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Alterations in Energy Metabolism, Neuroprotection and Visual Signal Transduction in the Retina of Parkinsonian, MPTP-Treated Monkeys

Parkinson disease is mainly characterized by the degeneration of dopaminergic neurons in the central nervous system, including the retina. Different interrelated molecular mechanisms underlying Parkinson disease-associated neuronal death have been put forward in the brain, including oxidative stress and mitochondrial dysfunction. Systemic injection of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to monkeys elicits the appearance of a parkinsonian syndrome, including morphological and functional impairments in the retina. However, the intracellular events leading to derangement of dopaminergic and other retinal neurons in MPTP-treated animal models have not been so far investigated. Here we have used a comparative proteomics approach to identify proteins differentially expressed in the retina of MPTP-treated monkeys. Proteins were solubilized from the neural retinas of control and MPTP-treated animals, labelled separately with two different cyanine fluorophores and run pairwise on 2D DIGE gels. Out of >700 protein spots resolved and quantified, 36 were found to exhibit statistically significant differences in their expression levels, of at least ±1.4-fold, in the parkinsonian monkey retina compared with controls. Most of these spots were excised from preparative 2D gels, trypsinized and subjected to MALDI-TOF MS and LC-MS/MS analyses. Data obtained were used for protein sequence database interrogation, and 15 different proteins were successfully identified, of which 13 were underexpressed and 2 overexpressed. These proteins were involved in key cellular functional pathways such as glycolysis and mitochondrial electron transport, neuronal protection against stress and survival, and phototransduction processes. These functional categories underscore that alterations in energy metabolism, neuroprotective mechanisms and signal transduction are involved in MPTP-induced neuronal degeneration in the retina, in similarity to mechanisms thought to underlie neuronal death in the Parkinson’s diseased brain and neurodegenerative diseases of the retina proper.

Introduction
Parkinson disease (PD) is one of the most common progressive neurodegenerative disorders in our society, affecting over 1% of the aged people in Western countries and with a current estimated prevalence in the world of around 4.5 million persons [1]. Collected under the term parkinsonism, the symptoms exhibited by patients with this disease include resting tremor, rigidity, postural instability and bradykinesia [2]. The causal origin of PD is unknown in most cases, and treatments available today, based on the administration of levodopa and dopamine analogues, although substantially ameliorate clinical symptoms, do not alter the course of disease [3]. This is characterized by the massive, progressive and irreversible degeneration of dopaminergic cell bodies located in the midbrain substantia nigra and their axon terminals in the striatum, and by their cytoplasmic accumulation of typical protein fibrillar inclusions, named Lewy bodies [2,4]. This leads to a decrease in the substantia nigra of the levels of Tyr hydrolase (TH), the characteristic enzyme of dopaminergic neurons, and a decrease of the neurotransmitter dopamine in the striatum. Although the etiology of parkinsonism is still largely unknown, it is nowadays considered as a multifactorial disease beyond any doubt. Hence, although considerable progress has been made on the identification of genes associated with the development of parkinsonism, the action of environmental factors, mostly unknown, is recognized to be the cause of most cases of common (sporadic or idiopathic) PD [1,5,6]. In this context, some neurotoxins are known to induce the selective destruction of dopaminergic neurons, such as 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6hydroxydopamine and a variety of metals and pesticides, such as rotenone, whose administration to monkeys and rodents has allowed the generation of animal models of idiopathic PD [7][8][9] that add up to available rodent genetic models [10]. Extensive research on these experimental systems together with available PD patients has led to envision a series of molecular mechanisms as accounting for the pathophysiology of PD, including oxidative stress, mitochondrial dysfunction and excitotoxic damage [5,6,11,12], among others including mutations in genes encoding proteins with a neuroprotective role, such as parkin [2,5]. In this context lies the repeated finding in PD patients of decreased activity of complex I of the electron transport chain and, recently, of mitochondrial DNA deletions [13]. Hence, the chemical inhibition of complex I by systemically-administered MPTP, 6-hydroxydopamine or rotenone recapitulates dopaminergic neuron degeneration and associated neurological disability in experimental animals [6,7]. Also, the proposed prooxidant intracellular status generated by a cytoplasmic accumulation of dopamine, resulting from its defective vesicular storage, has been postulated to lead dopaminergic neurons to apoptosis [11,14].
The mammalian retina is the tissue with the most active metabolism in the organism and hence exhibiting the highest O 2 consumption. Any impairment in mitochondrial activity, induced for instance by the above neurotoxic agents, is thus expected to greatly affect the retina as part of the central nervous system (CNS). A growing body of experimental evidence has accumulated concerning visual dysfunction in the retina of parkinsonian patients including loss of visual acuity, contrast sensitivity, colour discrimination and motion perception [15][16][17]. The relevance of macaques treated with MPTP [18,19] or 6-hydroxydopamine [20] as a model of parkinsonism in the retina was initially supported by a series of studies highlighting a number of dysfunctional features detectable by electroretinogram and visual evoked potential recordings, which are similar to those reported in PD patients. These deficits have been correlated by our group with a series of morphological alterations occurring in the retina of MPTPtreated monkeys, where loss of TH immunoreactivity in the substantia nigra was accompanied by the degeneration of both retinal dopaminergic neurons and their postsynaptic AII amacrine cells, together with synapses formed among these neuronal subtypes in the inner plexiform layer [21]. More recently, we have also characterized the cellular and functional impairments taking place in the retina of rotenone-treated rats, where damage extended not only to dopaminergic neurons, but also to photoreceptors and their synaptic connectivity [22]. Deficiencies occurring at the molecular level in the parkinsonian retina are, however, essentially unknown, as are the particular mechanisms by which MPTP or rotenone elicits the degeneration of dopaminergic cells and other retinal neurons. In this work we have investigated the alterations in the retinal proteome taking place in the MPTP-treated monkey model of PD, in order to provide insight into the molecular mechanisms underlying neuronal degeneration in the retina under parkinsonism. Most of the proteins identified as differentially expressed in parkinsonian monkeys were found to be downregulated, and their functions were related to cellular energy metabolism, neuroprotection against stress and visual signal transduction.

Biological Material and Ethics Information
All studies were carried out in accordance with the guidelines promulgated by the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes of the Council of Europe (no. 123; June 15th, 2006), and following the Code of Ethics of the European Directive 2010/63/EU and the U.S. National Institutes of Health (NIH). All protocols and animal handling procedures used were approved by the bioethics research committee of the Universidad de Murcia [23]. Long-tailed macaques (Macaca fascicularis) were imported from R.C. Hartelust B.V. (Tilburg, the Netherlands) and housed at the animal care facility of the Universidad de Murcia under supervision by veterinarians and technicians skilled in the health care and maintenance of non-human primates. The animals were maintained in cages of 65 x 75 x 95 cm in a primate house under temperature (25°C) and humidity (50%) controlled conditions and a 12 h light/12 h dark cycle (lights on at 8:30 AM), and they had free access to special food (Masuri primate diet; Scientific Dietary Services, UK), fresh fruit and water. Even though animals were housed in partners, the arrangement of cages allowed each individual to have visual contacts and interaction with monkeys housed in adjacent cages. Animals used in the present work had not been utilized for any prior research.
Experiments were performed on three adult macaques (4-6 kg; both sexes) that were rendered parkinsonian upon treatment with MPTP hydrochloride (Sigma; 0.3 mg/kg i.v. for several months, 1 injection every 2 weeks). Monkeys were injected in the saphenous vein under gentle restraint and were never given levodopa or dopaminergic agonists. The number of MPTP doses administered was adjusted according to each animal's susceptibility to this compound, until a stable parkinsonian syndrome was reached [21,23]. Three additional, age-matched monkeys were kept without injections under the same conditions as the three treated, and used as control subjects. The total dose of MPTP and the time necessary to obtain sustained parkinsonian features ranged from 20 to 24 mg and from 26 to 28 weeks, respectively. This protocol represents a reproducible MPTP cumulative dosing regimen that leads to the first appearance of parkinsonian clinical signs after 6 ± 1 injections. Animal behaviour was assessed both in their home cages and in special observation cages for filming. One special cage, measuring 1 m 3 , was designed so that an independent camera located in front of the cage, in a special space separated from the animals with methacrylate, could perform independent recordings from each animal. The parkinsonian syndrome was independently analyzed by several scientists accustomed to evaluating motor disabilities in monkeys, which was carried out by coaxing the animals to perform various tasks upon offering them appetizing fruits. The degree of parkinsonism was scored twice weekly, over a 10 min observation period with respect to control animals, on the basis of a validated parkinsonian macaque clinical motor scale ranging from 0-2 (normal) to 25 (maximum severity) [24]. This scale covers eight features of motor disability induced by MPTP: spontaneous activity (0-5), bradykinesia (0-3), tremor duration (0-3), tremor intensity (0-3), posture (0-3), balance (0-2), freezing (0-3) and feeding (0-3).
No animal was sacrificed solely for the purpose of the research reported here, but instead other body parts were harvested by different researchers for a range of unrelated experiments, and their suffering was minimized at all times during this procedure [23]. The monkeys were anesthetized with ketamine (10 mg/kg i.m.) and then administered a lethal injection of pentobarbital (50 mg/kg i.p.). Eyeballs were then enucleated, and the neural retina was dissected free from the retinal pigment epithelium, frozen in liquid N 2 for transport to the Universidad de Alicante and stored at -80°C until use.

Protein Extraction and Processing
Retinal tissue samples were thawed at room temperature and homogenized in lysis buffer (20 mM Hepes pH 7.9, 10% glycerol, 10 mM KCl, 0.4 M NaCl, 0.1% Nonidet P-40 and 2 mM DTT) supplemented with protease inhibitors (1 mM PMSF, 10 μM leupeptin, 0.3 μM aprotinin, 100 μM benzamidine). After incubation on ice for 15-20 min with occasional gentle shaking, samples were centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant, containing solubilized proteins, was aliquoted and stored at -80°C. Protein concentration was quantified spectrophotometrically at 595 nm using the Bradford reagent (Sigma) and BSA as a standard.
Solubilized proteins (200 μg for analytical gels, 900 μg for preparative gels) were preincubated in 0.2 mg/l sodium deoxycholate (Sigma) for 15 min on ice. Then, they were precipitated by addition of trichloroacetic acid to 6% and incubation for 30 min on ice, and recovered by centrifugation at 16,000×g for 15 min at 4 °C. The pellet obtained was washed twice with 1.5 ml of 80% acetone (precooled at -20°C), centrifuging each time at 16,000×g for 15 min at 4 °C. Then the pellet was dried for 15 min at room temperature, and dissolved by vigorous stirring in 100 μl of 2D lysis buffer composed of 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and 30 mM Tris base. The proteins were purified with the 2-D Clean-Up kit from GE Healthcare (Buckinghamshire, UK) and finally dissolved in 40 μl of 2D lysis buffer by vigorous shaking and sonication for 10 min. Protein concentration was quantified using the RC-dC Protein Assay from Bio-Rad (Hercules, CA).

Fluorescent Labelling of Proteins
The pH of samples (50 μg of protein) for DIGE was adjusted to 8.5-9.0 by adding 1-2 μl of 100 mM NaOH. Then cyanine fluorescent dyes (GE Healthcare) were conjugated to solubilized proteins via N-hydroxysuccinimidyl linkages following the manufacturer's minimal labelling protocol. Briefly, the parkinsonian or control protein sample was taken to a volume of 18 μl with 2D lysis buffer and labelled alternately with Cy3 (green) or Cy5 (red) by addition of 1 μl of 0.4 mM fluorophore in N,N-dimethylformamide. Separately, an internal standard was prepared consisting of a pool of identical amounts of protein from the six monkey retinal samples labelled with Cy2. Reactions were carried out for 30 min on ice in the dark, and quenched by addition of 1 μl of 10 mM Lys and further 10 min incubation. For each gel, 50 μg of control group sample and 50 μg of MPTP group sample, each labelled with a different dye, was mixed with 50 μg of Cy2-labelled internal standard, to obtain a 65 μl mixture containing 150 μg of protein.
Then DIGE sample buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 2% IPG buffer pH 4-7 (GE Healthcare) and 130 mM DTT was added to a final volume of 130 μl, and the mixture was incubated on ice for further 15 min. To avoid biasing due to the dye, half of the samples of each experimental group were labelled with Cy3 and the other half with Cy5 [25].

2D Gel Electrophoresis
For IEF in the first dimension 18 cm-long Immobiline DryStrips (GE Healthcare) were used, containing immobilized ampholytes forming a linear pH 4-7 gradient. The strips were rehydrated in 350 μl of a buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 2% IPG buffer pH 4-7, 18 mM DTT and traces of bromphenol blue, for 16 h at room temperature covered with mineral oil. The samples were applied onto the strips following a cup loading protocol, and IEF was performed at room temperature in an Ettan IPGphor II unit (GE Healthcare) at 50 mA per IPG strip, according to the following program: fast gradient to 300 V for 3 h, linear gradient to 1,000 V for 6 h, linear gradient to 8,000 V for 3 h, and fast gradient to 8,000 V for 4 h, totalling 40,000 V·h at the end of the run.
After IEF, the IPG strips were incubated with shaking for 15 min in equilibration buffer I (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl pH 8.8, 1% DTT), and then for further 15 min in equilibration buffer II (buffer I in which DTT was replaced by 1.25% iodoacetamide). Thereafter, they were individually applied onto the surface of a vertical 12.5% polyacrylamide gel and sealed with 0.5% agarose dissolved in electrophoresis buffer (25 mM Tris-HCl pH 8.3, 192 mM Gly, 0.1% SDS) with traces of bromphenol blue. SDS-PAGE Broad Range or All Blue Precision Plus Protein (Bio-Rad) molecular weight standards were loaded by each end of the strip, and the second-dimension electrophoresis was carried out in a DALTsix Ettan unit (GE Healthcare) at a constant current of 10 mA for 14-16 h, or 40 mA for 4-5 h.

Gel Scanning and Staining
Following electrophoresis, signals from the three fluorophores in each gel were sequentially scanned on a Typhoon 9410 fluorescence imager (GE Healthcare) at a photomultiplier voltage of 500 V and a pixel size of 100 μm (254 dpi resolution). For Cy3, excitation and emission wavelengths used were 532 and 580 nm, respectively, and for Cy5 633 and 670 nm. Cy2 images were obtained at wavelengths of 488 and 520 nm.
DIGE gels were stained with silver nitrate, essentially as previously described [26]. Preparative gels were stained with colloidal Coomassie Blue according to established procedures [27], with some modifications. Briefly, gels were fixed in 50% ethanol and 2% phosphoric acid for 3 h and, after washing with milliQ H 2 O, incubated for 1 h in staining solution (3% phosphoric acid, 35% methanol, 100 mM ammonium sulfate). Then Coomassie Brilliant Blue G-250 solution in methanol (USB, Cleveland, OH) was added to a concentration of 0.65 g/l, and the gels were incubated for 14-16 h at room temperature. After destaining with milliQ H 2 O, they were stored in 5% acetic acid at 4 °C. Gels were digitized on an UVIdoc Documentation System (UVItec, Cambridge, UK).

MS Analyses and Database Search for Protein Identification
With the aim of identifying the proteins detected in the retina as differentially expressed in control vs. MPTP-treated monkeys, the spots were manually excised from Coomassie Blue-stained preparative polyacrylamide gels using a manual spot picker (Gel Company, Tübingen, Germany) with a 1.5 mm diameter picker head, and processed for trypsin digestion by using an Investigator Progest protein workstation (Genomic Solutions, Cambridgeshire, UK). The protein spots were washed sequentially in this system with 25 mM ammonium bicarbonate and then with H 2 O, and thereafter subjected to reduction with 10 mM DTT and alkylation with 100 mM iodoacetamide in 50 mM ammonium bicarbonate. The proteins were then digested with modified porcine trypsin from Promega (Madison, WI), in 25 mM ammonium bicarbonate for 6-7 h at 37 °C. The resulting peptides were sequentially extracted with ammonium bicarbonate, then with 70% acetonitrile, and finally with 1% formic acid.
For MALDI-TOF MS analysis, the peptides were dried under vacuum in a SpeedVac system for at least 1 h at 60°C. Then they were purified and concentrated on Zorbax 300SB-C18 silica gel tips (Agilent Technologies, Santa Clara, CA), and redissolved in 5 μl of a 0.1% trifluoroacetic acid: acetonitrile mixture (2:1). One μl of sample was spotted onto a MALDI plate (MTP AnchorChip 400/384 TF, from Bruker Daltonics, Bremen, Germany) together with 1 μl of 2,5-dihydroxybenzoic acid matrix dissolved in the same trifluoroacetic acid: acetonitrile mixture. Prior to analysis, the sample was allowed to dry on the matrix and an external calibration of the MALDI-TOF Autoflex mass spectrometer was performed using the PeptideMix peptide standard (both from Bruker Daltonics). The peptides in each sample were ionized with a 337-nm N 2 laser, and mass spectra were acquired in the positive ion mode at a reflectron voltage of 20 kV by averaging 30 laser shots. The range of masses considered was 800-3500 Da, suitable to cover in general the majority of peptides from trypsin digestion.
For LC-MS/MS, tryptic peptides were SpeedVac-dried as above and redissolved in 15 μl of 0.1% formic acid. LC-MS/MS analysis was performed using an Agilent 1100 nano-highperformance liquid chromatography system coupled to a mass spectrometer equipped with an XCTplus ion trap and an electrospray ionization source (Agilent Technologies). After concentration and desalting of peptides by employing a Zorbax 300SB-C18 column (0.3 mm × 5 mm, 5 μm) at a flow rate of 0.3 μl/min, they were separated on a Zorbax 300SB-C18 analytical column (75 μm × 15 cm, 3.5 μm) using a linear gradient of 5-45% acetonitrile in 0.1% formic acid at a constant flow of 0.3 μl/min, for 50 min. For acquisition of mass and mass/mass spectra, the parameters previously indicated [28] were used, with some modifications. Briefly, the spectra were obtained using the standard enhanced and UltraScan modes, at 26,000 and 8,100 m/z per second respectively. Parameters included a ionization potential of 1.8 kV and an ICC smart target (number of ions in the trap before acquisition) of 500,000 or a maximum accumulation time of 150 ms. MS/MS spectra analyses were performed using automated switching with a preference for +2 charged ions, a threshold of 105 counts and a 1.3 kV fragmentation amplitude.
Peptide mass fingerprints obtained from MALDI-TOF MS analysis were used to interrogate the NCBInr polypeptide database by employing the Mascot search engine [29]. Parameters were configured as follows: maximum trypsin missed cleavages, 1; fixed modification, Cys carbamidomethylation; variable modification, Met oxidation; peptide mass tolerance, ±100 ppm; mass values, monoisotopic, and peptide charge state, 1+. Molecular weight search (Mowse) scores were calculated by comparison of search results against estimated random match population, and reported as -10 × log (P) where P is the absolute probability [30]. Only those identifications that met the following criteria were considered as valid: significant (>80) Mowse score, ≥4 peptides identified and sequence coverage >25%.
The MS/MS spectra obtained from LC-MS/MS analysis were used to search the Swiss-Prot polypeptide database with the Spectrum Mill Proteomics Workbench software rev. A.03.03 (Agilent Technologies). Parameters were set as: maximum missed cleavages, 2; fixed modification, Cys carbamidomethylation; variable modifications, Met oxidation, Asn deamidation and pyro-Glu formation; and fragment mass tolerance, ±0.7 and ±0.6 Da for parental and fragment ions, respectively. Only those identifications that met the following criteria were considered as valid: scores given by the Spectrum Mill software, >8 to each peptide and >20 to the protein, and sequence determined for at least 2 different peptides. The automatic validation of each protein and peptide was verified manually to avoid false positives. The raw data (mzXML) and peak lists (*. PKL) are available as a 1.4 Gb compressed file upon request to the corresponding author.

Statistical and Quantitative Analyses
The spot staining pattern of DIGE gels was analyzed using the Progenesis SameSpots software v. 3.0 (Nonlinear Dynamics, Newcastle, UK). Equivalent spots across gels were matched, and images from control and parkinsonian monkeys were separately grouped. Spots outlined by the program were also visually inspected and manually edited when appropriate. The amount of protein present in each spot was densitometrically measured and its volume was calculated by integration of the optical density over the spot's area. Then normalized volumes (NV) were obtained by dividing it by the total volume over the whole set of gel spots (yielded by the internal standard, labelled with Cy2), and the NV values of each spot were separately averaged for control and MPTPtreated monkeys. The fold change in protein expression was calculated as the ratio between the higher and the lower of each pair of values, and a minus sign (-) was added when the lower was that of the treated group. The processing of numerical data was performed using Microsoft Excel, and statistical analyses were carried out using the Prism v. 5 program from GraphPad Software (San Diego, CA). Significance of differences between treatments was evaluated using the non-parametric Student's t-test with a confidence interval of 95%. In DIGE experiments the differences were additionally evaluated by analysis of variance (ANOVA), and only spots yielding a P value lower than 0.05 in both tests were considered for further analysis.
Autoradiography films from Western blots were digitized using an ImageScanner in transparency mode at 300 dpi and 16 bits grey scale, and image acquisition was performed using the LabScan v. 5.0 software (both from GE Healthcare). Densitometric quantitation of protein bands was accomplished using ImageJ 1.42q [33], and values obtained for each protein were normalized to β-actin levels. For each quantified protein we analyzed the results of at least two independent experiments, each containing a minimum of three biological replicates.

Results
Three eyes were used for this study each from a different macaque that had been chronically treated with MPTP and who had developed a stable and persistent parkinsonian syndrome, from moderate to severe as judged from its motor alterations. Two of them exhibited a severe parkinsonism (motor disability, 16/25) and the third moderate parkinsonian symptoms (motor disability, 13/25). These monkeys displayed bradykinesia/ akinesia, rigidity, freezing phenomena, action tremor and paradoxical kinesias, as well as abnormal vertical and horizontal saccadic ocular movements. They also showed postural disturbances (trunk and limb flexion) and balance alterations with occasional falls. Three additional, age-matched monkeys kept without injections under the same conditions as the three treated were used as control subjects (motor disability, 0-2/25). After sacrifice, their eyes were enucleated and their neural retinas dissected. Monkeys treated in the same fashion and exhibiting comparable motor scores displayed a substantial reduction of TH immunoreactivity in both the substantia nigra and the retina, resulting in the degeneration of dopaminergic cell bodies and dendritic plexi in both tissues [21].

Analysis of Differential Protein Expression
Differences in the abundance of particular proteins in the retina of parkinsonian vs. control monkeys were screened by means of 2D DIGE technology. A preliminary 2D gel was run in the pH range 3-10, where most detected proteins were observed to fall within the pI interval 4-7. Therefore, this pI range was subsequently used for the first-dimension IEF. For the second dimension regular SDS-PAGE was carried out at a fixed, 12.5% polyacrylamide concentration. Thereafter, the 2D gels were scanned with a Typhoon 9410 Variable Mode Imager and the spot maps obtained were bioinformatically analyzed using the Progenesis SameSpots software. A representative DIGE gel is shown in Figure 1, where separate images corresponding to the proteins from a control parkinsonian monkey retina (labelled in green) and from an MPTP-treated subject (labelled in red) are shown in Figure 1A and 1B, respectively. The merged image is shown in Figure 1C, where yellow spots represented proteins with similar expression levels in both samples and were thus not considered.
Over 700 discrete spots were clearly resolved on 2D DIGE gels. The electrophoretic pattern obtained was consistent for retinal extracts from individuals of the same group (i.e. control or MPTP-treated monkeys), although a certain variability was observed attributable to the fact that the animals studied were not isogenic. A bioinformatic analysis revealed that quantitative differences existed for a total of 36 spots between the two experimental groups, whose positions in 2D gels are numerically labelled in Figure 1C. These spots were selected for their variation exceeding an arbitrary threshold of 1.4-fold increase (40%) or decrease (29%) in their expression levels and for differences being concluded to be statistically significant from both the Progenesis SameSpots software and Student's t-test. In particular, 34 spots exhibited a decrease of expression above 1.4-fold in parkinsonian monkeys and 2

Identification of Differentially-Expressed Proteins
From preparative 2D gels stained with colloidal Coomassie Blue a series of proteins were recovered that exhibited altered amounts in the parkinsonian monkey retina when compared to controls. A total of 29 spots were selected on the basis of their unequivocal visualization and localization in the preparative gel, and following excision and trypsin digestion they were subjected to MS analysis. Eighteen spots were successfully identified by means of peptide mass fingerprinting and/or MS/MS search analyses by interrogation of the NCBInr and Swiss-Prot polypeptide databases, respectively, and the characteristics of the corresponding proteins are summarized in Table 2. In instances where these databases did not contain any matching M. fascicularis protein sequence (most cases), the identification was carried out on the basis of its homology to the human (Homo sapiens), rhesus macaque (Macaca mulatta) or chimpanzee (Pan troglodytes) ortholog sequence. Table 2 includes, in addition to protein names, their accession numbers in the corresponding database (NCBInr and/or Swiss-Prot) and the species to whom such sequence belongs. Also indicated for each protein in Table 2 are its pI and Mr values, both experimental (calculated from its migration position in 2D gels) and theoretical (from its amino acid sequence in the database), together with the MS method(s) allowing its identification. Some spot pairs corresponded to the same protein, in particular 3 and 115 (S-arrestin), 13 and 18 (DDAH1), and 20 and 237 (γ-enolase), which represented isoelectric variants derived from proteolytic processing or other post-translational modifications changing their intrinsic charge and/or mass. For some of the identified proteins significant differences were found between their experimental and theoretical pI (e.g. spots 1 and 37) or Mr (e.g. spots 13 and 20) values, which could be attributed to the polypeptide excised from the gel harbouring covalent modifications occurring post-translationally (e.g. phosphorylation, glycosylation, etc.) or "accidentally" during sample handling (e.g. oxidation), or else to its encoding mRNA undergoing alternative splicing. For identifications carried out by peptide mass fingerprinting followed by database screening using the Mascot search engine, Table 2 shows each protein's Mowse score, taken as a confidence index, the number of peptides whose mass was represented in the fingerprint, and the sequence coverage. Mowse scores for this group of proteins ranged between 133 and 294, the sequence coverage between 53 and 73%, and the number of matching peptides between 15 and 21. Table S1 gives for each protein spot the number of peptide masses obtained by MALDI-TOF used for database screening, the number of masses matching amino acid segments of the identified protein, and sequences of the latter. For identifications carried out by LC-MS/MS, the score yielded by the Spectrum Mill software is given in Table 2, together with the percentage of sequence coverage and the number of identified peptides. For this group of proteins, which constituted the vast majority, the MS/MS search scores varied between 17.43 and 378.20, the sequence coverage between 6 and 58% and the number of peptides between 2 and 21. Those with the lowest values for these three parameters, i.e. spots no. 20 and 34, were identified by de novo sequencing using the Sherenga algorithm [34], illustrated in Figure S1. The sequences of the different peptides identified in each protein spot by means of LC-MS/MS or de novo sequencing using Sherenga are shown in Table S2. The corresponding score, charge, scored peak intensities (SPI) and monoisotopic mass of each identified peptide precursor are also indicated in Table  S2.
In total, a number of 15 different polypeptides were identified, whose differential expression levels are illustrated in Figure 2. These proteins are involved in a variety of functions within the cell. Table 3 summarizes, for each protein, its encoding gene and its accession number in the Gene Ontology (GO) database, where data on biological processes, cellular components and molecular functions associated with every known protein are compiled [35]. These are as follows: Spot no. 1, whose expression levels were decreased (by 1.93-fold) in the retina of MPTP-treated monkeys compared to control animals (Table 1; Figure 2), was identified as αenolase, encoded by the ENO1 gene. This is a ubiquitous protein in adult tissues which participates in a variety of biological processes, such as growth control, hypoxia tolerance and allergy, but whose main function is to act as a glycolytic enzyme (Table 3) catalyzing the conversion of 2-phospho-Dglycerate into phosphoenolpyruvate.
Spot no. 2 (which exhibited a 2.97-fold reduction of staining intensity in the parkinsonian retina) was found to correspond to b. The isoelectric point (pI) and relative molecular mass (Mr) are also given, both experimental and theoretical, together with the MS method(s) used for its identification.
c. The MOWSE score is indicated together with the fraction (%) of protein sequence covered by the identified peptides and the number of these represented in the mass fingerprint.
d. The corresponding score is indicated together with the fraction (%) of sequence and the number of peptides identified.  the β subunit of ATP synthase F 1 component, encoded by the ATP5B gene. This is a constituent protein of mitochondrial complex V, i.e. the terminal complex in the electron transport chain, whose well known function is to carry out ATP synthesis from ADP by using the proton-motive force generated across the mitochondrial inner membrane. Adjacent spots numbered 3 and 115 (see Figure 1C) were ascribed to presumable isoelectric variants of S-arrestin (showing content decreases of 2.82-and 2.17-fold, respectively). This is the product of the SAG gene, constituting a major component of the outer segments of rod photoreceptors where it participates in desensitization of the phototransduction cascade [36]. This is exerted through Sarrestin ability to bind and inhibit phosphorylated, lightactivated rhodopsin, thereby preventing activation of cGMP phosphodiesterase mediated by transducin [36].
Spot no. 5 (whose normalized volume was increased by 2.52-fold in the parkinsonian retina) corresponded to βcrystallin B2, the product of CRYBB2. Crystallins, the majoritary structural components of the eye lens, are a superfamily of oligomer-forming proteins whose function is to increase the refractive index of the lens while maintaining its transparency. Yet, an additional role of these proteins in retinal neuron survival is becoming increasingly clear [37,38].
Spot no. 7 (displaying a 2.47-fold reduction in the MPTPtreated retina) was identified as the heat shock 70 kDa protein 8 (HSC70), also called heat shock cognate 71 kDa protein. This is the product of the HSPA8 gene, which codes for a constitutively-expressed, cytosolic chaperone belonging to the Hsp70 family responsible for the correct folding and assembly of nascent polypeptides and degradation of misfolded proteins by chaperone-mediated autophagy [37]. Spot 12 was ascribed to stathmin, the product of the STMN1 gene (presenting at lower levels, i.e. a 1.61-fold reduction, in treated monkeys), which is involved in cytoskeleton dynamics by preventing the assembly and promoting the disassembly of microtubules in a phosphorylation-regulated manner [39].
Spots 13 and 18 (showing a decrease in their normalized volumes by 2.10-and 1.99-fold, respectively) corresponded to the product of the DDAH1 gene, N G , N G -dimethylarginine dimethylaminohydrolase-1. This enzyme hydrolyzes both asymmetric N G , N G -dimethyl-L-arginine (ADMA) and N Gmonomethyl-L-arginine (MMA), two competitive inhibitors of all isoforms of nitric oxide synthase (NOS), to yield dimethylamine and L-citrulline, which no longer inhibit NOS [40]. The DDAH1 enzyme thus represents a mechanism for modulation of NO synthesis in both physiological and pathological states.
Spots 20 and 237 were also underexpressed in the retina of parkinsonian macaques (by 1.95-and 1.51-fold respectively) and were identified as pertaining to γ-enolase, a neuronspecific protein encoded by ENO2. Like the α isoenzyme above, γ-enolase participates in glycolysis by catalyzing the conversion of 2-phospho-D-glycerate into phosphoenolpyruvate. However, it also has neurotrophic and neuroprotective properties on neurons, promoting their survival in culture [41].
Spot 21 (whose intensity was found diminished 1.94-fold in the treated monkey retina) corresponded to the 78 kDa glucose-regulated protein (GRP78 or BiP), the product of the HSPA5 gene. This is a chaperone mainly located at the endoplasmic reticulum (ER) that, alike Hsc70, belongs to the Hsp70 protein family. GRP78 not only is involved in correct protein folding and the assembly of protein complexes in the ER lumen, but also exhibits anti-apoptotic functions in neurons [42]. a. Refers to that indicated in Figure 1C.
b. The protein pertaining to each spot and the name of its encoding gene are indicated.
c. Accession number(s) for each protein and most relevant associations found in the GO database in relation with the scope of this article.
MS analysis allowed the identification of spot 31 (reduced by 1.37 fold in MPTP monkeys) as the GAPDH gene-encoded glyceraldehyde 3-phosphate dehydrogenase. This is a NAD +dependent, relevant enzyme in the glycolytic pathway that catalyzes the phosphorylation-coupled oxidation of Dglyceraldehyde 3-phosphate to yield 1,3-bisphosphoglycerate. However, GAPDH has roles additional to its classical function as a glycolytic enzyme, also acting as a mediator of apoptosis promoted by cytotoxic stressors in neurons [43] and bearing a modulatory function of microtubule organization and dynamics [44].
Spot 33 was found to represent inorganic pyrophosphatase (showing a 1.67-fold in its normalized volume in the retina of parkinsonian vs. control individuals), coded for by the PPA1 gene. This is a relevant enzyme in phosphate metabolism carrying out the hydrolysis of pyrophosphate (PPi) to yield two inorganic phosphate (Pi) molecules.
Spot 34 (whose intensity in the MPTP-treated retina was 1.65-fold lower than normal) was identified as calbindin D28k, the product of CALB1. This is a ubiquitous, Ca 2+ -binding protein with a relevant role in modulation of cytosolic Ca 2+ levels and a proposed neuroprotective role [45,46].
The second protein found overexpressed in parkinsonian individuals (by 1.41-fold) was that represented by spot 37, which was ascribed by MS analysis to nucleoside diphosphate kinase B (NDPK B), encoded by the NME2 gene. This is a cytoplasmic enzyme involved in the synthesis of nucleoside triphosphates, catalyzing the conversion of XTP plus YDP into XDP plus YTP (where X and Y are nucleosides other than adenosine) through formation of a His-phosphorylated enzyme intermediary form, although it may also act as a transcriptional activator [47]. In the retina it provides a key role in recycling GTP for phototransduction [48].
Spot 42 (which exhibited a volume reduction by 1.56-fold in the MPTP-treated retina) was found to correspond to the heme-containing, 5A subunit of cytochrome c oxidase (COX), encoded by COX5A. This is a constituent protein of mitochondrial complex IV, i.e. the terminal enzyme of the electron respiratory chain whose well known function is the transfer of electrons from cytochrome c to O 2 .
Finally, spot 91 was identified as γ-synuclein, encoded by the SNCG gene (and whose levels turned out to be 1.56-fold underexpressed in the treated monkeys). This protein plays a role in neurofilament network integrity and is thought to modulate axonal architecture during development and in the adult stage [49,50].

Validation of Differentially-Expressed Proteins
Once analyzed and identified a series of proteins that showed differential expression levels between parkinsonian monkeys treated with the neurotoxin MPTP and controls, we proceeded to validate the reliability of our proteomic approach by Western blotting. With this purpose, three proteins were selected for the sake of being involved in phototransduction (Sarrestin and NDPK B) or being a good marker for cone photoreceptors in primates (calbindin) [51], and their levels detected in retinal extracts from parkinsonian and control monkeys are shown in Figure 3A. The expression fold changes shown in Figure 3B were calculated as the ratio between the MPTP-treated and control normalized values. We confirmed that levels of S-arrestin and calbindin were reduced, to 55.4% and 53.4% with respect to controls, respectively, which was coherent with decreases determined by DIGE. Also in keeping with the latter, NDPK B levels were found increased in the PD retina by Western blotting, by 7.84 fold as compared to untreated subjects. These experiments were indicative that DIGE combined with MS yielded results in the same direction than immunoblotting regarding differential expression of proteins in the retina of MPTP-treated monkeys.

Discussion
Proteomics approaches have been previously used to address molecular alterations associated with a number of neurodegenerative disorders, including PD [6,14,[52][53][54][55], Alzheimer disease (AD) [56][57][58] and Down syndrome [56], as well as those accompanying aging [59], in the brains of both human patients and animal models. Proteomic comparative analyses have also been undertaken on the retinas of human patients and animal models of Leber congenital amaurosis [60], age-related macular degeneration (AMD) [61][62][63], glaucoma [64] and diabetic retinopathy (DR) [65][66][67][68]. However, research at the proteome level of molecular alterations associated with PD in the retina of human or parkinsonian experimental animals has not been conducted so far. We have undertaken in this work a proteomic analysis of molecular alterations present in the retina of monkeys treated with the proneurotoxin MPTP, an animal model of parkinsonism where our group has previously described the existence of significant retinal impairments at the morphological and synaptic levels [21]. Proteins found in this work to undergo a decrease (most cases) or increase in their abundance in this animal model can be grouped into three main functional categories, as follows.

Enzymes Involved in Energy Metabolism
We have determined in the retina of parkinsonian monkeys a lower abundance of enzymes involved in glycolysis, such as GAPDH and α-and γ-enolases. A reduced content of GAPDH has also been reported in the retina of streptozotocin-treated, DR rats [65]. Regarding enolases, the α polypeptide is expressed in most tissues, while the γ subunit is present exclusively in neurons, where functional enolase dimers mainly consist of γ/γ homodimers and α/γ heterodimers [69,70]. γ-Enolase plays roles additional to its function as a glycolytic enzyme, exhibiting neurotrophic and neuroprotective effects on cultured neocortical, mesencephalic and spinal cord neurons [41,71]. Alterations in γ-enolase levels have been reported in the cerebral cortex of mice with a KO mutation in the Park2 gene, encoding parkin [55]. Here it is relevant to emphasize that loss-of-function mutations in the human PARK2 gene are associated with autosomal recessive PD with juvenile onset [72]. Regarding the retina, α-enolase is found underexpressed at the onset of human AMD [61], and in aged rats this enzyme is irreversibly inactivated by the covalent addition of 4hydroxynonenal (4HNE) [73], a reactive aldehyde derived from polyunsaturated fatty acid peroxidation that also accumulates in the brain of Park2 KO mice [14]. Both α-and γ-enolases undergo nitrosative modification as well in the AD human brain [74]. Therefore, oxidation or nitrosylation of these enzymes in the CNS could bring about an impairment in glycolysis-coupled energy production, with especially harming effects in retinal neurons given their high energetic demand. Our observation of a decrease in levels of GAPDH together with those of α-and γenolases suggests the operation in the retina of a feed-back negative regulatory mechanism of glycolysis in response to blockade of mitochondrial complex I. Other glycolytic enzymes downregulated in the pathological retina are aldolase C at the onset of AMD [61] and triose phosphate isomerase in alloxantreated, DR model rats [67], while underexpressed Krebs cycle enzymes include pyruvate dehydrogenase in the midbrain of Park2 KO mice [14] and rotenone-treated rats [75]. In keeping with our hypotesis, it has been previously proposed that neurons respond to induced inhibition of mitochondrial respiration and increasing of reactive oxygen species (ROS) by actively downregulating glycolysis (through the action of the ubiquitin-proteasome pathway), giving priority to the consumption of glucose to maintain an antioxidant status over its use to fulfill their bioenergetic requirements [76].
We have also observed a reduction in the content of the 5A subunit of COX and β subunit of ATP synthase in the retina of parkinsonian monkeys, two enzymes that are well known constituents of mitochondrial complexes IV and V, respectively. A reduced activity or presence of these or other mitochondrial respiratory chain components has been reported in the brains of idiopathic PD, AD and Down syndrome cases [56,[77][78][79], which has been correlated with a reduced respiratory capacity of mitochondria from Park2 KO mice [14]. In the same context, the contents of both α and β subunits of ATP synthase F 1 complex are decreased in the retinal pigment epithelium of AMD patients [63], as it is the case for the d subunit of the F 0 complex (H + channel) in the neural retina of DR rats [67]. Taken together, these data underscore that MPTP-induced mitochondrial complex I dysfunction can lead to alterations in the levels of other components of the respiratory chain, in analogy to what occurs in PD [6,75,80] and other neurodegenerative diseases affecting the brain and/or the retina. In a related fashion, an additional enzyme whose levels we have found decreased in the parkinsonian monkey retina is inorganic pyrophosphatase 1 (PPA1), which is responsible for modulating Pi levels and is intimately linked to cell survival [81,82]. Given that its activity is coupled to reactions in which PPi is released, and that it provides a thermodynamic pull for many biosynthetic reactions, its lower levels found in our experimental system are consistent with a situation in which the production of ATP is decreased due to the MPTP-provoked mitochondrial dysfunction.

Neuroprotective Mechanisms
We have detected alterations in the expression of a series of proteins directly or indirectly related to neuronal protection against a variety of stresses, either endogenous or exogenous, in the retina of MPTP-treated monkeys. First, we have found underexpressed two members of the 70 kDa family of heat shock proteins (HSP70), namely HSC70 and GRP78, compared to control individuals. HSC70 is a molecular chaperone that functions to maintain protein homeostasis in cells under normal and stress conditions [37]. Its pattern of distribution in the rat retina includes all retinal layers, except photoreceptor outer segments [83], and its expression levels have been found by proteomic analysis diminished in the striatum and cortex of Park2 KO mice [55]. As well, a significant mRNA underexpression of its encoding gene, HSPA8, occurs with age in the primate retina [84]. It follows that its ameliorated levels found in PD or aging could underlie the neurodegeneration occurring in these conditions, as proposed for both the brain [58] and the retina [84]. On the other hand, the intravitreal injection of HSC70 is protective against acute light-induced damage in rats, increasing the number of surviving photoreceptors [85]. Relatedly, GRP78 is a rough-ER chaperone working in the unfolded-protein response to prevent ER stress-induced cell death in the brain [86,87] and retinal [88] neurons, a phenomenon associated with a number of neurodegenerative diseases [37] including retinal disorders [59,89]. GRP78 is expressed in photoreceptors, including their outer segments [90,91], and attenuated levels of this protein have been documented in the brain of PD patients [86] and the cortex of Park2 KO mice [55], this pointing out to the dysfunction of a neuroprotective mechanism additional to HSC70 in our MPTP-treated monkeys.
We have detected DDAH1 levels to be lower in the retina of PD macaques in this work, while DDAH2 has been found downregulated in the DR rat retina [67]. These are monomeric hydrolases expressed in the brain and other organs able to degrade ADMA and MMA, two methylated L-arginine derivatives which act as endogenous inhibitors of NOS, thereby contributing to regulated NO levels in numerous disease states [40]. Hence, a relationship exists between NO and protein misfolding in neurodegenerative disorders [92] such as PD, where nitrosative stress very often accompanies oxidative stress [6,12,93]. In this context, levels of proteins protective against the latter are diminished in the brains of PD patients and Park2 KO mice, together with a reduced antioxidant capacity and ability to respond to ROS generation [14,52]. In a related fashion, parkin is found S-nitrosylated in the brains of parkinsonian animal models and patients, this resulting in inhibition of its ubiquitin E3 ligase activity and neuroprotective function [93][94][95]. As well, TH itself is known to undergo Tyr nitration in MPTP-treated mice, also resulting in its inactivation [96]. Therefore, the decreased DDAH1 levels in the retina of our parkinsonian monkeys should result in lower NO levels, and thus be oriented at counteracting MPTP-derived nitrosative stress consequences, in the same line of thought as proposed for dopaminergic neurons of baboons [97] and mice [98,99] brains, and for DDAH2 downregulation in the DR rat retina [67].
As a difference with other proteins above, β-crystallin B2 was found overexpressed in the retina of parkinsonian monkeys. α-, β-and γ-crystallins, in addition to being the main structural constituents of the lens, are present in the neural retina and brain, and their additional role as oxidative stress-inducible chaperones of the small HSP family is especially clear for αcrystallins [38,100]. The localization of β-crystallin B2 in the mouse retina includes the inner segments of photoreceptors, some cells of the inner nuclear layer and ganglion cells [101]. Overexpression of β-crystallin B2 has also been reported in the retinas of rats suffering DR [68,102], optic nerve physical damage [103] or normal aging [73]. Other α-, β-and/or γcrystallins also become upregulated in the retina of several genetic rodent models of retinitis pigmentosa [104,105] and DR model rats [66][67][68]102], and under light-induced retinal degeneration [105], as well as in the human AD brain [57]. All these investigations have led to the view that crystallins could act, not only as chaperones, but also as factors promoting the regrowth of neuronal processes, thereby participating in axonal regeneration upon tissue damage [14,37,103]. Even, their level increases (especially of α-crystallins) have been correlated with the rate of photoreceptor loss [105].
Stathmin, encoded by the STMN1 gene, belongs to a family of microtubule-destabilizing proteins with an important role in cytoskeleton reorganization taking place during neuronal differentiation in development and after lesion in adulthood [39,106]. It is thus involved in modulating axon integrity in the nervous system, in the context of retinal plasticity and in regeneration processes. The levels of this protein appear reduced in the brains of patients with AD [107] and Down syndrome [108], as well as in the AMD retinal proteome [61]. Stathmin decrease in the PD monkey retina could either reflect the failure of a system in charge of maintaining microtubule stability and axonal integrity in the retina, such dysfunction contributing to neuronal degeneration. On the other hand, since stathmin is a microtubule-depolymerizing protein, its reduction could be aimed at counteracting neuronal morphology derangement, as it could also be the case for GAPDH [44]. Further research is needed to discern between these hypotheses.
Calbindin is a multifunctional cytoplasmic protein crucial in the modulation of cytosolic Ca 2+ levels and intracellular signalling. It has been found that dopaminergic neurons that express calbindin in the substantia nigra are spared from degeneration in PD patients and MPTP-treated monkeys and mice [45], and levels of this protein are reduced in the brain of old mice [59]. This indicates that dysregulation of Ca 2+ levels could be one of the mechanisms underlying brain and retinal neurodegeneration associated with parkinsonism, and points out a likely preventive role of calbindin in this context. In a related fashion, the levels of another potentiallyneuroprotective Ca 2+ -binding protein, the neuron-specific calretinin, are diminished in the cortex of Park2 KO mice [55] and in the retina of AMD patients [61].
Last in this group of neuroprotective proteins, γ-synuclein was also found underexpressed in the retina in our PD monkey model. This is a cytoplasmic protein whose function in the retina is poorly understood, and that is mainly contained in some ganglion cells and in optic nerve fibres [109]. It has been proposed to play a role as a retinal chaperone, working to restore the correct folding of anomalous proteins and prevent their aggregation [110]. In this context, γ-synuclein has shown some neuroprotective effects on a photoreceptor cell line expressing a retinitis pigmentosa-causative dominant allele of the rhodopsin gene, P23H [111], encoding a misfolded, cytotoxic variant of this protein.

Visual Signal Transduction
S-Arrestin, also known as rod arrestin, is one of the most abundant proteins in the outer segments, where it participates in the regeneration phase of the phototransduction cascade with restoration of high cGMP levels [36]. It binds to photoactivated, phosphorylated rhodopsin, thereby preventing its interaction with transducin and ensuing activation of cGMP phosphodiesterase 6B (PDE6B) [48]. Therefore, the attenuated S-arrestin expression we have found in MPTP monkeys would act in the sense of keeping PDE6B active and, consequently, Na + and Ca 2+ cGMP-gated channels closed, with ensuing cell hyperpolarization and cessation of Glu release in photoreceptor axon terminals. Somewhat surprisingly, interaction of S-arrestin with α-enolase has been recently reported to modulate the activity of the latter, in the sense of reducing its catalytic rate [112]. Conversely, and more in line with our results, mutations in the SAG gene have been associated with Oguchi disease, a variant of retinitis pigmentosa [113].
We have also found that NDPK B is overexpressed in the retina of parkinsonian monkeys. This protein, among other functions, provides the necessary intracellular GTP for transducin and guanylate cyclase activities. Two hexameric isoforms of this enzyme, A and B, are present in the retina [48], of which the B isoform is known to form a complex with the Gβγ heterodimer of various G proteins, including transducin. Actually, NDPK B and transducin copurify from rod outer segment membranes [114], and transducin is long known to mediate interaction of NDPK (A and B) with (bleached) rod outer segment membranes [115]. Furthermore, transducin Gβγ is likely to be a substrate for His phosphorylation by NDPK B in the retina [116]. Our results thus suggest that in parkinsonian monkeys an increased activation of transducin could take place independently of photoactivated rhodopsin in an NDPK Bmediated fashion, this enhancing PDE6B activity and thereby decreasing cGMP intracellular levels. The NDPK B increase would thus act in the same direction as S-arrestin underexpression, i.e. contributing to keep Na + and Ca 2+ cGMPgated channels closed. Very interestingly, the NDPK Bencoding mRNA is overexpressed in the degenerating retina of the rd/rd mouse, a retinitis pigmentosa model lacking PDE6B [104].
The alteration in S-arrestin and NDPK B levels reflects, in our belief, a protective response by photoreceptors to mitochondrial damage. Since NDPK B appears to be a stressinducible protein, its rise together with α-and β-crystallin upregulation may be a component of the stress response to photoreceptor loss and altered retinal architecture [104]. In this picture, Na + /Ca 2+ channel closure would be aimed at preventing a sustained elevation of intracellular Ca 2+ , which would trigger apoptotic death of photoreceptors [117], as well as at avoiding the energy expenditure required to maintain the dark current [118]. In fact, such high energetic demand could not be fulfilled by mitochondria in a situation of depressed metabolism plus oxidative stress, where the additionally superimposed calbindin downregulation would hamper the buffering of such Ca 2+ increase [119].

Conclusions
This study is the first in providing direct evidence of molecular alterations in the retina associated with PD, obtained from a proteomics approach. Proteins found with altered levels can be classified as related to: i) impairment of energy metabolism (particularly concerning the glycolytic pathway and mitochondrial ATP synthesis); ii) neuronal protection against various stresses; and iii) visual signal transduction. Most of these alterations act in the sense of potentiating neuroprotective mechanisms aimed at counteracting MPTP oxidative damage and/or promoting cell survival. The downregulated levels found for proteins crucial for glycolysis and mitochondrial electron transport chain reflect, in our view and in consistency with previous proteomic studies on parkinsonian human and animal-model brains, a feed-back cellular response to mitochondrial complex I inactivation oriented to diminish the global rate of cellular energy metabolism and/or the excess production of ROS ensuing mitochondrial dysfunction. Other alterations occurred in the sense of ameliorating nitrosative stress, increasing chaperone activity, counteracting morphological degeneration or lowering the phototransduction rate itself. Nevertheless, some decreases of protein expression (such as the lower presence of HSC70, GRP78, calbindin and γ-synuclein, and as discussed above for stathmin) may simply reflect a failure or dysregulation of protective neuronal systems. This would be a direct consequence of the intracellularly generated oxidative stress and subsequent intracellular damage, similar to that seen in the aging process [84], in cells that have irreversibly entered the apoptotic way and thereby contributing to its progression. This is consistent with the finding that in the human PD substantia nigra alterations in the mRNA levels of some genes do accelerate cell death activity, whereas other retard or attempt to compensate this process [120], as also concluded from other proteomics studies tackling neurodegenerative diseases. It follows that a complex set of mechanisms including neuroprotective responses as well as neuronal death appears to be involved in PD onset or progression, whose interplay should require a good number of further studies to be clarified.
Loss of dopaminergic amacrines and their postsynaptic cells in the peripheral retina of MPTP-treated monkeys has been suggested to impair the scotopic visual pathway [21]. However, recent studies on PD patients indicate that foveal vision is impaired in humans, as evidenced by thinning of the inner retina in the macular region detected by optical coherence tomography (OCT) [15,17,121], which together with loss of electrical activity recorded in the fovea reflects a deficit in the photopic pathway [122][123][124]. Differences in the retinal proteomes of monkeys treated with the parkinsonism inducer MPTP compared to control animals point out to important functional groupings (energy metabolism, protein folding vs. degradation, stress response, neuronal survival vs. apoptosis, signal transduction pathways, cytoskeletal dynamics and others) and particular protein actors that have been implicated as well in the pathogenesis of a number of CNS disorders in humans and animal models, some of which also affect the retina. It can thus be inferred that common intracellular agents and molecular mechanisms, whose involvement in neurodegenerative processes in the brain is increasingly known, are implicated as well in retinal degenerations, including PD-derived derangement of retinal neurons and retina-specific diseases. Further studies are needed to more exactly dilucidate the role of each of them in retinal neurodegeneration in monkeys treated with MPTP, and in terms of PD in general, and to unravel the levels at which they are interrelated. This information could eventually be useful to develop pharmacotherapies targeting fundamental biochemical defects aimed at retarding disease progression and/or alleviating neurodegeneration symptoms in the the retina and brain. Figure S1. MS/MS spectrum of a peptide identified by de novo sequencing. The fragmentation spectrum of a calbindin precursor peptide (spot no. 34) is shown, which was identified by de novo sequencing using the Sherenga algorithm. Its amino acid sequence obtained from database search using the Spectrum Mill software is indicated at the top of the image (MSTag), and that interpreted by the Sherenga algorithm is given below (upper and lower graphs, respectively). Graphs show the relative intensity (%) of each peptide fragment plotted as a function of its m/z value, indicated above its corresponding peak. (TIF)


extfieldsofstudy: Biology Medicine

text:
The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine

The Human Gene Mutation Database (HGMD®) is a comprehensive collection of germline mutations in nuclear genes that underlie, or are associated with, human inherited disease. By June 2013, the database contained over 141,000 different lesions detected in over 5,700 different genes, with new mutation entries currently accumulating at a rate exceeding 10,000 per annum. HGMD was originally established in 1996 for the scientific study of mutational mechanisms in human genes. However, it has since acquired a much broader utility as a central unified disease-oriented mutation repository utilized by human molecular geneticists, genome scientists, molecular biologists, clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals, bioinformatics and personalized genomics. The public version of HGMD (http://www.hgmd.org) is freely available to registered users from academic institutions/non-profit organizations whilst the subscription version (HGMD Professional) is available to academic, clinical and commercial users under license via BIOBASE GmbH.

Introduction
The Human Gene Mutation Database (HGMD Ò ) represents an attempt to collate all known gene lesions responsible for causing human inherited disease together with disease-associated/functional polymorphisms that have been published in the peer-reviewed literature. These data comprise single base-pair substitutions in coding, regulatory and splicing-relevant (both intronic and exonic) regions of human nuclear genes, as well as micro-deletions and micro-insertions, combined micro-insertions/micro-deletions (indels) of 20 bp or less, repeat variations, gross lesions (deletions, insertions and duplications of greater than 20 bp, up to and including a single characterized gene or group of contiguous genes that are directly involved in the aetiology of the disease/phenotype) and complex rearrangements (including inversions, translocations and complex indels). Mutation data are summarized in Table 1. HGMD does not include either somatic or mitochondrial mutations, which are well covered by COSMIC (Forbes et al. 2011) andMitoMap (Ruiz-Pesini et al. 2007), respectively. HGMD also does not attempt to provide comprehensive coverage of pharmacological variants (except for those variants where evidence supporting a functional impairment has been provided); such variants are covered by PharmGKB (Thorn et al. 2010). Finally, HGMD is not a general genetic variation database; users interested in this type of variant should visit dbSNP (Sherry et al. 2001) or the Exome Variant Server (http://evs.gs. washington.edu/EVS/). HGMD was originally established for the scientific study of mutational mechanisms in human genes causing inherited disease (Cooper et al. 2010), but has since acquired a much broader utility as a central unified repository for germ-line disease-related functional variation. It is now routinely accessed and utilized by next generation sequencing (NGS) project researchers, human molecular geneticists, molecular biologists, clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals, bioinformatics and personalized genomics.
HGMD is available in two versions: one public, one obtainable by subscription. The public version of HGMD (http://www.hgmd.org) is freely available to registered users from academic institutions/non-profit organizations. This version is, however, maintained in a basic form that is only updated twice per annum, is permanently 3 years out of date, and does not contain any of the additional annotations or extra features present in HGMD Professional (such as GRCh37/hg19 genomic chromosomal coordinates, HGVS nomenclature and additional literature references, see Table 2). The Professional version is available to both commercial and academic/non-profit users via subscription from BIOBASE GmbH (http://www.biobase-international. com).

Acquisition of mutation data
All HGMD mutation data are manually curated from the scientific literature. Identification of relevant literature reports is carried out via a combination of manual journal screening and automated procedures. The database currently contains mutation entries obtained from over 41,000 primary and 15,000 additional (supplementary) literature reports published in more than 1,950 different journals. Of [10,000 identified articles screened for mutation data during 2012, 35 % contained novel mutation data, 29 % contained additional useful information (e.g. in vitro functional data or further clinical or phenotypic information) and were therefore cited as additional references, whilst the remaining 36 % of articles contained no novel mutation data or supporting information to warrant their inclusion as either primary or supplementary references in HGMD. The number of articles screened by HGMD is increasing on a yearly basis; however, we impose no prior limit upon the number of articles we include as supplementary references for a given mutation.
For *4 % of all the missense/nonsense mutations reported in the literature during 2012, it was necessary for the HGMD Curators to contact the original authors to obtain correction and/or clarification of the nature or precise location of the mutations in question. However, only half of the mutations that required author contact were satisfactorily resolved by these means, leading to their inclusion in HGMD; the *2 % of unresolved missense/ nonsense mutations will not be entered into HGMD unless or until the nature or precise location of the mutation(s) in question is determined to the satisfaction of the HGMD curators. Such data (currently 366 entries) are, however, retained indefinitely by HGMD as part of a ''Bad Bank'' of inadequately described mutations.

Classes of variant listed in HGMD
There are six different classes of variant present in HGMD (Figs. 1, 2). Disease-causing mutations (DM) are entered ? HGMD Public is updated on a 6-monthly basis * Download customers only into HGMD where the authors of the corresponding report(s) have demonstrated that the reported mutation(s) are involved in conferring the associated clinical phenotype upon the individuals concerned. The DM classification may, however, also appear with a question mark (DM?), denoting a probable/possible pathological mutation, reported to be pathogenic in the corresponding report, but where (1) the author has indicated that there may be some degree of uncertainty; (2) the HGMD curators believe greater interpretational caution is warranted; or (3) subsequent evidence has appeared in the literature which has called the putatively deleterious nature of the variant into question. Disease-associated polymorphisms (DP) are entered into HGMD where there is evidence for a significant association with a disease/clinical phenotype along with additional evidence that the polymorphism is itself likely to be of functional relevance (e.g. as a consequence of genic/ genomic location, evolutionary conservation, transcription factor binding potential, etc.), although there may be no direct evidence (e.g. from an expression study) of a functional effect. Functional polymorphisms (FP) are included in HGMD where the reporting authors have shown that the polymorphism in question exerts a direct functional effect (e.g. by means of an in vitro reporter gene assay or alternatively by protein structure, function or expression studies), but with no disease association reported as yet. Disease-associated polymorphisms with supporting functional evidence (DFP) must meet both of the above criteria in that the polymorphism should not only be reported to be significantly associated with disease but should also display evidence of being of direct functional relevance.
Copy number variations (CNVs) represent an important subset of potentially functional disease-associated variation. While HGMD does not wish to replicate the excellent curatorial work of other resources (e.g. the Database of Genomic Variants http://dgv.tcag.ca/dgv/app/home, DECIPHER http://decipher.sanger.ac.uk/ and Copy Number Variation in Disease http://202.97.205.78/CNVD/), we are nevertheless interested in including such variants (as gross deletions or duplications) if they meet certain criteria. Therefore, HGMD will include such variations if they have been shown to be both of functional significance and associated with disease, and involve a single characterized Hum Genet (2014) 133:1-9 3 gene that has itself been directly implicated in the disease association. Such variants would be entered under one of the above-mentioned polymorphism categories, depending upon the supporting evidence provided by the authors of the original reporting article.
In the opinion of the HGMD curators, the polymorphism data present in HGMD should be viewed with a considerable degree of caution owing to (1) the possibility that the observed disease association may be simply due to a linkage disequilibrium effect rather than a bona fide underlying functional mechanism and (2) the fact that in vitro studies are not invariably accurate indicators of in vivo functionality (Cirulli and Goldstein 2007;Dimas et al. 2009).
Finally, frameshift or truncating variants (FTV) are polymorphic or rare variants reported in the literature that are predicted to truncate or otherwise alter the length of the gene product (i.e. a stop-gain, stop-loss or frameshift variant) but with no disease association reported as yet. Most known FTVs have been identified during the course of large-scale genome/exome screening studies (involving either patient panels or apparently healthy individuals from the general population). They may be considered to represent either latent protein deficiencies or, potentially, heterozygous carrier states for recessive disorders. Coverage of FTVs is far from being comprehensive at this juncture, and it remains unclear what proportion will turn out to be clinically significant.
The HGMD curators have adopted a policy of continual reassessment of the curated content within the database. If and when additional and important new information pertaining to a specific mutation entry becomes available (e.g. questionable pathogenicity, confirmed pathogenicity, additional clinical or laboratory phenotypes, population frequency data, supporting functional studies, etc.), then the mutation entry may be revised or even re-categorized. Alternatively, a comment or additional reference may be added in order to communicate this new information to users. Where new information becomes available which suggests that a given disease-causing mutation (DM) is likely to be of questionable pathological relevance or possibly a neutral polymorphism (on the basis of additional case reports, genome/population screening studies, presence in dbSNP with reliable population frequency data, etc.), it may be flagged with a question mark (DM?) or even removed from the database entirely if it turns out to have been erroneously included ab initio. In a recent recuration exercise, a total of 539 mutations were re-examined due to their presence in the 1000 Genomes Project dataset at a frequency of [1 %. Of the total re-examined, 33 mutations were removed from HGMD, 109 were recategorized and 220 had additional comments or references added to further justify their inclusion in HGMD ). One reason why some HGMD-listed mutations are often to be found among 1000 Genomes Project data is that many pathogenic lesions are found quite frequently in the population at large (Nishiguchi and Rivolta 2012;Andreasen et al. 2013;Lazarin et al. 2013;Cooper et al. 2013). In addition to internal curation, users of HGMD Professional may utilize a feedback function in order to inform the HGMD curators of relevant new or missing information, to request corrections or to ask for the reclassification or removal of a listed variant.
Most of the clinical phenotypes attributed to DMs in HGMD represent individually rare conditions that are generally regarded as monogenic diseases. However, it is important to note that HGMD also considers a few silent protein deficiencies or biochemical phenotypes (e.g. butrylcholinesterase deficiency, reduced oxygen affinity haemoglobin, etc.) to be worthy of inclusion since they are potentially disease-relevant (even if they are relatively common in the general population); such variants may well be assigned to the DM category.
For individual mutations in HGMD, the provision of zygosity information (heterozygous, homozygous or compound heterozygous) has not been attempted. Reasons for this include (1) the fact that this information is not always unambiguously provided in the corresponding article; (2) the possibility that a given mutation may be pathogenic irrespective of the zygosity in which it is found; (3) the clinical consequences of zygosity may often be modified by other genetic variants either in cis or in trans and (4) the general phenomenon of variable or reduced penetrance which ensures that the genotype is not invariably predictive of the phenotype . Thus, information pertaining to zygosity would not always be helpful or informative with regard to ascertaining or predicting the clinical phenotype, and indeed might even prove inaccurate or misleading.
HGMD users should not assume that just because a mutation is labelled ''DM'', that it automatically follows that the mutation is known or believed to be pathogenic in all individuals harbouring it (i.e. that the mutation exhibits 100 % penetrance). This is not invariably going to be the case and many ''disease-causing mutations'' will display reduced or variable penetrance for a variety of different reasons (reviewed by Cooper et al. 2013). Indeed, next generation sequencing programmes (such as the 1000 Genomes Project) are now identifying considerable numbers of ''DM'' mutations in apparently healthy individuals (MacArthur et al. 2012;. Such lesions should not automatically be regarded as being clinically irrelevant because it is quite possible that they represent low-penetrance, mild or late onset, or more complex disease susceptibility alleles, as opposed to neutral variants ).
It has always been HGMD policy to enter a variant into the database even if its pathological relevance may be questionable (while indicating this fact wherever feasible to our users), rather than run the risk of inadvertently excluding a variant that may be directly (or indirectly) relevant to disease. We have taken several steps to highlight such equivocation in HGMD, viz. the recent introduction of the DM? variant class, a dbSNP 1000 Genomes frequency flag (to highlight HGMD variants that are also present in dbSNP, with allele frequency information included; see below) and the provision of additional literature citations where the pathogenicity of the variant may have been subsequently either questioned or confirmed. This latter point is particularly pertinent in the clinical setting, where a greater burden of proof may be required for use in diagnostic and predictive medicine, and when considering the return of incidental findings to patients after testing (Green et al. 2012(Green et al. , 2013Ng et al. 2013;Gonsalves et al. 2013).

HGMD Professional
HGMD Professional has been developed to serve as the subscription version of HGMD, and is available to both commercial and academic customers under license from BIOBASE GmbH. HGMD Professional allows access to up-to-date mutation data with a quarterly release cycle; this version is therefore essential for checking the novelty of newly found mutations. HGMD Professional contains many features not available in the free public version (Table 2). More powerful search tools in the form of an expanded search engine with full text Boolean searching are provided. A batch search mode has recently been developed to allow users to search HGMD using gene (e.g. OMIM IDs) and variant (e.g. dbSNP IDs) oriented lists. Users can employ these tools to perform additional searches for gene-specific (e.g. chromosomal locations, gene names/aliases and gene ontology), mutation-specific (e.g. chromosomal coordinates, HGVS nomenclature, dbSNP ID) or citation-specific (e.g. first author, publication year, PubMed ID) information. The provision of chromosomal coordinates (hg19) for the vast majority of our nucleotide substitutions (98.7 % coverage) and other micro-lesions (97.3 % coverage) has made HGMD an invaluable tool for the large-scale analysis of NGS datasets such as the 1000 Genomes Project Genomes Project Consortium 2010. Additional information is also provided on a mutation-specific basis including curatorial comments pertaining to particular mutations (for example, if the mutation data presented required in-house correction in relation to the data presented in the original publication [5-10 % of entries], or if the clinical phenotype is associated with a more complex, i.e. a digenic or SNP incis inheritance pattern), additional reports comprising functional characterisation, further phenotypic information, comparative biochemical parameters, evolutionary conservation and SIFT (Sim et al. 2012) and MutPred (Li et al., 2009) predictions. These additional annotations are updated on a regular basis.
Recently, HGMD clinical phenotypes have been annotated against the Unified Medical Language System (UMLS) using a combination of manual curation and natural language processing. The UMLS is a comprehensive collection of biomedical concepts and the relationships between them (http://www.nlm.nih.gov/research/umls/). These UMLS mappings provide users with a more accurate and expanded phenotype search. Thus, searches using alternative disease names will return the same result-set, e.g. a search for ''breast cancer'' would yield identical results to a search for ''malignant breast tumour''. In addition, utilizing the UMLS allows for powerful semantic searching (e.g. searches for all mutations linked to blood disorders or all immune disorders).
Another new feature involves the highlighting of HGMD entries where the pathogenicity of the variant may have been cast into doubt by virtue of its allele frequency. HGMD Professional now displays a frequency flag when a listed variant is also found in dbSNP, and population frequency data from the 1000 Genomes Project are also provided. HGMD data have also recently been made available in Variant Call Format or VCF (Danecek et al. 2011), which will facilitate the comparison of HGMD with large NGS datasets. In addition to searching and viewing mutation data in a variety of ways, users of HGMD Professional may utilize a new feedback facility to submit corrections to the database curators or to request additional features.
HGMD Professional also contains an Advanced Search suite which has been designed to enhance mutation searching, viewing and retrieval. Two of the main types of mutation in HGMD (single-nucleotide substitutions and micro-lesions) can be interrogated with this toolset. Datasets for more than one mutation type may be combined (for example, micro-deletions, micro-insertions and indels) to enable more powerful searching across comparable types of mutation. When using the Advanced Search, users can tailor their queries with more specific criteria, including functional profile (e.g. in vitro and in silico characterized transcription factor binding sites, post-translational modifications, microRNA binding sites, upstream ORFs, and catalytic residues, see Fig. 3); amino-acid change; nucleotide substitution; size and/or sequence composition of micro-deletions, micro-insertions or indels; pre-or userdefined sequence motifs (both those created and those abolished by the mutation); dbSNP number; keywords Hum Genet (2014) 133:1-9 5 found in the article title or abstract. Results returned by the Advanced Search can be downloaded as tab-delimited text or a genome browser track, ready to be used in different applications. The Advanced Search also includes a batch mode called ''Mutation Mart'' to query HGMD via multiple identifiers including dbSNP, Entrez gene (http://www. ncbi.nlm.nih.gov/gene) and PubMed. HGMD Professional is available to subscribers either as an online only package or in downloadable form enabling users to incorporate HGMD data into their local variant analysis pipelines (http://www.biobase-international.com).

Other variant databases
Several other databases are available that attempt to record disease-causing or disease-associated (i.e. pathogenic) variation. These include the Online Mendelian Inheritance in Man, OMIM (http://www.omim.org/; Amberger et al. 2009), ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/), dbSNP (http://www.ncbi.nlm.nih.gov/SNP/; Sherry et al. 2001) and an assorted collection of locus-specific mutation databases (LSDBs) (http://www.hgvs.org/dblist/glsdb. html). OMIM does not provide statistics for allelic variants on its website; however, 22,901 germline OMIM variants appear to have been added to ClinVar, which itself contains a total of 25,375 pathogenic and probable pathogenic germline variants, while dbSNP contains 23,973 pathogenic or probable pathogenic germline variants (all databases were accessed July 10th 2013). Owing to the highly dispersed nature of the LSDBs and the potential for duplication between databases, accurate statistics with regard to like-for-like bona fide germline disease-causing (not merely neutral) variation is difficult to obtain. Since OMIM only records a limited number of variants per gene, and ClinVar is still in its infancy, HGMD is the only database of human pathological mutations that approaches comprehensive coverage of the peer-reviewed literature (Peterson et al. 2013). Since ClinVar and the LSDBs contain unpublished (non-peer reviewed) mutation data, the question has arisen as to whether HGMD should also include these data (Patrinos et al. 2012). However, several obstacles have been encountered by the LSDBs, including serious problems pertaining to data quality as well as issues of data provenance and consent. HGMD has therefore taken the decision not to include such data at this time.

How HGMD is utilized
Registered users of the public HGMD website currently number in excess of 60,000. Users may not download HGMD data in their entirety. However, mutation data may be made available at the discretion of the curators for noncommercial research purposes. Potential collaborators who wish to access HGMD data in full are required to sign a confidentiality agreement. HGMD data have been used to perform an extensive series of meta-analyses on different types of gene mutation causing human inherited disease. These studies have helped to improve our understanding of mutational spectra and the molecular mechanisms underlying human inherited disease . They have served to demonstrate not only that human gene mutation is an inherently non-random process but also that the nature, location and frequency of different types of mutation are shaped in large part by the local DNA sequence environment . HGMD data have been used extensively in several international collaborative research projects including the 1000 Genomes Project Genomes Project 2010, where a surprising number of HGMD variants were found in apparently healthy individuals. They have also been used in the comparative analysis of several orthologous genomes including gorilla (Scally et al. 2012), cynomolgus and Chinese macaque (Yan et al. 2011), Rhesus macaque (Rhesus Macaque Genome Sequencing and Analysis Consortium 2007) and rat (Rat Genome Sequencing Project Consortium 2004), in which many apparently disease-causing mutations in human were found as wild type ('compensated mutations').
In a clinical setting, HGMD is widely utilized by many groups in ongoing NGS diagnostic (Johnston et al. 2012, Calvo et al. 2012, Bell et al. 2011) and human genome sequencing (Tong et al. 2010;Kim et al. 2009) programmes. HGMD has also been used by a number of different groups to aid the development of post-NGS variant interpretation algorithms including MutPred (Li et al. 2009), PROVEAN (Choi et al. 2012), CAROL (Lopes et al. 2012), CRAVAT , NEST ) and FATHMM (Shihab et al. 2013). Finally, HGMD has been used as a resource for structural biologists in the reconstruction of protein interaction networks Guo et al. 2013). A more complete list of articles which have utilized HGMD data or expertise in their production can be found on the HGMD website (http://www.hgmd.cf.ac.uk/docs/articles.html).

Data sharing
A limited HGMD data set, containing both chromosomal coordinates and HGMD identifiers, has been made available via academic data exchange programmes to the Gen2Phen project (Webb et al. 2011), the European Bioinformatics Institute (EBI)/Ensembl (Flicek et al. 2013) and the University of California, Santa Cruz (UCSC) (Meyer et al. 2013) and may be viewed in these projects' respective genome browsers. Data from HGMD Professional have additionally been made available to HGMD subscribers via Genome Trax TM (BIOBASE GmbH) and Alamut (Interactive Biosoftware), but are also accessible as part of the HGMD Professional stand-alone package (BIOBASE GmbH). Allowing free access to the bulk of the mutation data present in HGMD, while generating sufficient income from its commercial distribution to support its maintenance and expansion, represents a business model that should maximize the availability of HGMD at the same time as ensuring its long-term sustainability. Although we are necessarily obliged to be prudent with regard to data sharing with public data repositories, we have always taken the view that making as much data as possible publicly available is generally beneficial to both HGMD and its users worldwide.

Future plans
The provision of chromosomal coordinates for the vast majority of coding region micro-lesions in HGMD is now complete. Expanding this provision to include microlesions in non-coding regions and the gross and complex lesion dataset (where feasible) is a high priority, as is expanding the provision of genomic coordinates to include popularly utilized NGS formats such as General Feature Format (GFF) (http://www.sanger.ac.uk/resources/ software/gff/) and BED format, to complement the recently added HGMD Variant Call Format (VCF) (Danecek et al. 2011). Mutations will also be mapped to the new genome build (GRCh38) in due course. A listing of removed variants will be implemented as time allows. Provision of genomic reference sequences based on the NCBI RefSeqGene project (Pruitt et al. 2009), links to available protein structures and homology models, and mapping HGMD phenotypes to the Human Phenotype Ontology (HPO) are also regarded as priorities.
In its current state of development, HGMD provides the user with a unique resource that can be utilized not only to obtain evidence to support the pathological authenticity and/ or novelty of detected gene lesions and to acquire an overview of the mutational spectra for specific genes, but also as a knowledgebase for use in the bioinformatics and whole genome screening projects that underpin personalized genomics.
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