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INTRODUCTION The cerebral cortex underlies our complex cognitive capabilities. Variations in human cortical surface area and thickness are associated with neurological, psychological, and behavioral traits and can be measured in vivo by magnetic resonance imaging (MRI). Studies in model organisms have identified genes that influence cortical structure, but little is known about common genetic variants that affect human cortical structure. RATIONALE To identify genetic variants associated with human cortical structure at both global and regional levels, we conducted a genome-wide association meta-analysis of brain MRI data from 51,665 individuals across 60 cohorts. We analyzed the surface area and average thickness of the whole cortex and 34 cortical regions with known functional specializations. RESULTS We identified 369 nominally genome-wide significant loci ( P < 5 × 10 −8 ) associated with cortical structure in a discovery sample of 33,992 participants of European ancestry. Of the 360 loci for which replication data were available, 241 loci influencing surface area and 66 influencing thickness remained significant after replication, with 237 loci passing multiple testing correction ( P < 8.3 × 10 −10 ; 187 influencing surface area and 50 influencing thickness). Common genetic variants explained 34% (SE = 3%) of the variation in total surface area and 26% (SE = 2%) in average thickness; surface area and thickness showed a negative genetic correlation ( r G = −0.32, SE = 0.05, P = 6.5 × 10 −12 ), which suggests that genetic influences have opposing effects on surface area and thickness. Bioinformatic analyses showed that total surface area is influenced by genetic variants that alter gene regulatory activity in neural progenitor cells during fetal development. By contrast, average thickness is influenced by active regulatory elements in adult brain samples, which may reflect processes that occur after mid-fetal development, such as myelination, branching, or pruning. When considered together, these results support the radial unit hypothesis that different developmental mechanisms promote surface area expansion and increases in thickness. To identify specific genetic influences on individual cortical regions, we controlled for global measures (total surface area or average thickness) in the regional analyses. After multiple testing correction, we identified 175 loci that influence regional surface area and 46 that influence regional thickness. Loci that affect regional surface area cluster near genes involved in the Wnt signaling pathway, which is known to influence areal identity. We observed significant positive genetic correlations and evidence of bidirectional causation of total surface area with both general cognitive functioning and educational attainment. We found additional positive genetic correlations between total surface area and Parkinson’s disease but did not find evidence of causation. Negative genetic correlations were evident between total surface area and insomnia, attention deficit hyperactivity disorder, depressive symptoms, major depressive disorder, and neuroticism. CONCLUSION This large-scale collaborative work enhances our understanding of the genetic architecture of the human cerebral cortex and its regional patterning. The highly polygenic architecture of the cortex suggests that distinct genes are involved in the development of specific cortical areas. Moreover, we find evidence that brain structure is a key phenotype along the causal pathway that leads from genetic variation to differences in general cognitive function. Identifying genetic influences on human cortical structure. ( A ) Measurement of cortical surface area and thickness from MRI. ( B ) Genomic locations of common genetic variants that influence global and regional cortical structure. ( C ) Our results support the radial unit hypothesis that the expansion of cortical surface area is driven by proliferating neural progenitor cells. ( D ) Cortical surface area shows genetic correlation with psychiatric and cognitive traits. Error bars indicate SE. IMAGE CREDITS: (A) K. COURTNEY; (C) M. R. GLASS

Abstract The behavioural variant of frontotemporal dementia (bvFTD) is a frequent cause of early-onset dementia. The diagnosis of bvFTD remains challenging because of the limited accuracy of neuroimaging in the early disease stages and the absence of molecular biomarkers, and therefore relies predominantly on clinical assessment. BvFTD shows significant symptomatic overlap with non-degenerative primary psychiatric disorders including major depressive disorder, bipolar disorder, schizophrenia, obsessive-compulsive disorder, autism spectrum disorders and even personality disorders. To date, ∼50% of patients with bvFTD receive a prior psychiatric diagnosis, and average diagnostic delay is up to 5–6 years from symptom onset. It is also not uncommon for patients with primary psychiatric disorders to be wrongly diagnosed with bvFTD. The Neuropsychiatric International Consortium for Frontotemporal Dementia was recently established to determine the current best clinical practice and set up an international collaboration to share a common dataset for future research. The goal of the present paper was to review the existing literature on the diagnosis of bvFTD and its differential diagnosis with primary psychiatric disorders to provide consensus recommendations on the clinical assessment. A systematic literature search with a narrative review was performed to determine all bvFTD-related diagnostic evidence for the following topics: bvFTD history taking, psychiatric assessment, clinical scales, physical and neurological examination, bedside cognitive tests, neuropsychological assessment, social cognition, structural neuroimaging, functional neuroimaging, CSF and genetic testing. For each topic, responsible team members proposed a set of minimal requirements, optimal clinical recommendations, and tools requiring further research or those that should be developed. Recommendations were listed if they reached a ≥ 85% expert consensus based on an online survey among all consortium participants. New recommendations include performing at least one formal social cognition test in the standard neuropsychological battery for bvFTD. We emphasize the importance of 3D-T1 brain MRI with a standardized review protocol including validated visual atrophy rating scales, and to consider volumetric analyses if available. We clarify the role of 18F-fluorodeoxyglucose PET for the exclusion of bvFTD when normal, whereas non-specific regional metabolism abnormalities should not be over-interpreted in the case of a psychiatric differential diagnosis. We highlight the potential role of serum or CSF neurofilament light chain to differentiate bvFTD from primary psychiatric disorders. Finally, based on the increasing literature and clinical experience, the consortium determined that screening for C9orf72 mutation should be performed in all possible/probable bvFTD cases or suspected cases with strong psychiatric features.

Abstract X-linked chronic granulomatous disease (X-CGD) is an immune deficiency that results from mutations in the gp91phox subunit of the phagocyte NADPH-oxidase. We have developed a modified RD114-pseudotyped simian immunodeficiency virus (SIVmac) vector encoding human gp91phox and report here that this vector efficiently targets peripheral blood-mobilized CD34+ hematopoietic stem cells (PBSCs) from X-CGD patients, correcting the oxidase deficient phenotype. The majority of lentiviral gene transfer studies have relied on human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope. Because HIV is a human pathogen, there are theoretical benefits to considering other types of lentivectors engineered from viruses less pathogenic for humans such as SIV-based vectors. In addition, the VSV-G envelope has some toxicity for cells, limiting the titers of vector that can be used. RD114/TR-pseudotyped SIV gp91phox encoding vector particles were transiently produced following transfection of 293T cells and concentrated by high-speed centrifugation achieving stock titers of 1.5 × 10E7 infectious units/ml. PBSCs from two patients with X-CGD (P1 and P2) were cultured and transduced (multiplicity of infection 2–3) overnight four times before transplantation into sublethally irradiated NOD/SCID mice on day 5 of culture. Ex vivo transduction efficiency was 40.5% and 46%, respectively. Cells maintained in culture showed a progressive decrease in marking over the first 9 days, after which marking stabilized at 22.7% (P1) and 27.1% (P2) out to day 26 of culture with an averaged copy number of 2.2–2.4 per transduced cell at that time. Compared to healthy control, 18–19% of myeloid colonies derived from patient CD34+ cells stained positive in the nitroblue tetrazolium (NBT) test. Liquid cell cultures at 20 days were analyzed for production of reactive oxidative species using the flow cytometry dihydrorhodamine 123 (DHR) assay. Cells from P1 demonstrated 15.3% and from P2 9.1% of the activity of healthy cells. At 6 weeks after transplant, analysis of mouse bone marrow (mBM) samples revealed human cell engraftment levels of 36.8–74% (CD45+ cells). Compared to analysis of mice transplanted with healthy human PBSCs, 10.5% (P1) and 7.3% (P2) of the myeloid cells (CD13+) developing from X-CGD CD34+ cells in the chimeric mBM expressed gp91phox transgene. Human CD34+ cells were isolated from mBM and grown in liquid culture or plated for colony assays. 2.1% (P1) and 1.2% (P2) of cultured cells were gp91phox positive at day 14 with an averaged copy number of 2.3–5.2 per marked cell. 4.8% and 3.6% of colonies derived from the same cells were positive in the NBT test. DHR assay at day 21 of culture demonstrated 2.7% (P1) and 2.0% (P2) of the activity of healthy cells. Linear amplification-mediated PCR retrieved multiple unique vector insertion sites in CD34+ cells from both patients isolated from chimeric mBM, without predominance of any one clone suggesting polyclonal marking. In conclusion, these data suggest that modified RD114-pseudotyped SIVmac-based vectors may be suitable for hematopoietic gene therapy for CGD and represent an alternative to HIV-1 based vectors. Studies are ongoing to further improve vector production titers to achieve higher transduction levels of PBSCs.

Abstract X-linked severe combined immunodeficiency (XSCID) is characterized by profound immunodeficiency (dysfunctional B cells; absence of T and NK cells) and early mortality, caused by mutations in the IL2RG gene encoding the common gamma chain (γc) of receptors for interleukins (IL)-2,-4,-7,-9,-15 and -21. The standard therapy for XSCID in infants is a T cell depleted bone marrow transplant with either none or very modest conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution that may be limited to only the T cell lineage. Ex vivo autologous stem cell gene therapy has emerged as an alternate treatment capable of achieving substantial to complete immune reconstitution in infants without a sibling donor. However, some children have developed lymphocytic leukemia, which appears in part to be related to vector insertional mutagenesis. The gammaretroviruses currently in use have potent enhancers in the LTR and have a predilection to insert at the 5′ end of genes. Self-inactivating (SIN) lentivirus vectors may be advantageous because they do not show this property and can be constructed with internal promotors that have less enhancer activity together with insertion of insulators. We have constructed SIN simian immunodeficiency viral vectors (SIVmac) encoding human γc (hγc), with or without a double copy chicken insulator core element in the 3′ LTR. In addition we have pseudotyped the vector with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicity of the VSV-G envelope traditionally used with lentivectors. We have previously shown that it is possible to correct a dog model of XSCID using a RD114-pseudotyped gammaretroviral vector encoding the dog γc for in vivo gene therapy. Unlike the mouse model of XSCID which lacks B cells, the dog model more closely resembles the phenotype in humans. We decided to use our in vivo dog model for preclinical testing of safety and efficacy of our SIV hγc vectors. Viral particles were produced by transient transfection of 293T cells with a 4-plasmid system and concentrated by high-speed centrifugation. 30 mls (average 2.4–3×107 viral particles in total) was injected IV into 2–5 day-old pups. Transgene marking in blood lymphocytes was detected as early as 2 wks after treatment, increased within the first 6–8 wks and became relatively stable thereafter. The absolute lymphocyte count was normalized in one dog (injected on the second day after birth) by wk 6 (4000, 76% hγc+), and improved in two other dogs (422, 29% hγc+ at 18 wks; 498, 27% hγc+, at 16 wks) which had received the same amount of virus over 2 days. Furthermore, up to 5% of the myeloid lineage showed gene marking at 10–18 wks after viral delivery, indicating that early committed progenitors or HSCs had been transduced. It is noteworthy that our finding that the hγc can improve the disease phenotype in XSCID dogs makes it an excellent large animal model for preclinical evaluation of vectors. In summary, we have demonstrated that in vivo delivery of SIV lentiviral vectors expressing hγc efficiently reconstitute the T-lymphoid compartment in the XSCID canine model. We plan to monitor these dogs closely for potential adverse events.

ABSTRACT High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased β-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet β-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a β-cell-targeted gene transfer vector and determined the impact of β-cell-specific increase in GCK expression on β-cell function and glucose handling in vitro and in vivo. Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. β-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), β-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired β-cell GCK expression as an underlying mechanism for dysregulated β-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes. This article has an associated First Person interview with the first author of the paper.

Abstract Glucokinase (GCK) is crucial to regulating glucose metabolism in the liver and in pancreatic β-cells. There are two major GCK isoforms, hepatic and pancreatic GCKs, which differ only in exon 1. However, the functional differences between the two GCK isoforms remain poorly understood. Here, we used a β-cell-targeted gene transfer vector to determine the impact of isoform-specific GCK overexpression on β-cells in vitro and in vivo. We showed that pancreatic GCK had a nuclear localization signal unique to the pancreatic isoform, facilitating its nuclear distribution in β-cells. Despite the difference in subcellular distribution, overexpression of GCK isoforms similarly enhanced glucose uptake and β-cell proliferation in vitro. Overexpression of hepatic or pancreatic GCK also similarly enhanced β-cell proliferation in normal diet mice without affecting fasting glucose and intraperitoneal glucose tolerance tests (IPGTT). Our further study on human GCK sequences identified disproportional GCK amino acid variants in exon 1, while mutations linked to maturity onset diabetes of the young type 2 (MODY2) were disproportionally found in exons 2 through 10. Our results therefore indicate functional conservation between the two major GCK isoforms despite their distinct subcellular distribution.

AbstractBackgroundDeleterious mutations in PRF1 result in lethal, childhood disease, familial hemophagocytic lymphohistiocytosis type 2 (FHL 2). However, not all mutations in PRF1 are deleterious and result in FHL 2. Currently, these nondeleterious mutations are being investigated in the onset of numerous disorders, such as lymphomas and diabetes. Yet, there is still an overwhelmingly large amount of PRF1 mutations that are not associated with disease.MethodsWe conducted a post hoc analysis of the PRF1 mutations in the coding region using the recently published Exome Aggregation Consortium genomes, Leiden Open Variation Database, NCBI SNP database, and primary literature to better understand PRF1 variation in the human population.ResultsThis study catalogs 460 PRF1 mutations in the coding region, and demonstrates PRF1 is more variant then previously predicted. We identify key PRF1 mutations with high allelic frequency and are only found in certain populations. Additionally, we define PRF1 SNVs are geographically distributed.ConclusionsThis study concludes with a novel hypothesis that nondeleterious mutation in PRF1, which decreases perforin expression and/or activity, may be an example of selective advantage in the context of environmental stressors prevalent near the equator. Our studies illustrate how perforin deficiency can be protective from injuries resulting in blood–brain barrier (BBB) disruption.

SummaryThis paper is a result of work of the AES/ILAE Translational Task Force of the International League Against Epilepsy. The aim is to provide acceptable standards and interpretation of results of electrophysiological depth recordings in vivo in control rodents.