We previously showed that heterozygous loss-of-function mutations in GRN are an important cause of FTLD, explaining up to 25% of familial patients with the FTLD-U pathological subtype. In this study we focused our attention on the role of the common genetic variant rs5848, located in the 3′-UTR of GRN. This was stimulated by our observation of a deviation from the expected HWE for rs5848 in an extensive Mayo Clinic FTLD patient series, due to a selective increase in the TT genotype frequency in patients compared with control individuals. We hypothesized that if rs5848 is a true genetic risk factor for FTLD, the genetic association would be strongest in a homogeneous population of patients with FTLD-U pathology. To test this hypothesis, we performed genetic association analyses in a pathology-confirmed FTLD-U series derived from the MCJ brain bank and confirmed the genetic risk associated with rs5848. In our series, the OR to develop FTLD-U for carriers homozygous for the minor T-allele of rs5848 compared with homozygous C-allele carriers was 3.18 (95% CI: 1.50–6.73; Padjusted = 0.003). Detailed genetic analyses of GRN and its flanking genomic regions further showed multiple haplotype backgrounds for the risk T-allele of rs5848 and did not reveal other genetic variants or haplotypes that were more strongly associated with FTLD-U, favoring rs5848 as the functional variant underlying the association.
We determined that rs5848 is located in the 3′-UTR of GRN
within a predicted binding site for the human specific miRNA miR-659. miRNAs are a widely distributed class of non-coding RNAs that play an integral role in gene regulation by binding to partially complementary sites in the 3′-UTR of target mRNA transcripts, thereby inducing translational repression (20
). Using in silico
analyses we predicted a stronger binding (reducing GRN expression) of miR-659 to the GRN 3′-UTR containing the risk T-allele compared with the wild-type C-allele of rs5848.
To support the hypothesis that rs5848 is indeed a functional variant regulating GRN expression, we determined GRN expression levels in brain extracts derived from FTLD-U rs5848 CC and TT carriers by western blot analyses, ELISA and immunohistochemistry. These in vivo analyses confirmed the functional capacity of rs5848. Compared with CC carriers, a statistically significant ~30% decrease in GRN protein levels was observed in FTLD-U rs5848 TT carriers. Furthermore, intermediate levels of GRN protein were observed in FTLD-U patients heterozygous for rs5848 supporting a dose-dependent decrease of GRN protein levels with each T-allele. As expected from a translational suppression of GRN by miRNAs, GRN mRNA expression levels were not significantly different between FTLD-U rs5848 CC and TT carriers.
Using cell-based systems we further demonstrated that miR-659 binds to the predicted binding site in the 3′-UTR of GRN and is able to suppress GRN expression in vitro. To determine the differential regulation of GRN expression resulting from the wild-type C-allele or risk T-allele of rs5848, we generated luciferase reporter constructs expressing firefly luciferase under the regulation of the GRN 3′-UTR carrying both rs5848 alleles. Our data suggested that miR-659 binds more efficiently to the risk T-allele than the wild-type C-allele, as demonstrated by the dose-dependent translational inhibition of the luciferase reporter (representing GRN protein) from the T-allele construct at low miRNA doses ranging from 5 to 100 pm (Fig. E). No translational repression was observed using the wild-type C-allele constructs, further supporting the differential effect of rs5848 (Fig. F). Together with the confirmation of positive expression of miR-659 in human brain, these results strongly support the functional potential of the miR-659/GRN mRNA complex. Of note, neither the binding site of miR-659 in the 3′-UTR of GRN, nor the gene encoding miR-659 on human chromosome 22q13, are conserved in other vertebrate or invertebrate species, prohibiting the modeling of the regulation of GRN by miR-659 in non-human systems.
If rs5848 increases FTLD-U risk by miRNA-mediated translational repression of GRN, one would expect a significant proportion of homozygous T-allele carriers to develop a pathological FTLD-U phenotype resembling GRN
loss-of-function mutation carriers. Indeed, histological subtyping of FTLD-U patients from our cohort, blinded to their rs5848 genotype status, revealed that >65% of patients homozygous for the risk T-allele (10/15 patients) had FTLD-U consistent with Mackenzie type 1 (12
). In addition, compared with the other rs5848 genotype groups, the frequency of lentiform NIIs was significantly higher in homozygous T-allele carriers (P
= 0.02). Finally, MND pathology, which is rare or absent in GRN
loss-of-function mutation carriers, was only present in one of the 15 FTLD-U patients homozygous for the risk T-allele (6.6%). Re-analyses of the genetic association study excluding patients with a pathological diagnosis of FTD- MND increased the OR for homozygous T-allele carriers to 3.76 (95% CI: 1.69–8.39; Padjusted
= 0.001). The strong similarity in pathological presentation between GRN
loss-of- function mutation carriers and patients homozygous for the rs5848 T-allele provides additional support for the hypothesis that rs5848 increases FTLD-U risk by reducing the expression of GRN.
In 2 years, 57 different pathogenic GRN
loss-of-function mutations have been reported in 160 genealogically unrelated FTLD families (Alzheimer Disease and Frontotemporal Dementia Mutation Database, http://www.molgen.ua.ac.be/FTDmutations/
). The majority of these mutations are nonsense, frameshift and splice-site mutations that introduce a premature stop codon leading to the degradation of mutant RNA by the process of nonsense-mediated decay and the subsequent loss of 50% functional GRN (15
). The identification in this study of homozygosity of the T-allele of rs5848 as a major risk factor for FTLD-U, represents yet another way in which genetic variation in GRN
may lead to FTLD-U via a decrease in the levels of functional GRN. In this case, haploinsufficiency is predicted to result from increased suppression of GRN translation through altered miRNA regulation. Our findings suggest a scenario whereby a decrease in GRN expression below a critical threshold results in the development of FTLD-U. While a single GRN
null allele can sufficiently reduce GRN expression levels to cause FTLD-U, two copies of the risk T-allele of rs5848 are required (but may not be sufficient) to reduce the GRN expression below this critical threshold. We propose that a homozygous state of the risk T-allele at rs5848 combined with other environmental and/or genetic factors, will ultimately determine which individuals develop FTLD-U. In this respect, the association of rs5848 with FTLD-U is similar to many other neurodegenerative diseases where genetic variability at the same loci implicated in Mendelian pathogenic forms of the disease predispose to sporadic forms of the disease (24
). Whether rs5848 may also contribute to the development of other TDP43-proteinopathies (such as amyotrophic lateral sclerosis) or may determine the presence of TDP-43 pathology in more common neurodegenerative conditions such as AD (30
) needs further investigation.
The assumption of a role for miRNA pathways in neurodegeneration is intriguing and has been previously suggested based on experiments in mice, flies and cultured neurons in which the enzyme Dicer (required for miRNA maturation) was genetically inactivated (31
). A role for specific miRNAs in the development of common neurodegenerative diseases has further been suggested by comparing changes in miRNA expression profiles in brain samples derived from patients and controls (32
). Based on the possible role of miRNA dysregulation of GRN in FTLD presented in this study and the knowledge that overexpression of neurodegenerative disease proteins, such as the amyloid precursor protein in AD (35
) and α-synuclein in Parkinson’s disease (36
), is sufficient to cause disease, we speculate that regulatory mutations affecting the interaction between miRNAs and their targets may present a common mechanism underlying complex neurodegenerative disorders.