The range of mutations identified in GRN
over the past 2 years established GRN haploinsuffiency as the uniform disease mechanism underlying FTLD in GRN
mutation carriers (Gijselinck et al.
). Microarray data further showed significantly reduced GRN mRNA expression in peripheral blood in GRN
loss-of-function mutation carriers compared to non-mutation carriers (Coppola et al.
). These findings raised the important question as to whether expression of GRN in plasma could predict GRN
mutation status and could be used as a biological marker to identify GRN
To answer this question, we first studied a large consecutive series of clinical FTLD patients ascertained at Mayo Jacksonville, for which DNA and plasma samples were available. Direct sequencing analyses of GRN
identified six different GRN
loss-of-function mutations in nine FTLD patients explaining the disease in 4.1% (9/219) of the population and 5.7% (5/87) of FTLD patients with a positive family history. This GRN
mutation frequency is very similar to our previously reported mutation frequency of 4.8% in an unbiased subpopulation of US FTLD patients (Gass et al.
mutations included typical nonsense and frameshift mutations as well as the previously reported p.A9D mutation in the signal peptide sequence of GRN, and a partial deletion of GRN
exon 11, which extends from 15-bp upstream of exon 11 to 177-bp in exon 11 and is predicted to result in an in-frame deletion of exon 11 (p.A472_Q548del) (Gass et al.
; Mukherjee et al.
; Pickering-Brown et al.
; Kelley et al.
; Spina et al.
). GRN expression studies in the complete FTLD cohort using a GRN ELISA demonstrated that plasma GRN levels were strongly reduced in all loss-of-function GRN
mutation carriers compared to non-GRN
mutation carriers (P
< 0.001). Even though we observed a wide range in GRN expression in FTLD patients and control individuals, all mutation carriers showed significantly reduced GRN levels to about one third of the levels observed in non-GRN
carriers and control individuals. In our series, a plasma GRN cut-off value of 112 ng/ml distinguished all GRN
mutation carriers from non-GRN
carriers and we therefore expect it to have close to 100% sensitivity and specificity in future samples. These results are in line with a recently published study by Ghidoni et al.
) who established that in an Italian FTLD population a GRN cut-off level of 110.9 ng/ml was 92.8% specific and 100% sensitive to identify GRN
mutations. However, since there were only eight GRN
mutation carriers in our FTLD population, further carriers will need to be assessed to more definitively decide on the optimal cut-off; in the meantime it may be prudent to use a higher threshold than 112 ng/ml to be more certain of a high sensitivity, without materially lowering specificity. For example, examining we see that a cut-off value of 135 ng/ml has a lower confidence limit for sensitivity of >99%, and this is achieved with an estimated specificity of 97.7%.
Interestingly, we showed strongly reduced GRN expression in patients carrying all types of loss-of-function GRN mutations, including patient NGR019 carrying the p.A9D signal peptide mutation and patient NGR247 carrying the partial GRN deletion, p.A472_Q548del. We also confirmed similar low levels of GRN in a French patient with clinical Parkinson's disease carrying a near complete deletion in GRN.
Next, by analysing unaffected relatives carrying GRN mutations, we showed that the GRN reduction is independent of the disease status, suggesting that the GRN ELISA could also function as a diagnostic tool to identify asymptomatic GRN mutation carriers.
sequencing analyses in our cohort of FTLD patients also identified four different GRN
missense mutations with unknown pathogenic nature in six FTLD patients. The plasma GRN levels in these missense mutation carriers generally supported the previously predicted effect on GRN
function based on in silico
structural protein modeling analyses (Brouwers et al.
; Gijselinck et al.
). Mutation p.C139R was predicted to be likely pathogenic because of a destabilizing effect on the granulin-fold by disrupting one of the cysteine disulfide bridges. This mutation had been previously reported in an Italian early-onset familial FTD patient and a Belgian late-onset Alzheimer's disease patient, while absent in >900 control individuals that were analysed by us and others (Bernardi et al.
; Brouwers et al.
). We observed this mutation in a familial FTLD patient diagnosed with FA at the age of 76 years. His problem began with language difficulty including expression and comprehension. Sentence repetition was preserved. Picture description was normal except for some mild anomia. MRI showed mild non-asymmetrical atrophy. PET analyses showed hypometabolism in the left parietotemporal area and neuropsychometric testing indicated deficits in language, frontal function and preserved memory. In line with the predictions, low amounts of GRN were measured in the plasma sample of this patient (124 ng/ml), supporting the pathogenic potential of this mutation through a partial loss of GRN function. In contrast, all four patients carrying missense mutations p.A324T and p.R433W, which were predicted by in silico
analyses to be tolerated, and both carriers of the silent GRN
mutations (p.H447 and p.P578) showed GRN plasma levels within the normal range. Although we cannot exclude the possibility that these mutations abolish GRN function without reducing the overall expression of GRN, these findings suggested that plasma GRN levels may be a valuable tool in predicting the pathogenic potential of GRN
mutations. We propose that individuals carrying intermediate levels of GRN (~110–140 ng/ml) undergo additional genetic testing to determine the presence of potential pathogenic GRN
Previous studies have reported clinical Alzheimer's disease diagnoses in patients carrying loss-of-function mutations in GRN
(Boeve, 2007; Kelley et al.
). In a clinicopathological study, we performed on the most common GRN
mutation worldwide, p.R493X, 10% of the mutation carriers received a primary clinical diagnosis of Alzheimer's disease and memory impairment was the second most common initial clinical symptom, affecting 30% of the mutation carriers (Rademakers et al.
). In this study, using our GRN ELISA as a screening tool, we analysed 72 patients with clinically diagnosed early-onset probable Alzheimer's disease for mutations in GRN
and identified one patient (1.4% of the population) carrying a loss-of-function mutation. If a disease modifying treatment for either Alzheimer's disease or FTLD becomes available in the future, then, based on the finding above and knowing that that FTLD usually starts early, one could make the case that it would be reasonable to screen early-onset probable Alzheimer's disease cases with a plasma measure for GRN.
In our study, plasma GRN levels were ~75% reduced in mutation carriers with respect to non-GRN
patients and controls, which is significantly more than expected based on the haploinsufficiency disease mechanism. A similar reduction in GRN expression was recently reported in an Italian series of GRN
mutation carriers (Ghidoni et al.
). In contrast, only 35–50% reductions in GRN mRNA levels have been reported in GRN
mutation carriers (Baker et al.
). These findings, together with the fact that we established that only full-length GRN and no granulins or intermediate GRN fragments can be detected by our ELISA, predict an unbalanced GRN metabolism in GRN
mutation carriers, whereby the processing of GRN into granulins is increased. This would suggest that patients with GRN
mutations may specifically lack full-length GRN, while maintaining normal levels of granulins. This is an interesting finding which may have implications for future therapeutic strategies, especially since GRN and the proteolytically derived granulin fragments each have distinct biological properties, e.g. GRN functions as a growth factor or anti-inflammatory agent, while granulin peptides boost inflammation (Zhu et al.
; He and Bateman, 2003
; Ahmed et al.
). In future experiments, detailed biochemical analysis of the secretion and proteolytic processing of GRN in mutation carriers and control individuals will be essential.
In conclusion, this large study of GRN expression in patients with FTLD, Alzheimer's disease and control individuals provides strong support for the use of a GRN ELISA as a reliable and inexpensive tool to identify GRN mutation patients and carriers. Moreover, compared to classical mutation screening of GRN by sequencing analyses, a GRN ELISA has the important advantage of identifying individuals carrying all types of mutations including GRN deletions.