Comprehensive genetic screening of early-onset dementia patients in an Austrian cohort-suggesting new disease-contributing genes.

Early-onset dementia (EOD), with symptom onset before age 65, has a strong genetic burden. Due to genetic and clinical overlaps between different types of dementia, whole-exome sequencing (WES) has emerged as an appropriate screening method for diagnostic testing and novel gene-finding approaches. We performed WES and C9orf72 repeat testing in 60 well-defined Austrian EOD patients. Seven patients (12%) carried likely disease-causing variants in monogenic genes, PSEN1, MAPT, APP, and GRN. Five patients (8%) were APOE4 homozygote carriers. Definite and possible risk variants were detected in the genes TREM2, SORL1, ABCA7 and TBK1. In an explorative approach, we cross-checked rare gene variants in our cohort with a curated neurodegeneration candidate gene list and identified DCTN1, MAPK8IP3, LRRK2, VPS13C and BACE1 as promising candidate genes. Conclusively, 12 cases (20%) carried variants relevant to patient counseling, comparable to previously reported studies, and can thus be considered genetically resolved. Reduced penetrance, oligogenic inheritance and not yet identified high-risk genes might explain the high number of unresolved cases. To address this issue, we provide complete genetic and phenotypic information (uploaded to the European Genome-phenome Archive), enabling other researchers to cross-check variants. Thereby, we hope to increase the chance of independently finding the same gene/variant-hit in other well-defined EOD patient cohorts, thus confirming new genetic risk variants or variant combinations.

C9orf72 repeat length might influence clinical sub-phenotypes in dementia patients.

BACKGROUND: C9orf72 repeat expansions have been observed in a wide variety of neurodegenerative disorders. The cut-off between normal and pathogenic alleles is not well established as repeat sizing methods are often semi-quantitative. However, intermediate alleles might influence disease prevalence and phenotype, as seen for other repeat expansion disorders. We aimed to further delineate the prevalence of small, intermediate and expanded C9orf72 alleles and elucidate their potential influence on the disease phenotype. METHODS: DNA derived from patients (n = 1804) and healthy individuals (n = 643) was obtained from multiple collectives in Austria. Genotyping was performed using a two-step PCR assay followed by Southern blotting. RESULTS: 3.4% of clinically diagnosed frontotemporal dementia (FTD; n = 5/147) cases and 0.8% of clinically diagnosed Alzheimer's disease (AD; n = 5/602) cases were carriers of a pathological C9orf72 repeat expansion. A significantly earlier disease onset was detected in expansion carriers compared to non-carriers in the FTD and AD cohorts (median 50 years, range 39-64 vs. median 64 years, range 36-92, p = 0.018 and median 63 years, range 54-71 vs. median 74 years, range 45-92, p = 0.006, respectively). C9orf72 intermediate alleles were significantly associated with cerebellar symptoms (p = 0.0004) and sensory deficits in the dementia cohort (p = 0.01). CONCLUSIONS: C9orf72 repeat expansion carriers showed earlier disease onset compared to non-carriers with clinical diagnosis of FTD and AD. Furthermore, C9orf72 intermediate repeats might modify the phenotypic expression in dementia.

Diagnostic exome sequencing in non-acquired focal epilepsies highlights a major role of GATOR1 complex genes.

BACKGROUND: The genetic architecture of non-acquired focal epilepsies (NAFEs) becomes increasingly unravelled using genome-wide sequencing datasets. However, it remains to be determined how this emerging knowledge can be translated into a diagnostic setting. To bridge this gap, we assessed the diagnostic outcomes of exome sequencing (ES) in NAFE. METHODS: 112 deeply phenotyped patients with NAFE were included in the study. Diagnostic ES was performed, followed by a screen to detect variants of uncertain significance (VUSs) in 15 well-established focal epilepsy genes. Explorative gene prioritisation was used to identify possible novel candidate aetiologies with so far limited evidence for NAFE. RESULTS: ES identified pathogenic or likely pathogenic (ie, diagnostic) variants in 13/112 patients (12%) in the genes DEPDC5, NPRL3, GABRG2, SCN1A, PCDH19 and STX1B. Two pathogenic variants were microdeletions involving NPRL3 and PCDH19. Nine of the 13 diagnostic variants (69%) were found in genes of the GATOR1 complex, a potentially druggable target involved in the mammalian target of rapamycin (mTOR) signalling pathway. In addition, 17 VUSs in focal epilepsy genes and 6 rare variants in candidate genes (MTOR, KCNA2, RBFOX1 and SCN3A) were detected. Five patients with reported variants had double hits in different genes, suggesting a possible (oligogenic) role of multiple rare variants. CONCLUSION: This study underscores the molecular heterogeneity of NAFE with GATOR1 complex genes representing the by far most relevant genetic aetiology known to date. Although the diagnostic yield is lower compared with severe early-onset epilepsies, the high rate of VUSs and candidate variants suggests a further increase in future years.

Correction to: The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

An amendment to this paper has been published and can be accessed via the original article.

The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

BACKGROUND: In the past urine was considered sterile. Through the introduction of next generation sequencing, it has become clear that a urinary microbiome exists. Acute kidney injury (AKI) represents a major threat to kidney transplant recipients. Remarkable changes in the urinary metabolome occur during AKI, which may influence the urinary microbiome. To our knowledge, this is the first study that examines the urinary microbiome in renal transplant recipients (RTX) and non-transplant recipients (nRTX) at time of AKI. METHODS: In this cross-sectional pilot-study the urinary microbiome of 21 RTX and 9 nRTX with AKI was examined. Clean catch morning urine samples were obtained from all patients on the first day of AKI diagnosis. AKI was defined according to KDIGO guidelines. Urinary microbiota and the urinary metabolome during AKI were assessed in one patient. 16S rRNA sequencing was performed. Sequences were processed using UPARSE-pipeline for operational taxonomic units (OTU) and taxon finding. RESULTS: We successfully extracted and sequenced bacterial DNA from 100% of the urine samples. All 30 patients revealed at least 106,138 reads. 319 OTU and 211 different genera were identified. The microbiotic diversity richness in the RTX group was no different from the nRTX group. Eighteen genera were solely present in nRTX and 7 in RTX. CONCLUSIONS: The urinary microbiome at time of AKI showed different bacterial genera in RTX compared to nRTX. The nRTX group exhibited no different diversity to the RTX group. Irrespective of the status of a previous renal transplantation, the urinary microbiome comprised > 210 different genera. An intraindividual change in microbiota diversity and richness was observed in one study patient during recovery from AKI.

The Parkinson's disease VPS35[D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.

Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.

Rare variants in γ-aminobutyric acid type A receptor genes in rolandic epilepsy and related syndromes.

OBJECTIVE: To test whether mutations in γ-aminobutyric acid type A receptor (GABAA -R) subunit genes contribute to the etiology of rolandic epilepsy (RE) or its atypical variants (ARE). METHODS: We performed exome sequencing to compare the frequency of variants in 18 GABAA -R genes in 204 European patients with RE/ARE versus 728 platform-matched controls. Identified GABRG2 variants were functionally assessed for protein stability, trafficking, postsynaptic clustering, and receptor function. RESULTS: Of 18 screened GABAA -R genes, we detected an enrichment of rare variants in the GABRG2 gene in RE/ARE patients (5 of 204, 2.45%) in comparison to controls (1 of 723, 0.14%; odds ratio = 18.07, 95% confidence interval = 2.01-855.07, p = 0.0024, pcorr = 0.043). We identified a GABRG2 splice variant (c.549-3T>G) in 2 unrelated patients as well as 3 nonsynonymous variations in this gene (p.G257R, p.R323Q, p.I389V). Functional assessment showed reduced surface expression of p.G257R and decreased GABA-evoked currents for p.R323Q. The p.G257R mutation displayed diminished levels of palmitoylation, a post-translational modification crucial for trafficking of proteins to the cell membrane. Enzymatically raised palmitoylation levels restored the surface expression of the p.G257R variant γ2 subunit. INTERPRETATION: The statistical association and the functional evidence suggest that mutations of the GABRG2 gene may increase the risk of RE/ARE. Restoring the impaired membrane trafficking of some GABRG2 mutations by enhancing palmitoylation might be an interesting therapeutic approach to reverse the pathogenic effect of such mutants.

A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease.

To identify rare causal variants in late-onset Parkinson disease (PD), we investigated an Austrian family with 16 affected individuals by exome sequencing. We found a missense mutation, c.1858G>A (p.Asp620Asn), in the VPS35 gene in all seven affected family members who are alive. By screening additional PD cases, we saw the same variant cosegregating with the disease in an autosomal-dominant mode with high but incomplete penetrance in two further families with five and ten affected members, respectively. The mean age of onset in the affected individuals was 53 years. Genotyping showed that the shared haplotype extends across 65 kilobases around VPS35. Screening the entire VPS35 coding sequence in an additional 860 cases and 1014 controls revealed six further nonsynonymous missense variants. Three were only present in cases, two were only present in controls, and one was present in cases and controls. The familial mutation p.Asp620Asn and a further variant, c.1570C>T (p.Arg524Trp), detected in a sporadic PD case were predicted to be damaging by sequence-based and molecular-dynamics analyses. VPS35 is a component of the retromer complex and mediates retrograde transport between endosomes and the trans-Golgi network, and it has recently been found to be involved in Alzheimer disease.

Elevated urine BMP phospholipids in LRRK2 and VPS35 mutation carriers with and without Parkinson's disease.

Elevated urine bis(monoacylglycerol)phosphate (BMP) levels have been found in gain-of-kinase function LRRK2 G2019S mutation carriers. Here, we have expanded urine BMP analysis to other Parkinson's disease (PD) associated mutations and found them to be consistently elevated in carriers of LRRK2 G2019S and R1441G/C as well as VPS35 D620N mutations. Urine BMP levels are promising biomarkers for patient stratification and potentially target engagement in clinical trials of emerging targeted PD therapies.

Replication study of multiple sclerosis (MS) susceptibility alleles and correlation of DNA-variants with disease features in a cohort of Austrian MS patients.

We performed a replication study in 883 Austrian multiple sclerosis (MS) patients and 972 control individuals for 25 previously risk-associated loci (39 SNPs). Two loci, rs1109670 (DDEF2/MBOAT2, p < 0.02) and rs16914086 (TBC1D2, p < 0.05), are replicated here for the first time. Furthermore, we tested all 39 SNPs for association with age at disease onset and measures of disease severity. We observed a trend for association of rs3135388 (HLA-DRB1*1501, p < 0.01), rs7090530 (IL2RA, p < 0.026) and rs1841770 (ZIC1, p < 0.017) with a younger age at MS onset and of rs12044852 (CD58, p < 0.035) with shorter time to reach EDSS6.

Role of LINGO1 polymorphisms in Parkinson's disease.

A clinical overlap between Parkinson's disease (PD) and essential tremor (ET) has prompted a discussion whether these conditions share common genetic susceptibility factors. Recently, the first genome-wide association study in ET revealed a significant association with a variant in the LINGO1 gene. LINGO1 has also been demonstrated to play a role in the survival of dopaminergic neurons in an animal model of PD, and therefore constitutes a potential candidate gene for PD. In this study, SNPs rs9652490, rs11856808, and rs7177008 of LINGO1 were genotyped in a total of 694 Austrian subjects (349 PD, 345 controls). No association could be found between genotype or allele counts and PD. Neither did a subgroup analysis in tremor-dominant patients with PD reveal a significant association. This study on LINGO1-variants in PD argues against a major role of LINGO1 gene variations for PD.

TPP2 mutation associated with sterile brain inflammation mimicking MS.

OBJECTIVE: To ascertain the genetic cause of a consanguineous family from Syria suffering from a sterile brain inflammation mimicking a mild nonprogressive form of MS. METHODS: We used homozygosity mapping and next-generation sequencing to detect the disease-causing gene in the affected siblings. In addition, we performed RNA and protein expression studies, enzymatic activity assays, immunohistochemistry, and targeted sequencing of further MS cases from Austria, Germany, Canada and Jordan. RESULTS: In this study, we describe the identification of a homozygous missense mutation (c.82T>G, p.Cys28Gly) in the tripeptidyl peptidase II (TPP2) gene in all 3 affected siblings of the family. Sequencing of all TPP2-coding exons in 826 MS cases identified one further homozygous missense variant (c.2027C>T, p.Thr676Ile) in a Jordanian MS patient. TPP2 protein expression in whole blood was reduced in the affected siblings. In contrast, TPP2 protein expression in postmortem brain tissue from MS patients without TPP2 mutations was highly upregulated. CONCLUSIONS: The homozygous TPP2 mutation (p.Cys28Gly) is likely responsible for the inflammation phenotype in this family. TPP2 is an ubiquitously expressed serine peptidase that removes tripeptides from the N-terminal end of longer peptides. TPP2 is involved in various biological processes including the destruction of major histocompatibility complex Class I epitopes. Recessive loss-of-function mutations in TPP2 were described in patients with Evans syndrome, a rare autoimmune disease affecting the hematopoietic system. Based on the gene expression results in our MS autopsy brain samples, we further suggest that TPP2 may play a broader role in the inflammatory process in MS.

Lack of association between ABCC2 gene variants and treatment response in epilepsy.

AIM: The aim of this study was to replicate a previously reported association between drug resistance in epilepsy patients and the 24C>T variant of the ABCC2 gene that codes for the drug efflux transporter MRP2. PATIENTS & METHODS: We genotyped 381 Caucasian epileptic patients (337 drug resistant and 44 drug responsive) and 247 healthy controls for the ABCC2 gene -24C>T polymorphism (rs717620) and two other nearby SNPs in linkage disequilibrium (1249G>A and 3972C>T). Genotype, allele and three-SNP-haplotype frequencies were compared between groups. Patients were further stratified into four groups according to their degree of drug resistance (as measured by seizure frequency under medication) to perform regression analysis against genotypes and haplotpyes. RESULTS: We detected no significant differences in the distribution of any of the tested alleles, genotypes or haplotypes between the investigated groups. Neither was there an association between genotypes or haplotypes and degree of drug resistance. This study was adequately powered to detect genotype relative risks of above two. CONCLUSION: Although adequately powered to detect the previously reported effect size and although our definition of drug resistance, following the International League Against Epilepsy guidelines, was slightly stricter than in the original study, we failed to confirm an association between the 24C>T variant in the ABCC2 gene and drug resistance in epilepsy.

Variant in the sequence of the LINGO1 gene confers risk of essential tremor.

We identified a marker in LINGO1 showing genome-wide significant association (P = 1.2 x 10(-9), odds ratio = 1.55) with essential tremor. LINGO1 has potent, negative regulatory influences on neuronal survival and is also important in regulating both central-nervous-system axon regeneration and oligodendrocyte maturation. Increased axon integrity observed in Lingo1 mouse [corrected] knockout models highlights the potential role of LINGO1 in the pathophysiology of ET [corrected]

A novel LRRK2 mutation in an Austrian cohort of patients with Parkinson's disease.

To investigate the frequency of mutations in the Leucine-Rich Repeat Kinase 2 gene (LRRK2) in a sample of Austrian Parkinson's disease (PD) patients, we sequenced the complete coding region in 16 patients with autosomal dominant PD. Furthermore, we sequenced exons 31, 35, and 41 additionally in 146 patients with idiopathic PD and 30 patients with dementia with Lewy bodies. Furthermore, all 192 patients were screened for 21 putative LRRK2 mutations. While the most common mutation G2019S and the risk variant G2385R were not found in our samples, we detected a novel missense mutation (S973N) in a patient with familial, late-onset and dopa-responsive PD.

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen.

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.