Abstract
Mutations in progranulin gene (GRN) are a common cause of autosomal dominant frontotemporal lobar degeneration syndromes and are associated with a wide phenotypic heterogeneity. The majority of genetic defects in GRN consists of loss-of-function mutations, causing haploinsufficiency, and is associated with extremely low plasma progranulin levels. Herein, we describe a patient who developed language dysfunctions and memory disturbances at 63 years of age. Considering the early onset and the positive family history (sister aged 50 with non-fluent/agrammatic variant of primary progressive aphasia, father with behavioral disturbances in his sixties), a genetic analysis was carried out, showing the presence of a novel mutation [g.9543delA (IVS3-2delA)] in a predicted splicing site of GRN. Her progranulin plasma levels were under the reference threshold, as in her sister, thus supporting the causative role of the new variant. The same genetic mutation was confirmed by sequencing in her sister. Results described enlarge current knowledge on genetic causes of the disease and clinical characteristics of carriers.
Keywords
INTRODUCTION
Mutations in progranulin gene (GRN) account for about 10% of familial cases of frontotemporal dementia (FTD) [1]. The gene is located on chromosome 17q21 and is composed by 13 exons encoding for progranulin, a cysteine-rich growth factor which is secreted and cleaved in several fragments (granulins). It is expressed in multiple tissues of the body and it has pleiotropic actions, including functions related to neural proliferation, survival, and inflammation. The majority of genetic defects in GRN consists of loss-of- function mutations, causing haploinsufficiency, and are associated with TAR DNA-binding protein (TDP)-43 inclusions (FTLD-TDP) [2]. A growing body of evidence suggests low progranulin levels in plasma could be used to predict GRN mutation status in patients with symptoms encompassing the FTD spectrum, as well as in their asymptomatic family members [3, 4].
The clinical presentation in GRN mutation is extremely heterogeneous as regards symptoms, disease duration, and age of onset. The penetrance of GRN mutations is incomplete and this condition is probably due to the presence of other genes influencing the manifestation of the disease. Here, we describe a novel mutation in GRN, in a putative splice site, in the proband and her sister, both with non-fluent/agrammatic variant of primary progressive aphasia (naPPA) [6, 7].
CASE REPORT
A female, 63 year old patient, developed language dysfunctions and memory deficits (Fig. 1, I:3). Her family history was positive for dementia, as her father developed bizarre behaviors and language dysfunctions at 65 years of age and died at age 69. In addition, a sister of the patient was diagnosed with naPPA in her forties (Fig. 1, I:7). The family history was also positive for psychiatric disturbances (suicide attempts, alcohol abuse, violent behavior). The collection of anamnestic information revealed the presences of hypertension, hypercholesterolemia, and asthma. During the medical evaluation, she showed no significant abnormalities. The neurological examination was unremarkable. She showed anosognosia for her cognitive deficits.
On the Mini-Mental State Examination, she performed 16/30, suggesting a moderate dementia. Neuropsychological assessment showed pathological performances in language domain (such as phonemic and semantic fluencies, visual naming), executive functions nonverbal intelligence, constructive praxia, and the ability to resist to the effects of irrelevant interferences. Selective attention, visuo-spatial memory, verbal working memory, immediate and delayed verbal and visual memory were within normal range. Daily living functional activities were fully preserved (ADL 6/6; IADL 8/8). The language functions were impaired with the presence of frequent anomic pauses, circumlocutions, errors of omission, neologisms, and semantic replacements. The presence of difficulties in spontaneous speech with agrammatism, impaired comprehension of complex sentences, and spared object knowledge, supported the diagnosis of naPPA [6, 7]. Repetition was notimpaired.
EEG showed the presence of slow waves (theta) in left temporal region.
Magnetic resonance imaging (MRI) showed leukoencephalopathy involving semioval centers without ischemic lesions or lacunae.
[18F]-fludeoxyglucose positron emission tomography (FDG-PET) showed brain glucose hypometabolism involving bilateral superior, inferior, and middle frontal gyri (L >R), left sublobar lentiform gyrus (putamen), left sublobar insula, left parietal postcentral gyrus, and left anterior cingulate and bilateral cingulate gyrus (p = 0.01, Fig. 2). This brain metabolic profile was in line with imaging diagnostic criteria for nonfluent variant of naPPA [6].
Cerebrospinal fluid biomarkers were in the normality range (amyloid beta = 890 pg/ml; tau = 200 pg/ml; Ptau = 35 pg/ml) [8].
The patient met the criteria for naPPA [6, 7], presenting some similarities with the distinct phenotype of progranulin-associated PPA proposed previously [9].
METHODS AND RESULTS
Progranulin plasma levels, evaluated through an ELISA kit (Adipogene, Korea) after the ethical committee approval and proband’s consent, was 9 ng/ml, largely below normality threshold indicated by the manufacturer (>61 ng/ml).
Direct sequencing failed to detect mutations known at that time (http://www.molgen.vib-ua.be/), but a novel deletion, namely g.9543delA (relative to nt1 in NG 007886.1) in the 3’ splice site of intron 3 was found (IVS3-2delA, Fig. 3). Sequence alignment by Ensembl Genome Browser (http://www.ensembl.org/index.html) showed that the region affected by the mutation is highly conserved in vertebrates (Fig. 4).
In silico analysis with Spliceport (http://spliceport.cbcb.umd.edu/) predicted that the wild type sequence is localized in a possible donor splice site, which is abolished by the deletion.
We had the opportunity to collect blood samples of her sister, who, at the age of 50, was institutionalized in akinetic mutism (Fig. 1, I:7), after being diagnosed with naPPA in her forties. Progranulin plasma levels were 22 ng/ml and the same genetic defect was found at sequencing, thus confirming the segregation of the new variant with the disease.
mRNA relative expression analysis in peripheral blood mononuclear cells (PBMC) from the proband (Fig. 1, I:3) showed a 50% reduction as compared with non-carriers (Fig. 5; details of methods in the Supplementary Material).
The expansion in chromosome 9 Open Reading Frame 72 (C9ORF72) was absent in both sisters.
DISCUSSION
Here, we described the novel GRN mutation (g.9543delA) in the 3’ splice site of intron 3 (IVS3-2delA), associated with naPPA. The segregation of the variant with the disease supports the hypothesis of a causative role. The deletion is localized in a region recognized as splicing domain, and it is predicted to abolish a splice site. Moreover, the observation that the sequence is very conserved among vertebrates further supports this hypothesis.
Regarding the clinical phenotype, both the proband and her sister developed mainly language and abstract reasoning disturbances, with initial preserved functional activity of daily living, as well as did their father. Differently, the age at onset and severity of the disease was quite heterogeneous among family members. Notably, some family members had psychiatric problems, therefore we analyzed the C9ORF72, known to be associated with psychiatric manifestations (see [10] for review), which was absent. Progranulin plasma levels were almost undetectable, once more supporting the evidence that plasma progranulin evaluation could be a very good biomarker for identifying potential carriers.
In line with previous data [8], CSF biomarkers were normal, including tau protein, which is supposed to reflect the degree of neurodegeneration.
mRNA relative levels in PBMC were reduced of about 50%, in line with previous results obtained also in brain [11, 12]. Despite this, plasma progranulin levels were extremely low, possibly due to a number of mechanisms, including mRNA translation inhibition or altered balance between progranulin and granulins. Nevertheless, in vitro studies would be needed to better characterize the molecular alterations (i.e., exon-skipping or frameshift) associated with this mutation.
In conclusions, although further studies on other family members will be needed to demonstrate the segregation of the genetic defect with the disease, the evidence of very low plasma progranulin levels together with the in silico prediction strongly support the hypothesis that the variant we described is pathogenic.
