Coadministration of CH31 Broadly Neutralizing Antibody Does Not Affect Development of Vaccine-Induced Anti-HIV-1 Envelope Antibody Responses in Infant Rhesus Macaques.

Prevention of mother-to-child transmission (MTCT) is an indispensable component in combatting the global AIDS epidemic. A combination of passive broadly neutralizing antibody (bnAb) infusion and active vaccination promises to provide protection of infants against MTCT from birth through the breastfeeding period and could prime the immune system for lifelong immunity. In this study, we investigate the impact of a single infusion of CD4 binding site (CD4bs) bnAb administered at birth on de novo antibody responses elicited by concurrent active HIV envelope vaccination. Four groups of infant macaques received active immunizations with subunit Env protein or modified vaccinia Ankara (MVA)-vectored Env and subunit Env protein, with or without a single intravenous coadministration of CH31 bnAb at birth. Vaccinated animals were monitored to evaluate binding and functional antibody responses elicited by the active vaccinations. Despite achieving plasma concentrations that were able to neutralize tier 2 viruses, coadministration of CH31 did not have a large impact on the kinetics, magnitude, specificity, or avidity of vaccine-elicited binding or functional antibody responses, including epitope specificity, the development of CD4bs antibodies, neutralization, binding to infected cells, or antibody-dependent cell-mediated cytotoxicity (ADCC). We conclude that infusion of CD4bs bnAb CH31 at birth does not interfere with de novo antibody responses to active vaccination and that a combination of passive bnAb infusion and active HIV-1 Env vaccination is a viable strategy for immediate and prolonged protection against MTCT.IMPORTANCE Our study is the first to evaluate the impact of passive infusion of a broadly neutralizing antibody in newborns on the de novo development of antibody responses following active vaccinations in infancy. We demonstrated the safety and the feasibility of bnAb administration to achieve biologically relevant levels of the antibody and showed that the passive infusion did not impair the de novo antibody production following HIV-1 Env vaccination. Our study paves the way for further investigations of the combination strategy using passive plus active immunization to provide protection of infants born to HIV-1-positive mothers over the entire period of risk for mother-to-child transmission.

Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis.

OBJECTIVE: Hippocampal sclerosis is the most common neuropathologic finding in cases of medically intractable mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations, and to shed light on the transcriptional changes associated with hippocampal sclerosis. METHODS: RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. RESULTS: Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes up-regulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. SIGNIFICANCE: By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology.

Exome sequencing followed by large-scale genotyping fails to identify single rare variants of large effect in idiopathic generalized epilepsy.

Idiopathic generalized epilepsy (IGE) is a complex disease with high heritability, but little is known about its genetic architecture. Rare copy-number variants have been found to explain nearly 3% of individuals with IGE; however, it remains unclear whether variants with moderate effect size and frequencies below what are reliably detected with genome-wide association studies contribute significantly to disease risk. In this study, we compare the exome sequences of 118 individuals with IGE and 242 controls of European ancestry by using next-generation sequencing. The exome-sequenced epilepsy cases include study subjects with two forms of IGE, including juvenile myoclonic epilepsy (n = 93) and absence epilepsy (n = 25). However, our discovery strategy did not assume common genetic control between the subtypes of IGE considered. In the sequence data, as expected, no variants were significantly associated with the IGE phenotype or more specific IGE diagnoses. We then selected 3,897 candidate epilepsy-susceptibility variants from the sequence data and genotyped them in a larger set of 878 individuals with IGE and 1,830 controls. Again, no variant achieved statistical significance. However, 1,935 variants were observed exclusively in cases either as heterozygous or homozygous genotypes. It is likely that this set of variants includes real risk factors. The lack of significant association evidence of single variants with disease in this two-stage approach emphasizes the high genetic heterogeneity of epilepsy disorders, suggests that the impact of any individual single-nucleotide variant in this disease is small, and indicates that gene-based approaches might be more successful for future sequencing studies of epilepsy predisposition.

Rare deletions at 16p13.11 predispose to a diverse spectrum of sporadic epilepsy syndromes.

Deletions at 16p13.11 are associated with schizophrenia, mental retardation, and most recently idiopathic generalized epilepsy. To evaluate the role of 16p13.11 deletions, as well as other structural variation, in epilepsy disorders, we used genome-wide screens to identify copy number variation in 3812 patients with a diverse spectrum of epilepsy syndromes and in 1299 neurologically-normal controls. Large deletions (> 100 kb) at 16p13.11 were observed in 23 patients, whereas no control had a deletion greater than 16 kb. Patients, even those with identically sized 16p13.11 deletions, presented with highly variable epilepsy phenotypes. For a subset of patients with a 16p13.11 deletion, we show a consistent reduction of expression for included genes, suggesting that haploinsufficiency might contribute to pathogenicity. We also investigated another possible mechanism of pathogenicity by using hybridization-based capture and next-generation sequencing of the homologous chromosome for ten 16p13.11-deletion patients to look for unmasked recessive mutations. Follow-up genotyping of suggestive polymorphisms failed to identify any convincing recessive-acting mutations in the homologous interval corresponding to the deletion. The observation that two of the 16p13.11 deletions were larger than 2 Mb in size led us to screen for other large deletions. We found 12 additional genomic regions harboring deletions > 2 Mb in epilepsy patients, and none in controls. Additional evaluation is needed to characterize the role of these exceedingly large, non-locus-specific deletions in epilepsy. Collectively, these data implicate 16p13.11 and possibly other large deletions as risk factors for a wide range of epilepsy disorders, and they appear to point toward haploinsufficiency as a contributor to the pathogenicity of deletions.

Expansion of the Parkinson disease-associated SNCA-Rep1 allele upregulates human alpha-synuclein in transgenic mouse brain.

Alpha-synuclein (SNCA) gene has been implicated in the development of rare forms of familial Parkinson disease (PD). Recently, it was shown that an increase in SNCA copy numbers leads to elevated levels of wild-type SNCA-mRNA and protein and is sufficient to cause early-onset, familial PD. A critical question concerning the molecular pathogenesis of PD is what contributory role, if any, is played by the SNCA gene in sporadic PD. The expansion of SNCA-Rep1, an upstream, polymorphic microsatellite of the SNCA gene, is associated with elevated risk for sporadic PD. However, whether SNCA-Rep1 is the causal variant and the underlying mechanism with which its effect is mediated by remained elusive. We report here the effects of three distinct SNCA-Rep1 variants in the brains of 72 mice transgenic for the entire human SNCA locus. Human SNCA-mRNA and protein levels were increased 1.7- and 1.25-fold, respectively, in homozygotes for the expanded, PD risk-conferring allele compared with homozygotes for the shorter, protective allele. When adjusting for the total SNCA-protein concentration (endogenous mouse and transgenic human) expressed in each brain, the expanded risk allele contributed 2.6-fold more to the SNCA steady-state than the shorter allele. Furthermore, targeted deletion of Rep1 resulted in the lowest human SNCA-mRNA and protein concentrations in murine brain. In contrast, the Rep1 effect was not observed in blood lysates from the same mice. These results demonstrate that Rep1 regulates human SNCA expression by enhancing its transcription in the adult nervous system and suggest that homozygosity for the expanded Rep1 allele may mimic locus multiplication, thereby elevating PD risk.

IL28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis C.

UNLABELLED: Genetic variation in the IL28B (interleukin 28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C (CHC) who were treated with peginterferon-α and ribavirin. We hypothesized that IL28B polymorphism is associated with intrahepatic expression of interferon-stimulated genes (ISGs), known to influence treatment outcome. IL28B genotyping (rs12979860) and whole-genome RNA expression were performed using liver biopsies from 61 North American patients with CHC. After correction for multiple testing (false discovery rate < 0.10), 164 transcripts were found to be differentially expressed by IL28B-type. The interferon signaling pathway was the most enriched canonical pathway differentially expressed by IL28B-type (P < 10(-5)), with most genes showing higher expression in livers of individuals carrying the poor-response IL28B-type. In 25 patients for which treatment response data were available, IL28B-type was associated with SVR (P = 0.0054). ISG expression was also associated with SVR; however, this was not independent of IL28B-type. Analysis of miR-122 expression in liver biopsies showed reduced miR-122 levels associated with poorer treatment outcome, independently of IL28B-type. No association was observed between IL28B-type and levels of liver IL28B or IL28A messenger RNA expression. IL28B protein sequence variants associated with rs12979860 were therefore investigated in vitro: no differences in ISG induction or inhibition of HCV replication were observed in Huh7.5 cells. CONCLUSION: The good response IL28B variant was strongly associated with lower level ISG expression. The results suggest that IL28B genotype may explain the relationship between hepatic ISG expression and HCV treatment outcome, and this is independent of miR-122 expression. IL28B-type was not associated with intrahepatic IL28B messenger RNA expression in vivo. Further investigation of the precise molecular mechanism(s) by which IL28B genetic variation influences HCV outcomes is warranted.

Tissue-specific genetic control of splicing: implications for the study of complex traits.

Numerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes.

Host determinants of HIV-1 control in African Americans.

We performed a whole-genome association study of human immunodeficiency virus type 1 (HIV-1) set point among a cohort of African Americans (n = 515), and an intronic single-nucleotide polymorphism (SNP) in the HLA-B gene showed one of the strongest associations. We use a subset of patients to demonstrate that this SNP reflects the effect of the HLA-B*5703 allele, which shows a genome-wide statistically significant association with viral load set point (P = 5.6 x 10(-10)). These analyses therefore confirm a member of the HLA-B*57 group of alleles as the most important common variant that influences viral load variation in African Americans, which is consistent with what has been observed for individuals of European ancestry, among whom the most important common variant is HLA-B*5701.

Postmortem delay has minimal effect on brain RNA integrity.

The Bryan Alzheimer Disease Research Center obtains postmortem human brain tissue from patients with Alzheimer disease (AD) and cognitively normal control subjects for molecular and genetic research programs. A growing body of research suggests that variations in gene transcript levels may contribute to the onset and progression of disease. Identifying how the regulation of gene expression may affect AD requires the use of high-quality mRNA from banked human brains. The present study was conducted to establish the quality and suitability of available banked brain tissue for future gene expression studies. We chose 32 AD cases with Braak stage IV, V, or VI. These AD cases were matched to 36 normal control cases by age and sex when possible. Multiple regions from each brain were sampled, including frontal cortex, temporal cortex, occipital cortex, and cerebellum. Hippocampus was also available for study from 14 control cases. A comparison of several antemortem and postmortem variables, such as postmortem interval, agonal state, ventricular cerebrospinal fluid pH, and cause of death were analyzed. RNA was isolated from at least 1 area from every brain and most brains yielded intact RNA from all regions tested. Analysis of the clinical variables did not reveal any features that correlated with the ability to recover intact mRNA. We conclude that undegraded mRNA may be isolated from most brain regions many hours postmortem and that neither the pH of ventricular fluid nor postmortem interval is predictive of mRNA integrity.

Somatic uniparental disomy of Chromosome 16p in hemimegalencephaly.

Hemimegalencephaly (HME) is a heterogeneous cortical malformation characterized by enlargement of one cerebral hemisphere. Somatic variants in mammalian target of rapamycin (mTOR) regulatory genes have been implicated in some HME cases; however, ∼70% have no identified genetic etiology. Here, we screened two HME patients to identify disease-causing somatic variants. DNA from leukocytes, buccal swabs, and surgically resected brain tissue from two HME patients were screened for somatic variants using genome-wide genotyping arrays or sequencing of the protein-coding regions of the genome. Functional studies were performed to evaluate the molecular consequences of candidate disease-causing variants. Both HME patients evaluated were found to have likely disease-causing variants in DNA extracted from brain tissue but not in buccal swab or leukocyte DNA, consistent with a somatic mutational mechanism. In the first case, a previously identified disease-causing somatic single nucleotide in MTOR was identified. In the second case, we detected an overrepresentation of the alleles inherited from the mother on Chromosome 16 in brain tissue DNA only, indicative of somatic uniparental disomy (UPD) of the p-arm of Chromosome 16. Using methylation analyses, an imprinted locus on 16p spanning ZNF597 was identified, which results in increased expression of ZNF597 mRNA and protein in the brain tissue of the second case. Enhanced mTOR signaling was observed in tissue specimens from both patients. We speculate that overexpression of maternally expressed ZNF597 led to aberrant hemispheric development in the patient with somatic UPD of Chromosome 16p possibly through modulation of mTOR signaling.

Genetic regulation of alpha-synuclein mRNA expression in various human brain tissues.

Genetic variability across the SNCA locus has been repeatedly associated with susceptibility to sporadic Parkinson's disease (PD). Accumulated evidence emphasizes the importance of SNCA dosage and expression levels in PD pathogenesis. However whether genetic variability in the SNCA gene modulates the risk to develop sporadic PD via regulation of SNCA expression remained elusive. We studied the effect of PD risk-associated variants at SNCA 5' and 3'regions on SNCA-mRNA levels in vivo in 228 human brain samples from three structures differentially vulnerable to PD pathology (substantia-nigra, temporal- and frontal-cortex) obtained from 144 neurologically normal cadavers. The extensively characterized PD-associated promoter polymorphism, Rep1, had an effect on SNCA-mRNA levels. Homozygous genotype of the 'protective', Rep1-259 bp allele, was associated with lower levels of SNCA-mRNA relative to individuals that carried at least one copy of the PD-risk associated alleles, amounting to an average decrease of approximately 40% and >50% in temporal-cortex and substantia-nigra, respectively. Furthermore, SNPs tagging the SNCA 3'-untranslated-region also showed effects on SNCA-mRNA levels in both the temporal-cortex and the substantia-nigra, although, in contrast to Rep1, the 'decreased-risk' alleles were correlated with increased SNCA-mRNA levels. Similar to Rep1 findings, no difference in SNCA-mRNA level was seen with different SNCA 3'SNP alleles in the frontal-cortex, indicating there is brain-region specificity of the genetic regulation of SNCA expression. We provide evidence for functional consequences of PD-associated SNCA gene variants in disease relevant brain tissues, suggesting that genetic regulation of SNCA expression plays an important role in the development of the disease.

Gamma protocadherin expression in the embryonic chick nervous system.

Protocadherin gamma (pcdh-gamma) family expression was examined in the embryonic chick central nervous system by in situ hybridization. Transcripts were visualized in discrete regions of fore-, mid-, and hindbrain at stages 23 and 25 and in spinal cord and optic lobe at stages 27 and 43, respectively. Results suggest that pcdh-gamma may function cooperatively with other cell adhesion molecules in neuronal differentiation and establishment of neural networks in several areas of the developing brain, particularly regions involved in visual processing.