Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Here, we have performed a prospective nationwide genetic association study using the Human Exome BeadChip and identified gene variants in encoding dynactin 4 (DCTN4), retinoic acid early transcript 1E (RAET1E), and V-akt murine thymoma viral oncogene homolog 3 (AKT3) to be associated with unfavourable outcome in patients with pneumococcal meningitis. No clinical replication cohort is available, so we validated the role of one of these targets, AKT3, in a pneumococcal meningitis mouse model. Akt3 deficient mice had worse survival and increased histopathology scores for parenchymal damage (infiltration) and vascular infiltration (large meningeal artery inflammation) but similar bacterial loads, cytokine responses, compared to wild-type mice. We found no differences in cerebrospinal fluid cytokine levels between patients with risk or non-risk alleles. Patients with the risk genotype (rs10157763, AA) presented with low scores on the Glasgow Coma Scale and high rate of epileptic seizures. Thus, our results show that AKT3 influences outcome of pneumococcal meningitis.

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease with no available therapy. Components of the innate immune system are activated in the spinal cord and central nervous system of ALS patients. Studies in the SOD1(G93A) mouse show deposition of C1q and C3/C3b at the motor end-plate before neurological symptoms are apparent, suggesting that complement activation precedes neurodegeneration in this model. To obtain a better understanding of the role of complement at the motor end-plates in human ALS pathology, we analyzed post-mortem tissue of ALS donors for complement activation and its regulators.

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) of the biliary tree and pancreas is difficult to distinguish from sclerosing cholangitis and biliary/pancreatic malignancies (CA). An accurate noninvasive test for diagnosis and monitoring of disease activity is lacking. We demonstrate that dominant IgG4(+) B-cell receptor (BCR) clones determined by next-generation sequencing accurately distinguish patients with IgG4-associated cholangitis/autoimmune pancreatitis (n = 34) from those with primary sclerosing cholangitis (n = 17) and CA (n = 17). A novel, more affordable, and widely applicable quantitative polymerase chain reaction (qPCR) protocol analyzing the IgG4/IgG RNA ratio in blood also achieves excellent diagnostic accuracy (n = 125). Moreover, this qPCR test performed better than serum IgG4 levels in sensitivity (94% vs. 86%) and specificity (99% vs. 73%) and correlates with treatment response (n = 20).

Small virus-derived interfering RNAs (viRNAs) play an important role in antiviral defence in plants, insects and nematodes by triggering the RNA interference (RNAi) pathway. The role of RNAi as an antiviral defence mechanism in mammalian cells has been obscure due to the lack of viRNA detection. Although viRNAs from different mammalian viruses have recently been identified, their functions and possible impact on viral replication remain unknown. To identify viRNAs derived from HIV-1, we used the extremely sensitive SOLiD(TM) 3 Plus System to analyse viRNA accumulation in HIV-1-infected T lymphocytes. We detected numerous small RNAs that correspond to the HIV-1 RNA genome. The majority of these sequences have a positive polarity (98.1%) and could be derived from miRNAs encoded by structured segments of the HIV-1 RNA genome (vmiRNAs). A small portion of the viRNAs is of negative polarity and most of them are encoded within the 3'-UTR, which may represent viral siRNAs (vsiRNAs). The identified vsiRNAs can potently repress HIV-1 production, whereas suppression of the vsiRNAs by antagomirs stimulate virus production. These results suggest that HIV-1 triggers the production of vsiRNAs and vmiRNAs to modulate cellular and/or viral gene expression.

Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum.

The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αβ-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective β-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/β-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.

Pontocerebellar hypoplasia (PCH) represents a group of recessive developmental disorders characterized by impaired growth of the pons and cerebellum, which frequently follows a degenerative course. Currently, there are 10 partially overlapping clinical subtypes and 13 genes known mutated in PCH. Here, we report biallelic TBC1D23 mutations in six individuals from four unrelated families manifesting a non-degenerative form of PCH. In addition to reduced volume of pons and cerebellum, affected individuals had microcephaly, psychomotor delay, and ataxia. In zebrafish, tbc1d23 morphants replicated the human phenotype showing hindbrain volume loss. TBC1D23 localized at the trans-Golgi and was regulated by the small GTPases Arl1 and Arl8, suggesting a role in trans-Golgi membrane trafficking. Altogether, this study provides a causative link between TBC1D23 mutations and PCH and suggests a less severe clinical course than other PCH subtypes.

Serial Analysis of Gene Expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the evaluation of these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce the need for duplicate experiments and facilitate a more extensive exchange of data within the research community. This requires a measure for the correspondence of the different gene expression platforms. To date, a number of cross-platform evaluations (including a few studies using SAGE and Affymetrix GeneChips) have been conducted showing a variable, but overall low, concordance. This study evaluates these overall measures and introduces the between-ratio difference as a concordance measure pergene.

The dystrobrevin-binding protein 1 (DTNBP1) gene is a susceptibility gene for schizophrenia. There is growing evidence that DTNPB1 contributes to intelligence and cognition. In this study, we investigated association between single nucleotide polymorphisms (SNPs) in the DTNBP1 gene and intellectual functioning in patients with a first episode of schizophrenia or related psychotic disorder (first-episode psychosis, FEP), their healthy siblings, and unrelated controls.

Listeria monocytogenes is a Gram-positive bacterium that can be found in a broad range of environments, including soil, food, animals, and humans. L. monocytogenes can cause a foodborne disease manifesting as sepsis and meningo-encephalitis. To evaluate signals of selection within the core genome of neuroinvasive L. monocytogenes strains, we sequenced 122 L. monocytogenes strains from cerebrospinal fluid (CSF) of Dutch meningitis patients and performed a genome-wide analysis using Tajima's D and ω (dN/dS). We also evaluated the residual variation intolerance score (RVIS), a computationally less demanding methodology, to identify loci under selection. Results show that the large genetic distance between the listerial lineages influences the Tajima's D and ω (dN/dS) outcome. Within genetic lineages we detected signals of selection in 6 of 2327 loci (<1%), which were replicated in an external cohort of 105 listerial CSF isolates from France. Functions of identified loci under selection were within metabolism pathways (lmo2476, encoding aldose 1-epimerase), putative antimicrobial resistance mechanisms (lmo1855, encoding PBPD3), and virulence factors (lmo0549, internalin-like protein; lmo1482, encoding comEC). RVIS over the two genetic lineages showed signals of selection in internalin-like proteins loci potentially involved in pathogen-host interaction (lmo0549, lmo0610, and lmo1290). Our results show that RVIS can be used to detect bacterial loci under selection.

The most common genetic risk factor for Parkinson's disease known is a damaging variant in the GBA1 gene. The entire GBA1 gene has rarely been studied in a large cohort from a single population. The objective of this study was to assess the entire GBA1 gene in Parkinson's disease from a single large population.

Fibroblasts from a patient carrying a heterozygous 18bp deletion in exon 8 of the ACVRL1 gene (c.1120del18) were reprogrammed using episomal vectors. The in-frame deletion in ACVRL1 causes the loss of 6 amino acids of the protein, which is associated with Hereditary Hemorrhagic Telangiectasia (HHT) type 2 (Letteboer et al., 2005). CRISPR-Cas9 editing was used to genetically correct the mutation in the induced pluripotent stem cells (iPSCs). The top5-predicted off-target sites were not altered. Patient and isogenic iPSCs showed high pluripotent marker expression, in vitro differentiation capacity into all three germ layers and displayed a normal karyotype. The obtained isogenic pairs will enable proper in vitro disease modelling of HHT (Roman and Hinck, 2017).

The Khorana score is a validated tool to identify cancer patients at higher risk of venous thromboembolism (VTE).

The characteristics of pneumococcal carriage vary between infants and adults. Host immune factors have been shown to contribute to these age-specific differences, but the role of pathogen sequence variation is currently less well-known. Identification of age-associated pathogen genetic factors could leadto improved vaccine formulations. We therefore performed genome sequencing in a large carriage cohort of children and adults and combined this with data from an existing age-stratified carriage study. We compiled a dictionary of pathogen genetic variation, including serotype, strain, sequence elements, single-nucleotide polymorphisms (SNPs), and clusters of orthologous genes (COGs) for each cohort - all of which were used in a genome-wide association with host age. Age-dependent colonization showed weak evidence of being heritable in the first cohort (h2 = 0.10, 95% CI 0.00-0.69) and stronger evidence in the second cohort (h2 = 0.56, 95% CI 0.23-0.87). We found that serotypes and genetic background (strain) explained a proportion of the heritability in the first cohort (h2serotype = 0.07, 95% CI 0.04-0.14 and h2GPSC = 0.06, 95% CI 0.03-0.13) and the second cohort (h2serotype = 0.11, 95% CI 0.05-0.21 and h2GPSC = 0.20, 95% CI 0.12-0.31). In a meta-analysis of these cohorts, we found one candidate association (p=1.2 × 10-9) upstream of an accessory Sec-dependent serine-rich glycoprotein adhesin. Overall, while we did find a small effect of pathogen genome variation on pneumococcal carriage between child and adult hosts, this was variable between populations and does not appear to be caused by strong effects of individual genes. This supports proposals for adaptive future vaccination strategies that are primarily targeted at dominant circulating serotypes and tailored to the composition of the pathogen populations.

Neisseria meningitidis causes sepsis and meningitis in humans. It has been suggested that pathogen genetic variation determines variance in disease severity. Here we report results of a genome-wide association study of 486 N. meningitidis genomes from meningococcal meningitis patients and their association with disease severity. Of 369 meningococcal meningitis patients for whom clinical data was available, 44 (12%) had unfavorable outcome and 24 (7%) died. To increase power, thrombocyte count was used as proxy marker for disease severity. Bacterial genetic variants were called as k-mers, SNPs, insertions and deletions and clusters of orthologous genes (COGs). Population-level meningococcal genetic variation did not explain variance in disease severity (unfavorable outcome or thrombocyte count) in this cohort (h2 = 0.0%; 95% confidence interval: 0.0-0.9). Genetic variants in the bacterial uppS gene represented the top signal associated with thrombocyte count (p-value = 9.96e-07) but this did not reach statistical significance. We did not find an association between previously published variants in lpxL1, fHbp, and tps genes and unfavorable outcome or thrombocyte count. A power analysis based on simulated phenotypes based on real genetic data from 880 N. meningitidis genomes showed that we would be able to detect a continuous phenotype with h2 > = 0.5 with the population size available in this study. This rules out a major contribution of pathogen genetic variation to disease severity in meningococcal meningitis, and shows that much larger sample sizes are required to find specific low-effect genetic variants modulating disease outcome in meningococcal meningitis.

CRB1 gene mutations can cause early- or late-onset retinitis pigmentosa, Leber congenital amaurosis, or maculopathy. Recapitulating human CRB1 phenotypes in animal models has proven challenging, necessitating the development of alternatives. We generated human induced pluripotent stem cell (iPSC)-derived retinal organoids of patients with retinitis pigmentosa caused by biallelic CRB1 mutations and evaluated them against autologous gene-corrected hiPSCs and hiPSCs from healthy individuals. Patient organoids show decreased levels of CRB1 and NOTCH1 expression at the retinal outer limiting membrane. Proximity ligation assays show that human CRB1 and NOTCH1 can interact via their extracellular domains. CRB1 patient organoids feature increased levels of WDFY1+ vesicles, fewer RAB11A+ recycling endosomes, decreased VPS35 retromer complex components, and more degradative endolysosomal compartments relative to isogenic control organoids. Taken together, our data demonstrate that patient-derived retinal organoids enable modeling of retinal degeneration and highlight the importance of CRB1 in early endosome maturation receptor recycling in the retina.

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Early recognition of the pathogen and subsequent innate immune response play a vital role in disease susceptibility and outcome. Genetic variations in innate immune genes can alter the immune response and influence susceptibility and outcome of meningitis disease. Here we conducted a sequencing study of coding regions from 46 innate immune genes in 435 pneumococcal meningitis patients and 416 controls, to determine the role of genetic variation on pneumococcal meningitis susceptibility and disease outcome. Strongest signals for susceptibility were rs56078309 CXCL1 (p=4.8e-04) and rs2008521 in CARD8 (p=6.1e-04). For meningitis outcome the rs2067085 in NOD2 (p=5.1e-04) and rs4251552 of IRAK4 were the strongest associations with unfavorable outcome (p=6.7e-04). Haplotype analysis showed a haplotype block, determined by IRAK4 rs4251552, significantly associated with unfavorable outcome (p=0.004). Cytokine measurements from cerebrospinal fluid showed that with the IRAK4 rs4251552 G risk allele had higher levels of IL-6 compared to individuals with A/A genotype (p=0.04). We show that genetic variation within exons and flanking regions of 46 innate immunity genes does not yield significant association with pneumococcal meningitis. The strongest identified signal IRAK4 does imply a potential role of genetic variation in pneumococcal meningitis.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease in which motor neurons in the brain and spinal cord degenerate by largely unknown mechanisms. ALS is familial (FALS) in 10% of cases, and the inheritance is usually dominant, with variable penetrance. Mutations in copper/zinc super oxide dismutase (SOD1) are found in 20% of familial and 3% of sporadic ALS cases. Five families with ALS and frontotemporal dementia (ALS-FTD) are linked to 9q21, whereas one family with pure ALS is linked to 18q21. We identified two large European families with ALS without SOD1 mutations or linkage to known FALS loci and conducted a genomewide linkage screen using 400 microsatellite markers. In both families, two-point LOD scores >1 and a haplotype segregating with disease were demonstrated only across regions of chromosome 16. Subsequent fine mapping in family 1 gave a maximum two-point LOD score of 3.62 at D16S3137 and a three-point LOD score of 3.85 for markers D16S415 and D16S3137. Haplotype analysis revealed no recombination > approximately 30 cM, (flanking markers at D16S3075 and D16S3112). The maximum two-point LOD score for family 2 was 1.84 at D16S415, and the three-point LOD score was 2.10 for markers D16S419 and D16S415. Definite recombination occurred in several individuals, which narrowed the shared haplotype in affected individuals to a 10.1-cM region (flanking markers: D16S3396 and D16S3112). The region shared by both families on chromosome 16q12 corresponds to approximately 4.5 Mb on the Marshfield map. Bioinformatic analysis of the region has identified 18 known genes and 70 predicted genes in this region, and sequencing of candidate genes has now begun.