Association of Polygenic Risk Score With Lifetime Risk of Developing Multiple Sclerosis in a Population-Based Birth-Year Cohort.

BACKGROUND AND OBJECTIVES: More than 200 genetic variants have been associated with multiple sclerosis (MS) susceptibility. However, it is unclear to what extent genetic factors influence lifetime risk of MS. Using a population-based birth-year cohort, we investigate the effect of genetics on lifetime risk of MS. METHODS: In the Project Y study, we tracked down almost all persons with MS (pwMS) from birth year 1966 in the Netherlands. As control participants, we included non-MS participants from the Project Y cohort (born 1965-1967 in the Netherlands) and non-MS participants from the Amsterdam Dementia Cohort born between 1963 and 1969. Genetic variants associated with MS were determined in pwMS and control participants using genotyping or imputation methods. Polygenic risk scores (PRSs) based on variants and weights from the largest genetic study in MS were calculated for each participant and assigned into deciles based on the PRS distribution in the control participants. We examined the lifetime risk for each decile and the association between PRS and MS disease variables, including age at onset and time to secondary progression. RESULTS: MS-PRS was calculated for 285 pwMS (mean age 53.0 ± 0.9 years, 72.3% female) and 267 control participants (mean age 51.8 ± 3.2 years, 58.1% female). Based on the lifetime risk estimation, we observed that 1:2,739 of the women with the lowest 30% genetic risk developed MS, whereas 1:92 of the women with the top 10% highest risk developed MS. For men, only 1:7,900 developed MS in the lowest 30% genetic risk group, compared with 1:293 men with the top 10% genetic risk. The PRS was not significantly associated with age at onset and time to secondary progression in both sexes. DISCUSSION: Our results show that the lifetime risk of MS is strongly influenced by genetic factors. Our findings have the potential to support diagnostic certainty in individuals with suspected MS: a high PRS could strengthen a diagnosis, but especially a PRS from the lowest tail of the PRS distribution should be considered a red flag and could prevent misdiagnosing conditions that mimic MS.

The SORL1 p.Y1816C variant causes impaired endosomal dimerization and autosomal dominant Alzheimer's disease.

Truncating genetic variants of SORL1, encoding the endosome recycling receptor SORLA, have been accepted as causal of Alzheimer's disease (AD). However, most genetic variants observed in SORL1 are missense variants, for which it is complicated to determine the pathogenicity level because carriers come from pedigrees too small to be informative for penetrance estimations. Here, we describe three unrelated families in which the SORL1 coding missense variant rs772677709, that leads to a p.Y1816C substitution, segregates with Alzheimer's disease. Further, we investigate the effect of SORLA p.Y1816C on receptor maturation, cellular localization, and trafficking in cell-based assays. Under physiological circumstances, SORLA dimerizes within the endosome, allowing retromer-dependent trafficking from the endosome to the cell surface, where the luminal part is shed into the extracellular space (sSORLA). Our results showed that the p.Y1816C mutant impairs SORLA homodimerization in the endosome, leading to decreased trafficking to the cell surface and less sSORLA shedding. These trafficking defects of the mutant receptor can be rescued by the expression of the SORLA 3Fn-minireceptor. Finally, we find that iPSC-derived neurons with the engineered p.Y1816C mutation have enlarged endosomes, a defining cytopathology of AD. Our studies provide genetic as well as functional evidence that the SORL1 p.Y1816C variant is causal for AD. The partial penetrance of the mutation suggests this mutation should be considered in clinical genetic screening of multiplex early-onset AD families.

Genome wide association study of clinical duration and age at onset of sporadic CJD.

Human prion diseases are rare, transmissible and often rapidly progressive dementias. The most common type, sporadic Creutzfeldt-Jakob disease (sCJD), is highly variable in clinical duration and age at onset. Genetic determinants of late onset or slower progression might suggest new targets for research and therapeutics. We assembled and array genotyped sCJD cases diagnosed in life or at autopsy. Clinical duration (median:4, interquartile range (IQR):2.5-9 (months)) was available in 3,773 and age at onset (median:67, IQR:61-73 (years)) in 3,767 cases. Phenotypes were successfully transformed to approximate normal distributions allowing genome-wide analysis without statistical inflation. 53 SNPs achieved genome-wide significance for the clinical duration phenotype; all of which were located at chromosome 20 (top SNP rs1799990, pvalue = 3.45x10-36, beta = 0.34 for an additive model; rs1799990, pvalue = 9.92x10-67, beta = 0.84 for a heterozygous model). Fine mapping, conditional and expression analysis suggests that the well-known non-synonymous variant at codon 129 is the obvious outstanding genome-wide determinant of clinical duration. Pathway analysis and suggestive loci are described. No genome-wide significant SNP determinants of age at onset were found, but the HS6ST3 gene was significant (pvalue = 1.93 x 10-6) in a gene-based test. We found no evidence of genome-wide genetic correlation between case-control (disease risk factors) and case-only (determinants of phenotypes) studies. Relative to other common genetic variants, PRNP codon 129 is by far the outstanding modifier of CJD survival suggesting only modest or rare variant effects at other genetic loci.

Connecting dementia risk loci to the CSF proteome identifies pathophysiological leads for dementia.

Genome-wide association studies have successfully identified many genetic risk loci for dementia, but exact biological mechanisms through which genetic risk factors contribute to dementia remains unclear. Integrating CSF proteomic data with dementia risk loci could reveal intermediate molecular pathways connecting genetic variance to the development of dementia. We tested to what extent effects of known dementia risk loci can be observed in CSF levels of 665 proteins (proximity extension-based (PEA) immunoassays) in a deeply-phenotyped mixed-memory clinic cohort (n=502, mean age (sd) = 64.1 [8.7] years, 181 female [35.4%]), including patients with Alzheimer's disease (AD, n=213), dementia with Lewy bodies (DLB, n=50) and frontotemporal dementia (FTD, n=93), and controls (n=146). Validation was assessed in independent cohorts (n=99 PEA platform, n=198, MRM-targeted mass spectroscopy and multiplex assay). We performed additional analyses stratified according to diagnostic status (AD, DLB, FTD and controls separately), to explore whether associations between CSF proteins and genetic variants were specific to disease or not. We identified four AD risk loci as protein quantitative trait loci (pQTL): CR1-CR2 (rs3818361, P=1.65e-08), ZCWPW1-PILRB (rs1476679, P=2.73e-32), CTSH-CTSH (rs3784539, P=2.88e-24) and HESX1-RETN (rs186108507, P=8.39e-08), of which the first three pQTLs showed direct replication in the independent cohorts. We identified one AD-specific association between a rare genetic variant of TREM2 and CSF IL6 levels (rs75932628, P = 3.90e-7). DLB risk locus GBA showed positive trans effects on seven inter-related CSF levels in DLB patients only. No pQTLs were identified for frontotemporal dementia, either for the total sample as for analyses performed within FTD only. pQTL variants were involved in the immune system, highlighting the importance of this system in the pathophysiology of dementia. We further identified pQTLs in stratified analyses for AD and DLB, hinting at disease-specific pQTLs in dementia. Dissecting the contribution of risk loci to neurobiological processes aids in understanding disease mechanisms underlying dementia.

Interest in genetic susceptibility testing and disclosure of AD dementia risk in cognitively normal adults: a survey study.

BACKGROUND: Apolipoprotein-E (APOE) genetic testing for Alzheimer's disease is becoming more important as clinical trials are increasingly targeting individuals carrying APOE-ε4 alleles. Little is known about the interest in finding out one's genetic risk for Alzheimer's disease in the general population. Our objective was to examine this in a sample of cognitively normal (CN) adults within a population-based online research registry with the goal to implement APOE-ε4 status for trial recruitment. METHODS: An online survey was completed by 442 CN participants between the age of 49 and 75 years (56% female) from the Dutch Brain Research Registry. The survey assessed interest in participation in research into, and disclosure of, genetic risk for dementia. The survey assessed interest in participation in research into, and disclosure of, genetic risk for dementia and knowing their genetic risk in different hypothetical risk scenarios (10%, 30%, and 50% genetic risk for dementia at age 85, corresponding to APOEε2/ε2 or ε2/ε3, APOEε3/ε4 or ε2ε4, and APOE-ε4/ε4 genotypes). Cochran's Q and post hoc McNemar tests were used to analyse differences in frequencies across scenarios. RESULTS: Most participants were interested in participating in research into and disclosure of their genetic risk (81%). The most reported reason was to contribute to scientific research (94%). Interest was higher in males, whilst lower-educated participants were more often undecided. When provided with different risk scenarios, interest in knowing their risk was somewhat higher in the scenarios with higher risk, i.e. in the 50% (79%) compared to the 10% scenario (73%;χ2(2) = 7.98; p = .005). Most individuals expected they would share their genetic risk with close relatives (77-89%), would participate in medication trials (79-88%), and would make long-term arrangements, e.g. retirement, health care, will (69-82%), with larger proportions for scenarios with higher hypothetical genetic risk. CONCLUSIONS: Our findings indicate that the vast majority of CN adults participating in a research registry expresses interest in AD genetic risk research and disclosure. Interest in genetic risk disclosure is higher in scenarios corresponding to the APOE-ε4 genotype. This suggests APOE-ε4 screening within an online research registry is potentially a well-received method to accelerate inclusion for trials.

Quantitative trait loci mapping of circulating metabolites in cerebrospinal fluid to uncover biological mechanisms involved in brain-related phenotypes.

Genomic studies of molecular traits have provided mechanistic insights into complex disease, though these lag behind for brain-related traits due to the inaccessibility of brain tissue. We leveraged cerebrospinal fluid (CSF) to study neurobiological mechanisms in vivo , measuring 5,543 CSF metabolites, the largest panel in CSF to date, in 977 individuals of European ancestry. Individuals originated from two separate cohorts including cognitively healthy subjects (n=490) and a well-characterized memory clinic sample, the Amsterdam Dementia Cohort (ADC, n=487). We performed metabolite quantitative trait loci (mQTL) mapping on CSF metabolomics and found 126 significant mQTLs, representing 65 unique CSF metabolites across 51 independent loci. To better understand the role of CSF mQTLs in brain-related disorders, we performed a metabolome-wide association study (MWAS), identifying 40 associations between CSF metabolites and brain traits. Similarly, over 90% of significant mQTLs demonstrated colocalized associations with brain-specific gene expression, unveiling potential neurobiological pathways.

Amyloid-ß and APOE genotype predict memory decline in cognitively unimpaired older individuals independently of Alzheimer's disease polygenic risk score.

BACKGROUND: What combination of risk factors for Alzheimer's disease (AD) are most predictive of cognitive decline in cognitively unimpaired individuals remains largely unclear. We studied associations between APOE genotype, AD-Polygenic Risk Scores (AD-PRS), amyloid-ß pathology and decline in cognitive functioning over time in a large sample of cognitively unimpaired older individuals. METHODS: We included 276 cognitively unimpaired older individuals (75 ± 10 years, 63% female) from the EMIF-AD PreclinAD cohort. An AD-PRS was calculated including 83 genome-wide significant variants. The APOE gene was not included in the PRS and was analyzed separately. Baseline amyloid-ß status was assessed by visual read of [18F]flutemetamol-PET standardized uptake value images. At baseline and follow-up (2.0 ± 0.4 years), the cognitive domains of memory, attention, executive function, and language were measured. We used generalized estimating equations corrected for age, sex and center to examine associations between APOE genotype and AD-PRS with amyloid-ß status. Linear mixed models corrected for age, sex, center and education were used to examine associations between APOE genotype, AD-PRS and amyloid-ß status, and their interaction on changes in cognitive functioning over time. RESULTS: Fifty-two participants (19%) had abnormal amyloid-ß, and 84 participants (31%) carried at least one APOE ε4 allele. APOE genotype and AD-PRS were both associated with abnormal amyloid-ß status. Increasingly more risk-full APOE genotype, a high AD-PRS and an abnormal amyloid-ß status were associated with steeper decline in memory functioning in separate models (all p ≤ 0.02). A model including 4-way interaction term (APOE×AD-PRS×amyloid-ß×time) was not significant. When modelled together, both APOE genotype and AD-PRS predicted steeper decline in memory functioning (APOE ß(SE)=-0.05(0.02); AD-PRS ß(SE)=-0.04(0.01)). Additionally, when modelled together, both amyloid-ß status and AD-PRS predicted a steeper decline in memory functioning (amyloid-ß ß(SE)=-0.07(0.04); AD-PRS ß(SE)=-0.04(0.01)). Modelling both APOE genotype and amyloid-ß status, we observed an interaction, in which APOE genotype was related to steeper decline in memory and language functioning in amyloid-ß abnormal individuals only (ß(SE)=-0.13(0.06); ß(SE)=-0.22(0.07), respectively). CONCLUSION: Our results suggest that APOE genotype is related to steeper decline in memory and language functioning in individuals with abnormal amyloid-ß only. Furthermore, independent of amyloid-ß status other genetic risk variants contribute to memory decline in initially cognitively unimpaired older individuals.

Genome-wide meta-analysis for Alzheimer's disease cerebrospinal fluid biomarkers.

Amyloid-beta 42 (Aß42) and phosphorylated tau (pTau) levels in cerebrospinal fluid (CSF) reflect core features of the pathogenesis of Alzheimer's disease (AD) more directly than clinical diagnosis. Initiated by the European Alzheimer & Dementia Biobank (EADB), the largest collaborative effort on genetics underlying CSF biomarkers was established, including 31 cohorts with a total of 13,116 individuals (discovery n = 8074; replication n = 5042 individuals). Besides the APOE locus, novel associations with two other well-established AD risk loci were observed; CR1 was shown a locus for Aß42 and BIN1 for pTau. GMNC and C16orf95 were further identified as loci for pTau, of which the latter is novel. Clustering methods exploring the influence of all known AD risk loci on the CSF protein levels, revealed 4 biological categories suggesting multiple Aß42 and pTau related biological pathways involved in the etiology of AD. In functional follow-up analyses, GMNC and C16orf95 both associated with lateral ventricular volume, implying an overlap in genetic etiology for tau levels and brain ventricular volume.

Mapping the genetic landscape of early-onset Alzheimer's disease in a cohort of 36 families.

BACKGROUND: Many families with clinical early-onset Alzheimer's disease (EOAD) remain genetically unexplained. A combination of genetic factors is not standardly investigated. In addition to monogenic causes, we evaluated the possible polygenic architecture in a large series of families, to assess if genetic testing of familial EOAD could be expanded. METHODS: Thirty-six pedigrees (77 patients) were ascertained from a larger cohort of patients, with relationships determined by genetic data (exome sequencing data and/or SNP arrays). All families included at least one AD patient with symptom onset <70 years. We evaluated segregating rare variants in known dementia-related genes, and other genes or variants if shared by multiple families. APOE was genotyped and duplications in APP were assessed by targeted test or using SNP array data. We computed polygenic risk scores (PRS) compared with a reference population-based dataset, by imputing SNP arrays or exome sequencing data. RESULTS: In eight families, we identified a pathogenic variant, including the genes APP, PSEN1, SORL1, and an unexpected GRN frameshift variant. APOE-ε4 homozygosity was present in eighteen families, showing full segregation with disease in seven families. Eight families harbored a variant of uncertain significance (VUS), of which six included APOE-ε4 homozygous carriers. PRS was not higher in the families combined compared with the population mean (beta 0.05, P = 0.21), with a maximum increase of 0.61 (OR = 1.84) in the GRN family. Subgroup analyses indicated lower PRS in six APP/PSEN1 families compared with the rest (beta -0.22 vs. 0.10; P = 0.009) and lower APOE burden in all eight families with monogenic cause (beta 0.29 vs. 1.15, P = 0.010). Nine families remained without a genetic cause or risk factor identified. CONCLUSION: Besides monogenic causes, we suspect a polygenic disease architecture in multiple families based on APOE and rare VUS. The risk conveyed by PRS is modest across the studied families. Families without any identified risk factor render suitable candidates for further in-depth genetic evaluation.

New insights into the genetic etiology of Alzheimer's disease and related dementias.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele.

Cerebrospinal fluid tau levels are associated with abnormal neuronal plasticity markers in Alzheimer's disease.

BACKGROUND: Increased total tau (t-tau) in cerebrospinal fluid (CSF) is a key characteristic of Alzheimer's disease (AD) and is considered to result from neurodegeneration. T-tau levels, however, can be increased in very early disease stages, when neurodegeneration is limited, and can be normal in advanced disease stages. This suggests that t-tau levels may be driven by other mechanisms as well. Because tau pathophysiology is emerging as treatment target for AD, we aimed to clarify molecular processes associated with CSF t-tau levels. METHODS: We performed a proteomic, genomic, and imaging study in 1380 individuals with AD, in the preclinical, prodromal, and mild dementia stage, and 380 controls from the Alzheimer's Disease Neuroimaging Initiative and EMIF-AD Multimodality Biomarker Discovery study. RESULTS: We found that, relative to controls, AD individuals with increased t-tau had increased CSF concentrations of over 400 proteins enriched for neuronal plasticity processes. In contrast, AD individuals with normal t-tau had decreased levels of these plasticity proteins and showed increased concentrations of proteins indicative of blood-brain barrier and blood-CSF barrier dysfunction, relative to controls. The distinct proteomic profiles were already present in the preclinical AD stage and persisted in prodromal and dementia stages implying that they reflect disease traits rather than disease states. Dysregulated plasticity proteins were associated with SUZ12 and REST signaling, suggesting aberrant gene repression. GWAS analyses contrasting AD individuals with and without increased t-tau highlighted several genes involved in the regulation of gene expression. Targeted analyses of SNP rs9877502 in GMNC, associated with t-tau levels previously, correlated in individuals with AD with CSF concentrations of 591 plasticity associated proteins. The number of APOE-e4 alleles, however, was not associated with the concentration of plasticity related proteins. CONCLUSIONS: CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and blood-brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF proteomic profiles.

Association of low-frequency and rare coding variants with information processing speed.

Measures of information processing speed vary between individuals and decline with age. Studies of aging twins suggest heritability may be as high as 67%. The Illumina HumanExome Bead Chip genotyping array was used to examine the association of rare coding variants with performance on the Digit-Symbol Substitution Test (DSST) in community-dwelling adults participating in the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. DSST scores were available for 30,576 individuals of European ancestry from nine cohorts and for 5758 individuals of African ancestry from four cohorts who were older than 45 years and free of dementia and clinical stroke. Linear regression models adjusted for age and gender were used for analysis of single genetic variants, and the T5, T1, and T01 burden tests that aggregate the number of rare alleles by gene were also applied. Secondary analyses included further adjustment for education. Meta-analyses to combine cohort-specific results were carried out separately for each ancestry group. Variants in RNF19A reached the threshold for statistical significance (p = 2.01 × 10-6) using the T01 test in individuals of European descent. RNF19A belongs to the class of E3 ubiquitin ligases that confer substrate specificity when proteins are ubiquitinated and targeted for degradation through the 26S proteasome. Variants in SLC22A7 and OR51A7 were suggestively associated with DSST scores after adjustment for education for African-American participants and in the European cohorts, respectively. Further functional characterization of its substrates will be required to confirm the role of RNF19A in cognitive function.

BDNF-Met polymorphism and amyloid-beta in relation to cognitive decline in cognitively normal elderly: the SCIENCe project.

Brain-derived neurotrophic factor (BNDF) plays a role in synapse integrity. We investigated in 398 cognitively normal adults (60±8years, 41% female, MMSE=28±1) the joint association of the Val66Met polymorphism of the BDNF gene (Met+/-) and plasma BDNF levels and abnormal cerebrospinal fluid (CSF) amyloid-beta status (A+/-) with cognitive decline and dementia risk. Age-, sex- and education-adjusted linear mixed models showed that compared to Met-A-, Met+A+ showed steeper decline on tests of global cognition, memory, language, attention and executive functioning, while Met-A+ showed steeper decline on a smaller number of tests. There were no associations between Met+A- and cognitive decline. Cox models showed that compared to Met-A-, Met+A+ participants were at increased risk of dementia (HR=8.8, 95%CI: 2.8-27.9), as were Met-A+ participants (HR=6.5, 95%CI: 2.2-19.5). Lower plasma BDNF was associated with an increased risk of progression to dementia in the A+ participants. Our results imply that Met-carriage on top of amyloid-beta pathology might increase rate of cognitive decline to dementia.

Differences in Sex Distribution Between Genetic and Sporadic Frontotemporal Dementia.

BACKGROUND: Reported sex distributions differ between frontotemporal dementia (FTD) cohorts. Possible explanations are the evolving clinical criteria of FTD and its subtypes and the discovery of FTD causal genetic mutations that has resulted in varying demographics. OBJECTIVE: Our aim was to determine the sex distribution of sporadic and genetic FTD cases and its subtypes in an international cohort. METHODS: We included 910 patients with behavioral variant frontotemporal dementia (bvFTD; n = 654), non-fluent variant primary progressive aphasia (nfvPPA; n = 99), semantic variant primary progressive aphasia (svPPA; n = 117), and right temporal variant frontotemporal dementia (rtvFTD; n = 40). We compared sex distribution between genetic and sporadic FTD using χ2-tests. RESULTS: The genetic FTD group consisted of 51.2% males, which did not differ from sporadic FTD (57.8% male, p = 0.08). In the sporadic bvFTD subgroup, males were predominant in contrast to genetic bvFTD (61.6% versus 52.9% males, p = 0.04). In the other clinical FTD subgroups, genetic cases were underrepresented and within the sporadic cases the sex distribution was somewhat equal. CONCLUSION: The higher male prevalence in sporadic bvFTD may provide important clues for its differential pathogenesis and warrants further research.

Risk of dementia in APOE ε4 carriers is mitigated by a polygenic risk score.

INTRODUCTION: We investigated relationships among genetic determinants of Alzheimer's disease (AD), amyloid/tau/neurodegenaration (ATN) biomarkers, and risk of dementia. METHODS: We studied cognitively normal individuals with subjective cognitive decline (SCD) from the Amsterdam Dementia Cohort and SCIENCe project. We examined associations between genetic variants and ATN biomarkers, and evaluated their predictive value for incident dementia. A polygenic risk score (PRS) was calculated based on 39 genetic variants. The APOE gene was not included in the PRS and was analyzed separately. RESULTS: The PRS and APOE ε4 were associated with amyloid-positive ATN profiles, and APOE ε4 additionally with isolated increased tau (A-T+N-). A high PRS and APOE ε4 separately predicted AD dementia. Combined, a high PRS increased while a low PRS attenuated the risk associated with ε4 carriers. DISCUSSION: Genetic variants beyond APOE are clinically relevant and contribute to the pathophysiology of AD. In the future, a PRS might be used in individualized risk profiling.

Genome-wide association study of frontotemporal dementia identifies a C9ORF72 haplotype with a median of 12-G4C2 repeats that predisposes to pathological repeat expansions.

Genetic factors play a major role in frontotemporal dementia (FTD). The majority of FTD cannot be genetically explained yet and it is likely that there are still FTD risk loci to be discovered. Common variants have been identified with genome-wide association studies (GWAS), but these studies have not systematically searched for rare variants. To identify rare and new common variant FTD risk loci and provide more insight into the heritability of C9ORF72-related FTD, we performed a GWAS consisting of 354 FTD patients (including and excluding N = 28 pathological repeat carriers) and 4209 control subjects. The Haplotype Reference Consortium was used as reference panel, allowing for the imputation of rare genetic variants. Two rare genetic variants nearby C9ORF72 were strongly associated with FTD in the discovery (rs147211831: OR = 4.8, P = 9.2 × 10-9, rs117204439: OR = 4.9, P = 6.0 × 10-9) and replication analysis (P < 1.1 × 10-3). These variants also significantly associated with amyotrophic lateral sclerosis in a publicly available dataset. Using haplotype analyses in 1200 individuals, we showed that these variants tag a sub-haplotype of the founder haplotype of the repeat expansion that was previously found to be present in virtually all pathological C9ORF72 G4C2 repeat lengths. This new risk haplotype was 10 times more likely to contain a C9ORF72 pathological repeat length compared to founder haplotypes without one of the two risk variants (~22% versus ~2%; P = 7.70 × 10-58). In haplotypes without a pathologic expansion, the founder risk haplotype had a higher number of repeats (median = 12 repeats) compared to the founder haplotype without the risk variants (median = 8 repeats) (P = 2.05 × 10-260). In conclusion, the identified risk haplotype, which is carried by ~4% of all individuals, is a major risk factor for pathological repeat lengths of C9ORF72 G4C2. These findings strongly indicate that longer C9ORF72 repeats are unstable and more likely to convert to germline pathological C9ORF72 repeat expansions.