Jasper Van Dongen

A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study

BACKGROUND Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are extremes of a clinically, pathologically, and genetically overlapping disease spectrum. A locus on chromosome 9p21 has been associated with both disorders, and we aimed to identify the causal gene within this region. METHODS We studied 305 patients with FTLD, 137 with ALS, and 23 with concomitant FTLD and ALS (FTLD-ALS) and 856 controls from Flanders (Belgium); patients were identified from a hospital-based cohort and were negative for mutations in known FTLD and ALS genes. We also examined the family of one patient with FTLD-ALS previously linked to 9p21 (family DR14). We analysed 130 kbp at 9p21 in association and segregation studies, genomic sequencing, repeat genotyping, and expression studies to identify the causal mutation. We compared genotype-phenotype correlations between mutation carriers and non-carriers. FINDINGS In the patient-control cohort, the single-nucleotide polymorphism rs28140707 within the 130 kbp region of 9p21 was associated with disease (odds ratio [OR] 2·6, 95% CI 1·5-4·7; p=0·001). A GGGGCC repeat expansion in C9orf72 completely co-segregated with disease in family DR14. The association of rs28140707 with disease in the patient-control cohort was abolished when we excluded GGGGCC repeat expansion carriers. In patients with familial disease, six (86%) of seven with FTLD-ALS, seven (47%) of 15 with ALS, and 12 (16%) of 75 with FTLD had the repeat expansion. In patients without known familial disease, one (6%) of 16 with FTLD-ALS, six (5%) of 122 with ALS, and nine (4%) of 230 with FTLD had the repeat expansion. Mutation carriers primarily presented with classic ALS (10 of 11 individuals) or behavioural variant FTLD (14 of 15 individuals). Mean age at onset of FTLD was 55·3 years (SD 8·4) in 21 mutation carriers and 63·2 years (9·6) in 284 non-carriers (p=0·001); mean age at onset of ALS was 54·5 years (9·9) in 13 carriers and 60·4 years (11·4) in 124 non-carriers. Postmortem neuropathological analysis of the brains of three mutation carriers with FTLD showed a notably low TDP-43 load. In brain at postmortem, C9orf72 expression was reduced by nearly 50% in two carriers compared with nine controls (p=0·034). In familial patients, 14% of FTLD-ALS, 50% of ALS, and 62% of FTLD was not accounted for by known disease genes. INTERPRETATION We identified a pathogenic GGGGCC repeat expansion in C9orf72 on chromosome 9p21, as recently also reported in two other studies. The GGGGCC repeat expansion is highly penetrant, explaining all of the contribution of chromosome 9p21 to FTLD and ALS in the Flanders-Belgian cohort. Decreased expression of C9orf72 in brain suggests haploinsufficiency as an underlying disease mechanism. Unidentified genes probably also contribute to the FTLD-ALS disease spectrum. FUNDING Full funding sources listed at end of paper (see Acknowledgments).

Investigating the role of rare heterozygous TREM2 variants in Alzheimer's disease and frontotemporal dementia

Homozygous mutations in exon 2 of TREM2, a gene involved in Nasu-Hakola disease, can cause frontotemporal dementia (FTD). Moreover, a rare TREM2 exon 2 variant (p.R47H) was reported to increase the risk of Alzheimers disease (AD) with an odds ratio as strong as that for APOEε4. We systematically screened the TREM2 coding region within a Belgian study on neurodegenerative brain diseases (1216 AD patients, 357 FTD patients, and 1094 controls). We observed an enrichment of rare variants across TREM2 in both AD and FTD patients compared to controls, most notably in the extracellular IgV-set domain (relative risk = 3.84 [95% confidence interval = 1.29-11.44]; p = 0.009 for AD; relative risk = 6.19 [95% confidence interval = 1.86-20.61]; p = 0.0007 for FTD). None of the rare variants individually reached significant association, but the frequency of p.R47H was increased ~ 3-fold in both AD and FTD patients compared to controls, in line with previous reports. Meta-analysis including 11 previously screened AD cohorts confirmed the association of p.R47H with AD (p = 2.93×10(-17)). Our data corroborate and extend previous findings to include an increased frequency of rare heterozygous TREM2 variations in AD and FTD, and show that TREM2 variants may play a role in neurodegenerative diseases in general.

C9orf72 G4C2 repeat expansions in Alzheimer's disease and mild cognitive impairment.

C9orf72 G4C2 repeat expansion is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Its role in Alzheimers disease (AD) is less clear. We assessed the prevalence of G4C2 pathogenic repeat expansions in Flanders-Belgian patients with clinical AD or mild cognitive impairment (MCI). In addition, we studied the effect of non-pathogenic G4C2 repeat length variability on susceptibility to AD, and on AD cerebrospinal fluid (CSF) biomarker levels. A pathogenic repeat expansion was identified in 5 of 1217 AD patients (frequency <1%). No pathogenic expansions were observed in patients with MCI (n = 200) or control individuals (n = 1119). Nonpathogenic repeat length variability was not associated with AD, risk of conversion to AD in MCI individuals, or CSF biomarker levels. We conclude that pathogenic C9orf72 G4C2 repeat expansions can be detected in clinical AD patients and could act as a contributor to AD pathogenesis. Non-pathogenic repeat length variability did not affect risk of AD or MCI, nor AD biomarker levels in CSF, indicating that C9orf72 is not a direct AD risk factor.

A 22-single nucleotide polymorphism Alzheimer's disease risk score correlates with family history, onset age, and cerebrospinal fluid Aβ42

The ability to identify individuals at increased genetic risk for Alzheimers disease (AD) may streamline biomarker and drug trials and aid clinical and personal decision making.

Complement receptor 1 coding variant p.Ser1610Thr in Alzheimer's disease and related endophenotypes

We previously described an intragenic functional copy number variation (CNV) in complement receptor 1 (CR1) that is associated with Alzheimer disease (AD) risk. A recent study, however, reported a rare CR1 coding variant p.Ser1610Thr (rs4844609) associated with AD susceptibility, explaining the effect of genome wide association (GWA) top single nucleotide polymorphism rs6656401. We assessed the role of the Ser1610Thr variant in AD pathogenesis and the effect on AD-related endophenotypes in a Flanders-Belgian cohort. We evaluated whether this rare variant rather than the CR1 CNV could explain the association of CR1 in our population. The Ser1610Thr variant was not associated with AD, memory impairment, total tau, amyloid β(1-42) or tau phosphorylated at threonine 181 levels. It did not explain (part of) the association of genome wide association top single-nucleotide polymorphisms rs3818361/rs6656401, nor of the CR1 CNV, with AD in our cohort, whereas the CR1 CNV and rs3818361/rs6656401 represented the same association signal. These findings question a role for the Ser1610Thr variant in AD risk and related endophenotypes, and reaffirm our previous observation that the CR1 CNV could be the true functional risk factor explaining the association between CR1 and AD.

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.

An intronic VNTR affects splicing of ABCA7 and increases risk of Alzheimer’s disease

Mutations leading to premature termination codons in ATP-Binding Cassette Subfamily A Member 7 (ABCA7) are high penetrant risk factors of Alzheimer’s disease (AD). The influence of other genetic variants in ABCA7 and downstream functional mechanisms, however, is poorly understood. To address this knowledge gap, we investigated tandem repetitive regions in ABCA7 in a Belgian cohort of 1529 AD patients and control individuals and identified an intronic variable number tandem repeat (VNTR). We observed strong association between VNTR length and a genome-wide associated signal for AD in the ABCA7 locus. Expanded VNTR alleles were highly enriched in AD patients [odds ratio = 4.5 (1.3–24.2)], and VNTR length inversely correlated with amyloid β1–42 in cerebrospinal fluid and ABCA7 expression. In addition, we identified three novel ABCA7 alternative splicing events. One isoform in particular—which is formed through exon 19 skipping—lacks the first nucleotide binding domain of ABCA7 and is abundant in brain tissue. We observed a tight correlation between exon 19 skipping and VNTR length. Our findings underline the importance of studying repetitive DNA in complex disorders and expand the contribution of genetic and transcript variation in ABCA7 to AD.

DELETERIOUS ABCA7 MUTATIONS CONTRIBUTE TO EARLY-ONSET ALZHEIMER’S DISEASE AND ARE SUBJECT TO TRANSCRIPT RESCUE MECHANISMS

Background:Genes that are expressed in a cell-specific fashion in the human brain were identified from transcriptome-wide (RNAseq) expression measures of purified cell populations (Zhang Y. et al, Neuron, 2016). Leveraging these findings, we estimated the relative proportions of cell-types in a temporal cortex (TCX), tissue-based, transcriptome dataset (RNAseq: Allen M. et al, Sci Data, 2016) of 268 subjects with a pathological diagnosis of Alzheimer’s disease (AD; N1⁄480), progressive supranuclear palsy (PSP; N1⁄482), pathologic aging (PA; N1⁄430) or normal controls (CON; N1⁄476), to identify disease relevant changes and cell-specific vulnerabilities. Methods:We generated expression residuals, adjusting for appropriate covariates, for cell–specific genes (Zhang. Y et al, Neuron 2016) expressed in our dataset (neurons1⁄4415; astrocytes1⁄4234; oligodendroglia1⁄4117; microglia1⁄4352, endothelia1⁄498 genes). We used principal components analysis to generate a surrogate variable (signed eigenvector 1: EV1) representing cell-specific transcripts for each cell type. Each cell-specific EV1 was used as a quantitative variable to test for association with diagnosis, age, sex or APOEε4 genotype (linear regression, R software). Pairwise correlations were tested between all cell-specific EV1 levels (Pearson correlation, R). Analyses were done on the complete cohort, adjusting for diagnosis, and for each individual diagnostic group. We also performed a genome-wide association study (GWAS; linear regression, PLINK) for each cellspecific EV1 on the complete cohort. Results: Significant positive correlations (r2>0.5; p<2.2E-16) were identified between endothelial EV1 and astrocyte or microglia EV1 levels. Neuronal EV1 was negatively correlated with EV1 levels for all other cell types. These correlations were similar across the diagnostic groups. EV1 for astrocytes, microglia, and endothelia were higher, and for neurons lower, in AD subjects compared to other groups, with similar trends observed also for female sex and APOEε4. GWAS did not identify genome-wide significant variants (p<1E08), although suggestive associations include variants at the TMEM106B locus with astrocytes (p1⁄49.0E-07), and an intergenic locus (Chr2) flanked by NXPH2 and LRP1B(p1⁄42.1E-08) with microglia. Conclusions:We have identified brain cell type variability across different diagnosis groups and genetic associations that may influence these. These findings imply specific vulnerabilities for different cell types. We are performing similar analyses in cerebellum, and additional human and mouse brain transcriptome datasets.

TRANSCRIPTOME ANALYSIS IN BLOOD AND BRAIN IDENTIFIES GENE EXPRESSION REGULATION AND CORRESPONDING QUANTITATIVE TRAIT LOCI IN ALZHEIMER’S DISEASE

Background: TDP-43 (transactive response DNA-binding protein 43kDa) proteinopathy is closely associated with Alzheimer’s disease (AD), and affects clinical outcome in AD. TMEM106B rs1990622is a risk allele for TDP-43 proteinopathy, and its implications in clinical presentations of AD remains to be elucidated. Methods: Our subjects are from Religious Orders Study and the Rush Memory and Aging Project (ROS-MAP), two communitybased longitudinal cohort studies of older adults. Our analyses included 841 subjects with genotyping, cognitive, and pathology data (beta-amyloid, paired helical filament tau, and TDP-43 stage). Cognitive decline was defined as the slope of change in composite cognitive z-score in a general linear mixed model, controlling for age at enrollment, sex, and education. The imputed dosage of TMEM106B rs1990622 was tested for its association with cognitive decline after adjusting for AD pathology. Then, TDP-43 was included in the model, and a subsequent mediation analysis (quasi-Bayesian Monte Carlo method with 10,000 simulations) was done to evaluate whether the TMEM106Bvariant’s effect was mediated by TDP-43 proteinopathy. Of note, all analyses were adjusted for study cohort (ROS vs MAP), genotyping platform, and first three principal components from the genotype covariance matrix. Results:TMEM106B rs1990622was associated with more rapid cognitive decline when controlled for the AD pathology (EE1⁄4-0.0098, 95% confidence interval (CI) -0.018 to -0.0016, p1⁄40.020). When TDP-43 stage was also controlled, the association of rs1990622 with cognitive decline loses statistical significance (p1⁄40.078). A mediation analysis shows that about 15% of the association between rs1990622 and more rapid cognitive decline is mediated by more advanced TDP-43 stages (average causal mediation effect p<0.0001). Conclusions: TMEM106B rs1990622, a known TDP-43 proteinopathy risk variant, is associated with more rapid cognitive decline in people with similar degree of AD pathology, and this association is mediated by more advanced TDP-43 stages. Nonetheless, the association of rs1990622 with more rapid cognitive decline in AD is only partially explained by the link between rs1990622 and more advanced TDP-43 stages.