Enrico Saracchi

Analysis of vesicular monoamine transporter 2 polymorphisms in Parkinson’s disease

Generation of reactive oxygen species during dopamine (DA) oxidation could be one of the factors leading to the selective loss of nigral dopaminergic neurons in Parkinson’s disease (PD). Vesicular monoamine transporter type 2 (VMAT2) proteins in nerve terminals uptake dopamine into synaptic vesicles, preventing its cytoplasmic accumulation and toxic damage to nigral neurons. Polymorphisms in VMAT2 gene and in its regulatory regions might therefore serve as genetic risk factors for PD. In the present study, we have analyzed 8 single-nucleotide polymorphisms (SNPs) located within/around the VMAT2 gene for association with PD in an Italian cohort composed of 704 PD patients and 678 healthy controls. Among the 8 SNPs studied, only the 2 located within the promoter region (rs363371 and rs363324) were significantly associated with PD. In the dominant model, odds ratios were 0.72 (95% confidence interval [CI]: 0.6–0.9, p < 0.005) for rs363371 and 0.76 (95% CI: 0.6–0.9, p = 0.01) for rs363324; in the additive model, odds ratios were 0.78 (95% CI: 0.65–0.94, p = 0.008) for rs363371 and 0.85 (95% CI: 0.7–20.92, p = 0.04) for rs363324. There were no significant relationships between the remaining SNPs (rs363333, rs363399, rs363387, rs363343, rs4752045, and rs363236) and the risk of sporadic PD in any genetic model. This study adds to the previous evidence suggesting that variability in VMAT2 promoter region may confer a reduced risk of developing PD, presumably via mechanisms of gene overexpression.

Reduced expression of the chaperone-mediated autophagy carrier hsc70 protein in lymphomonocytes of patients with Parkinson's disease

Chaperone-mediated autophagy (CMA) impairment is recognized to play a pathogenetic role in Parkinsons disease (PD). A reduced expression of lysosomal-associated membrane protein (lamp) 2A and heat shock cognate (hsc) 70 protein, the two key regulators of CMA, has been reported in brains of PD patients. To verify the existence of a possible systemic CMA dysfunction in PD, in this study the expression of hsc70 and lamp2A was assessed in peripheral blood mononuclear cells (PBMC) of patients with sporadic PD and compared to healthy subjects. The expression of myocyte enhancer factor 2D (MEF2D), a transcriptional factor implicated in neuronal survival and specific substrate of CMA, was also evaluated. Protein and gene expression was assessed by Western blot and real-time PCR, respectively, in PBMC obtained from 53 sporadic PD patients and 53 healthy subjects. A significant reduction of hsc70 levels was observed in PBMC of PD patients, both under basal conditions and after autophagy induction obtained with serum deprivation. No difference emerged in lamp2A and MEF2D expression between patients and controls. No influence of the clinical characteristics of patients emerged on hsc70, lamp2A and MEF2D expression. These results, despite being not suggestive of the existence of a CMA impairment in PBMC of PD patients, identify a systemic hsc70 reduction in PD patients. Further studies on specific mechanisms and biological significance of such alteration are needed to corroborate this finding that could lead to the identification of a new trait biomarker for PD.

TREX1 C-terminal frameshift mutations in the systemic variant of retinal vasculopathy with cerebral leukodystrophy

AbstractRetinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset disorder caused by C-terminal heterozygous frameshift (fs) mutations in the human 3′–5′ DNA exonuclease TREX1. Hereditary systemic angiopathy (HSA) is considered a variant of RVCL with systemic involvement of unknown genetic cause, described in a unique family so far. Here we describe the second case of RVCL with systemic involvement, characterized by cerebral calcifications and pseudotumoral lesions, retinopathy, osteonecrosis, renal and hepatic failure. The genetic screening of TREX1 in this patient revealed the novel heterozygous T270fs mutation on the C-terminal region. On the same gene, we found the V235fs mutation, formerly shown in RVCL, in one patient previously reported with HSA. These mutations lead to important alterations of the C-terminal of the protein, with the loss of the transmembrane helix (T270fs) and the insertion of a premature stop codon, resulting in a truncated protein (V235fs). Functional analysis of T270fs-mutated fibroblasts showed a prevalent localization of the protein in the cytosol, rather than in the perinuclear region. RVCL with systemic involvement is an extremely rare condition, whose diagnosis is complex due to multiorgan manifestations, unusual radiological and histopathological findings, not easily attributable to a single disease. It should be suspected in young adults with systemic microangiopathy involving retina, liver, kidney, bones and brain. Here we confirm the causative role played by TREX1 autosomal dominant fs mutations disrupting the C-terminal of the protein, providing a model for the study of stroke in young adults.

Fahr's disease linked to a novel SLC20A2 gene mutation manifesting with dynamic aphasia

Background: Idiopathic basal ganglia calcification (IBGC), also known as Fahrs disease, is a rare disorder characterized by widespread cerebral calcifications, an autosomal dominant pattern of inheritance and clinical and genetic heterogeneity. The recently identified IBGC gene, SLC20A2, encodes for type III sodium-dependent phosphate transporter 2 and its loss-of-function mutations may lead to the regional accumulation of inorganic phosphate in the brain, causing calcium phosphate deposition. Objective: To describe the clinical, neuroimaging and genetic findings in an Italian family with IBGC. Methods: The family members underwent clinical and radiological examination in order to diagnose IBGC according to standard criteria and screening for SLC20A2 gene mutations. The affected subjects also underwent neuropsychological longitudinal assessments and functional neuroimaging investigations. Results: The 2 affected family members harbored a novel missense mutation, G1618A, in the SLC20A2 gene, leading to gly540-to-arg (G540R) substitution in a highly conserved residue. This is the first SLC20A2 gene mutation associated with familial IBGC reported in the Italian population and is damaging according to all prediction programs. In the index case we observed a fair correlation between cortical areas with no calcifications but with significant hypometabolism at [18F]FDG-PET (inferior frontal premotor cortex) and the neuropsychological picture dominated by dynamic aphasia and buccofacial apraxia. Conclusion: These findings expand the catalog of SLC20A2 mutations identified to date and add dynamic aphasia to the spectrum of neuropsychological deficits reported in IBGC, supporting the use of functional neuroimaging studies for better investigation of genotype-phenotype correlations.

Novel neurofibromatosis type 2 mutation presenting with status epilepticus

Neurofibromatosis type 2 (NF2) is a dominantly inherited syndrome caused by mutations of the tumour-suppressor NF2, which encodes the merlin protein. Mutations are associated with a predisposition to development of benign tumours in the central nervous system. Even though cerebral cortical lesions are frequently associated with seizures, epilepsy is rarely described in NF2. Here, we describe an adult case of NF2 in which the onset of symptoms was characterised by status epilepticus. In this patient, we identified the novel c.428_430delCTTdel mutation in NF2, involving the amino-terminal FERM domain, which is fundamental for the correct tumour suppressor function of the protein. Bioinformatic analyses revealed an important structural perturbation of the FERM domain, with a predicted impairment of the anti-tumour activity.

A novel heterozygous SETX mutation in a patient presenting with chorea and motor neuron disease

Extensive CSF and blood tests were normal except for elevated serum alpha-fetoprotein (AFP) levels (25 ng/l, n.v. 7 ng/l). Nerve conduction stud-ies showed a motor axonal neuropathy; needle EMG examination demonstrated diffuse fi brillations and enlarged polyphasic action potentials in limb and bulbar muscles. MRI studies revealed slight wide-spread supratentorial atrophy and marked thinning of the cervical spinal cord predominantly involving the C4 – C5 segments, without signal abnormalities (Figure 1A). Cerebral FDG-PET showed mild hypo-metabolism of frontoparietal and anterior temporal lobes, with subtle signs of frontal dysfunction at neu-ropsychological evaluation. The patient died from respiratory failure two years after fi rst examination. No autopsy could be performed. Over the disease course, genetic testing for HD, DRPLA, FRDA1, SCA1-2-7-17 and FALS-related genes (

A case of Leber hereditary optic neuropathy plus dystonia caused by G14459A mitochondrial mutation

Leber hereditary optic neuropathy (LHON) is a maternally inherited disease characterized by subacute, painless and symmetric visual loss, which usually develops during young adult life. Males are four to five times more likely than females to be affected. Mutations in the mitochondrial genes that encode subunits of NADH dehydrogenase (ND genes) are known to be associated with LHON. Three major mtDNA point mutations (G3460A in ND1, G11778A in ND4, T14484C in ND6) are responsible for more than 90 % of LHON cases, while only 5–10 % of patients harbor different uncommon pathogenic mutations. In addition to the classic phenotype, characterized by subacute, progressive non-syndromic visual loss, some patients present visual loss combined with other neurological features, this condition being named ‘‘Leber plus’’ syndrome. In particular, LHON plus dystonia (LDYT, OMIM 500001) is characterized by a variable combination of progressive generalized dystonia and visual loss, occasionally accompanied by pyramidal tract signs and intellectual impairment. This rare condition can be caused by various mitochondrial mutations, among which the G14459A is the most frequently observed: it was first described in a Hispanic-American family with LDYT in 1994 [1], and since then it has been identified in various independent pedigrees presenting not only with LDYT, but also with isolated pediatric-onset dystonia, non-syndromic LHON, Leigh or Leigh-like syndrome or in clinically asymptomatic individuals [2]. Here, we describe the case of a male patient who developed left foot dystonic posture rapidly extending to the ipsilateral arm, with progressive gait disturbances, when he was 4-year old. His family history was unremarkable for neurological diseases. At that time the following tests gave normal results: blood copper, blood ceruloplasmin, urinary copper excretion, urinary organic acids, serum carnitine and acylcarnitines, analysis of amino acids and phenylalanine loading test; a brain MRI revealed bilateral symmetric T2-weighted hyperintensities of putamina and caudate nuclei. Suspecting a mitochondrial disease, a muscle biopsy was performed: neither mitochondrial proliferation, nor lipid accumulation or ragged red fibers were detected, and the activities of the respiratory chain complexes were normal. Mitochondrial DNA analysis did not reveal macrodeletions or NARP mutations. Moreover, genetic tests for DYT1, HD, SCA3 were negative, and no clinical improvement was observed after a levo-dopa challenge. In the following years, the clinical picture developed to a progressive generalized dystonia, involving the bulbar and axial muscles, leading to anarthria and severe scoliosis. Cognitive function was spared, and ophthalmologic examination was within normal limits. At 16 years of age the patient underwent GPi deep brain stimulation, without any significant improvement of dystonia. At age 17 he was referred to us for neurologic evaluation secondary to a 3-month history of painless bilateral visual loss associated with dyschromatopsia. Opthalmological examination revealed a visual acuity 1/10 in both eyes; the fundus oculi E. Saracchi (&) J. C. DiFrancesco L. Brighina L. Marzorati N. A. Curtò C. Ferrarese Department of Neurology, S. Gerardo Hospital, via Pergolesi 33, 20052 Monza (MB), Italy e-mail: enricosaracchi@yahoo.it

Association analysis of PARP1 polymorphisms with Parkinson's disease.

Alpha-synuclein accumulation in intracellular inclusions, oxidative stress and microglia-mediated inflammation in the substantia nigra are crucial events in the pathogenesis of Parkinson’s disease (PD). Poly (ADP-ribose) polymerase-1 (PARP1), a DNA-binding enzyme and transcriptional regulator, plays an important role in modulating the cellular response to oxidative stress, inflammatory stimuli, and in apoptotic cell death. Inhibition of PARP1 results in significant neuroprotection in PD animal models; moreover PARP1 has a physiological role in the regulation of alpha-synuclein expression. A previous study had demonstrated that variants located within the PARP1 gene promoter reduce the risk of PD and delay the disease age at onset. In light of these data, we carried out an association study to investigate whether variability within this gene is associated with PD risk and disease age at onset in an Italian cohort composed of 600 PD patients and 592 healthy controls. To this purpose, we used a comprehensive tag SNP approach spanning the entire gene and the upstream and downstream regions. We did not detect any significant association of the PARP1 gene with PD either at genotypic or haplotypic level; none of the 11 genotyped SNPs was significantly associated with PD age at onset. We conclude that, despite previous evidence, PARP1 is not a susceptibility gene for PD in our population.

Cerebellar hematoma in a carrier of the A3243G MELAS mutation

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is often related to the A3243G mutation of the mitochondrial DNA, accounting for about 80% of typical cases, with a relevant phenotypic variability. Hemorrhagic strokes are quite unusual in these patients. A case of recurrent brain hematomas has been reported in a MELAS patient carrying the G13513A mutation, with clinical onset at age 31 [1]. Another fatal case of intracerebral hemorrhage was reported in a MERRF/MELAS overlap syndrome due to a T8356C mutation [2]. Finally, a putaminal hemorrhagic stroke was described in a MERRF patient carrying the A8344G mutation, with clinical onset at age 26 [3]. Here, we report the case of a 37-year-old male arriving to our attention because of a two-week history of headache associated with nausea, vomiting, and dizziness. A brain MRI scan showed a left hemispheric cerebellar hemorrhage at a stage compatible with the onset of the symptoms. The patient neither had history of arterial hypertension, any hemorrhagic risk-factor, diabetes, nor was taking any medication. A few years before, he was found to be asymptomatic carrier of the A3243G mitochondrial DNA mutation (heteroplasmy confirmed on a blood sample), since a MELAS diagnosis was made on his younger brother expressing a typical phenotype. On current examination, our patient presented slight left ataxic hemiparesis and hypoesthesia. A cerebral angiography failed to show any vascular abnormality. Since our patient was carrier of a MELAS mutation, we decided to assess its putative clinical burden. An EEG showed generalized electrogenetic dysfunction with a single generalized epileptic spike discharge; EMG suggested slight myopathic signs; mild perceptive bilateral deafness was also present. A venous lactic acid test with compression of the right arm was significant (baseline values 2.9 mmol/L; increase up to 7.5 mmol/L after 3 min; NV 0.44–2.22 mmol/L). No hearth conduction defects were present. A week later, on a control CT scan, progressive blood reabsorption was noted and basal ganglia calcifications were not observed; the headache had improved and the neurological examination was normal. Iizuka et al. [4] demonstrated cortical laminar necrosis and focal HMPAO SPET hyperperfusion during subacute stages of stroke-like episodes; moreover, they reported several cases of microhemorrhages and at least one case of intracortical gyral hemorrhage, suggesting that stroke-like episodes are characterized by increased capillary permeability and focal hyperemia [5]. Consistently, vasogenic edema has been reported during stroke-like episodes [6]. Anyhow, in our case, the aspect of the lesion does not suggest the hemorrhagic transformation of a stroke-like episode; this also considering that the hematoma was confined in a specific vascular territory, which is not a characteristic of stroke-like episodes [4]. Conversely, we cannot exclude a hemorrhagic coincidental event in a patient carrying the A3243G mutation with a subclinical MELAS phenotype. However, mitochondrial angiopathy in E. Saracchi L. Tremolizzo J. C. DiFrancesco L. Brighina G. Costantino B. Frigeni M. Brioschi M. L. Piatti L. Fumagalli L. Marzorati N. A. Curto C. Ferrarese Department of Neurology, S. Gerardo Hospital, Monza, Italy