Tim De Pooter

Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21.

Frontotemporal dementia (FTD) with ubiquitin-immunoreactive neuronal inclusions (both cytoplasmic and nuclear) of unknown nature has been linked to a chromosome 17q21 region (FTDU-17) containing MAPT (microtubule-associated protein tau). FTDU-17 patients have consistently been shown to lack a tau-immunoreactive pathology, a feature characteristic of FTD with parkinsonism linked to mutations in MAPT (FTDP-17). Furthermore, in FTDU-17 patients, mutations in MAPT and genomic rearrangements in the MAPT region have been excluded by both genomic sequencing and fluorescence in situ hybridization on mechanically stretched chromosomes. Here we demonstrate that FTDU-17 is caused by mutations in the gene coding for progranulin (PGRN), a growth factor involved in multiple physiological and pathological processes including tumorigenesis. Besides the production of truncated PGRN proteins due to premature stop codons, we identified a mutation within the splice donor site of intron 0 (IVS0 + 5G > C), indicating loss of the mutant transcript by nuclear degradation. The finding was made within an extensively documented Belgian FTDU-17 founder family. Transcript and protein analyses confirmed the absence of the mutant allele and a reduction in the expression of PGRN. We also identified a mutation (c.3G > A) in the Met1 translation initiation codon, indicating loss of PGRN due to lack of translation of the mutant allele. Our data provide evidence that PGRN haploinsufficiency leads to neurodegeneration because of reduced PGRN-mediated neuronal survival. Furthermore, in a Belgian series of familial FTD patients, PGRN mutations were 3.5 times more frequent than mutations in MAPT, underscoring a principal involvement of PGRN in FTD pathogenesis.

α-Synuclein promoter confers susceptibility to Parkinson's disease

Familial Parkinsons disease (PD) has been linked to missense and genomic multiplication mutations of the α‐synuclein gene (SNCA). Genetic variability within SNCA has been implicated in idiopathic PD in many populations. We now confirm and extend these findings, within a Belgian sample, using a high‐resolution map of genetic markers across the SNCA locus. Our study implicates the SNCA promoter in susceptibility to PD, and more specifically defines a minimum promoter haplotype, spanning approximately 15.3kb of sequence, which is overrepresented in patients. Our findings represent a biomarker for PD and may have implications for patient diagnosis, longitudinal evaluation, and treatment. Ann Neurol 2004;56:591–595

Neuronal inclusion protein TDP-43 has no primary genetic role in FTD and ALS.

The nuclear TAR DNA binding protein (TDP-43) is deposited in ubiquitin-positive inclusions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two clinicopathologically overlapping neurodegenerative diseases. In this study we excluded mutations and copy number variations in the gene encoding TDP-43 (TARDBP) from an extended series of 173 FTD and 237 ALS patients. Further, we did not identify association of common genetic variants in these patients. Our data implicate that TDP-43 has no primary genetic role in the pathophysiological mechanisms underlying central nervous system neurodegeneration in these diseases.

Linkage and Association Studies Identify a Novel Locus for Alzheimer Disease at 7q36 in a Dutch Population-Based Sample

We obtained conclusive linkage of Alzheimer disease (AD) with a candidate region of 19.7 cM at 7q36 in an extended multiplex family, family 1270, ascertained in a population-based study of early-onset AD in the northern Netherlands. Single-nucleotide polymorphism and haplotype association analyses of a Dutch patient-control sample further supported the linkage at 7q36. In addition, we identified a shared haplotype at 7q36 between family 1270 and three of six multiplex AD-affected families from the same geographical region, which is indicative of a founder effect and defines a priority region of 9.3 cM. Mutation analysis of coding exons of 29 candidate genes identified one linked synonymous mutation, g.38030G-->C in exon 10, that affected codon 626 of the PAX transactivation domain interacting protein gene (PAXIP1). It remains to be determined whether PAXIP1 has a functional role in the expression of AD in family 1270 or whether another mutation at this locus explains the observed linkage and sharing. Together, our linkage data from the informative family 1270 and the association data in the population-based early-onset AD patient-control sample strongly support the identification of a novel AD locus at 7q36 and re-emphasize the genetic heterogeneity of AD.

Founder mutation p.R1441C in the leucine-rich repeat kinase 2 gene in Belgian Parkinson's disease patients

We determined the prevalence of mutations in two major functional domains of the leucine-rich repeat kinase 2 gene (LRRK2) in Belgian Parkinsons disease (PD) patients (N=304) of which 18.1% were familial PD patients. Ten patients were heterozygous for five different missense mutations (3.29%) of whom six carried the same mutation p.R1441C (1.97%). All six p.R1441C carriers were familial PD patients explaining 10.7% of familial PD in the Belgian patient group. Moreover, they shared a common disease haplotype of 21 consecutive markers in a region of 438 kb, suggesting that they are distant descendants of a single common ancestor. Clinically, p.R1441C carriers had typical levodopa-responsive parkinsonism with tremor as the most common presenting feature. Their age at onset was highly variable and ranged from 39 to 73 years, suggesting the influence of modifying factors. The remaining four patients were heterozygous each for a novel missense mutation located in the Roc or kinase domain. The pathogenic nature of these mutations remains to be determined, though we have genetic evidence that at least some represent rare but benign variants rather than causal mutations. The latter observation indicates that prudence is needed in diagnostic testing of LRRK2 in PD patients. Functional data should underlie a conclusion on the pathogenic nature of some mutations that have not been conclusively linked to disease.

Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome

We sequenced the Yoruban NA19240 genome on the long read sequencing platform Oxford Nanopore PromethION for benchmarking and evaluation of recently published aligners and structural variant calling tools. In this work, we determined the precision and recall, present high confidence and high sensitivity call sets of variants and discuss optimal parameters. The aligner Minimap2 and structural variant caller Sniffles are both the most accurate and the most computationally efficient tools in our study. We describe our scalable workflow for identification, annotation, and characterization of tens of thousands of structural variants from long read genome sequencing of an individual or population. By discussing the results of this genome we provide an approximation of what can be expected in future long read sequencing studies aiming for structural variant identification.

Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION.

Tandem repeats (TRs) can cause disease through their length, sequence motif interruptions, and nucleotide modifications. For many TRs, however, these features are very difficult - if not impossible - to assess, requiring low-throughput and labor-intensive assays. One example is a VNTR in ABCA7 for which we recently discovered that expanded alleles strongly increase risk of Alzheimer’s disease. Here, we investigated the potential of long-read whole genome sequencing to surmount these challenges, using the high-throughput PromethION platform from Oxford Nanopore Technologies. To overcome the limitations of conventional base calling and alignment, we developed an algorithm to study the TR size and sequence directly on raw PromethION current data. We report the long-read sequencing of multiple human genomes (n = 11) using only a single sequencing run and flow cell per individual. With the use of fresh DNA extractions, DNA shearing to approximately 20kb and size selection, we obtained an average output of 70 gigabases (Gb) per flow cell, corresponding to a 21x genome coverage, and a maximum yield of 98 Gb (30x genome coverage). All ABCA7 VNTR alleles, including expansions up to 10,000 bases, were spanned by long sequencing reads, validated by Southern blotting. Classical approaches of TR length estimation suffered from low accuracy, low precision, DNA strand effects and/or inability to call pathogenic repeat expansions. In contrast, our novel NanoSatellite algorithm, which circumvents base calling by using dynamic time warping on raw PromethION current data, achieved more than 90% accuracy and high precision (5.6% relative standard deviation) of TR length estimation, and detected all clinically relevant repeat expansions. In addition, we identified alternative TR sequence motifs with high consistency, allowing determination of TR sequence and distinction of VNTR alleles with homozygous length. In conclusion, we validated the robustness of single-experiment whole genome long-read sequencing on PromethION, a prerequisite for application of long-read sequencing in the clinic. In addition, we outperformed Southern blotting, enabling improved characterization of the role of expanded ABCA7 VNTR alleles in Alzheimer’s disease, and opening new opportunities for TR research.

Human structural variation identified by Oxford Nanopore PromethION

1 Chaisson, Mark J. P., Ashley D. Sanders, Xuefang Zhao, Ankit Malhotra, David Porubsky, Tobias Rausch, Eugene J. Gardner, et al. 2017. “Multi-Platform​​ Discovery​​ Of​​ Haplotype-Resolved Structural​​ Variation​​ In​​ Human​​ Genomes.” bioRxiv. 2 De Coster, Wouter, Svenn D’Hert, Darrin T. Schultz, Marc Cruts, and Christine Van Broeckhoven. 2018. “NanoPack: Visualizing and Processing Long Read Sequencing Data.” Bioinformatics Introduction The majority of the structural variants in the genome, defined as changes in copy number or location of elements > 50 bp, remain hidden with currently dominant technologies. Long read sequencing has the advantage of a higher mappability, the ability to span breakpoints and align uniquely to repetitive sequences. For benchmarking and evaluation of tools we have sequenced the Yoruban reference genome NA19240, part of the HapMap and 1000 genomes project and well characterized using modern technologies1, allowing independent validation of our findings. We reached 258 gigabase or 80x coverage and our data is publicly available on ENA (PRJEB26791). Disclaimer: we have received consumables from ONT for this project and have been reimbursed for presenting at conferences.

O2-05-02: VCP mutation in frontotemporal lobar degeneration with frequent TDP-43–positive intranuclear inclusions

chymotrypsin (ACT), complement factors, and cytokines with the plaques led to the hypothesis that these inflammatory proteins play a role in the development of AD pathology. Studies from our lab and others have shown that ACT induces accelerated plaque formation and cognitive deficit in APP transgenic mice. Recently we showed that ACT also induces tau hyperphorylation and these tau epitopes are similar to the ones that are associated with neurofibrillary tangles. Methods: In vitro studies with ACT were done in cultured cortical neurons. Tau phosphorylation in cells and sections were determined by immunohistochemistry. In vivo studies were done in mice expressing APP, human tau or ACT transgenes. ACT infusion was done using alzet pump. Results: To determine whether the effect of ACT on tau hyperphosphorylation was A-beta dependent, we performed experiments in cultured neurons from APP knockout mice. We found that ACT induces tau hyperphosphorylation in these neurons suggesting that the effect of ACT is not A-beta dependent. To study the in vivo effect of ACT, it was infused into the hippocampus on one hemisphere of the mouse brain while the other hemisphere received artificial CSF for the same period of time. After two weeks of infusion the mice were sacrificed and tau phosphorylation determined using specific P-tau antibodies. We found that the hemisphere that received ACT showed an induction of tau hyperphosphorylation compared to the hemisphere that received artificial CSF. This further confirms that inflammatory proteins, namely ACT induces tangle pathology. To further understand the mechanisms involved in ACT’s effect on tau, transgenic mice overexpressing human tau in a mouse tau null background (htau) were crossed with human ACT expressing mice. Preliminary studies suggest that mice expressing htau/ACT show increased tau hyperphosphorylation compared to age-matched htau only mice. Conclusions: ACT induces tau hyperphosphorylation in vitro and in vivo independent of A-beta. htau/ACT mice are excellent models for studying age-dependent changes in inflammation-induced neurodegeneration as well as assessing the efficacy of anti-inflammatory drugs.