713 participants with ADHD and 1529 controls from 23 sites with a median age of 14 years (range 4-63 years). The volumes of the accumbens (Cohens d=-0·15), amygdala (d=-0·19), caudate (d=-0·11), hippocampus (d=-0·11), putamen (d=-0·14), and intracranial volume (d=-0·10) were smaller in individuals with ADHD compared with controls in the mega-analysis. There was no difference in volume size in the pallidum (p=0·95) and thalamus (p=0·39) between people with ADHD and controls. Exploratory lifespan modelling suggested a delay of maturation and a delay of degeneration, as effect sizes were highest in most subgroups of children (<15 years) versus adults (>21 years): in the accumbens (Cohens d=-0·19 vs -0·10), amygdala (d=-0·18 vs -0·14), caudate (d=-0·13 vs -0·07), hippocampus (d=-0·12 vs -0·06), putamen (d=-0·18 vs -0·08), and intracranial volume (d=-0·14 vs 0·01). There was no difference between children and adults for the pallidum (p=0·79) or thalamus (p=0·89). Case-control differences in adults were non-significant (all p>0·03). Psychostimulant medication use (all p>0·15) or symptom scores (all p>0·02) did not influence results, nor did the presence of comorbid psychiatric disorders (all p>0·5). INTERPRETATION With the largest dataset to date, we add new knowledge about bilateral amygdala, accumbens, and hippocampus reductions in ADHD. We extend the brain maturation delay theory for ADHD to include subcortical structures and refute medication effects on brain volume suggested by earlier meta-analyses. Lifespan analyses suggest that, in the absence of well powered longitudinal studies, the ENIGMA cross-sectional sample across six decades of ages provides a means to generate hypotheses about lifespan trajectories in brain phenotypes. FUNDING National Institutes of Health.

Improved Detection of Common Variants Associated with Schizophrenia and Bipolar Disorder Using Pleiotropy-Informed Conditional False Discovery Rate

Several lines of evidence suggest that genome-wide association studies (GWAS) have the potential to explain more of the “missing heritability” of common complex phenotypes. However, reliable methods to identify a larger proportion of single nucleotide polymorphisms (SNPs) that impact disease risk are currently lacking. Here, we use a genetic pleiotropy-informed conditional false discovery rate (FDR) method on GWAS summary statistics data to identify new loci associated with schizophrenia (SCZ) and bipolar disorders (BD), two highly heritable disorders with significant missing heritability. Epidemiological and clinical evidence suggest similar disease characteristics and overlapping genes between SCZ and BD. Here, we computed conditional Q–Q curves of data from the Psychiatric Genome Consortium (SCZ; n = 9,379 cases and n = 7,736 controls; BD: n = 6,990 cases and n = 4,820 controls) to show enrichment of SNPs associated with SCZ as a function of association with BD and vice versa with a corresponding reduction in FDR. Applying the conditional FDR method, we identified 58 loci associated with SCZ and 35 loci associated with BD below the conditional FDR level of 0.05. Of these, 14 loci were associated with both SCZ and BD (conjunction FDR). Together, these findings show the feasibility of genetic pleiotropy-informed methods to improve gene discovery in SCZ and BD and indicate overlapping genetic mechanisms between these two disorders.

Genetic sharing with cardiovascular disease risk factors and diabetes reveals novel bone mineral density loci

Bone Mineral Density (BMD) is a highly heritable trait, but genome-wide association studies have identified few genetic risk factors. Epidemiological studies suggest associations between BMD and several traits and diseases, but the nature of the suggestive comorbidity is still unknown. We used a novel genetic pleiotropy-informed conditional False Discovery Rate (FDR) method to identify single nucleotide polymorphisms (SNPs) associated with BMD by leveraging cardiovascular disease (CVD) associated disorders and metabolic traits. By conditioning on SNPs associated with the CVD-related phenotypes, type 1 diabetes, type 2 diabetes, systolic blood pressure, diastolic blood pressure, high density lipoprotein, low density lipoprotein, triglycerides and waist hip ratio, we identified 65 novel independent BMD loci (26 with femoral neck BMD and 47 with lumbar spine BMD) at conditional FDR < 0.01. Many of the loci were confirmed in genetic expression studies. Genes validated at the mRNA levels were characteristic for the osteoblast/osteocyte lineage, Wnt signaling pathway and bone metabolism. The results provide new insight into genetic mechanisms of variability in BMD, and a better understanding of the genetic underpinnings of clinical comorbidity.

Genetic pleiotropy between multiple sclerosis and schizophrenia but not bipolar disorder: differential involvement of immune-related gene loci

Converging evidence implicates immune abnormalities in schizophrenia (SCZ), and recent genome-wide association studies (GWAS) have identified immune-related single-nucleotide polymorphisms (SNPs) associated with SCZ. Using the conditional false discovery rate (FDR) approach, we evaluated pleiotropy in SNPs associated with SCZ (n=21 856) and multiple sclerosis (MS) (n=43 879), an inflammatory, demyelinating disease of the central nervous system. Because SCZ and bipolar disorder (BD) show substantial clinical and genetic overlap, we also investigated pleiotropy between BD (n=16 731) and MS. We found significant genetic overlap between SCZ and MS and identified 21 independent loci associated with SCZ, conditioned on association with MS. This enrichment was driven by the major histocompatibility complex (MHC). Importantly, we detected the involvement of the same human leukocyte antigen (HLA) alleles in both SCZ and MS, but with an opposite directionality of effect of associated HLA alleles (that is, MS risk alleles were associated with decreased SCZ risk). In contrast, we found no genetic overlap between BD and MS. Considered together, our findings demonstrate genetic pleiotropy between SCZ and MS and suggest that the MHC signals may differentiate SCZ from BD susceptibility.

Genome-wide analyses for personality traits identify six genomic loci and show correlations with psychiatric disorders.

Personality is influenced by genetic and environmental factors and associated with mental health. However, the underlying genetic determinants are largely unknown. We identified six genetic loci, including five novel loci, significantly associated with personality traits in a meta-analysis of genome-wide association studies (N = 123,132–260,861). Of these genome-wide significant loci, extraversion was associated with variants in WSCD2 and near PCDH15, and neuroticism with variants on chromosome 8p23.1 and in L3MBTL2. We performed a principal component analysis to extract major dimensions underlying genetic variations among five personality traits and six psychiatric disorders (N = 5,422–18,759). The first genetic dimension separated personality traits and psychiatric disorders, except that neuroticism and openness to experience were clustered with the disorders. High genetic correlations were found between extraversion and attention-deficit–hyperactivity disorder (ADHD) and between openness and schizophrenia and bipolar disorder. The second genetic dimension was closely aligned with extraversion–introversion and grouped neuroticism with internalizing psychopathology (e.g., depression or anxiety).

Identifying common genetic variants in blood pressure due to polygenic pleiotropy with associated phenotypes

Blood pressure is a critical determinant of cardiovascular morbidity and mortality. It is affected by environmental factors, but has a strong heritable component. Despite recent large genome-wide association studies, few genetic risk factors for blood pressure have been identified. Epidemiological studies suggest associations between blood pressure and several diseases and traits, which may partly arise from a shared genetic basis (genetic pleiotropy). Using genome-wide association studies summary statistics and a genetic pleiotropy-informed conditional false discovery rate method, we systematically investigated genetic overlap between systolic blood pressure (SBP) and 12 comorbid traits and diseases. We found significant enrichment of single nucleotide polymorphisms associated with SBP as a function of their association with body mass index, low-density lipoprotein, waist/hip ratio, schizophrenia, bone mineral density, type 1 diabetes mellitus, and celiac disease. In contrast, the magnitude of enrichment due to shared polygenic effects was smaller with the other phenotypes (triglycerides, high-density lipoproteins, type 2 diabetes mellitus, rheumatoid arthritis, and height). Applying the conditional false discovery rate method to the enriched phenotypes, we identified 62 loci associated with SBP (false discovery rate <0.01), including 42 novel loci. The observed polygenic overlap between SBP and several related disorders indicates that the epidemiological associations are not mediated solely via lifestyle factors but also reflect an etiologic relation that warrants further investigation. The new gene loci identified implicate novel genetic mechanisms related to lipid biology and the immune system in SBP.

Genetic overlap between Alzheimer's Disease and Parkinson's Disease at the MAPT locus

We investigated the genetic overlap between Alzheimer’s disease (AD) and Parkinson’s disease (PD). Using summary statistics (P-values) from large recent genome-wide association studies (GWAS) (total n=89 904 individuals), we sought to identify single nucleotide polymorphisms (SNPs) associating with both AD and PD. We found and replicated association of both AD and PD with the A allele of rs393152 within the extended MAPT region on chromosome 17 (meta analysis P-value across five independent AD cohorts=1.65 × 10−7). In independent datasets, we found a dose-dependent effect of the A allele of rs393152 on intra-cerebral MAPT transcript levels and volume loss within the entorhinal cortex and hippocampus. Our findings identify the tau-associated MAPT locus as a site of genetic overlap between AD and PD, and extending prior work, we show that the MAPT region increases risk of Alzheimer’s neurodegeneration.

Restricted ethnic diversity in human embryonic stem cell lines

To the Editor: Human pluripotent stem cells (hPSCs) have the capacity to self-renew indefinitely and to differentiate into a wide array of cell types, which make them a potential source of essentially unlimited quantities of differentiated cells for basic and clinical research. The tremendous self-renewal of hPSCs might lead one to conclude that a small number of cell lines would be sufficient to meet all needs. However, it is becoming increasingly clear that the genetic background of human cell lines can have significant effects on experimental results. Although hundreds of associations between individual alleles and specific traits or diseases are discovered each year, a full understanding of the relationships between genetic variation and cellular or organismal phenotype is still remote1. The HapMap project2, along with many other studies, has made it clear that there are large differences in the allelic frequencies for many single-nucleotide polymorphisms (SNPs) among different ethnicities. Ethnicity can serve as a proxy for genetic variation to ensure diversity in genetic backgrounds in a study population. The availability of hPSCs from a variety of ethnic backgrounds would ensure the generalizability of results as well as increasing the likelihood that specific alleles or combinations of alleles of interest will be available for study. This is particularly important for the use of hPSCs and hPSC-derived cells for drug screening and toxicity studies. Several idiosyncratic drug effects have been attributed to specific genetic variations3, and the efficacy and toxicity of numerous drugs are presumed to be influenced by genetic factors. On the clinical side, hPSCs and their derivatives are potential sources for cell therapy, and recipients of cell transplants are more likely to find immunologic matches with donors who share their ethnic background. However, because most hPSC lines have been generated from de-identified material, the ethnic backgrounds of the donors are not known. We determined the ethnic origin of 47 human embryonic stem cell (hESC) lines (including 42 standard hESC lines and 5 parthenote hESC lines), 5 hiPSC lines and 58 non-pluripotent samples (19 blood and tissue samples and 39 primary cell lines). Although this study was not intended to be comprehensive, we included hESC lines derived in a variety of geographical locations in order to sample the spectrum of ethnic diversity present in hESC lines in general (Supplementary Table 1). Using genome-wide SNP genotyping and Bayesian analysis of population structure (BAPS; see Supplementary Methods), we determined that the ethnic origins of the hESC lines included in this study were quite restricted (Fig. 1, Supplementary Table 1). The large majority of hESC lines (43 of 47) were of European and East Asian ethnicity. The diversity of the hiPSC lines and nonpluripotent cells is also shown (Fig. 1, Supplementary Fig. 1). We note that the location of derivation of the hESC lines is not necessarily indicative of their ethnic origin. For instance, four of the five hESC lines described in one landmark publication4 were derived in the United States (Wisconsin) but reportedly5 came from blastocysts transported from Israel by collaborators. Our results on three of these lines indicate that they show a genetic profile similar to those of people from the region around Adygea (WA01), Tuscany (WA09) and Tuscany/Palestine (WA07), with indication of enzymatic activity3. We targeted residues Asp154, Glu155, Lys136 and Ser287 based on previous experiments showing increases in stability and/or enzymatic activity from mutations at these sites2,3. Mutations at Pro220 were tested based on a report of increased activity with alterations at this residue5; however, we were unable to find substitutions at Pro220 that increased activity or red-shift. The end result of this selection process was the variant RLuc/E155G/D162E/A164R/ L165I/M185V/Q235A/S287A, which we denote RLuc7-521. We used purified protein to assess the bioluminescence emission spectrum, enzymatic activity and quantum yield of the new variant (Supplementary Table 1). RLuc7-521’s signal was increased 1.6-fold on a photons per second per mole basis, and its emission spectra (Fig. 1a) showed a 40-nm red-shift compared to RLuc with both coelenterazine and the substrate analog coelenterazine-v. RLuc7-521’s quantum yield of 3.9 ± 0.1%, though higher than that of RLuc8.6535 (3.1 ± 0.2%), was lower than that of RLuc (5.3 ± 0.1%), indicating that further improvements may be achievable through additional mutagenesis. We tested RLuc7-521 as a mammalian reporter gene in cell culture and observed a nearly twofold improvement in light output compared to RLuc, and an almost identical intracellular stability (Fig. 1b). We also tested RLuc7-521 for small-animal imaging by injecting cells transiently transfected with RLuc or RLuc7-521 into mice. Western analysis of cells before injection showed equivalent levels of luciferase protein expression between conditions (Supplementary Fig. 2). Subsequent imaging of the mice showed a 3.3-fold increase in signal output for RLuc7-521 (Fig. 1c), a reflection of the improved light output of RLuc7-521 combined with reductions in signal attenuation by tissue for this red-shifted luciferase. In summary, RLuc7-521 is a seven-mutation variant of RLuc that shows a green-peaked (521-nm) emission spectrum, an identical intracellular stability, and at least a twofold increase in signal, with even greater signal gains in small-animal imaging due to decreased attenuation of its red-shifted photons. This new variant represents a direct replacement of Renilla luciferase for reporter-gene applications as it retains the temporal relationship between gene activation and luciferase activity while providing greater sensitivity.