No Evidence of PGRN or MAPT Gene Dosage Alterations in a Collection of Patients with Frontotemporal Lobar Degeneration

Abstract

Background/Aims:

Alterations in gene dosage have recently been associated with neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, and deletions of the progranulin (PGRN) locus were recently described in patients with frontotemporal lobar degeneration (FTLD). FTLD is a genetically complex neurodegenerative disorder with mutations in the PGRN and the microtubule-associated protein tau (MAPT) genes being the most common known causes of familial FTLD. In this study, we investigated 39 patients with FTLD, previously found negative for mutations in PGRN and MAPT, for copy number alterations of these 2 genes.

Methods:

Gene dosage analysis of PGRN and MAPT was performed using multiplex ligation-dependent probe amplification.

Results:

We did not identify any PGRN or MAPT gene dosage variations in the 39 FTLD patients investigated. Conclusion: We therefore conclude that alterations in gene copy number of PGRN and MAPT are not a cause of disease in this collection of FTLD patients.

Introduction

Frontotemporal lobar degeneration (FTLD) is a term used to describe a clinically and neuropathologically heterogeneous group of neurodegenerative disorders with disease onset often before 65 years of age. There are 3 different clinical subtypes of FTLD [1]. Frontotemporal dementia (FTD) is the most common clinical presentation, characterised predominantly by early behavioural and personality changes. FTLD can also present as semantic dementia or progressive non-fluent aphasia, characterised primarily by changes in language function. The neuropathological characteristics of FTLD include severe degeneration of the frontal and temporal lobes. At the histological level, FTLD can be divided into 2 major subtypes [2]. Many cases are characterised by intraneuronal protein aggregates consisting of the microtubule-associated protein tau (MAPT). However, the majority of cases lack tau-positive deposits, and instead show intraneuronal inclusions immunoreactive for ubiquitin and the TAR DNA-binding protein 43 (TDP-43). FTLD is a genetically heterogeneous disorder with several genetic factors contributing to the disease, and a positive family history is found in up to 40% of cases [3, 4, 5]. Mutations were first discovered in the MAPT gene in patients with FTD and associated parkinsonism [6, 7, 8]. The MAPT mutation cases are neuropathologically characterised by intraneuronal accumulation of insoluble hyperphosphorylated tau protein. In addition to FTLD, mutations in the MAPT gene can result in phenotypes resembling progressive supranuclear palsy (PSP) or corticobasal degeneration (CBD) [9, 10, 11, 12, 13, 14, 15], 2 neurodegenerative disorders showing a clinical and neuropathological overlap with FTLD. More recently, mutations in the progranulin (PGRN) gene, located in close proximity of the MAPT locus on chromosome 17q21, have been reported to cause FTLD [16, 17]. Patients with PGRN mutations often present as FTD, progressive non-fluent aphasia or corticobasal syndrome, and are neuropathologically characterised by ubiquitin- and TDP-43-positive intraneuronal inclusions [18]. Most of the described PGRN mutations create premature termination codons in the PGRN mRNA sequence, predicted to result in degradation of the mutant mRNA by nonsense-mediated decay and subsequent loss of functional PGRN protein [16, 17], Hemizygous deletions of the PGRN gene have also been reported in patients with FTLD, in further support of haploinsufficiency as the pathogenic mechanism in these patients [19, 20]. In this study, we set out to investigate the prevalence of PGRN gene deletions in 39 patients with FTLD. We also hypothesised that duplications of the MAPT gene could lead to tau-positive FTLD through increased production of tau protein. Therefore, MAPT gene duplications were investigated in the same set of patients. Due to the clinicopathological overlap of FTLD with PSP and CBD, we also wanted to investigate if MAPT gene duplications could be a cause of disease in these disorders. We therefore included 20 neuropathological cases with PSP as well as 5 cases with CBD in the MAPT gene dosage analysis.

Subjects and Methods

Subjects

The PGRN and MAPT gene dosage analysis included 39 FTLD cases. All patients investigated had previously been analysed and found negative for mutations in the PGRN and MAPT genes. Twenty-five of the cases were clinically diagnosed cases from Sweden, and 7 of these had a known family history of disease. The Swedish familial FTLD samples had also been found negative for mutations in the genes for TDP-43, charged multivesicular body protein 2B, valosin-containing protein, amyloid precursor protein, presenilin 1 and α-synuclein. The remaining 14 FTLD cases originated from the USA, and had been neuropathologically verified at Massachusetts General Hospital (MGH) (table 1). Four of these had been described as ubiquitin- and TDP-43 positive, and 10 displayed tau-positive pathology (table 1). At least 2 of these had a known family history of disease. The MAPT gene dosage investigation also included neuropathologically verified cases with PSP (20 patients) and CBD (5 patients) from MGH (table 1). The study was approved by the Ethical Committees of Uppsala University, Uppsala, Sweden and MGH, Boston, Mass., USA.

Table 1.

Patients included in the PGRN and MAPT gene dosage analysis

Diagnosis	Patients	Clinical diagnosis	Neuropathological diagnosis		MLPA probe mix
			THP-43	Tau
FTLD	39	25	4	10	PGRN/MAPT
PSP	20			20	MAPT
CBD	5			5	MAPT

Multiplex Ligation-Dependent Probe Amplification

Multiplex ligation-dependent probe amplification (MLPA) analysis of genomic DNA from the FTLD cases was carried out using the P275-B1 MAPT-PGRN kit (MRC-Holland, Amsterdam, The Netherlands). The kit contains 32 probes, including 5 PGRN probes (located in exons 1, 3, 6, 10 and 12), 13 MAPT probes (located in exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14), and 14 probes located within other genes on chromosome 17q21. The P275-B1 MAPT-PGRN kit also includes 7 control fragments for identification of insufficient amounts of DNA sample and for verification of successful denaturation and ligation of the MLPA reaction, as well as 10 reference probes in order to ensure that the expected copy number of these sequences can be detected in both control and patient samples. The PSP and CBD cases were analysed using the P275 MAPT-17q21 kit (MRC-Holland), including 13 MAPT probes, 1 PGRN probe, 14 probes located within genes in close proximity of the MAPT gene, 7 control fragments and 9 reference probes. The MLPA reactions were carried out in duplicates according to the manufacturer's instructions using 50 ng of DNA per reaction. Two unrelated genomic DNA control samples were included for each run. The resulting products were analysed on ABI 3,700 (Applied Biosystems, Foster City, Calif., USA) and the MLPA data were analysed using the GeneMarker program v1.6 (Softgenetics, State Collage, Pa., USA).

Results

The MLPA method was used to examine 39 FTLD samples for dosage alterations of the PGRN and MAPT genes. The PSP and CBD samples were examined for MAPT gene copy variations only. The MLPA technique allows identification of both deletions and multiplications of individual exons as well as entire genes. When analysing the amount of products generated from the MLPA of the PGRN and MAPT probes in the FTLD samples, all probes displayed relative amount values within the threshold levels of 0.75–1.25 when compared to reference probes. This indicates a normal copy number of these 2 genes in the investigated samples (data not shown). Furthermore, the MLPA analysis could not find evidence of alterations in copy number of single or multiple exons of the MAPT gene in samples from 20 PSP and 5 CBD patients included in the study (data not shown).

Discussion

Genomic dosage alterations due to multiplications or deletions have in the past few years been found to occur much more frequently than previously thought, and in some cases these structural variants represent pathogenic changes involved in genetic disease [21]. Recently, PGRN locus deletions causing haploinsufficiency were described in patients with FTLD [19, 20]. However, our analysis of 39 FTLD patients did not find evidence of any PGRN gene deletions. Although our study was limited by a relatively small number of cases, some of the samples showed clear hereditary indications and have been screened and found negative for mutations in genes previously implicated in FTLD, thus presenting good candidates for other genetic disease causes. The results from the present investigation indicate that PGRN gene deletions are a rare cause of FTLD, in agreement with results from previous studies [19, 20, 22, 23, 24]. PGRN gene deletions being a rare cause of FTLD is perhaps somewhat unexpected, given that most described PGRN gene mutations cause nonsense-mediated decay and haploinsufficiency, which effectively mimics the consequences of a gene deletion. However, although genomic dosage alterations are more common than previously assumed, deletions are believed to be less abundant within genes than within other genomic regions, which might explain the rare occurrence of PGRN gene deletions [25]. Recently, gene multiplications were identified as a rare cause of Alzheimer's disease and Parkinson's disease, 2 neurodegenerative diseases characterised by protein aggregation, demonstrating that increases in gene dosage can be a pathogenic event subsequently leading to protein deposition and neurodegeneration [26, 27, 28]. It is therefore possible that tau-positive FTLD could be caused by a similar mechanism through a change in dosage levels of tau protein. This is supported by the fact that an overexpression of tau in transgenic mouse models recapitulates key features of FTLD, including tau deposition [29, 30, 31]. The 17q21.31 chromosome region is genetically complex, and both inversions and deletions in this region have been identified [32]. The gene deletions found in the 17q21.31 region include the MAPT locus, and have been described in several patients with mental retardation [33, 34, 35, 36]. We were unable to find evidence of MAPT gene duplications in our set of FTLD patients (including 10 patients with known tau-positive pathology). Previous studies have also failed to find duplications of the MAPT gene in patients with FTLD [37, 38]. If MAPT gene duplications exist, they are most likely not a common cause of FTLD. Moreover, we found no evidence for MAPT gene duplications in the PSP and CBD cases examined here. However, studies involving larger patient cohorts will be necessary to rule out the possibility of MAPT gene dosage alterations as a cause of PSP and CBD. In conclusion, our analysis did not identify any PGRN or MAPT gene dosage alterations in the 39 FTLD patients investigated. However, our study was limited by a relatively small number of patients and additional studies are needed to elucidate the importance of alterations in gene copy number of PGRN and MAPT in FTLD and associated disorders.