The discovery of mutations in PGRN
causing FTLD has been a major breakthrough in the field. Twenty-three percent of the FTLD cases with positive family history and 5% to 10% of the total FTLD cases are caused by PGRN
mutations according to a recent large-scale study.11
Unlike the other FTLD-causing mutations reported in MAPT
, mutations in PGRN
act through a haploinsufficiency mechanism, resulting in reduced PGRN
expression levels in mutation carriers. This suggests that alternative strategies aimed at quantifying the transcript levels can be effective in patient screening.
Peripheral blood is a widely used source of DNA. Systematic evaluation of comparability of gene expression in blood and brain has shown that whole-blood gene expression profile shares significant similarities with that of multiple central nervous system tissues12
; therefore it may provide a suitable surrogate for gene expression in central nervous system disease in some cases as well.13
For example, real-time quantitative PCR on peripheral leukocytes has been proposed as an alternative assay for molecular diagnosis in Friedreich's ataxia, as levels of frataxin are reduced in peripheral blood of patients with this neurodegenerative disease,14
and microarray studies on peripheral lymphoblasts or peripheral blood have identified candidate genes relevant for AD,15
We found that PGRN
is highly expressed in peripheral blood, confirming initial reports of high expression levels in rat lymphocytes.18
This high blood level is consistent with the proposed role for progranulin in wound repair and/or inflammation.19
High expression in peripheral blood allows reliable quantification of mRNA levels using microarray and real-time qPCR. We screened 143 patients and control subjects and identified reduced levels of PGRN
mRNA, compatible with haploinsufficiency, in 1 out of 43 (2%) patients with clinical diagnosis of FTLD and in 1 unaffected control subject. This warrants a wide screening for progranulin mutations (including patients with family history negative for dementia) and supports gene quantification as a valid approach, an alternative to direct sequencing.
We confirmed the presence of pathogenic PGRN mutations in both subjects with reduced levels of PGRN mRNA by direct sequencing. The observed reduced mRNA levels support nonsense-mediated decay as the most likely pathogenetic mechanism and constitutes a proof of principle that it is possible to screen patients for PGRN haploinsufficiency on peripheral total blood samples, without deriving cell lines, by using widely available gene quantification methods. In addition, this method would identify reduced PGRN levels caused by out-of-frame single exon deletions and duplications, as well as microdeletions, which are usually not detected through regular sequencing. However, the possibility that reduced PGRN levels can be related to active regulation of the gene has to be considered. For example, we observed a trend for PGRN increase with age in control subjects (data not shown), and other factors might be involved in regulating PGRN levels. Interestingly, three subjects (one FTLD patient, one CBS patient, and one control subject) showed reduced (60%) levels of progranulin, but tested negative for mutations in PGRN, suggesting that it might be worthwhile to investigate whether progranulin levels are contributing factors in the pathogenesis/clinical course of FTLD/CBS.
The gene expression profile in the two subjects with progranulin mutations is distinct from other FTLD patients. Although suggestive that specific pathogenetic mechanisms can be involved in this genetic form of FTLD, this observation is based only on two subjects and warrants extended gene expression studies involving a larger number of patients.
We reported increased mRNA PGRN
levels in peripheral blood of patients with clinical diagnosis of AD, a difference that remains after the exclusion of outliers. This is the first report of quantitative assessment of PGRN
expression in AD patients. Intense progranulin staining of senile plaques and microglia in AD pathology has been reported,6
and an increase of PGRN
mRNA was also reported in spinal cords of amyotrophic lateral sclerosis patients in an early microarray study.20
This is intriguing and suggests the idea that, whereas decreased levels cause FTLD, increased levels of this gene are associated with distinct, but related neurodegenerative conditions such as AD and amyotrophic lateral sclerosis. Given the proposed roles of PGRN
, this increase could be related to neuroinflammation associated with granulin peptides and/or a repair strategy. However, this finding needs to be confirmed on a larger series with neuropathological assessment.
In conclusion, quantification of PGRN levels in peripheral blood samples is a valid and effective strategy for large-scale first screening in patients with dementia and related conditions. Expression data in AD patients suggest that PGRN may play a role in AD pathogenesis.