Volume 36, Issue 4 p. 1034-1038
Letters: New Observations
Open Access

Expanding the Spectrum of AP5Z1-Related Hereditary Spastic Paraplegia (HSP-SPG48): A Multicenter Study on a Rare Disease

Marianthi Breza MD, MSc

Corresponding Author

Marianthi Breza MD, MSc

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

M.B., J.H., V.C., G.K., H.H., and G.S. contributed equally to this work.

Correspondence to: Dr. Marianthi Breza, 1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, 72-74 Vas. Sophias Ave, 11528 Athens, Greece; E-mail: [email protected]

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Jennifer Hirst PhD

Jennifer Hirst PhD

Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom

M.B., J.H., V.C., G.K., H.H., and G.S. contributed equally to this work.

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Viorica Chelban MD, MSc

Viorica Chelban MD, MSc

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

Department of Neurology and Neurosurgery, Institute of Emergency Medicine, Chisinau, Republic of Moldova

M.B., J.H., V.C., G.K., H.H., and G.S. contributed equally to this work.

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Guillaume Banneau MD, PhD

Guillaume Banneau MD, PhD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

Département de Génétique Médicale, Institut Fédératif de Biologie, Toulouse, France

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Laurène Tissier MD

Laurène Tissier MD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

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Bophara Kol MD

Bophara Kol MD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

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Thomas Bourinaris MD

Thomas Bourinaris MD

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

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Samia A. Said MD

Samia A. Said MD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

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Yann Péréon MD

Yann Péréon MD

Department of Clinical Neurophysiology, Reference centre for NMD, CHU Nantes, Place Alexis-Ricordeau, Nantes, France

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Anna Heinzmann MD

Anna Heinzmann MD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

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Rabab Debs MD

Rabab Debs MD

Department of Clinical Neurophysiology, APHP, Pitié-Salpêtrière Hospital, Paris, France

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Raul Juntas-Morales MD

Raul Juntas-Morales MD

Département de Neurologie, Centre Hospitalier Universitaire de Montpellier, Montpellier, France

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Victoria G. Martinez MD

Victoria G. Martinez MD

Département de Neurologie, Centre Hospitalier Universitaire de Montpellier, Montpellier, France

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Jean P. Camdessanche MD

Jean P. Camdessanche MD

Département de Neurologie, Centre Hospitalier Universitaire de Montpellier, Montpellier, France

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Clarisse Scherer-Gagou MD

Clarisse Scherer-Gagou MD

Département de Neurologie, Centre Hospitalier Universitaire de Montpellier, Montpellier, France

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Jean-Médard Zola MD

Jean-Médard Zola MD

Département de Neurologie, Centre Hospitalier Universitaire de Montpellier, Montpellier, France

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Alkyoni Athanasiou-Fragkouli MSc

Alkyoni Athanasiou-Fragkouli MSc

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

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Stephanie Efthymiou MSc

Stephanie Efthymiou MSc

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

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George Vavougios MD, PhD

George Vavougios MD, PhD

Department of Neurology, Athens Naval Hospital, Athens, Greece

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Georgios Velonakis MD, PhD

Georgios Velonakis MD, PhD

2nd Department of Radiology, National and Kapodistrian University of Athens, Medical School, Attikon Hospital, Athens, Greece

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Maria Stamelou MD, PhD

Maria Stamelou MD, PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

Parkinson's Disease and Movement Disorders Department, Hygeia Hospital, Athens, Greece

School of Medicine, European University of Cyprus, Nicosia, Cyprus

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John Tzartos MD, PhD

John Tzartos MD, PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Constantin Potagas MD, PhD

Constantin Potagas MD, PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Thomas Zambelis MD, PhD

Thomas Zambelis MD, PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Caterina Mariotti MD, PhD

Caterina Mariotti MD, PhD

Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Instituto Neurologico Carlo Besta, Milan, Italy

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Craig Blackstone MD, PhD

Craig Blackstone MD, PhD

Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA

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Jana Vandrovcova PhD

Jana Vandrovcova PhD

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

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Theodoros Mavridis MD

Theodoros Mavridis MD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Chrisoula Kartanou MSc

Chrisoula Kartanou MSc

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Leonidas Stefanis MD, PhD

Leonidas Stefanis MD, PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Nicholas Wood MD, PhD

Nicholas Wood MD, PhD

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

Neurogenetics Laboratory, National Hospital for Neurology and Neurosurgery, London, United Kingdom

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Georgia Karadima PhD

Georgia Karadima PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

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Eric LeGuern MD, PhD

Eric LeGuern MD, PhD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

Sorbonne Université, Institut du Cerveau, INSERM, CNRS, CHU Pitié-Salpêtrière, Paris, France

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Georgios Koutsis MD, PhD

Georgios Koutsis MD, PhD

1st Department of Neurology, Eginition Hospital, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

M.B., J.H., V.C., G.K., H.H., and G.S. contributed equally to this work.

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Henry Houlden MD, PhD

Henry Houlden MD, PhD

Department of Neuromuscular Disease, Institute of Neurology, University College London, London, United Kingdom

Neurogenetics Laboratory, National Hospital for Neurology and Neurosurgery, London, United Kingdom

M.B., J.H., V.C., G.K., H.H., and G.S. contributed equally to this work.

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Giovanni Stevanin PhD

Giovanni Stevanin PhD

Département de génétique, Hôpital Pitié-Salpêtrière, Assistance Publique – Hôpitaux de Paris, Sorbonne Université, Paris, France

Sorbonne Université, Institut du Cerveau, INSERM, CNRS, CHU Pitié-Salpêtrière, Paris, France

PSL Research University, EPHE, Neurogenetics Team, Paris, France

M.B., J.H., V.C., G.K., H.H., and G.S. contributed equally to this work.

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First published: 05 February 2021
Citations: 9

Relevant conflicts of interest/financial disclosures: M.B. has received a research grant from the European Academy of Neurology (EAN) and travel grants from Merck, Teva, Genesis Pharma, Pfizer, and Novartis. G.K. has received research grants from Genesis Pharma and Teva and consultation fees, advisory boards, and honoraria from Genzyme, Genesis Pharma, Teva, and Novartis. J.T. has shares in a diagnostic laboratory (Tzartos Neurodiagnostics) in Athens. All other authors report no disclosures relevant to the manuscript.

Biallelic mutations in AP5Z1 are known to cause a rare, autosomal-recessive, complex form of hereditary spastic paraplegia (HSP) referred to as SPG48 (MIM#613647).1 To date, only 11 SPG48 patients have been reported. The clinical spectrum of SPG48 is complex and heterogeneous, presenting with neuropathy, ataxia, dystonia, and parkinsonism in addition to spastic paraplegia (SP). AP5Z1 codes for the ζsubunit of the AP-5 complex, implicated in vesicular-mediated intracellular sorting and trafficking of cargo proteins1 - Functional studies demonstrate the accumulation of multilamellar structures (endolysosomes) in SPG48 skin fibroblasts.2

Here, we screened 2035 HSP patients from 3 tertiary centers (Athens, University of Athens [UOA]; London, University College London; and Paris, Paris, Sciences & Lettres [PSL], Assistance Publique-Hôpitaux de Paris [APHP]) for mutations in AP5Z1 and performed functional studies in 2 cases with pathogenic variants in AP5Z1. We also present a literature review for AP5Z1 cases, a pathway analysis, and follow-up data on previously reported patients, where available (Supplementary Material 1, 4, and 5).

In total, 9 patients from 8 unrelated families carrying biallelic pathogenic variants in AP5Z1 were identified (Fig. 1; Supplementary Material 1). We show that AP5Z1-related disease usually presents with a combination of late-onset SP (mean: 54.3 ± 5.3 years) and axonal neuropathy. Other frequent clinical features in our cohort were urinary incontinence, hearing loss, and visual impairment. Interestingly, 1 patient had epileptic seizures. Brain magnetic resonance imaging (MRI) was available for 6 patients. Leukoencephalopathy and thinning of the corpus callosum (TCC) were present in 1 patient and “ears of the lynx” sign, a “moth-eaten” appearance of the basal ganglia, and TCC in another (Fig. 2). The remaining 4 patients had normal MRI.

Details are in the caption following the image
Schematic representation of AP5Z1 with novel SPG48 variants identified in this study depicted above (top—red), whereas previously reported SPG48 variants are indicated below the schematic (bottom—blue). Among the 9 variants identified in this study, 5 were homozygous and 4 were compound heterozygous in 2 patients. They were all novel except 1 reported by Słabicki et al (2010). All variants were leading to a premature stop codon and further mRNA degradation by the nonsense-mediated mRNA decay; 2 were nonsense, 5 were leading to frameshifts, and 2 were splice site variants. [Color figure can be viewed at wileyonlinelibrary.com]
Details are in the caption following the image
Brain magnetic resonance imaging (MRI) findings of the SPG48 patients. (A–E) Brain MRI of patient G; (F–H) Brain MRI of patient F5; (A, B) Axial T2 FLAIR MRI brain: bifrontal and periventricular white-matter hyperintensities with incipient “ears of the lynx” and dilated Sylvian fissures are noted; (C) Axial T2-weighted MRI brain: a “moth-eaten” appearance of the basal ganglia is observed; (D, E) Sagittal T1 and T2 MRI brain showing characteristic thinning of corpus callosum; (F, G) Axial T2 FLAIR and T2-weighted MRI brain showing diffuse, symmetrical leukoencephalopathy and “moth-eaten” appearance of the basal ganglia; (H) Sagittal T1 MRI brain showing thinning of corpus callosum.

Our extended analysis of all SPG48 patients identified in the literature (Supplementary Material 1) shows that 22 AP5Z1 variants are linked to SPG48 worldwide, including the 8 newly reported here. Follow-up data were obtained from 3 SPG48 patients, all of whom had a disease duration of ≥10 years. The progression of the disease was slow (Supplementary Material 5), with the phenotype consisting of predominantly severe lower-limb spasticity with patients becoming wheelchair-bound after 10 years from onset.

AP5Z1 protein levels in patient fibroblasts were reduced to undetectable levels, correlating with an accumulation of lysosomal-associated membrane protein 1 (LAMP1)-positive structures and a 25% deficit in recycling between endosomes and Golgi (Fig. 3, Fig. 4, Supplementary Material 6). This was also supported by functional enrichment analyses which pinpointed that within the network of HSP-associated genes mediating membrane trafficking, AP5Z1 relates to endosomal trafficking, determining the fate of sorting endosomes toward lysosomal fusion, the plasma membrane, or the trans-Golgi network (Supplementary Material 4).

Details are in the caption following the image
Functional studies on SPG48 patient fibroblasts. (A) Whole-cell Western blots of patient-derived fibroblast lines (denoted by their mutation) including controls, loaded at equal protein levels and probed with antibodies against SPG11, SPG15, AP5Z1, AP1G1 (AP1G1 is used as a loading control). Note the loss of AP5Z1; (B) Immunofluorescence microscopy of SPG48 patient-derived fibroblasts doubled labeled with antibodies against EEA1 to mark early endosomes and LAMP1 to mark late endosomes/lysosomes. In AP5Z1-deficient patient lines, there is an increase in the brightness and size of the LAMP1-positive puncta. Scale bar = 20 μm; (C) Quantification of the increase in LAMP1 spot intensity was performed for 2 independent SPG48 patient-derived fibroblast lines using Volocity. At least 20 cells were quantified per condition. Data show mean of 3 independent experiments and results of a 2-tailed t test: (*P < 0.05; **P < 0.01). [Color figure can be viewed at wileyonlinelibrary.com]
Details are in the caption following the image
Functional studies on SPG48 patient fibroblasts. (A) Immunofluorescence microscopy of patient-derived fibroblasts and treated with monensin for 90 minutes followed by washout for 2.25 hours. In AP5Z1-deficient patient lines, there is reduced retrieval of GOLIM4 back to the juxtanuclear region (where GM130 is located) compared to healthy control patient lines. Scale bar: 20 μm.; (B) Quantification of the retrieval defect (the reduction in the level of colocalization of GOLIM4 and GM130) was performed for 2 independent SPG48 patient-derived fibroblast lines using Pearson's correlation coefficient. At least 20 cells were quantified per condition. Data show mean of 3 independent experiments and results of a 2-tailed Mann-Whitney U test: **P < 0.01. [Color figure can be viewed at wileyonlinelibrary.com]

In this study we expanded the phenotypic and genotypic spectrum of SPG48 showing that SPG48 is a slowly progressing, late-onset, complicated HSP manifesting with SP, axonal neuropathy, cognitive impairment in line with the SPG48 patients reported so far [and in Refs 1 and 3] and, interestingly, epileptic seizures (patient G, Supplementary Material 3). Epileptic seizures have not been previously reported in SPG48; however, they are well described in other HSP subtypes, such as SPG11 and SPG15, which are functionally related to SPG483 and in many lysosomal storage diseases. Indeed, our functional studies on SPG48 cell lines confirm defects in endosome and lysosome homeostasis. We also confirm here previously described neuroimaging findings (“ears of the lynx” sign, TCC, and white matter lesions) in a subgroup of patients.

To date, no specific therapies are approved for HSP. Of note, treatment strategies are proposed in complex forms of HSP such as cholesterol-lowering agents for HSP-CYP7B1 (SPG5A), as CYP7B1 gene is involved in the degradation of cholesterol into primary bile acids.4 A randomized-controlled trial showed that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in the serum of SPG5 patients5 and evolocumab (PCSK9 inhibitor) is currently evaluated in a phase 2 clinical trial (NCT04101643). In addition, “tideglusib” (GSK3β inhibitor) was tested on iPSC neuronal lines of an SPG11 patient and decreased cell death.6

Our study strengthens the evidence supporting autophagic dysfunction as one of the underlying molecular pathways in HSP7 and further expands the phenotypic spectrum of AP5Z1-related SPG48.

Acknowledgments

We thank the participants and their families for their essential help with this work. The Greek family was recruited as part of the SYNaPS Study Group collaboration funded by the Wellcome Trust and strategic award (Synaptopathies) funding (WT093205 MA and WT104033AIA). This research was conducted as part of the Queen Square Genomics group at University College London, supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. This work was supported by the Wellcome Trust (Synaptopathies strategic award [104033]), a Wellcome Trust Multi-User Equipment Grant, Muscular Dystrophy UK (MD UK), MDC USA, and the Medical Research Council (MRC UK International Centre and project grants), the French Strumpell-Lorrain association, Spastic Paraplegia Foundation, the MRC (MR/S01165X/1, MR/S005021/1, G0601943), the National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetree Trust, Ataxia UK, MSA Trust, Brain Research UK, Sparks GOSH Charity, Muscular Dystrophy UK (MD UK), Muscular Dystrophy Association (MDA USA). European Union funding (grant 779257 Solve-RD from the Horizon 2020 Research and Innovation Programme) was provided to G.S. J.H. was supported by grants from the Wellcome Trust: 086598 and 214272 to Microsoft Research Cambridge. G.K. acknowledges support from Genesis Pharma (Grant Number 13044) special account for research grants for the National and Kapodistrian University of Athens.

    Author Roles

    Name Role Contribution
    Marianthi Breza Author Designed and conceptualized the study; acquisition of data; interpreted the data and analyzed the genetic and clinical data; and drafted the manuscript for intellectual content
    Jennifer Hirst Author Acquisition of data; performed functional studies; interpreted the data and analyzed the genetic data; and drafted the manuscript for intellectual content
    Viorica Chelban Author Acquisition of data; interpreted the data and analyzed the genetic and clinical data; performed molecular analysis; and drafted the manuscript for intellectual content
    Guillaume Banneau Author Acquisition of data; interpreted the data and analyzed the genetic and clinical data; drafted the manuscript for intellectual content; performed molecular analysis; and revised the manuscript for intellectual content
    Laurène Tissier Author Acquisition of data; interpreted the data and analyzed the genetic and clinical data; drafted the manuscript for intellectual content; performed molecular analysis; and revised the manuscript for intellectual content
    Bophara Kol Author Acquisition of data and revised the manuscript for intellectual content
    Thomas Bourinaris Author Acquisition of data; interpreted the data and analyzed the genetic and clinical data; drafted the manuscript for intellectual content; and revised the manuscript for intellectual content
    Samia Ait Said Author Acquisition of data and revised the manuscript for intellectual content
    Yann Péréon Author Acquisition of data and revised the manuscript for intellectual content
    Anna Heinzmann Author Acquisition of data and revised the manuscript for intellectual content
    Rabab Debs Author Acquisition of data and revised the manuscript for intellectual content
    Raul Juntas-Morales Author Acquisition of data and revised the manuscript for intellectual content
    Victoria Gonzalez Martinez Author Acquisition of data and revised the manuscript for intellectual content
    Jean Philippe Camdessanche Author Acquisition of data and revised the manuscript for intellectual content
    Clarisse Scherer-Gagou Author Acquisition of data and revised the manuscript for intellectual content
    Jean-Médard Zola Author Acquisition of data and revised the manuscript for intellectual content
    Alkyoni Athanasiou-Fragkouli Author Acquisition of data and revised the manuscript for intellectual content
    Stephanie Efthymiou Author Acquisition of data and revised the manuscript for intellectual content
    George Vavougios Author Acquisition of data; performed pathway analysis; and revised the manuscript for intellectual content
    Georgios Velonakis Author Acquisition of data; performed MRI analysis; and revised the manuscript for intellectual content
    Maria Stamelou Author Acquisition of data and revised the manuscript for intellectual content
    John Tzartos Author Acquisition of data and revised the manuscript for intellectual content
    Constantin Potagas Author Acquisition of data and revised the manuscript for intellectual content
    Thomas Zambelis Author Acquisition of data and revised the manuscript for intellectual content
    Caterina Mariotti Author Acquisition of data and revised the manuscript for intellectual content
    Craig Blackstone Author Acquisition of data and revised the manuscript for intellectual content
    Jana Vandrovcova Author Acquisition of data and revised the manuscript for intellectual content
    Theodoros Mavridis Author Acquisition of data and revised the manuscript for intellectual content
    Chrisoula Kartanou Author Acquisition of data; performed panel molecular testing; and revised the manuscript for intellectual content
    Leonidas Stefanis Author Acquisition of data and revised the manuscript for intellectual content
    Nicholas Wood Author Acquisition of data and revised the manuscript for intellectual content
    Georgia Karadima Author Acquisition of data; performed molecular testing; and revised the manuscript for intellectual content
    Eric LeGuern Author Acquisition of data and revised the manuscript for intellectual content
    Georgios Koutsis Author Acquisition of data; revised the manuscript for intellectual content; and supervision of the entire process
    Henry Houlden Author Acquisition of data; revised the manuscript for intellectual content; and supervision of the entire process
    Giovanni Stevanin Author Acquisition of data; revised the manuscript for intellectual content; and supervision of the entire process