The Limb girdle muscular dystrophies (LGMD) are a group of genetically inherited muscle diseases that lead to progressive weakness and wasting of the limb-girdle muscles.1,2 Axial, facial and/or respiratory muscles can be variably involved as well. Although the condition has been well characterized, clinical and genetic heterogeneity is observed in patients with LGMD in terms of age of onset, extent of muscle weakness and prognosis.1,2 Based on the mode of inheritance the LGMD can be subdivided in two main subtypes, autosomal dominant (LGMD D) or autosomal recessive (LGMD R).1,3

A systematic review, including studies from the USA, Asia, Europe, Africa and Oceania, estimated a combined worldwide combined prevalence of all muscular dystrophies between 3.8 and 26.8 per 100,000 inhabitants and 0.8–5.7 per 100,000 inhabitants for LGMD.4 LGMD 2A/R1 calpain3-related, LGMD 2B/R2 dysferlin-related and LGMD 2C/R3 γ-sarcoglycan-related are the most frequent LGMD subtypes.5,6

There is restricted data concerning the frequency of LGMD in Argentina. In a cohort of patients from Brazil, Argentina, Chile, Ecuador and Colombia with a clinically suspected limb-girdle syndrome studied for LGMD and Pompe Disease (PD), a molecular diagnosis was identified in 16% of the patients. LGMD 2B/R2 dysferlin-related and LGMD 2A/R1 calpain3-related were the most frequent molecular diagnosis in this group.7

PD is an autosomal recessive hereditary myopathy, characterized by a deficit of the lysosomal α-glucosidase acid (AGA) enzyme activity,8 which could be misdiagnosed as a LGMD. Based on the age of clinical presentation, the PD is classified into an infantile onset or late onset (LOPD), below or above 1 year old respectively.8In worldwide multicenteric genetic descriptive studies of LGMD (using next generation sequencing/whole exome sequencing) the frequency of PD ranged from 0.4 to 3%.7,9–11

The early recognition, accurate genetic diagnosis and speedy development of population registries for LGMD and PD is of outmost importance since they allow a comprehensive characterization of the disease, adequate family counseling, timely identification of patients and family care needs , appropriate medical follow-up, and access to approved therapies or participation in natural history studies or clinical trials.12–14

This study aims to describe the frequency and variant profile of LGMD and PD in a group of Argentinean patients presenting with proximal muscle weakness and studied with a genetic panel for LGMD and PD.

The samples from 472 patients were studied (258 males, mean age 39.0 ± 20.1 years old, range 1–81). The 82% of the population were older than 18 years old (Table 1).

Out of 472 patients, 272 (57.6%) had a genetic variant identified through the forementioned panel. One hundred and twenty three (123/472, 26.1%) patients showed at least one P/LP variant and 149 (149/472, 31.5%) at least one Variant of Uncertain Significance (VUS). A total of 176 P/LP variants and 179 VUS were identified (Table 1, Table 2 and Table 3). Overall, DYSF was the gene in which more genetic variants were identified (Fig. 1a).CAPN3 represented the gene with the highest frequency of P/LP variants and DYSF the one with the highest frequency of VUS (Fig. 1b and c). No novel variants were identified.

Fig 1.

Frequency of variants identified. Pie chart A shows the distribution by gene of all the variants (n = 355) identified in the population studied. Pie chart B shows the distribution by gene of the pathogenic/likely pathogenic (n = 176) variants identified in the population studied. Pie chart C shows the distribution by gene of the Variants of Unknown Significance identified in the population studied (n = 179).

AGA/GAA: Acid Alpha-Glucosidase, ANO5: Anoctamin 5, CAPN3: Calpain 3, CAV3: Caveolin 3, DYSF: Dysferlin, FKRP: Fukutin-Related Protein, HNRPDL: Heterogeneous Nuclear Ribonucleoprotein D Like, SGCA: Alpha Sarcoglycan, SGCB: Sarcoglycan Beta, SGCD: Sarcoglycan Delta, SGCG: Sarcoglycan Gamma, TCAP: Telethonin.

(0.28MB).

Of the 123 patients having at least one P/LP variant, a definitive molecular diagnosis of a recessive genetic muscular disease was confirmed in 51 (41.4%) which represented the 10.8% of the total population (Tables 1 and 4). Eighty-four percent (43/51) of the patients were compound heterozygous variants and 16% (8/51) were homozygous. The three most common identified disorders were LGMD 2A/R1 (CAPN3) in 3%, Pompe Disease (GAA) in 2.5% and LGMD 2B/R2 (DYSF) in 2.1% (Table 4). The 72 remaining patients presented heterozygous variants for autosomal recessive diseases and were confirmed as carriers.

Twenty-one patients presented at least one P/LP variant in the GAA gene. In 12 of them a definitive molecular diagnosis of PD was confirmed, being all compounds heterozygous. We further obtained a detailed medical history of each of the confirmed patients with PD. In 10 patients with PD a low blood enzymatic activity was already known by the primary clinician by the time of the genetic panel referral. Thus, in these 10 patients with PD the panel was requested to genetically confirm the diagnosis rather than for screening purposes. In the remaining two patients with PD, the suspected diagnosis was a LGMD and enzymatic studies were not requested before performing the genetic panel.

The remaining nine patients were heterozygous for a P/LP variant in the GAA gene. Seven of them had a normal MLPA and in the other two patients the study was not performed. Among the patients with a normal MLPA, the enzymatic activity was low in only two patients. The first patient was a 22-year-old man presenting with myalgia and carrying the variant c.-32-13 T > G. The second one, was a 21-year-old man presenting with hiperckemia and mild tongue and neck flexor weakness and carrying the variant c.2237G > A (p.Trp746Ter). Both patients had a first degree relative with a genetic and clinical diagnosis of PD, a maternal uncle in the former and the father of the patient in the latter.

This paper represents the first genetic multicenter study of LGMD and PD performed in Argentina and evaluated by a centralized single reference laboratory.

Next Generation Sequencing technology enabled a definitive molecular diagnosis of a LGMD in 10.8% of the population. This result was similar to those reported in Canada (15%,16 17%10) and in Latin American (16%).7 However, it was lower than the diagnostic yield reported in the USA (21%),9 Italy (23%17), Germany (33%)18 and in a multicenter study including 9 countries (24%).19 Differences among studies are most likely due to reasons such as: variations in the number of genes included in the panels, ranging from 12 genes in our study to 169 in others,19,25 as well as different inclusion criteria and ethnic backgrounds.

Calpain-3 (14/472 patients, 3%) was the most frequent LGMD identified in this study similarly to others.5,9,20,26 Nevertheless, a Latin American study,7 which included 50% of patients from Brazil, reported that the most frequent AR LGMD in this region was due to mutations in the DYSF gene. Likewise, two additional Brazilian studies identified dysferlin mutations as the most common cause of LGMD21,22 in that country. The difference in the LGMD distribution between neighbour countries might be due to historical reason since Argentina was an Spanish colony and had a remarkable immigration from mainly Italy and Spain at the end of the 19th century and the first decades of the 20th century,23 whereas Brazil had an important Portuguese immigration background. LGMD2B was identified as the most frequent LGMD in a Portuguese study24 as well.

The second most common identified myopathy in our study was PD (12/472, 2,5%). This frequency is similar to that reported on previous studies.10,17 After the genetic results, we were able to obtain clinical information from all PD patients identified and it became clear that 10/12 patients already had low GAA enzymatic levels which were not reported at the time of the genetic testing. Therefore, only 2/472 (0.4%) patients with no previous suspected diagnosis of PD were identified through this screening panel as was previously reported in Latin America (0.4%).7 This represented 3.9% (2/51) of all patients with a confirmed myopathy. Screening genetic studies searching for PD should carefully exclude patients with known low GAA enzymatic levels in order to inform a more accurate PD frequency (Table 5).5,8,11,14,15,19,20 In addition, two types of screening studies of PD are reported in the literature. In the first one, patients with a LGMD phenotype, hyperckemia or an unspecified myopathy were initially studied with enzymatic assays and the results were lately confirmed with a genetic technique. The reported PD frequency using this approach was between 1.7% and 4%.27–31 The second approach consists of prospective or retrospective genetic studies of PD in patients with variable inclusion criteria (Table 5) in the majority of which PD was identified in less than 1%. The inclusion of patients with family history or previous abnormal enzymatic levels could be opened to selection bias. In this study, as well as in the USA and Europe, the most common variant identified in the GAA gene was the c.-32-13 T > G intronic variant different from those reported in Asia-Pacific (c.2238G > C) and in the Middle East (c.1064 T > C).32,33

Seventy-two patients carrying a heterozygous pathogenic variant for an AR LGMD were identified. Nine patients had an heterozygous pathogenic variant in the GAA gene, among whom two had a low enzymatic essay despite the fact that a second variant in the GAA gene could not be identified through MLPA. This finding could be due to limitations of the study methodology used to detect intronic or cryptic gene variants. Although this finding did not modify the frequency of patients with a definitive diagnosis of PD in this study, as the molecular diagnostic criteria was not fulfilled by none of these two patients, they both showed mild phenotypes of PD. Patients with similar clinical and molecular findings were reported to respond to enzyme replacement therapy34 and should prompt further investigations to identify a second variant in the GAA gene that could explain the patients clinical features.

Limitations of this work include its retrospective nature with lack and a non-homogeneous collection of detailed clinical information of the majority of the patients, the use of a bounded genetic panel and the lack of family segregation analysis. Furthermore, patients below 1 year old were not included in the analysis. Finally, whole exome sequencing,11 whole genome sequencing and RNA-seq-based transcriptome analysis35 were not feasible. All these factors might have affected the frequency of the LGMD and PD identified in this study.

In conclusion, this study represents the first genetic characterization of LGMD and PD in Argentina. We identified a genetic muscular disorder in 10.8% of the investigated population. CAPN3 was the main LGMD, and a definitive molecular diagnosis of new PD cases was confirmed in 0.4% of the patients. Molecular tests are useful tools for a prompt diagnosis of muscular genetic disorders, allowing their early recognition, consideration for treatment and genetic counseling. Results of molecular studies need to be integrated with clinical and complementary assessments to reach a final diagnosis. Inclusion criteria for genetic screening evaluations of muscular disorders could critically affect the results, so, information regarding family history, previous abnormal muscle pathology or abnormal enzymatic assays for PD should be carefully excluded since they may lead to frequency inaccuracies. Further researches are needed to expand the characterization of the LGMD and PD in Argentina.

The authors and members of the Argentine Muscular Dystrophy Consortium have no conflicts of interest to declare.

This research has not received specific aid from agencies of the public sector, commercial sector or non-profit entities. No funding was received by any author and/or members of the Argentine Muscular Dystrophy Consortium in the preparation of data or the manuscript.

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Adriana Pinzone; MD, Centro de Especialidades Médicas Ambulatorias de Rosario (CEMAR), Santa Fe, Argentina; Agustín Jauregui, MD, Fundación Favaloro, Buenos Aires, Argentina; Alberto Alemán, MD, Instituto Médico de Alta Complejidad (IMAC), Salta, Argentina; Alejandro Rodríguez, MD, Instituto de Neurociencias Buenos Aires, Buenos Aires, Argentina; Alicia Cueto, MD, Instituto de Neurología de Bs As, Buenos Aires, Argentina; Ana Carolina Costi, MD, Hospital san Martín de la Plata, La Plata, Argentina; Andrea Chirino Misisian, MD, Sanatorio Allende, Córdoba, Argentina; Andrea Legarreta, MD, Hospital Ramon de Madariaga, Misiones Argentina; Andrea Pianesi, MD, Sanatorio Güemes, Buenos Aires, Argentina; Andrés Barboza, MD, Hospital Central de Mendoza, Mendoza, Argentina; Andrés Berardo, MD, Sanatorio Allende Córdoba Argentina; Aquiles Uccelli, MD, Hospital Presidente Perón, Buenos Aires, Argentina; Ariel Guzmán, MD, Hospital Público Materno Infantil, Salta, Argentina; Ariel Villagra Cocco, MD, Hospital San Roque Nuevo, Córdoba, Argentina; Barbara Masotto, Centro Nacional de Genética Médica, Buenos Aires, Argentina; Brenda Borrego Guerrero, MD, Sanatorio Tandil, Buenos Aires, Argentina; Bruno De Ambrosi, MD, Instituto de Investigaciones Neurológicas Dr. Raúl Carrea (FLENI), Buenos Aires, Argentina; Carolina Azcona, MD, Hospital Italiano, Buenos Aires, Argentina; Cecilia Fiore, MD, Hospital El Sauce, Mendoza, Argentina; Cecilia Montes, Hospital de Niños de la Santísima Trinidad, Córdoba, Argentina; Cecilia Quarracino, MD,  Instituto de Investigaciones Médicas Alfredo Lanari, Buenos Aires, Argentina; Claudia Verónica Guevara, MD, Private Clinic, Tucumán, Argentina; Consuelo Durand, MD, Laboratorio de Neuroquímica Dr. N.A. Chamoles, Buenos Aires, Argentina; Cristian Calandra, MD, Hospital El Cruce S.A.M.I.C  Buenos Aires, Argentina; Daniel Schonfeld, MD, Centro Diagnóstico San Jorge, Puerto Madryn, Chubut, Argentina; Daniela Giardino, MD, Unidad Asistencial por más Salud Dr Cesar Milstein, Buenos Aires, Argentina; Daniela Graci, MD, Hospital Presidente Nicolas Avellaneda, Tucumán, Argentina; Eduardo L De Vito, MD, PhD, Instituto de Investigaciones Médicas Alfredo Lanari, Buenos Aires, Argentina; Elisa Cisneros, MD, Hospital Churruca Visca, Buenos Aires, Argentina; Ernesto Fulgenzi, MD, Hospital Pirovano  Buenos; Esteban Calabrese, MD, Hospital Español de Rosario, santa Fe, Argentina; Eugenia Conti, MD, Hospital de Clínicas José de San Martin, Buenos Aires, Argentina; Evangelina Maldonado, MD, Hospital Nacional Posadas , Buenos Aires, Argentina; Facundo Heredia, MD, Hospital Británico, Buenos Aires, Argentina; Fátima Pantiu, MD, Hospital Británico, Buenos Aires, Argentina; Fernando Auvieux Arias, MD, Hospital de Alta Complejidad Pte J.D. Perón de la provincia de Formosa, Formosa, Argentina, Fernando Chloca, MD, Fundación Favaloro, Buenos Aires, Argentina; Fernando Racca, MD, Clínica Privada Independencia, Buenos Aires, Argentina; Florencia Aguirre, MD, Hospital Ramos Mejía, Buenos Aires, Argentina; Florencia Bevilacqua, MD, Centro Nacional de Genética Médica “Dr. Eduardo E. Castilla”, ANLIS Malbrán; Gabriel Hernán Capellino, MD, Centro Médico Roentegen, Córdoba, Argentina; Gonzalo Vidal, MD, Hospital Perrano, Chaco, Argentina; Graciela Arguello, MD, Private Clinic, Rio Gallegos, Argentina; Graciela Contreras, MD, Hospital Pedro de Elizalde, Buenos Aires, Argentina; Gregorio Rosario Pascual Abiusi, MD, Hospital Boulogne, Buenos Aires, Argentina; Guadalupe Bruera, MD, Hospital Privado de Rosario, Santa Fe, Argentina; Guillermo Berbotto, MD, Sanatorio Británico, Santa Fe, Argentina; Gustavo Da Prat, MD, Sanatorio de la Trinidad Mitre, Buenos Aires, Argentina; Gustavo Sabbatini, MD, Hospital Iturraspe, Santa Fe, Argentina; Horacio Sacristán, MD, Instituto Municipal de Rehabilitación "Dr Anselmo Marini", Buenos Aires, Argentina; Ivan Martos, MD, Private Clinic, Tierra del Fuego, Argentina; Javier Muntadas, MD, Hospital Italiano, Buenos Aires, Argentina; José Crespo, MD, Sanatorio Güemes, Buenos Aires, Argentina; José Siemsen, MD, Hospital Militar, Buenos Aires, Argentina; Juan Ignacio Rolón, MD, Hospital Alemán, Buenos Aires, Argentina; Laura Cassar, MD, Hospital Pediátrico Alexander Fleming, Mendoza, Argentina; Laura Failletaz, MD, Private Clinic, Buenos Aires, Argentina; Leandro Nicolas Borisonik, MD, Hospital 4 de Junio "Dr. Ramón Carrillo", Chaco, Argentina; Lilia Mesa, MD, Fundación Favaloro, Buenos Aires, Argentina; Lorena Fasulo, MD, Clínica San Lucas, Neuquén, Argentina; Luciana Estefanía Sosa, MD, Hospital A. Korn de La Plata, Buenos Aires, Argentina; Luciano Recchia, MD, Clínica Universitaria Reina Fabiola, Córdoba, Argentina; Lucila Lecchini, MD, Hospital Italiano, Buenos Aires, Argentina; Marcela García Erro, MD, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina; Marcela Mozzi, MD, Instituto de Neurología, Neuquén, Argentina; Marcelo Ruggiero, MD, Hospital Italiano, Buenos Aires, Argentina; María Belén Tillard, MD, Sanatorio de los Arcos, Buenos Aires; María del Carmen Martínez Perea, MD, MS, Hospital. B. Rivadavia, Buenos Aires, Argentina; María Fernanda Peralta, MD, Unité Pédiatrique Des Maladies Métaboliques, Centre Hospitalier Universitaire Vaudois, Lausanne, Swiss; María Alejandra Figueredo, MD, Hospital San Roque, La Plata, Buenos Aires, Argentina; María Inés Araoz, MD, Sanatorio Altos de Salta, Salta, Argentina; María Julia Papagno, MD, Private Clinic, Argentina; María Monserrat Lozano, MD, Hospital General de Agudos Eva Perón de San Martín, Buenos Aires, Argentina; María Soledad Monges, MD, Hospital 'Prof. Dr. Juan P. Garrahan, Buenos Aires, Argentina; María Verónica Iovanna, MD, Hospital Municipal del Niño de San Justo, La Matanza, Buenos Aires, Argentina; María Yamila Hassan, MD, Centro Neurológico Platense, Buenos Aires, Argentina; Mariana Lujan Rivira, MD, Hospital Presidente Juan Domingo Perón,  Formosa, Argentina; Mariano Borrelli, MD, Hospital Militar Central Cirujano Mayor Dr. Cosme Argerich, Buenos Aires, Argentina; Mariela Bettini, MD, Hospital Italiano, Buenos Aires, Argentina; Mariela Lucero, MD, Hospital Sor María Ludovica  La Plata, Buenos Aires, Argentina; Marina Szlago, MD, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina; Marisol Ferrua, MD, Clínica La pequeña Familia Junín, Buenos Aires, Argentina; Marta Medina, MD, Hospital Córdoba, Córdoba, Argentina; Martin Cedrolla, MD, NEURONEA, Corrientes, Argentina; Maximiliano Arana, MD, Hospital Eva Perón de San Martín, Buenos Aires, Argentina; Milagros Sussini, MD, Hospital Escuela, Corrientes, Argentina; Myrian Zudaire, MD, Hospital Dr. Alende, Mar del Plata, Buenos Aires, Argentina; Natalia Alejandra Lucero, MD, Servicio de Neurología del Hospital Nacional de Clínicas de Córdoba, Córdoba, Argentina; Natalia Falco, Centro Médico Santa Fe, Santa Fe, Argentina; Néstor David Genco, MD, Hospital Santa Isabel de Hungría, Mendoza, Argentina; Nicolas Emmanuel Rodríguez, MD, Private Clinic, Argentina; Noelia Klug, MD, Sanatorio San Geronimo, Santa Fe, Argentina; Omar Gerardi, MD, Hospital Italiano, Buenos Aires, Argentina; Pamela Seilikovich, MD, Sanatorio privado San Geronimo, Santa Fé; Paola Pivetta, MD, Complejo Medico Policial Churruca Visca, Buenos Aires, Argentina; Patricia Quaglio, MD, Laboratorio CIGEN, Rosario, Santa Fe, Argentina; Patricia Santoro, MD, Instituto de Investigaciones Médicas Alfredo Lanari, Buenos Aires, Argentina; Patricio Brand, MD, Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia (FLENI), Buenos Aires, Argentina; Paz Zuberbuhler, MD, Hospital General de Agudos Dr. Teodoro Álvarez, Buenos Aires, Argentina; Ricardo Alonso, MD, Sanatorio Güemes, Buenos Aires, Argentina; Rodrigo Santamarina, MD, Clínica Cuyo, Mendoza, Argentina; Rosana Ocampo, MD, Hospital Provincial de Rosario, Santa Fe, Argentina; Rossana Espíndola, MD, Hospital Dr. Ramón Madariaga, Buenos Aires, Argentina; Sara Regliner, MD, Sanatorio Juan XXIII, General Roca, Río Negro, Argentina; Sebastián Figueroa Bonaparte, MD, Hospital Universitario Privado de Córdoba, Córdoba, Argentina; Sebastián Ianardi, MD, Hospital Lagomaggiore, Mendoza, Argentina; Silvina Plati, MD, Hospital el Carmen, Mendoza; Silvina Rusconi, MD, Hospital Central San Isidro, Buenos Aires, Argentina; Soledad Smechow, CEINSA, Buenos Aires, Argentina; Sonia Tolosa, MD, Hospital Urquiza de Concepción del Uruguay, Entre Ríos, Argentina; Susana Liwacki, MD, Clínica Universitaria Reina Fabiola, Córdoba, Argentina; Úrsula Vanesa Paris, MD, Private Clinic, Buenos Aires, Argentina; Valeria Muro, MD, Hospital Británico, Buenos Aires, Argentina; Valeria Salutto, MD,  Instituto de Investigaciones Médicas Alfredo Lanari, Buenos Aires, Argentina; Vanina Solari, MD, Hospital Dr. Orlando Alassia  Santa Fe, Argentina; Verónica Díaz, MD, Hospital Ángel. C Padilla Tucumán, Argentina; Victoria Fernández, MD, Hospital Ramos Mejía, Buenos Aires, Argentina.