Alessio Di Fonzo

Comprehensive analysis of the LRRK2 gene in sixty families with Parkinson's disease

Mutations in the gene leucine-rich repeat kinase 2 (LRRK2) have been recently identified in families with Parkinsons disease (PD). However, the prevalence and nature of LRRK2 mutations, the polymorphism content of the gene, and the associated phenotypes remain poorly understood. We performed a comprehensive study of this gene in a large sample of families with Parkinsons disease compatible with autosomal dominant inheritance (ADPD). The full-length open reading frame and splice sites of the LRRK2 gene (51 exons) were studied by genomic sequencing in 60 probands with ADPD (83% Italian). Pathogenic mutations were identified in six probands (10%): the heterozygous p.G2019S mutation in four (6.6%), and the heterozygous p.R1441C mutation in two (3.4%) probands. A further proband carried the heterozygous p.I1371 V mutation, for which a pathogenic role could not be established with certainty. In total, 13 novel disease-unrelated variants and three intronic changes of uncertain significance were also characterized. The phenotype associated with LRRK2 pathogenic mutations is the one of typical PD, but with a broad range of onset ages (mean 55.2, range 38–68 years) and, in some cases, slow disease progression. On the basis of the comprehensive study in a large sample, we conclude that pathogenic LRRK2 mutations are frequent in ADPD, and they cluster in the C-terminal half of the encoded protein. These data have implications both for understanding the molecular mechanisms of PD, and for directing the genetic screening in clinical practice.

The Mitochondrial Disulfide Relay System Protein GFER Is Mutated in Autosomal-Recessive Myopathy with Cataract and Combined Respiratory-Chain Deficiency

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patients muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.

Adaptive deep brain stimulation in a freely moving parkinsonian patient

The future of deep brain stimulation (DBS) for Parkinsons disease (PD) lies in new closed‐loop systems that continuously supply the implanted stimulator with new settings obtained by analyzing a feedback signal related to the patients current clinical condition.1 The most suitable feedback for PD is subthalamic local field potential (LFP) activity recorded from the stimulating electrode itself.2, 3, 4 This closed‐loop technology known as adaptive DBS (aDBS) recently proved superior to conventional open‐loop DBS (cDBS) in patients with PD.2

The LRRK2 Arg1628Pro variant is a risk factor for Parkinson's disease in the Chinese population

The c.G4883C variant in the leucine-rich repeat kinase 2 (LRRK2) gene (protein effect: Arg1628Pro) has been recently proposed as a second risk factor for sporadic Parkinson’s disease in the Han Chinese population (after the Gly2385Arg variant). In this paper, we analyze the Arg1628Pro variant and the associated haplotype in a large sample of 1,337 Han subjects (834 patients and 543 controls) ascertained from a single referral center in Taiwan. In our sample, the Arg1628Pro allele was more frequent among patients (3.8%) than among controls (1.8%; p = 0.004, OR 2.13, 95% CI 1.29–3.52). Sixty heterozygous and two homozygous carriers of the Arg1628Pro variant were identified among the patients, of which only one was also a carrier of the LRRK2 Gly2385Arg variant. We also show that carriers of the Arg1628Pro variant share a common, extended haplotype, suggesting a founder effect. Parkinson’s disease onset age was similar in patients who carried the Arg1628Pro variant and in those who did not carry it. Our data support the contention that the Arg1628Pro variant is a second risk factor for Parkinson’s disease in the Han Chinese population. Adding the estimated effects of Arg1628Pro (population attributable risk [PAR] ~4%) and Gly2385Arg variants (PAR ~6%) yields a total PAR of ~10%.

SPG11: a consistent clinical phenotype in a family with homozygous spatacsin truncating mutation.

Hereditary spastic paraplegias (HSP) are a heterogeneous group of neurodegenerative disorders leading to progressive spasticity of the lower limbs. Here, we describe clinical and genetic features in an Italian family affected by autosomal recessive HSP (ARHSP) with mental impairment and thin corpus callosum (TCC). In both affected subjects, genetic analysis revealed the presence of a homozygous small deletion (733_734delAT) leading to a frameshift (M245VfsX) within the coding region of SPG11 gene, encoding spatacsin. This finding is the first independent confirmation that spatacsin loss of function mutations cause ARHPS-TCC.

Novel missense mutation and large deletion of GNE gene in autosomal-recessive inclusion-body myopathy

The UDP‐N‐acetylglucosamine 2‐epimerase/N‐acetylmannosamine kinase (GNE) gene is the causative gene for autosomal‐recessive hereditary inclusion‐body myopathy (h‐IBM). Two sisters affected with autosomal‐recessive h‐IBM were shown to be compound heterozygous for two novel GNE mutations: a large deletion involving exons 1—9, and a R162C amino acid change in the epimerase domain. This is the first deletion event observed in a GNE allele and expands the molecular pathogenesis of autosomal‐recessive h‐IBM. Muscle Nerve 28: 113–117, 2003

Autophagy in motor neuron disease: Key pathogenetic mechanisms and therapeutic targets

Autophagy is a lysosome-dependant intracellular degradation process that eliminates long-lived proteins as well as damaged organelles from the cytoplasm. An increasing body of evidence suggests that dysregulation of this system plays a pivotal role in the etiology and/or progression of neurodegenerative diseases including motor neuron disorders. Herein, we review the latest findings that highlight the involvement of autophagy in the pathogenesis of amyotrophic lateral sclerosis (ALS) and the potential role of this pathway as a target of therapeutic purposes. Autophagy promotes the removal of toxic, cytoplasmic aggregate-prone pathogenetic proteins, enhances cell survival, and modulates inflammation. The existence of several drugs targeting this pathway can facilitate the translation of basic research to clinical trials for ALS and other motor neuron diseases.

Unusual adult-onset Leigh syndrome presentation due to the mitochondrial m.9176T>C mutation.

Leigh syndrome (LS) is an incurable, nearly always fatal, neurodegenerative, pediatric disorder that results from respiratory chain failure. The most common mitochondrial DNA (mtDNA) mutations that result in LS are m.8993T→C/G and m.9176T→C/G, which were previously found in several patients with early-onset Leigh syndrome. Here, we describe clinical and molecular features of a novel pedigree, where LS developed in two siblings. The proband was a young woman with an unusual adult-onset LS. She harbored a homoplasmic m.9176T→C mutation, based on analysis of a muscle biopsy. In contrast, the brother died at a young age. This novel case report and literature review highlights the variability of phenotypic expression of the m.9176T→C mutation.

Lower motor neuron disease with respiratory failure caused by a novel MAPT mutation.

Objective: To investigate the molecular defect underlying a large Italian kindred with progressive adult-onset respiratory failure, proximal weakness of the upper limbs, and evidence of lower motor neuron degeneration. Methods: We describe the clinical features of 5 patients presenting with prominent respiratory insufficiency, proximal weakness of the upper limbs, and no signs of frontotemporal lobar degeneration or semantic dementia. Molecular analysis was performed combining linkage and exome sequencing analyses. Further investigations included transcript analysis and immunocytochemical and protein studies on established cell models. Results: Genome-wide linkage analysis showed an association with chromosome 17q21. Exome analysis disclosed a missense change in MAPT segregating dominantly with the disease and resulting in D348G-mutated tau protein. Motor neuron cell lines overexpressing mutated D348G tau isoforms displayed a consistent reduction in neurite length and arborization. The mutation does not seem to modify tau interactions with microtubules. Neuropathologic studies were performed in one affected subject, which exhibited α-motoneuron loss and atrophy of the spinal anterior horns with accumulation of phosphorylated tau within the surviving motor neurons. Staining for 3R- and 4R-tau revealed pathology similar to that observed in familial cases harboring MAPT mutations. Conclusion: Our study broadens the phenotype of tauopathies to include lower motor neuron disease and implicate tau degradation pathway defects in motor neuron degeneration.

Adaptive deep brain stimulation controls levodopa-induced side effects in Parkinsonian patients

The potential superior benefits of adaptive deep brain stimulation (aDBS) approaches compared to classical, constantparameters DBS were already proven by scientific evidence from different research groups. aDBS provides better symptoms control in Parkinson’s disease patients by adapting the stimulation parameters to the patient’s clinical state estimated through the analysis of subthalamic neuronal oscillations (ie, local field potentials) in the beta band (13-30 Hz). Because aDBS administration was never systematically assessed during prolonged stimulation sessions in more ecologic conditions, we tested unilateral aDBS delivered for 2 hours, with specific focus on the concurrent administration of levodopa treatment, in freely moving parkinsonian patients. We therefore randomly administered aDBS and cDBS through an external wearable prototype in 10 PD patients with DBS electrode implant in 2 different experimental sessions taking place the 5th and the 6th day after surgery (Fig. 1A). Each experimental session lasted 2 hours, during which the patient, after a baseline assessment (OFF DBS and OFF medication, stimOFF/medOFF), received both levodopa and stimulation (aDBS or cDBS), thus allowing one to study the interaction between electrical and pharmacological stimulation (ON DBS and ON medication, stimON/medON). The patient was blind to the type of DBS received during the session. The clinical effects were blindly evaluated through the UPDRS III (motor part) and the Unified Dyskinesia Rating Scale (UDysRS). According to the gold standard, the clinical assessment was performed by a blinded video rater (rigidity scores were excluded from the analysis). The total electrical energy delivered (TEED) was used for energy efficiency assessment and adverse events were collected for safety assessment. The clinical scores were not significantly different between the 2 experimental sessions at baseline (stimOFF/ medOFF UPDRS III, aDBS vs cDBS: 37.0 6 16.8 vs 36.6 6 16.2; F1,9 5 0.2, P > .05). When the patient was under the effect of both levodopa and DBS (stimON/medON), we observed a similar improvement on global motor symptoms regardless to the type of DBS (UPDRS III percent change from baseline, aDBS vs cDBS: 246.1% 6 10.5% vs 240.1% 6 17.5%; F1,9 5 0.6, P > .05; Fig. 1B). Conversely, in this condition, aDBS was more effective on dyskinesias than cDBS (UDysRS score, aDBS vs cDBS: 11.7 6 67 vs 15.0 6 8.7; F1,9 5 6.1, P 5 .02; Fig. 1C). These results were obtained with an average power saving of 73.6% 6 22.9% in aDBS compared with cDBS (mean TEED aDBS vs cDBS: 44.6 6 47.9 lW vs 158.7 6 69.7 lW; F1,8 5 30.4, P 5 .0005). Throughout the entire experiment, we did not observe any serious adverse event specifically linked to DBS. These results support the idea that aDBS, being effective, efficient, and safe, when administered concomitantly to levodopa could help clinicians limit the severity of side effects induced by the transient summation of DBS stimulation and pharmacological therapy. However, the acute experimental setting, characterized by a microlesional