ACTUAL RESEARCH LINES

Dr. Juan Jesús Vílchez

Chief of the Neurology Service of La Fe Hospital of Valencia

Dr. José Alcamí

Chief of the AIDS Immunopathology Unit of the Health Institute Carlos III– Madrid

Prof. Ruben Artero

Translational Genomics Group, Incliva Health Research Institute, Valencia

Dr. Rafael Vázquez

Research Group in Molecular, Cellular and Genomic Biomedicine, Health Research Institute La Fe, Valencia

Prof. Giovanna Cenacchi

Pathology and Subcellular Diagnose Laboratory, Bologna University

Prof. Corrado Angelini

Neurobiology Laboratory – San Camillo Hospital’s Foundation, IRCCS – SAN CAMILLO – VENEZIA LIDO

OTHER RESEARCHERS INVOLVED

RAMÓN MARTÍ

Head Neuromuscular And Mitochondrial Pathology, Vall d’Hebron, Barcelona

BJARNE UDD

Neuromuscular Research Center, Tampere University and University Hospital of Tampere

VINCENZO NIGRO

TIGEM (Telethon Institute of Genetics and Medicine), University at Campania, Naples, Italy

JULIEN OCHALA

Faculty of Life Sciences & Medicine, King’s College London

VINCENT MOULY

Institute of Myology, Sorbonne University, Paris

GREG TOWERS

Professor of Molecular Virology, University College London; Head of Research Group, UCL Division of Infection and Immunity

MARIA JUDIT MOLNAR

Institute of Genomic Medicine and Rare Disorders, Budapest, Hungary

ENDRE PÁL

Department of Neurology, University of Pécs, Medical School, Pécs, Hungary

ZEGER DEBYSER

Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Flanders, Belgium

1. In Spain

Since 2015, four investigation groups have decided to join efforts to study in a multidisciplinar project the protein Transportin 3, involved in the HIV infection and in a rare muscular disease (LGMD1F). The investigation project is called “Study of the Transportin 3 deficit. A common way in the treatment of LGMD1F muscular dystrophy and the infection by HIV”

The Alliance is leadered by Juan Jesús Vílchez Padilla, chief of the Neurology Service of La Fe Hospital of Valencia, Ramon Martí Seves, researcher of the neuromuscular and mitochondrial pathology Laboratory of the Vall d’Hebron Research Institute of Barcelona, Rubén Artero, researcher of the Translation Genomic Laboratory of the Sanitary Investigation Institute INCLIVA in the University of Valencia and José Alcamí chief of the AIDS Immunopathology Unit of the Health Institute Carlos III.

During 2017, Dr. Rafael Vázquez – Health Research Institute La Fe, Valencia, joined the team tp study the application of the CRISPR gene editing system to try to restore the function of the transportin 3.

The fact that the Transportin 3 gene (TNPO3), an importin involved in the transport of factors to the cell nucleus and in the infection by the AIDS virus, is the cause of the limb-girdle 1F type myopathy has opened this multidisciplinary investigation line that through the study of the molecular mechanisms by which this disease causes TNPO3 aims to open lines that allow both therapeutic treatment of myopathy LGMD1F and developing a new class of antiretroviral.

The groups of Vílchez and Martí evidenced that this disease is due to a microdeletion in the TNPO3 gene which generates an abnormal protein with a 15 amino acids extension. The pathophysiological mechanism by which this defect originates the myopathy is still unknown. In the laboratory of Alcamí it has been demonstrated not only the resistance to the infection in lymphocytes of patients with LGMD1F but also that the mutant protein is associated to the normal variant and blocks its function, which aggravates the impact of the defect which is produced in a single copy of the gene.

“It is a unique occasion” indicates Alcamí, because we are in front of the second genetic defect described – the first is the delta32 deletion in the CCR5 receptor of the HIV – that blocks the infection by HIV. By one side the cells of these patients allow us to study and understand an essential step in the infective cycle of the HIV which is bad known yet, and by the other side, we can use the HIV as a tool that allows us understand the molecular mechanism involved in the limb-girdle myopathy LGMD1F type”.

2. In Italy

In this context, it has been added an Italian investigation group, that will work together with the Spanish one integrating the results of both studies, formed by the professor Giovanna Cenacchi, from the Pathology and Subcellular Diagnose Laboratory – Biomedical and Neuromotor Sciences Department, at Bologna University, and by the Prof. Corrado Angelini, from the Neurobiology Laboratory – San Camillo Hospital’s Foundation, IRCCS, at Venecia Lido, with an investigation project entitled “Setting up of an in vitro cell culture system to investigate the role of troponin3 (TNPO3) in skeletal muscle.”

The aims of the present study is creating a new in vitro cell culture model and an overall view given by all the experiments that will take place in this project, which may lead to identify some alterations in protein expression that could be directly linked to a TNPO3 absence or mutation, and that could help to clarify the role of TNPO3 in muscle and that could lead also to a better understanding of its role in LGDM1F pathogenesis.

The first step to be taken in order to have the opportunity of put in practice the results of these studies towards a possible treatment of this pathology is to identify the pathogenetic mechanism, beginning from the Transportin 3 mutation, considering that to this day it has been identified the mutation but not yet clarified the subsequent mechanisms which causes the disease. The mutated transportin seems like not being capable of act correctly with “cargo” proteins, blocking the regular intranuclear transport of proteins involved in the RNA splicing mechanism, basic step in order to have a correct translation of the messenger RNA in the prothein synthesis. To do so, in this project there will be studied expressions from different proteins related with the splicing and the degradation of the protein in experimental standard conditions in differenced cellular lines and also in silencing conditions of the transportin in order to reproduce in vitro the situation assimilable to the pathologic one. All this, to try to comprehend mechanism that leads to the morphological picture described in the muscular biopsies. Once hypothesized the steps of the molecular mechanism it will be possible to interfere more specifically in their critical phases.

REPORT OF SITUATION

Let’s explain briefly which are the results acquired thanks to the investigation in the last few years.

In 2013 becomes discovered the gene which codifies wrongly our Transportin 3 protein, causes the Limb-girdle Muscular Dystrophy LGMD1F. Important conclusions of this investigation demonstrated that the same protein is involved in the AIDS virus transmission and that there are many similarities between this dystrophy and another rare muscular diseases. This means that the investigations about our muscular dystrophy helps, at the same time and in a world scale, on a research for a possible therapy against the AIDS and many other neuromuscular diseases which affect million people in all over the world.

RELATION OF OUR DISEASE WITH THE AIDS

¡Hemos puesto otra vez en marcha la investigación sobre LGMD1F!

Nosotros, como enfermos de esta distrofia muscular, estamos ya colaborando con estos investigadores desde hace años, proporcionando muestras de sangre y todo lo necesario para que estos laboratorios puedan realizar su trabajo.

Para la continuación y el desarrollo de estos estudios es necesario un suministro continuo de financiación , dado el gran número de investigadores e instrumentación necesarios, puesto que estamos en frente a mecanismos genéticos completamente nuevos que abrirían las puertas a la comprensión de muchas enfermedades y podrian llevar a la creación de una nueva técnica de terapia génica.

WHAT HAS BEEN DONE TILL NOW

After several years of study, visits and exams in Spain, the first publications about our 1F Limb-girdle Muscular Dystrophy were from 2000, with the article “Autosomal dominant limb-girdle muscular dystrophy. A large kindred with evidence for anticipation” (J. Gámez; C. Navarro; A.L. Andreu; J.M. Fernandez; L. Palenzuela; S. Tejeira; R. Fernández–Hojas; S. Schwartz; C. Karadimas; S. DiMauro; M. Hirano; C. Cervera) where through 5 muscular biopsies and the study of 61 members of our family it was discovered that suffered from a unknown muscular dystrophy. From it, the authors of this study did a full genomic scan to identify the locus of the disease. In 2003 they published the study “A novel autosomal dominant limb-girdle muscular dystrophy (LGMD 1F) maps to7q32.1-32.2” (L.Palenzuela; A.L.Andreu ; J.Gámez; M.R.Vilà; T.Kumimatsu; A.Meseguer; C.Cervera; I.Fernández Cadenas; P.F.M. Van der Ven; T.G.Nygaard; E.Bonilla; and M. Hirano) with which it was discovered the locus of the disease and it was detected that there was no defect at the Filamin C or its protein product, genetical defect already known inside of this cromosomic region. With this study, it was clarified that we were in front of a new autosomic limb-girdle dominant dystrophy yet to be discovered and it was given to it the name of LGMD1F, following the classification adopted before for the other limb-girdle muscular diseases discovered, where the 1 number means that is dominant and the F that is the sixth variant discovered.

Until 2011 there were no other publications directly about LGMD1F and our family, when two Italian affected of LGMD1F, members of the same Spanish family, did a new biopsy and got published the results “LGMD 1(F) – A pathogenetic hypothesis based on histopathology and ultrastructure” (G. Cenacchi, E. Peterle, L. Tarantino, V. Papa, M. Fanin, C. Angelini) in where apart from being described the morphological and ultrastructural situation, there were also hypothesized other encoding proteins of the actin as possible responsibles of the LGMD1F pathogenic mechanism.

In 2012 it was published the study “Next generation sequencing applications are ready for genetic diagnosis of muscular dystrophies” (M. Savarese, A. Torella, M. Mutarelli, M. Dionisi, T. Giugliano, G. Di Fruscio, M. Iacomino, A. Garofalo, S. Aurino, F. Del Vecchio Blanco, G. Piluso, L. Politano, M. Fanin, C. Angelini 5, V. Nigro) where it was said that it had been identified the alteration of the Transportin 3 (TNPO3) in the LGMD1F affected patients.

It followed another study in 2012 entitled “Ultrastructural changes in LGMD1F” (G.Cenacchi, E. Peterle, M. Fanin, V.Papa, R. Salaroli, C. Angelini) where it got evidenced that the morphological and ultrastructural data suggested, in the LGMD1F studied cases, a phenotypic myopathy similar to those described by the Z-disk diseases, which leaded to the hypothesis that the mutant protein in LGMD1F can cause a decomposition of the cytoskeletal net related with desmin.

In 2013 there were published two more studies about the discovering of the genetical alteration which caused 1F Limb-girdle Muscular Dystrophy – LGMD1F.

One Spanish: “Limb-girdle muscular dystrophy 1F is caused by a microdeletion in the transportin
3 gene” (Maria J. Melià, Akatsuki Kubota, Saida Ortolano, Juan J. Vilchez, Josep Gámez,Kurenai Tanji, Eduardo Bonilla, Lluis Palenzuela, Israel Fernández-Cadenas, Anna Pristoupilová, Elena Garcia-Arumí, Antoni L. Andreu , Carmen Navarro,Michio Hirano, Ramón Martí) where it was proclaimed the identification of the 1F limb-girdle muscular dystrophy mutation’s cause, in a single nucleotide heterozygote deletion (c.2771del) at the termination codon of the Transportin 3 (TNPO3) and which was not known for being a key factor in the HIV importation to nucleus process.

And an Italian one: “Next-Generation Sequencing Identifies Transportin 3 as the Causative Gene for
LGMD1F” (Annalaura Torella, Marina Fanin, Margherita Mutarelli, Enrico Peterle, Francesca Del Vecchio Blanco, Rossella Rispoli, Marco Savarese, Arcomaria Garofalo, Giulio Piluso, Lucia Morandi, Giulia Ricci, Gabriele Siciliano, Corrado Angelini, Vincenzo Nigro), where it was explained the identification of a shared variance in the LGMD1F affected apart from the heterozygote mutation on TNPO3 gene and that the mutant TNPO3 was located around the nucleus, but not inside. The genetic implication connected with the transport to the nucleus suggests a new pathologic mechanism which leads to the muscular dystrophy.

WHAT WE DO?

We have reactivated the investigation about LGMD1F!

We all affected from this dystrophy are already collaborating with these investigators since two years ago, providing them blood samples and all that is needed to help them.

In order to continue and develop this researches we need a continuous economical supply, given the great number of investigators and instrumentation needed, as is about the genetic mechanisms totally new that would open the door to the comprehension of many diseases and would achieve the application of new techniques of genetic therapy.

Our association has important function: besides of promoting the fund raising, it is in charge of channeling funds to the labs and coordinate these ongoing investigation lines, avoiding duplications in the studies that would carry to a useless waste of time and money. Specifically, in addition to put in contact the different investigators, we make easier the exchange of material for their investigations (like blood samples), so the several labs could always have the most updated information as possible.

We have direct contact with so much labs that are doing researches about us, in order to supervise the investments and the progress.

Our Association is constantly searching for funds and visibility with the aim of achieve the final objective which it was made for: to have the chance of continue these investigations and dispose a better future for millions of people.

The push for us to create this association was given to us when we saw that, thanks to the benevolence of some doctors and investigators, who in turn have stimulated and involved other labs and doctors that since now were not aware of our existence, advances are being done in the knowledge of our disease and for a possible future therapy. But due to the lack of funds, and regarding the historical moment that we are living, all this efforts could be done in vain.