Potential genetic modifiers of disease risk and age at onset in patients with frontotemporal lobar degeneration and GRN mutations: a genome-wide association study

Abstract

Background: Loss-of-function mutations in GRN cause frontotemporal lobar degeneration (FTLD). Patients with GRN mutations present with a uniform subtype of TAR DNA-binding protein 43 (TDP-43) pathology at autopsy (FTLD-TDP type A); however, age at onset and clinical presentation are variable, even within families. We aimed to identify potential genetic modifiers of disease onset and disease risk in GRN mutation carriers.

Methods: The study was done in three stages: a discovery stage, a replication stage, and a meta-analysis of the discovery and replication data. In the discovery stage, genome-wide logistic and linear regression analyses were done to test the association of genetic variants with disease risk (case or control status) and age at onset in patients with a GRN mutation and controls free of neurodegenerative disorders. Suggestive loci (p<1 × 10^-5) were genotyped in a replication cohort of patients and controls, followed by a meta-analysis. The effect of genome-wide significant variants at the GFRA2 locus on expression of GFRA2 was assessed using mRNA expression studies in cerebellar tissue samples from the Mayo Clinic brain bank. The effect of the GFRA2 locus on progranulin concentrations was studied using previously generated ELISA-based expression data. Co-immunoprecipitation experiments in HEK293T cells were done to test for a direct interaction between GFRA2 and progranulin.

Findings: Individuals were enrolled in the current study between Sept 16, 2014, and Oct 5, 2017. After quality control measures, statistical analyses in the discovery stage included 382 unrelated symptomatic GRN mutation carriers and 1146 controls free of neurodegenerative disorders collected from 34 research centres located in the USA, Canada, Australia, and Europe. In the replication stage, 210 patients (67 symptomatic GRN mutation carriers and 143 patients with FTLD without GRN mutations pathologically confirmed as FTLD-TDP type A) and 1798 controls free of neurodegenerative diseases were recruited from 26 sites, 20 of which overlapped with the discovery stage. No genome-wide significant association with age at onset was identified in the discovery or replication stages, or in the meta-analysis. However, in the case-control analysis, we replicated the previously reported TMEM106B association (rs1990622 meta-analysis odds ratio [OR] 0·54, 95% CI 0·46-0·63; p=3·54 × 10^-16), and identified a novel genome-wide significant locus at GFRA2 on chromosome 8p21.3 associated with disease risk (rs36196656 meta-analysis OR 1·49, 95% CI 1·30-1·71; p=1·58 × 10^-8). Expression analyses showed that the risk-associated allele at rs36196656 decreased GFRA2 mRNA concentrations in cerebellar tissue (p=0·04). No effect of rs36196656 on plasma and CSF progranulin concentrations was detected by ELISA; however, co-immunoprecipitation experiments in HEK293T cells did suggest a direct binding of progranulin and GFRA2.

Interpretation: TMEM106B-related and GFRA2-related pathways might be future targets for treatments for FTLD, but the biological interaction between progranulin and these potential disease modifiers requires further study. TMEM106B and GFRA2 might also provide opportunities to select and stratify patients for future clinical trials and, when more is known about their potential effects, to inform genetic counselling, especially for asymptomatic individuals.

Funding: National Institute on Aging, National Institute of Neurological Disorders and Stroke, Canadian Institutes of Health Research, Italian Ministry of Health, UK National Institute for Health Research, National Health and Medical Research Council of Australia, and the French National Research Agency.

Figure 1:. Manhattan plots of the case/control and age at onset analyses.

Negative log₁₀- transformed p-values are shown for each variant genotyped on the y axis in function of the chromosomal position on the x axis. The red line represents the genome-wide significant threshold (p=5×10⁻⁸). The blue line denotes suggestive associations with p<10^-5. Green dots represent the variants that were included in the design for follow-up in the replication stage. (A) case/controls analysis. (B) Age at onset analysis. Please note that at some loci a proxy of the top variant was selected for genotyping in the replication stage.

Figure 2:. GFRA2 genetic locus and expression studies.

(A) The GFRA2 locus zoom plot is presented on the top panel . Each dot represents a genotyped (triangle) or imputed (circle) variant. The purple dot is the most significant variant (rs36196656) among variants in the region. Dots are colored from red to blue according to the r² showing their degree of linkage disequilibrium with rs36196656 (grey color indicates an r² of zero). The blue line shows the estimated recombination rate. The bottom panel presents the GFRA2 gene and its three GFRA2 transcripts. Exons are represented as small black boxes and non-coding regions as straight line. The location of three variants in strong linkage disequilibrium (black arrows) with rs36196656 (red arrow) are represented as blue stars across the different GFRA2 transcripts. (B) Cerebellar mRNA expression level of GFRA2 transcripts stratified by rs36196656 genotype. All values are normalized to two reference genes and within each assay, expression levels are shown normalized to homozygous rs36196656-CC carriers. cM=centimorgan, Mb=megabase.

Figure 3:. Interaction of PGRN and GFRA2.

GFRA2 and PGRN immunoblots are displayed after immunoprecipitation with anti-GFRA2 antibody of cell lysates (A) of HEK293T cotransfected with untagged PGRN and untagged GFRA2 or vector control. Similarly, GFRA2 and PGRN immunoblots are displayed after immunoprecipitation with anti-PGRN antibody of cell lysates (B) of HEK293T co-transfected with untagged GFRA2 and untagged PGRN or vector control. IP=immunoprecipitation; IB=immunoblotting; 5% input=5% of the total amount of cell lysates used for immunoprecipitation.