Molecular characterization of novel progranulin (GRN) mutations in frontotemporal dementia

Abstract

Frontotemporal dementia (FTD) is a clinical term encompassing dementia characterized by the presence of two major phenotypes: 1) behavioral and personality disorder, and 2) language disorder, which includes primary progressive aphasia and semantic dementia. Recently, the gene for familial frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions (FTLD-U) linked to chromosome 17 was cloned. In the present study, 62 unrelated patients from the Washington University Alzheimer's Disease Research Center and the Midwest Consortium for FTD with clinically diagnosed FTD and/or neuropathologically characterized cases of FTLD-U with or without motor neuron disease (MND) were screened for mutations in the progranulin gene (GRN; also PGRN). We discovered two pathogenic mutations in four families: 1) a single-base substitution within the 3′ splice acceptor site of intron 6/exon 7 (g.5913A>G [IVS6-2A>G]) causing skipping of exon 7 and premature termination of the coding sequence (PTC); and 2) a missense mutation in exon 1 (g.4068C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (p.A9D). Functional analysis in mutation carriers for the splice acceptor site mutation revealed a 50% decrease in GRN mRNA and protein levels, supporting haploinsufficiency. In contrast, there was no significant difference in the total GRN mRNA between cases and controls NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript carrying the p.A9D mutation. Further, subcellular fractionation and confocal microscopy indicate that although the mutant protein is expressed, it is not secreted, and appears to be trapped within an intracellular compartment, possibly resulting in a functional haploinsufficiency.

Granulin mutations associated with frontotemporal lobar degeneration and related disorders: An update

“…A total of 27 other mutations were observed more than once (Table 2). For nine of these mutations (p.A9D, p.C31LfsX35, p.G35EfsX19, p.T52HfsX2, p.G79DfsX39, p.S226WfsX28, p.V200GfsX18, p.Q415X, and p.R418X) haplotype sharing analysis supported a common genetic origin for each of them (Table 2) Le Ber et al, 2007;Mukherjee et al, 2008;Beck et al, 2008]. The 34 remaining mutations were reported in a single family.…”

“…Families Dutch-III, Brescia-F071, Biv, and Seattle B segregated a frameshift mutation resulting in a PTC [Benussi et al, 2008;Bronner et al, 2007;Leverenz et al, 2007;Bruni et al, 2007]. In Family HDDD2, the disease was caused by a missense mutation (p.A9D) introducing a charged amino acid in the signal peptide, resulting in the mislocalization of the protein [Mukherjee et al, 2006[Mukherjee et al, , 2008, which was shown to lead to a decrease in secreted GRN protein [Mukherjee et al, 2008]. In the Kertesz family, GRN mutations were excluded [Bruni et al, 2007].…”

“…The loss of functional protein is mainly the result of loss of GRN transcript caused by NMD of transcripts containing PTCs (n 5 52) or by nuclear degradation of transcripts retaining the first intron due to splice-site mutations in intron 1 (n 5 2) Le Ber et al, 2007]. PTCs can be the result from nonsense mutations (n 5 12), splice-site mutations (n 5 10), and small insertions/deletions (n 5 30) Baker et al, 2006;Huey et al, 2006;Pickering-Brown et al, 2006Boeve et al, 2006;Masellis et al, 2006;Gass et al, 2006;Benussi et al, 2008;Bronner et al, 2007;Mesulam et al, 2007;Behrens et al, 2007;Leverenz et al, 2007;Bruni et al, 2007;Van Deerlin et al, 2007;Brouwers et al, 2007;Rademakers et al, 2007;Llado et al, 2007;Le Ber et al, 2007;Davion et al, 2007;Kelley et al, in press;Spina et al, 2007a;Mukherjee et al, 2008;Beck et al, 2008;Le Ber et al, 2008]. Second, loss of translation as a result of mutations affecting the Kozak sequence (n 5 4) Baker et al, 2006;Pickering-Brown et al, 2006;Boeve et al, 2006;Gass et al, 2006;Le Ber et al, 2008] and reduction of secreted protein due to missense mutations affecting the signal sequence (n 5 2) [Mukherjee et al, 2006;Gass et al, 2006;Kelley et al, in press;Mukherjee et al, 2008;Le Ber et al, 2008], results in reduced GRN.…”

Activity-dependent secretion of progranulin from synapses

“…However, a number of FTD-linked mutations that specifically lead to deficiencies in PGRN secretion have also been identified (Mukherjee et al, 2008;Shankaran et al, 2008;Wang et al, 2010). A further understanding of the regulation of PGRN secretion is therefore warranted.…”

Rescue of Progranulin Deficiency Associated with Frontotemporal Lobar Degeneration by Alkalizing Reagents and Inhibition of Vacuolar ATPase

“…Of the mutations reported to date (http:// www.molgen.vib-ua.be/FTDMutations/), most are loss-offunction mutations leading to GRN haploinsufficiency , which results in a severe reduction of GRN levels in tissues and biological fluids of patients (Ghidoni et al, 2008;Finch et al, 2009;Sleegers et al, 2009). Additionally, missense mutations (Schymick et al, 2007; Brouwers et al, 2008) lead to cytoplasmic missorting and degradation of GRN (Mukherjee et al, 2008;Shankaran et al, 2008) or to reduced secretion probably attributable to misfolding (Shanka- BafA1 but none of the tested protease inhibitors increase intracellular and secreted GRN levels. A, B, HeLa and SH-SY5Y cells were treated for 16 h with DMSO, BafA1 (50 nM), phenanthroline (10 mM), EDTA (15 mM), MG132 (10 M), epoxomicin (1 M), pepstatin A (1 M), E64 (10 M), antipain (5 M), ALLN (5 M), mix 1 (E64, leupeptin, and antipain), and mix 2 (mix 1 plus pepstatin A and ALLN).…”