Ana Gorostidi

The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson’s disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.

Assessing the role of the TREM2 p.R47H variant as a risk factor for Alzheimer's disease and frontotemporal dementia

A non-synonymous genetic rare variant, rs75932628-T (p.R47H), in the TREM2 gene has recently been reported to be a strong genetic risk factor for Alzheimers disease (AD). Also, rare recessive mutations have been associated with frontotemporal dementia (FTD). We aimed to investigate the role of p.R47H variant in AD and FTD through a multi-center study comprising 3172 AD and 682 FTD patients and 2169 healthy controls from Spain. We found that 0.6% of AD patients carried this variant compared to 0.1% of controls (odds ratio [OR] = 4.12, 95% confidence interval [CI] = 1.21-14.00, p = 0.014). A meta-analysis comprising 32,598 subjects from 4 previous studies demonstrated the large effect of the p.R47H variant in AD risk (OR = 4.11, 95% CI = 2.99-5.68, p = 5.27×10(-18)). We did not find an association between p.R47H and age of onset of AD or family history of dementia. Finally, none of the FTD patients harbored this genetic variant. These data strongly support the important role of p.R47H in AD risk, and suggest that this rare genetic variant is not related to FTD.

LRRK2 G2019S and R1441G mutations associated with Parkinson’s disease are common in the Basque Country, but relative prevalence is determined by ethnicity

Mutations in LRRK2 gene are the most frequent cause of Parkinson’s disease (PD) described, but their prevalence varies between populations. Patients, 418, with PD and 138 unrelated controls from the Basque Country were screened for LRRK2 G2019S and R1441G mutations. Of the patients, 3.82% were heterozygous carriers of G2019S and 13.15% of R1441G. G2019S frequency was higher in non-Basque population (6.0%), while R1441G was more common in Basque origin population (22.4%). Our conclusion is that both G2019S and R1441G mutations’ frequency varies markedly between Basque and non-Basque origin population reinforcing the importance of ethnicity consideration when establishing mutation prevalence.

Mutations in Progranulin Gene: Clinical, Pathological, and Ribonucleic Acid Expression Findings

BACKGROUND There is an increasing interest in the clinico-pathological correlation of mutations in progranulin (PGRN) and frontotemporal lobar degeneration (FTLD) complex diseases. We aim to study the PGRN expression variability in patients with different clinical features for a better understanding of its roles in FTLD disease. METHODS We sequenced the PGRN gene in 72 patients suffering from FTLD (25 familial and 47 sporadic cases) and in 24 asymptomatic at-risk relatives. We also analyzed PGRN expression in blood by quantitative real-time polymerase chain reaction from 37 patients, 8 asymptomatic mutation carriers, and 10 control subjects as well as in brain tissue from 16 patients and 9 control subjects. RESULTS Four novel mutations were associated with familial and sporadic FTLD and familial dementia associated with amyotrophic lateral sclerosis. We identified a close association between the IVS6-1G>A mutation in PGRN and corticobasal syndrome. Brain tissue was available for carriers of two of the four mutations (IVS6-1 G>A and P357HfsX3). Immunohistochemical analysis revealed ubiquitin- and TDP-43positive and tau/alpha-synuclein negative immunoreactive neuronal intranuclear inclusions. The relative expression of PGRN in the clinical sample was significantly lower in carriers of the IVS6-1 G>A than in control subjects. CONCLUSIONS Progranulopathies are a major cause of the main phenotypes included in the FTLD complex. According to our results, the level of expression of PGRN in blood could be a useful marker both for diagnostics of part of the spectrum of FTLD conditions and for monitoring future treatments that might boost the level of PGRN in this disorder.

Olfactory deficits and cardiac 123I‐MIBG in Parkinson's disease related to the LRRK2 R1441G and G2019S mutations

It has been proposed that olfactory tests and metaiodobenzylguanidine cardiac scintigraphy may help diagnose idiopathic Parkinsons disease in the premotor phase. However, it is not clear what value these tests have in all patients with Parkinsons disease and, particularly, in those who carry mutations in LRRK2. The objective was to analyze olfactory dysfunction and the changes in cardiac I‐metaiodobenzylguanidine uptake in patients with Parkinsons disease carrying the R1441G and G2019S mutations in LRRK2, and in patients with Parkinsons disease with no known mutations. Patients with Parkinsons disease were screened for R1441G and G2019S LRRK2 gene mutations and classified as LRRK2 mutation carriers or noncarriers. A total of 190 patients with Parkinsons disease (44 LRRK2 mutation carriers) were tested for olfactory dysfunction using the Brief Smell Identification Test. Cardiac 123I‐metaiodobenzylguanidine scintigraphy was performed on 90 patients with Parkinsons disease (27 LRRK2 mutation carriers). Thirty‐six percent of patients with LRRK2 mutations have hyposmia, compared to 75% of noncarrier patients with Parkinsons disease (P < .001). Sixty‐six percent of LRRK2 mutation carriers have low early metaiodobenzylguanidine uptake, compared to 86% of noncarriers (P = .048). Similarly, the heart/mediastinum ratio in delayed metaiodobenzylguanidine images appeared to differ between these groups of patients with Parkinsons disease, although these results did not reach statistical significance. The data obtained indicate that olfactory and cardiac impairment is less prevalent when Parkinsons disease is associated with mutations in LRRK2, although the underlying mechanisms for this difference remain unclear. Thus, such screening would be less useful to detect the premotor phase in asymptomatic relatives who carry mutations in LRRK2 than in cases not associated with LRRK2.

Neuropathology of Parkinson's Disease with the R1441G Mutation in LRRK2

We report the neuropathological findings in a patient with Parkinsons disease (PD) associated with Basque R1441G‐LRRK2/dardarin mutation. The patient was a man with disease onset at 68 years of age, with unilateral rest tremor; the Parkinsonism was well controlled with medication for 15 years. He died at the age of 86, after 18 years of evolution. The neuropathological examination disclosed mild neuronal loss in the substantia nigra pars compacta without α‐synuclein, tau, LRRK2, or ubiquitin cytoplasmic inclusions. Lewy bodies and Lewy neurites were absent. This is the first neuropathological study of PD associated with brain with the R1441G mutation in LRRK2.

A Novel PRNP Y218N Mutation in Gerstmann-Straussler-Scheinker Disease With Neurofibrillary Degeneration

Gerstmann-Sträussler-Scheinker (GSS) disease is a prion disease associated with prion protein gene (PRNP) mutations. We report a novel PRNP mutation (Y218N) associated with GSS disease in a pathologically confirmed case and in two other affected family members. The clinical features of these cases met criteria for possible Alzheimer disease and possible frontotemporal dementia. Neuropathologic analysis revealed deposition of proteinase K-resistant prion protein (PrPres), widespread hyperphosphorylated tau pathology, abnormal accumulation of mitochondria in the vicinity of PrPres deposits, and expression of mutant ubiquitin (UBB+1) in neurofibrillary tangles and dystrophic neurites. Prion protein immunoblotting using 3F4 and 1E4 antibodies disclosed multiple bands ranging from approximately 20 kd to 80 kd and lower bands of 15 kd and approximately 10 kd, the latter only seen after a long incubation. These bands were partially resistant to proteinase K pretreatment. This pattern differs from those seen in Creutzfeldt-Jakob disease andresembles those reported in other GSS cases. The approximately 10kd band was recognized with anti-PrP C-terminus antibodies but not with anti-N terminus antibodies, suggesting PrP truncation at the N terminal. This new mutation extends the list of known mutations responsible for GSS disease and reinforces its clinical heterogeneity. Genetic examination of the PRNP gene should be included in the workup of patients with poorly classifiable dementia.

Identification of a novel THAP1 mutation at R29 amino-acid residue in sporadic patients with early-onset dystonia.

Dystonia is a movement disorder characterized by involuntary, sustained muscular contractions affecting one or more sites of the body and abnormal postures. The clinical spectrum of dystonia is diverse: however, the most common form of the disease is primary torsion dystonia (PTD). PTD usually onsets in infancy or adolescence and affects the trunk, neck, or limbs. Two genes are associated with PTD, DYT1 (OMIM 128100; TOR1A) and DYT6 (OMIM 609520; THAP1). Mutations in THAP1 were, first, identified in families presenting with cranial or cervical dystonia that frequently progressed to involve other body regions, and later, in families presenting with early-onset generalized dystonia with prominent spasmodic dysphonia. Here, 24 patients presenting with either generalized (6) or cervical dystonia (18) were investigated for the presence of THAP1 mutations. The mean age at onset was 48 years (range 9–77 years). All patients provided written informed consent to participate in this study. Sequencing analysis of the coding region of the THAP1 gene revealed the presence of a novel c.86G>A transition resulting in p.Arg29Gln in two sporadic cases suffering from early-onset cervical dystonia and early-onset generalized dystonia, respectively. This novel mutation, located in exon 2 of the THAP1 gene, affects an amino-acid previously identified as mutated in families presenting with multifocal and generalized dystonia. Furthermore, R29 amino acid is conserved between species; suggesting, p.Arg29Gln is likely to be pathogenic. Although both p.Arg29Gln mutation carriers are from the same geographical region, there is no evidence of a common founder. A novel silent, mutation which resulted in c.81T>C transition causing p.Leu27Leu was also identified in a sporadic, late-onset dystonia patient presenting with involuntary movements in the neck at the age of sixty; however, its pathogenic role remains unclear. Both novel THAP1 mutations were absent in a Spanish series of 182 control chromosomes. We concluded that pathogenic mutations were only found in early-onset cases. THAP1 mutations occur throughout the entire gene; however, many seem to cluster in the THAP domain of the protein, including the novel THAP1 mutations described here (Fig. 1). It has been suggested that mutations in the THAP domain may reduce the DNA binding ability of THAP; therefore we speculate that mutations at R29 may also act via this mechanism. Clinical features of both patients carrying p.Arg29Gln mutation are described below. This case is a 55 years old man who, at the age of 9, presented with a right-sided torticollis which persisted for more than a year. A surgical intervention was given around his neck and in the right upper limb. Since then, no improvement was determined however the disease showed slow progression. At 54, he noted having cognitive changes and difficulties in speech and ingestion. There was no family history of movement disorders and his mother died with cognitive decline of late onset. Dystonia was mainly present in the face and proximal left upper limb. Left sided torticollis was found with a tendency of retrocollis and fluctuating involvement of both platysma. The trapezia and ECMs were hypertrophic. MRI brain was normal. The cervical spine showed mild disc protrusion at C5-C6 and C6-C7 without radicular involvements. To control the dystonia, increased dose of Botulinum toxin (Dysport), up to 700 units, was required. This case is a 33 years old man who, at the age of 14, presented with abnormal pain sensation in the left upper limb. He progressed to develop dystonic movements in the hand which usually presented as wrist flexion on the radial side and, occasionally, on the ulnar side. In addition, the left upper limb showed forced abduction. Dystonia has now spread to the left foot. His grandmother was diagnosed as having Parkinson’s disease and his great-aunt with possible blepharospasm. Brain MRI was normal and cervical spine showed minimal disc protrusion at C5-C6 without any compression noted. The arm abduction was controlled with botulinum toxin; however, dystonia remained uncontrolled and no additional medication was given. Serum copper, ceruloplasmin, calcium, phosphate, ferritin, and glucose were normal in both cases. To summarize, THAP1 mutations may be present in sporadic, early-onset, cervical, and generalized dystonia.

Analysis of the CHCHD10 gene in patients with frontotemporal dementia and amyotrophic lateral sclerosis from Spain

Sir, Recently, a study identified a mutation (c.176C>T, p.S59L) in the CHCHD10 gene as a cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD) in a large pedigree with mixed phenotypes encompassing ALS, FTD, cerebellar ataxia and mitochondrial myopathy (Bannwarth et al. , 2014).The same mutation was also found in a second kindred suffering from ALS, FTD and/or parkinsonian signs by the same authors. Additional mutations (p.R15L, p.P34S, p.G66V and p.P80L) have been subsequently reported in ALS and FTD with motor neuron disease (FTD-MND) patients (Chaussenot et al. , 2014; Johnson et al. , 2014; Muller et al. , 2014; Kurzwelly et al. , 2015; Ronchi et al. 2015; Chio et al. , 2015). More recently, exon 2 of the CHCHD10 gene has been sequenced in a cohort of ALS, FTD, Parkinsons disease and Alzheimer’s dementia, revealing two novel mutations (p.P23T and p.A35D) in two patients with FTD (Zhang et al. , 2015). Importantly, some of these screenings have not included neurologically healthy individuals from the same geographic origin (Bannwarth et al. , 2014; Johnson et al. , 2014; Muller et al. , 2014; Kurzwelly et al. , 2015) and therefore, the real allele diversity within CHCHD10 might have been missed. This could have important consequences in terms of establishing firm …

Penetrance in Parkinson's disease related to the LRRK2 R1441G mutation in the Basque country (Spain)†

The LRRK2 R1441G mutation was first identified in Basque families and it is responsible for 46% of familial Parkinsons disease (PD) and for 2.5% of sporadic PD in the PD population of Basque ascent. The aim of this study was to determine LRRK2 R1441G penetrance in PD in the Basque Country (Spain) to help in a more accurate genetic counseling. A total of 59 sibships containing 244 individuals, with a total of 40 PD‐affected relatives, were studied. Genetic testing for the R1441G mutation in the LRRK2 gene was performed in 133 individuals and was positive in 51% of them. Lifetime penetrance of R1441G mutations turned out to be 12.5% at 65 years to 83.4% at 80 years. No gender differences were found in penetrance.