Pruebas complementarias y técnicas de laboratorio: importancia para el cribado y diagnóstico de enfermedades neuromusculares raras de origen genético y utilidad en el apoyo a la clínica

PRUEBAS COMPLEMENTARIAS Y TÉCNICAS DE LABORATORIO: IMPORTANCIA PARA EL CRIBADO Y DIAGNÓSTICO DE ENFERMEDADES NEUROMUSCULARES RARAS DE ORIGEN GENÉTICO Y UTILIDAD EN EL APOYO A LA CLÍNICA Pilar Carbonell Corvillo TESIS DOCTORAL | 2022 Directoras: Carmen Paradas López Mª Dolores Jiménez Hernández Tutor: Pablo Mir Rivera UNIVERSIDAD DE SEVILLA PROGRAMA DE DOCTORADO EN BIOLOGÍA MOLECULAR, BIOMEDICINA E INVESTIGACIÓN CLÍNICA PRUEBAS COMPLEMENTARIAS Y TÉCNICAS DE LABORATORIO: IMPORTANCIA PARA EL CRIBADO Y DIAGNÓSTICO DE ENFERMEDADES NEUROMUSCULARES RARAS DE ORIGEN GENÉTICO Y UTILIDAD EN EL APOYO A LA CLÍNICA Pilar Carbonell Corvillo Directoras: Carmen Paradas López Mª Dolores Jiménez Hernández Tutor Pablo Mir Rivera UNIVERSIDAD DE SEVILLA PROGRAMA DE DOCTORADO EN BIOLOGÍA MOLECULAR, BIOMEDICINA E INVESTIGACIÓN CLÍNICA AÑO 2022 AGRADECIMIENTOS Este trabajo supone la consecución de un objetivo personal y académico-profesional a lo largo de bastantes años, en los que han sucedido los cambios y experiencias más significativos de mi vida. No ha sido fácil conseguirlo y para ello han sido fundamentales muchas personas. En primer lugar, mis padres, los que siempre me han enseñado que con esfuerzo, dedicación y constancia se alcanzan los objetivos. Mi hermano, mi admirador más fiel y mi apoyo constante en cualquier situación. Por supuesto, mi marido y mis hijos, ellos me han permitido conseguir esta meta, os pido perdón por el tiempo que os he quitado y los sofocos que os he dado. Somos un equipo y juntos conseguiremos lo que nos propongamos. Mis suegros, porque sin su ayuda hubiera sido imposible. Carmen Paradas, mi directora de tesis y mi consultora profesional, porque desde el principio de mi residencia en neurología fue una luz que siempre he intentado seguir y afortunadamente ella me ha querido guiar. Gracias por tu motivación y entusiasmo, por tu rigor científico, por continuar enseñándome, por tu ayuda constante en esta tesis o con cualquier duda de pacientes. Lola Jiménez, mi jefa y mi directora de tesis, que siempre ha confiado en mí, incluso sin conocerme. Pablo Mir, mi tutor, por su aprecio, su disponibilidad y sus enseñanzas. Eloy y Rai, los neuropatólogos por excelencia que son una pieza fundamental de nuestro trabajo diario y de esta tesis. Macarena Cabrera, el Dr. Laing y el Dr. Velayos que han permitido darle el fundamento genético a esta tesis. Los compañeros del IBIS: Fabiola, Emi, Antonio, Amalia que han colaborado y que tanto me han enseñado en el desarrollo de estos proyectos, permitiéndonos integrar lo clínico y lo básico. Maribel y Teresa, mis compañeras de Málaga, que son familia y que me dan los mejores consejos. Todos mis pacientes y familias porque sin ellos mi profesión no tendría sentido ni este trabajo hubiera existido. Muchas gracias a todos. ÍNDICE AGRADECIMIENTOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ABREVIATURAS Y ACRÓNIMOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 RESUMEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 INTRODUCCIÓN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 1 . Aspectos generales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2. Estudioselectrofisiológicos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Electromiografía de fibra aislada (SFEMG) ................................... 31 Estudio del sistema nervioso autónomo y de fibra fina ......................... 32 Estudio electrofisiológico en canalopatías musculares ......................... 32 3 . Resonanciamagnéticamuscular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4 . Biopsia muscular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5 . Estudios moleculares . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Secuenciación Sanger .................................................... 46 Next generation sequencing (NGS) ......................................... 47 Estudio de haplotipos .................................................... 48 Análisis de la patogenicidad: .............................................. 48 6 . Estudios funcionales in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 7 . MiopatíadistaldeLaing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 8 . Parálisisperiódicahipopotasémica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 9 . Coreoacantocitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 OBJETIVOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 OBJETIVO GENERAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 OBJETIVOS ESPECÍFICOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 CAPÍTULO 1 . A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 1 . METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Patients and study protocol ............................................... 63 Muscle biopsy .......................................................... 64 Muscle MRI ............................................................ 64 MYH7 genetic study ..................................................... 64 Haplotype analysis ....................................................... 64 In vivo functional study ................................................... 65 2 . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Clinical phenotype ....................................................... 65 Muscle histology ........................................................ 68 Imaging study ........................................................... 69 MYH7 mutation screening ................................................ 71 Haplotype analysis ....................................................... 71 In vivo functional analysis ................................................. 73 3 . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 CAPÍTULO 2 . REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 1 . METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Patients and study protocol ............................................... 81 Muscle MRI protocol ..................................................... 82 2 . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Clinical phenotype ....................................................... 82 Muscle MRI ............................................................ 85 Exercise testing ......................................................... 87 3 . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 CAPÍTULO 3 . NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES . . . . . . . . . . . . . . . . . . . . . 95 1 . METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Clinical phenotype ....................................................... 97 Electroneurography ...................................................... 98 Muscle MRI ............................................................ 98 Muscle biopsy .......................................................... 98 Chorein expression in muscle .............................................. 98 VPS13A genetic study .................................................... 99 2 . RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Clinical and laboratory findings ............................................ 99 Electroneurography ...................................................... 104 Muscle MRI ............................................................ 104 Muscle biopsy .......................................................... 105 Chorein expression in muscle .............................................. 111 Genetic analysis ......................................................... 112 3 . DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 DISCUSIÓN FINAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 CONCLUSIONES/CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 CONCLUSIONES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 CAPÍTULO 1 ............................................................ 123 CAPÍTULO 2 ............................................................ 123 CAPÍTULO 3 ............................................................ 124 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 CHAPTER 1 ............................................................. 125 CHAPTER 2 ............................................................. 125 CHAPTER 3 ............................................................. 126 APÉNDICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 REFERENCIAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 RESUMEN INTRODUCCIÓN La mayoría de las enfermedades neuromusculares raras de origen genético son muy invalidantes y tienen una gran repercusión en los familiares de los pacientes y su descendencia, por lo que el diagnóstico definitivo resulta esencial para poder advertir un pronóstico, aplicar una terapia u ofrecer un consejo genético. Sin embargo, la gran heterogeneidad clínica y genética que se relaciona con las enfermedades neuromusculares raras hace que el diagnóstico sea difícil y que se retrase con frecuencia. Disponer de pruebas o signos que nos permitan un diagnóstico precoz en estas enfermedades resulta de especial utilidad. El análisis de una posible relación genotipo-fenotipo es importante desde el punto de vista diagnóstico, especialmente cuando la expresión fenotípica es variable en una enfermedad. Los estudios genéticos de secuenciación de última generación han supuesto un gran avance en el diagnóstico genético definitivo, pero resulta primordial una orientación clínica adecuada, integrando los hallazgos clínicos, electrofisiológicos, radiológicos y patológicos. La miopatía distal de Laing es una enfermedad con herencia autosómica dominante debida a mutaciones en el gen MYH7, que asocia una gran heterogeneidad clínica, histológica y genética. Es conocido que las mutaciones que introducen prolina afectan a la estructura de la proteína, siendo probablemente muy patógenas. La resonancia magnética muscular y el test de ejercicio electrofisiológico se han descrito como técnicas útiles en la parálisis periódica hipopotasémica. Son bien conocidas las marcadas diferencias entre hombres y mujeres portadores de esta enfermedad, sin embargo, poco se conoce acerca de las pruebas complementarias en la población asintomática. La Coreoacantocitosis es una enfermedad debida a mutaciones en el gen VPS13A de herencia autosómica recesiva. La hiperCKemia es un hallazgo común en estos pacientes, en algún caso ha sido comunicada antes de cualquier otra manifestación clínica, sin embargo, hasta el momento no se ha descrito la posible afectación miopática específica asociada. OBJETIVOS OBJETIVO GENERAL Analizar el valor de las pruebas complementarias como herramientas de cribado en enfermedades musculares hereditarias concretas, y de las técnicas de laboratorio en el análisis de la patogenicidad de mutaciones nuevas. OBJETIVOS ESPECÍFICOS 1. Caracterización de la miopatía distal de Laing asociada a una nueva mutación c.4679G>C /p.R1560P en el gen MYH7 en dos familias no relacionadas de Andalucía. 2. Detectar los parámetros con mayor utilidad diagnóstica en el estudio de pacientes con parálisis periódica hipopotasémica familiar debida a mutación p.R528H en el gen CACNA1S . 3. Identificar el fenotipo completo de afectación neuromuscular en una serie de pacientes españoles con coreoacantocitosis genéticamente confirmada. MÉTODO • MiopatíadistaldeLaing:Estudiamos dos pacientes index y 24 miembros de dos familias aparentemente no relacionadas mediante el examen físico, análisis de creatinquinasa, resonancia magnética muscular, estudio genético y estudio de haplotipos. Realizamos estudios funcionales in vivo en un modelo de C . elegans con expresión transgénica de UNC-54::GFP. • Parálisisperiódicahipopotasémica:reclutamos 11 pacientes con parálisis periódica hipopotasémica pertenecientes a cuatro familias independientes, portadoras de la mutación p.R528H en el gen CACNA1S, incluyendo ocho varones y tres mujeres, junto con nueve controles de las mismas familias. La resonancia magnética muscular y el test electrofisiológico de ejercicio (análisis de la amplitud y área del potencial de acción muscular compuesto antes y varias veces después de un ejercicio corto de 10 segundos o largo de 5 minutos) fueron evaluados en esta cohorte homogénea. • Coreoacantocitosis: llevamos a cabo una descripción del fenotipo neuromuscular en una cohorte de diez pacientes pertenecientes a ocho familias con coreoacantocitosis genéticamente confirmada. Estudiamos a todos los pacientes mediante examen físico, nivel de creatinquinasa en suero, acantocitos en sangre periférica, electroneurograma, resonancia magnética muscular, biopsia muscular, expresión de coreina en músculo y estudio genético. RESULTADOS • MiopatíadistaldeLaing: 16 individuos fueron heterozigotos para la variante p.R1560P en el gen MYH7, no descrita previamente. Los pacientes presentaron el fenotipo clásico de miopatía distal de Laing, con primer dedo del pie caído como presentación inicial, incluso en pacientes levemente afectados que se declararon asintomáticos, aunque la debilidad para la flexión del cuello se objetivó como signo precoz en algunos casos infantiles. La resonancia magnética muscular mostró que el sartorio fue el primer músculo afectado, incluso en dos de los tres portadores asintomáticos. El estudio de haplotipo fue consistente con un ancestro común para las dos familias. Los análisis del fenotipo de motilidad en el modelo trasngénico de C . elegans indicó que altos niveles del ortólogo wild type, UNC-54, podrían ser perjudiciales en este modelo. La motilidad no es significativamente diferente entre los gusanos transgénicos expresando UNC-54(wt)::GFP o UNC-54(mut)::GFP. • Parálisisperiódicahipopotasémica:Todos los hombres portadores de la mutación fueron sintomáticos mientras que todas las mujeres portadoras en nuestra muestra eran asintomáticas. El test de ejercicio largo mostró un decremento tardío de la amplitud y área del potencial de acción muscular compuesto en todos los portadores independientemente de los síntomas, alcanzando una sensibilidad del 100% después de los 35 minutos post-ejercicio. La resonancia magnética muscular mostró un patrón consistente de atrofia y/o infiltración grasa afectando principalmente al compartimento posterior del muslo, con afectación precoz del músculo aductor magno y semimembranoso. El mismo patrón de distribución de la afectación muscular se observó tanto en sintomáticos como en asintomáticos, aunque con mayor gravedad en sintomáticos, estando relacionado con la edad. • Coreoacantocitosis: en el momento del estudio ocho pacientes presentaron el fenotipo clínico clásico de coreoacantocitosis con corea, discinesia o distonia, crisis epiléptica, trastorno psiquiátrico y arreflexia. Todos los pacientes mostraron niveles altos de creatinquinasa, siendo la hiperCKemia asintomática la primera y única manifestación en tres de ellos en el momento del estudio. Una polineuropatía sensitivomotora axonal fue el hallazgo más frecuente en la electroneurografía. La resonancia magnética muscular objetivó infiltración grasa precoz en el músculo gastrocnemio, independiente de la duración de la enfermedad. La biopsia muscular en músculos distales mostró un patrón mixto combinando hallazgos neurogénicos con alteraciones estructurales focales e inclusiones sarcoplásmicas, mientras que en los músculos proximales de los mismos pacientes no encontramos dichas alteraciones. CONCLUSIONES • Nuestros hallazgos apoyan la nueva variante p.R1560P en el gen MYH7 como mutación fundadora en Andalucía, y debería ser considerada como la primera mutación a estudiar en pacientes originarios del sur de España. • La debilidad para la flexión cervical, el primer dedo del pie caído y la degeneración precoz del músculo sartorio en la resonancia magnética muscular pueden ser útiles para detectar portadores asintomáticos de la mutación p.R1560P en el gen MYH7 . • C . Elegans no es el modelo de elección para el estudio in vivo de mutaciones en el gen MYH7 que causan fenotipos leves. • Nuestros hallazgos redefinen el test de ejercicio y los hallazgos de resonancia magnética muscular en pacientes con parálisis periódica hipopotasémica debida a la mutación p.R528H en el gen CACNA1S. • Los pacientes con parálisis periódica hipopotasémica asintomáticos presentan el mismo tipo de alteraciones electrofisiológicas y radiológicas que las descritas en pacientes sintomáticos, subrayando su valor como pruebas de screening. • Los hallazgos descritos en resonancia magnética muscular y en la biopsia muscular junto con la hiperCKemia, apoyan la existencia de alteraciones miopáticas en los pacientes con coreoacantocitosis, lo que podría ser útil para mejorar el diagnóstico precoz de estos pacientes. • El diagnóstico diferencial de una hiperCKemia asintomática debe incluir la coreoacantocitosis, lo que debe ser tenido en cuenta en el análisis de los estudios genéticos en estos pacientes, dada la gravedad del cuadro y sus implicaciones en cuanto al consejo genético. ABSTRACT INTRODUCTION Most of the rare neuromuscular diseases of genetic origin are very disabling and have a great impact on the relatives of the patients and their descendants, so that the definitive diagnosis is essential to be able to announce a prognosis, apply a therapy or offer genetic advice. However, the great clinical and genetic heterogeneity associated with neuromuscular diseases makes diagnosis difficult and frequently delayed. Having tests or signs that allow us to make an early diagnosis of these diseases is especially useful. The analysis of a possible genotype-phenotype relationship is important from the diagnostic point of view, especially when the phenotypic expression is variable in a disease. Next generation DNA sequencing has represented a great advance in definitive genetic diagnosis, but adequate clinical guidance is essential, integrating clinical, electrophysiological, radiological and pathological findings. Laing distal myopathy is an autosomal dominant disease due to mutations in the MYH7 gene, which associates great clinical, histological and genetic heterogeneity. It is known that mutations that introduce proline affect the structure of the protein and are probably highly pathogenic. Muscle magnetic resonance imaging and electrophysiological exercise testing have been described as useful techniques in hypokalemic periodic paralysis. The marked differences between men and women carriers of this disease are well known, however, little is known about complementary tests in the asymptomatic population. Choreoacanthocytosis is a disease due to mutations in the VPS13A gene with autosomal recessive inheritance. HyperCKemia is a common finding in these patients, in some cases it has been reported before any other clinical manifestation, however, to date the possible associated specific myopathic involvement has not been described. OBJECTIVES GENERAL OBJECTIVE Analyze the value of complementary tests as screening tools in specific hereditary muscle diseases, and of laboratory techniques in the analysis of the pathogenicity of new mutations. SPECIFIC OBJECTIVES 1. Characterization of Laing distal myopathy associated with a new mutation c.4679G>C /p.R1560P in the MYH7 gene in two unrelated families from Andalucía. 2. To detect the parameters with the greatest diagnostic utility in the study of patients with familial hypokalemic periodic paralysis due to the p.R528H mutation in the CACNA1S gene. 3. To identify the complete phenotype of neuromuscular involvement in a series of Spanish patients with genetically confirmed choreoacanthocytosis . METHODS • Laingdistalmyopathy:We studied two index patients and 24 family members from two apparently unrelated families by physical examination, serum creatine-kinase analysis, muscle magnetic resonance imaging, genetic study and haplotype study. We performed in vivo functional studies in a model of C .elegans with transgenic expression of UNC-54::GFP. • Hypokalemic periodic paralysis: We recruited 11 hypokalemic periodic paralysis patients from four independent families, harboring the frequent p.R528H mutation in the calcium channel gene CACNA1S, including eight male symptomatic and three female asymptomatic carriers, together with nine controls from the same families. Muscle magnetic resonance imaging and electrophysiological long-exercise test were evaluated in this homogeneous cohort. • Choreoacanthocytosis: we carried out a description of the neuromuscular phenotype in a cohort of ten patients belonging to eight families with genetically confirmed choreoacanthocytosis. We studied all patients by physical exam, serum creatine-kinase level, peripheral blood acanthocytes, electroneurography, muscle MRI, muscle biopsy, chorein expression in muscle, and genetic analysis. RESULTS • Laing distal myopathy: 16 individuals were heterozygous for the p.R1560P variant in the MYH7 gene, not previously described. Patients displayed the classic Laing distal myopathy phenotype, with hanging first toe as the initial presentation, even in mildly affected patients who declared themselves asymptomatic, although neck flexor weakness was revealed as early sign in some childhood cases. Muscle magnetic resonance imaging showed that sartorius was the first muscle affected, even in two of the three asymptomatic carriers. The haplotype study was consistent with a common ancestor for the two families. The analysis of the motility phenotype in transgenic C . elegans indicated that high levels of wild type UNC-54 could be harmful in this model . The motility does not significantly different between the transgenic worms expressing UNC-54(wt)::GFP or UNC-54(mut)::GFP. • Hypokalemic periodic paralysis: All male carriers of the mutation were symptomatic while all female carriers in our sample were asymptomatic. The long exercise test showed a delayed decrement of the compound muscle action potential amplitude and area, in all the carriers regardless of symptoms, becoming 100% sensitivity after 35 minutes post-exercise. Muscle magnetic resonance imaging showed a consistent pattern of atrophy and/or fatty infiltration involving mainly the posterior compartment of the thigh, with early involvement of the adductor magnus and semimembranous muscle. The same pattern of distribution of muscle involvement was observed in both symptomatic and asymptomatic patients, although with greater severity in symptomatic patients, being related to age. • Choreoacanthocytosis: At the time of the study, eight patients presented the classic choreoacanthocytosis clinical phenotype with chorea, dyskinesia or dystonia, epileptic seizure, psychiatric disorder and areflexia. All patients showed high creatine-kinase levels, being asymptomatic hyperCKemia the first and only manifestation in three of them at the time of the study. An axonal sensorymotor polyneuropathy was the most frequent finding in electroneurography. Muscle magnetic resonance imaging showed early fatty infiltration in the gastrocnemius muscle, regardless of the duration of the disease. Muscle biopsy in distal muscles showed a mixed pattern combining neurogenic findings with focal structural alterations and sarcoplasmic inclusions, while in the proximal muscles of the same patients we did not find such alterations. CONCLUSIONS • Our findings support the new p.R1560P variant in the MYH7 gene as a founder mutation in Andalucía, and should be considered as the first mutation to be studied in patients from Southern Spain. • Neck flexor weakness, hanging big toe and early sartorius muscle degeneration in muscle magnetic resonance imaging could be useful for detecting asymptomatic carriers of the p.R1560P mutation in the MYH7 gene. • C . elegans model might not be the model of choice to recapitulate in vivo mutations in human MYH7 that cause mild phenotypes. • Our findings redefine the exercise test and muscle MRI findings in patients with hypokalemic periodic paralysis due to the p.R528H mutation in the CACNA1S gene. • Patients with asymptomatic hypokalemic periodic paralysis present the same type of electrophysiological and radiological alterations as those described in symptomatic patients, highlighting their value as screening tools. • The findings described in muscle magnetic resonance imaging and muscle biopsy together with hyperCKemia, support the existence of myopathic alterations in choreoacanthocytosis patients, which could be useful to improve the early diagnosis of these patients. • The differential diagnosis of asymptomatic hyperCKemia should include choreoacanthocytosis, which should be taken into account in the analysis of genetic studies in these patients, given the severity of the condition and its implications in terms of genetic counseling. Es más sensible que la estimulación repetitiva porque la alteración de la unión neuromuscular que aún no induce bloqueo o decremento de las fibras musculares puede demostrar aumentos importantes en el jitter. De manera que en ocasiones se puede observar alteración significativa en el jitter sin que exista debilidad. Por ello esta prueba resulta especialmente útil en aquellos casos con sospecha de miastenia en los que los anticuerpos son negativos y la estimulación repetitiva no produce decremento. ESTUDIO DEL SISTEMA NERVIOSO AUTÓNOMO Y DE FIBRA FINA El sistema nervioso autónomo (SNA) y la sensibilidad térmica y algésica se transmiten a través de fibras finas y muy poco mielinizadas, por lo que pueden pasar desapercibidas para los estudios convencionales de ENG que estudia fundamentalmente fibras gruesas altamente mielinizadas. Las técnicas más utilizadas son: • Respuestasudomotorasimpáticorefleja: potencial eléctrico generado por la activación de las glándulas sudoríparas de la piel que implica una variación en la conductancia cutánea entre la piel con pelo y sin él ante un estímulo que implique un cambio emocional (estímulo nociceptivo, ruido, miedo, tos ) • QST(quantitativesensorytest):procedimientos psicofísicos de análisis de la percepción sensorial ante estímulos externos de intensidad variable y controlada. Los estímulos empleados pueden ser: mecánicos, eléctricos, vibratorios, punzantes, térmicos Para valorar la percepción del dolor y la temperatura se utiliza el termotest. • Variabilidad del intervalo R-R: los intervalos R-R en el electrocardiogrma son las diferencias de tiempo entre una contracción ventricular y la siguiente. Está influido por el SNA mediante el nervio vago. Alteraciones en la variabilidad sugieren disautonomía. • Sudoscan: mide la capacidad de las glándulas sudoríparas para liberar iones de cloruro en respuesta a un estímulo electroquímico en las palmas de las manos y las plantas de los pies, áreas con mayor densidad de glándulas sudoríparas. Los resultados de esta prueba son útiles como indicadores en pacientes con riesgo de disfunción autónoma mediante la medición de la conductancia electroquímica del sudor y la asimetría observada entre ambos lados. ESTUDIO ELECTROFISIOLÓGICO EN CANALOPATÍAS MUSCULARES Las canalopatías son enfermedades hereditarias debido a mutaciones en los canales iónicos o en las proteínas con las que interactúan y pueden afectar a un amplio rango de tejidos. Todas estas enfermedades tienen en común la alteración de las propiedades del potencial de acción o transmisión sináptica y como resultado los tejidos mantienen una tendencia aumentada a entrar o permanecer en un estado de excitabilidad alterado 11,12. Dentro de las canalopatías musculares, en términos generales y tradicionalmente, se puede distinguir entre tres formas distintas de miotonías: miotonía congénita (MC), paramiotonía congénita (PC) y miotonía agravada por potasio (MAP) y dos formas de parálisis periódicas: hiperpotasémica (HyperPP) e hipopotasémica (HypoPP), basadas en los cambios del nivel de potasio en sangre durante los ataques que estará elevado en la primera y disminuido en la segunda. Las consecuencias funcionales de las mutaciones en los canales iónicos sobre la excitabilidad de la membrana muscular pueden ser estudiadas por electromiografía directamente sobre los pacientes. Puesto que la debilidad en las canalopatías musculares puede ser desencadenada por ejercicio intenso, el uso de una contracción voluntaria fuerte y sostenida ha sido propuesto como test provocador para el diagnóstico. La respuesta muscular registrada en superficie tras un estímulo nervioso supramáximo es usada para monitorizar la actividad del sarcolema. El análisis de la amplitud del potencial de acción muscular compuesto (CMAP) antes y varias veces después de un ejercicio corto (10 segundos) o largo (5 minutos) nos aporta información sobre los cambios en el número de fibras musculares activadas y sobre su capacidad para despolarizar y repolarizar. Una disminución significativa en la amplitud del CMAP después de un test de ejercicio largo ha sido demostrada en aproximadamente el 70-80% de los pacientes con parálisis periódicas 13,14y en 17 y 33% de los pacientes con miotonía congénita y con paramiotonía congénita respectivamente 15,16.Ocasionalmente también se puede objetivar una paresia transitoria en los síndromes miotónicos después de un ejercicio corto. Es importante tener en cuenta que un ejercicio repetido mejora la rigidez muscular en la miotonía congénita pero no en la paramiotonía congénita, un ejercicio leve puede prevenir o retrasar ataques de debilidad mientras que un ejercicio intenso puede desencadenar crisis en las parálisis periódicas. El artículo más referente sobre estudio neurofisiológico en canalopatías musculares es el trabajo llevado a cabo por Fournier and col. 17, publicado en 2004 que representa el primer estudio electromiográfico en una serie larga de pacientes con canalopatías musculares identificadas genéticamente. En este estudio, exploraron un grupo de 51 pacientes portadores de mutaciones conocidas para los canales iónicos y asociadas con diferentes formas de parálisis periódicas o miotonía (incluyeron 6 pacientes con MC, 16 PC, 2 MAP, 6 hiperPP, 13 hipoPP-1, 2 hipoPP-2 y 6 casos con miotonía asociada a parálisis periódica). El estudio EMG les permitió establecer una relación entre los síndromes clínicos y la respuesta muscular eléctrica a los diferentes test evocadores (ejercicio corto repetido y ejercicio largo). Comparando los hallazgos del EMG y las respuestas al test de ejercicio corto y largo, muchos pacientes portadores de la misma mutación mostraron un patrón similar de anormalidades eléctricas musculares. De manera que definieron cinco patrones electrofisiológicos principales (Tabla 1), cada uno de los cuales corresponde a un grupo definido de mutaciones. Tabla 1 . Patrones electrofisiológicos en canalopatías musculares*. Fenotipoclínico PC MC Otras formas HyperPP HypoPP-1 MutaciónCanal T1313M R1448C Sodio Cloro G1306A 1693T Sodio T704M Sodio R528H Calcio Patrón electrofisiológico I II III IV V EMG (descargas miotónicas) ↑ ↑ ↑ No/raro No PAMC (trasejerciciocorto) Cambios en la amplitud tras el 1º test ↑/↓ ↓transitorio No ↑No Cambios en la amplitud tras el 2º y 3º test ↓gradual No No ↑ gradual No PAMC ( trasejerciciolargo) Cambio inmediato de amplitud ↓No/ leve↓No ↑No Cambio tardío de amplitud ↓No No ↓ ↓ Sensibilidad*(%) 100 83 63 83 84 EMG=electromiografía; PC: paramiotonía congenita; MC=miotonía congenital; pp=parálisis periódica; PAMC=potencial de acción muscular compuesto. *La sensibilidad de un patrón para el correspondiente grupo de mutaciones fue definido como el número de pacientes que cumplían el patrón versus el número total de pacientes portadores de la mutación. *Tabla modificada de Fournier and col17 . Centrándonos en los resultados obtenidos en las HypoPP en este mismo estudio, los pacientes portadores de la mutación R528H del canal del calcio se caracterizaron por una disminución tardía de la amplitud del PAMC después de un ejercicio largo sin cambios inmediatos después de un ejercicio corto o largo. Estos hallazgos fueron englobados en el patrón V, con una sensibilidad del 84%. Los dos pacientes que fueron estudiados con hipoPP-2 portadores de la mutación R672 en el gen SCN4A presentaron fenotipo IV y V, respectivamente. Además, un paciente con hipoPP-1 presentó un patrón normal junto con todos los sujetos controles estudiados. 3. RESONANCIA MAGNÉTICA MUSCULAR Los avances en las técnicas de imagen han conseguido que la resonancia magnética muscular (RMm) suponga una herramienta relevante y habitual en la evaluación de un paciente con patología neuromuscular, especialmente en aquellas con afectación muscular 18,19. De manera que actualmente la RMm es reconocida como una técnica útil para el diagnóstico y seguimien- to de las enfermedades musculares 20–22. Existen distintas secuencias que nos resultan útiles en la realización de una RMm para valoración muscular (Fig. 1 y 2): • Secuencia T1: permite identificar el infiltrado graso de los músculos. En esta secuencia tomando como referencia la intensidad del músculo sano, el interior de los vasos se observa hipointenso o negro y la grasa se observa hiperintensa o blanca. • Secuencia T2: no resulta útil en valoración de músculo, tanto la grasa como el agua se ven hiperintensas o de color blanco. • Secuencia de supresión grasa (STIR): se elimina la señal grasa sobre una imagen en T2, quedando una señal hiperintensa únicamente donde existe agua. Permite identificar el edema o la presencia de inflamación en el músculo. • Secuencia Dixon de 3 puntos: permite cuantificar de forma objetiva la grasa presente en el músculo mediante un software 23. • Espectroscopia de hidrógeno: se trata de una técnica que se está empezando a utilizar en ensayos clínicos, permite distinguir un pico de grasa y un pico de agua. Los estudios realizados han mostrado que la cantidad de grasa se correlaciona de forma significativa con las pruebas de función muscular en los pacientes con distrofias musculares 24. Figura 1 . Imagen de resonancia muscular ponderada en T1 . A) Músculo sano: en relación con el músculo, la grasa es hiperintensa y se ve de color blanco, mientras que el líquido es ligeramente hipointenso y se ve de color negro. B) Músculo patológico de paciente con miopatía: las secuencias en T1 permiten identificar de forma sencilla el infiltrado graso de los músculos afectos. Figura 2 . Imagen de resonancia muscular ponderada en STIR . En este tipo de secuencias resulta sencillo reconocer la presencia de agua libre que se observa hiperintensa con respecto al músculo. No existe unanimidad respecto al mejor protocolo de secuencias a realizar en el estudio de un paciente con resonancia muscular. Sin embargo, la mayoría de estudios se centran en el análisis de musculatura pélvica, de muslos y de piernas, aunque publicaciones de estudios de imagen muscular de cuerpo entero (whole body RMm) muestran utilidad para describir patrones más extensos y aumentar la especificad de la prueba. Los protocolos más difundidos se realizan en resonancias de 1.5 Teslas, incluyendo cortes axiales en T1 de las zonas a explorar y cortes con secuencia STIR, especialmente si se sospecha una miopatía inflamatoria 25. Para poder interpretar adecuadamente una RMm se debe identificar cada uno de los músculos. Existen atlas de RMm que permiten identificarlos de forma correcta y sencilla (Fig. 3-5). Figura3.Esquemaanatómicodelasregionesmáscomúnmenteestudiadasmedianteresonancia muscular centrada en pelvis . Figura4.Esquemaanatómicodelasregionesmáscomúnmenteestudiadasmedianteresonancia muscular centrada en muslos . Figura5.Esquemaanatómicodelasregionesmáscomúnmenteestudiadasmedianteresonancia muscular centrada en piernas . Para cuantificar el infiltrado graso de cada uno de los músculos, el método más utilizado es el de Mercuri 26. Se trata de una escala visual semicuantitativa que permite identificar de forma fácil el grado de infiltración en cada uno de los músculos (Fig. 6). Figura 6 . Escala de Mercuri . Representación visual de la escala de Mercuri en un corte axial en muslos (A) y piernas (B). La RMm resulta útil en la práctica clínica habitual por distintos motivos. Por ejemplo, a la hora de realizar una biopsia muscular para poder seleccionar el músculo adecuado, se aconseja un músculo en el que haya cambios pero no exista una infiltración grasa completa 27,28, aumentando de esta manera la rentabilidad de la biopsia 29. Diversos estudios han mostrado una buena correlación entre el grado de infiltración grasa en el músculo y la debilidad 30. Por este motivo, utilizamos la RMm para realizar el seguimiento de la evolución de la enfermedad o en los estudios de historia natural de las diferentes enfermedades musculares 20,31,32. (Fig. 7 y 8). Figura7*.Evolucióndelaafectaciónmuscularenmuslosenpacientescondistrofiadecinturas pordéficitdedisferlinaotipo2B(LGMD2B). Estas técnicas permiten detectar un gran número de mutaciones simultáneamente, reduciendo en gran medida el coste y el tiempo empleado. Al analizar los datos de estos estudios de secuenciación masiva, pueden producirse errores inherentes a la técnica, por lo que se recomienda la validación de los resultados mediante Sanger52. Otro aspecto relevante es que la NGS no puede detectar fácilmente eliminaciones o inserciones largas, variaciones de número de copias y expansiones repetidas o las contracciones. Por tanto, determinadas mutaciones que causan la distrofia miotónica, distrofia facioescapulohumeral, ataxias espinocerebelosas, CMT1A, entre otros, pueden no ser detectadas por NGS. Esta hace que la caracterización clínica sea primordial a la hora de guiar el estudio genético incluso cuando vaya a realizarse un NGS. ESTUDIO DE HAPLOTIPOS Un haplotipo es, en su sentido más general, un conjunto de variaciones de ADN a lo largo de un cromosoma que tienden a ser heredados juntos porque están muy próximos. El motivo por el cual se heredan juntos es porque al estar tan cerca no suele haber cruzamientos o recombinaciones entre estos marcadores, o diferentes polimorfismos, y por lo tanto tienden a ser transmitidas juntas a través de generaciones. Dada la alta variabilidad alélica en el genoma humano, la probabilidad de que dos individuos no relacionados presenten un mismo haplotipo, es prácticamente nula. Es por esto que el estudio de haplotipos se ha convertido en una herramienta útil en la determinación de relación génica entre individuos, y por lo tanto en el estudio del origen de mutaciones causantes de diversas enfermedades. De este modo, podemos decir que una mutación dada, presente en dos individuos aparentemente no relacionados entre sí tiene un mismo origen, si al realizar un estudio de haplotipos, el haplotipo relacionado con la mutación en uno de los individuos es igual al haplotipo relacionado con la mutación en el otro individuo. Los últimos estudios han defendido que los SNPs (polimorfismos de nucleótido simple) se heredan en grupos que se encuentran estrechamente relacionados en el ADN, en contraste con la tesis sostenida previamente, en la que se planteaba la segregación al azar debido a la recombinación genética. A este grupo de SNPs que se heredan en bloque, es a lo que hemos se denomina haplotipos. ANÁLISIS DE LA PATOGENICIDAD: Tanto si el estudio genético se realiza mediante secuenciación Sanger como por NGS es necesario analizar la patogenicidad de las variantes identificadas. Para ello es necesario: • Búsqueda de datos sobre la variante identificada en bases de datos como Locus-Specific Mutation Databases (LSDBs) o en Human Gene Mutation Database (HGMD) donde la información de estudios previos o resultados de laboratorios diagnósticos son incorporados a la base de datos de cada gen ( http://www.dmd.nl o http://www.biobase-in- ternational.com/product/hgmd ) • Cuando se trata de variantes no descritas, otras de las cosas a considerar es la prevalencia de esa variante en la población. Existen diferentes bases de datos disponibles online que recogen información de miles de exomas de controles ( http://www.ncbi. nlm.nih.gov/SNP o http://www.1000genomes.org o http://evs.gs.washington.edu/ EVS o http://exac.broadinstitute.org ) • Los predictores in silico utilizan diferentes algoritmos para inferir sobre las consecuencias funcionales de las mutaciones, incluyendo los cambios de splicing, modificaciones estructurales y bioquímicas ( http://www.mutationtaster.org o http://genetics.bwh. harvard.edu/pph2 o http://sift.jcvi.org o http://mutationassessor.org ). • El amplio conocimiento de los genes en concreto proporciona una información muy valiosa. Estar familiarizado con las mutaciones causantes de las enfermedades previamente descritas en un gen específico, los mecanismos patogénicos, los dominios estructurales, puntos calientes y cómo interactúan las mutaciones con la función de la proteína puede ser la clave para determinar la probabilidad de que una variante sea patógena. Por ejemplo, sabemos que las mutaciones missense que introducen una prolina en la cola de MYH7 son muy probablemente patógenas, pues la prolina interrumpe las estructuras helicoidales de la proteína y por tanto, afecta a la alineación de los filamentos gruesos e interfiere con la unión adecuada de la miosina 53. 6. ESTUDIOS FUNCIONALES IN VIVO Para una clasificación definitiva de una variante como causante de enfermedad, puede ser necesario estudios funcionales que prueban los efectos deletéreos de la mutación en asociación directa con la enfermedad. Dependiendo de la proteína y las mutaciones, diferentes evaluaciones con diferentes niveles de complejidad pueden ser apropiadas. No resulta una tarea fácil para cualquier laboratorio de investigación y mucho menos si tenemos en cuenta la cantidad de nuevas mutaciones y variantes que se detectan con la realización de NGS u otras técnicas de secuenciación. Establecer colaboraciones entre grupos clínicos expertos en enfermedades y grupos básicos con experiencia en modelos animales es ideal para poder profundizar en la patología subyacente a mutaciones asociadas a enfermedades neuromusculares. Los modelos animales son indispensables para identificar las bases genéticas y los mecanismos moleculares de las enfermedades raras, así como para comprender su fisiopatología, heterogeneidad clínica y correlaciones genotipo-fenotipo. De hecho, las enfermedades raras que tienen tan baja prevalencia y suelen ser complejas, son excelentes candidatas para modelos animales, particularmente en el contexto de animales genéticamente modificados 54. Sin embargo, debido a la escasez de información disponible sobre los modelos de enfermedades raras, uno de los principales problemas que obstaculizan la investigación traslacional es la elección del modelo en los estudios preclínicos, que en muchas ocasiones es incorrecta55. El ratón es el modelo animal más conocido y utilizado. En los últimos tiempos se está avanzando con otros modelos animales como el ratón Caenorhabditis elegans 56 y últimamente se están encontrando resultados muy exitosos en trabajos llevados a cabo en Drosophila 57 y Zebrafish 58. La modificación genética se puede realizar aplicando diferentes métodos: anulando o alterando ciertos genes presentes en un animal de manera que esta modificación se transmita a la descendencia, o bien transfiriendo genes a un animal desde la misma especie o de una especie diferente (modelos trasgénicos). Elegir el modelo animal y diseñar el experimento in vivo incluyendo la elección del método de modificación genética adecuado forma parte esencial de los estudios funcionales in vivo. 7. MIOPATÍA DISTAL DE LAING La MDL es una enfermedad con herencia autosómica dominante debida a mutaciones en el gen MYH7, que se caracteriza por una debilidad distal lentamente progresiva afectando al compartimento anterior de piernas y ocasionando el típico signo del “primer dedo del pie caído” 59. A pesar de ello, una gran variabilidad clínica y de fenotipos histológicos han sido ampliamente descritos en la MDL 50,60,61, y la misma mutación en el gen MYH7 puede causar diferentes fenotipos clínicos atendiendo a la edad de inicio, patrón de debilidad o severidad así como diferentes perfiles de afectación patológica, incluso en individuos de la misma familia 60. Respecto a los estudios de resonancia muscular en la MDL, el músculo tibial anterior es el músculo que más precoz y más severamente se afecta en prácticamente todos los casos publicados 62–64. Existe un estudio publicado con un análisis detallado del patrón radiológico en una cohorte de pacientes con MDL que describe conclusiones muy variables y no concluyentes respecto a los hallazgos en resonancia muscular salvo la precoz afectación del músculo tibial anterior 65. Las series más largas publicadas de pacientes con miopatía distal secundaria a mutaciones en el gen MYH7 describen una tasa muy alta de mutaciones de novo. Esta frecuencia tan alta de mutaciones esporádicas sugiere que pudiera deberse a una amplia distribución geográfica 65–67. Excepcionalmente, una mutación fundadora en el gen MYH, p.K1729del ha sido encontrada como causa de la mayoría de casos de MDL en la región de la Safor en España 62. La mayoría de las mutaciones están asociadas con el fenotipo clásico de MDL. Sin embargo, en la serie descrita por Fiorillo et al. 65 algunas característica clínicas atípicas fueron observadas ocasionando el retraso en el diagnóstico. Lamont et al. describieron en ocho de 15 familias estudiadas mutaciones missense que introducían una prolina en el gen MYH7 y se relacionaban con miopatía distal 67. Estas mutaciones afectaban a residuos de aminoácidos en todas la posiciones de los heptad repeatde la proteína lo que podría inducir modificaciones significativas en la estructura proteica, sugiriendo que las sustituciones de la prolina en cualquiera de estas posiciones tenía un efecto patógeno 68,69. En 1974, Sydney Brenner publicó su innovador trabajo sobre la caracterización genética de Caenorhabditis elegans 70 y, desde entonces, este nematodo se ha consolidado como un poderoso organismo modelo, generando no sólo hallazgos clave en muchas disciplinas biológicas diferentes, sino que también sirve como un valioso modelo preclínico para varias enfermedades humanas, incluidos los trastornos neuromusculares 71–74. En su estudio pionero, Brenner identificó y caracterizó varias clases de mutantes basándose en fenotipos visibles, siendo la serie genética más larga definida de mutantes con defectos de movimiento, denominados genéricamente como “no coordinados” (fenotipo unc) 70. Se demostró que las mutaciones en uno de esos genes, unc-54, causaba defectos en las células musculares de la pared corporal condicionando la parálisis del gusano y un trabajo posterior demostró que unc-54 codifica para una de las cuatro proteínas de la cadena pesada de la miosina del músculo esquelético en este organismo 70–75. De hecho unc-54 es uno de los genes más estudiados en C . elegans con cientos de mutantes disponibles y fenotipos que van desde defectos de motilidad muy leves hasta letalidad, debido al requisito de una proteína UNC-54 funcional para el ensamblaje de filamentos gruesos musculares 76–78. En definitiva, unc-54 es el gusano ortólogo del gen de la miosina humana MYH7, cuyas mutaciones provocan un grupo importante de enfermedades musculares con expresión clínica y morfológica variable según la isoforma mutada y el tipo y localización de la mutación 79. Debido a la alta conservación de la secuencia de aminoácidos entre el gusano UNC-54 y las proteínas MYH7 humanas (51 % de identidad y 71 % de similitud) así como su transparencia a lo largo del ciclo de vida, el modelo de C . elegans se ha utilizado con frecuencia para recrear las consecuencias fenotípicas de mutaciones en el gen MYH7 sobre la función y morfología de las células musculares. 8. PARÁLISIS PERIÓDICA HIPOPOTASÉMICA La HypoPP es una canalopatía muscular caracterizada por despolarización anormal del sarcolema muscular provocando episodios agudos de debilidad muscular flácida asociados a niveles bajos de potasio en sangre durante las crisis 11,80,81. Mutaciones de arginina en la región S4 del canal de calcio tipo-L (que codifica para la subunidad alfa del gen CACNA1S) o del canal de sodio voltaje dependiente (que codifica para la subunidad alfa del gen SCN4A) provocan el 90% de los casos de HypoPP 82–84. Estudios de correlación genotipo-fenotipo han mostrado una penetrancia incompleta en mujeres portadoras de la mutación p.R528H o p.H916Q en el gen CACNA1S, aunque el mecanismo subyacente está todavía por clarificar 82,85. Además, los test de ejercicio electrofisiológico en parálisis periódica familiares muestran diferentes patrones cuya sensibilidad diagnóstica depende del gen mutado, aunque el origen étnico y la frecuencia de los ataques puede influir en los resultados 13,14,17,86. La RMm es reconocida como una técnica útil para el diagnóstico y seguimiento de las enfermedades musculares heredadas 20,21, pero poco ha sido publicado sobre RMm en canalopatías, refiriéndose sobre todo a miotonías no distróficas 87–89. Con respecto a las HypoPP, algunas publicaciones muestran cambios en la RMm de estos pacientes 90,91, y recientemente ha sido publicado un inusual pero consistente patrón de RMm en una serie larga de pacientes con la mutación p.R528H en el gen CACNA1S. Este patrón muestra degeneración grasa en el glúteo mayor y en el compartimento posterior de muslos en la mayoría de pacientes con fenotipo de parálisis periódica, asociado a degeneración del músculo gastrocnemio medial y sóleo en los pacientes con afectación más severa y debilidad fija 92. 9. COREOACANTOCITOSIS Los síndromes de neuroacantocitosis (NA) son un grupo de enfermedades raras caracterizadas por trastornos neurológicos asociados a acantocitosis de glóbulos rojos (eritrocitos deformados como espinas). La coreoacantocitosis (ChAc) y el síndrome de Mc Leod (SML) son de las NA mejor conocidas. La ChAc es una enfermedad autósomica recesiva causada por mutaciones en el gen VPS13A que codifica para la proteína coreína 93,94. El SML está causado por mutaciones en el gen XK, que codifica un antígeno del sistema del grupo sanguíneo Kell y tiene un patrón de herencia ligado al X 95. Ambas entidades son definidas por acantocitos en sangre y diferentes alteraciones neurológicas, como trastornos del movimiento, epilepsia, deterioro cognitivo o trastorno de la conducta 96–98. Los pacientes con ChAc presentan una amplia variedad de síntomas en el curso de la enfermedad. Los síntomas iniciales pueden ser muy sutiles, pero la mayoría de los pacientes finalizan con corea severa, afectación cognitiva y trastornos psiquiátricos que producen una situación de completa dependencia y muerte. El diagnóstico precoz y el consejo genético en estos pacientes es muy importante para poder prevenir más casos. La acantocitosis de los hematíes es lo más común, con una variabilidad inter e intraindividual que puede variar entre 5% and 50% 96. Sin embargo, los acantocitos pueden aparecer tarde en el trascurso de la enfermedad 99, el porcentaje no se correlaciona con la severidad clínica 100, e incluso algunos casos de ChAc se han descritos sin acantocitos 101,102, lo que determina la dificultad y el retraso diagnóstico que suele relacionarse con las ChAc. Dulski et al. enfatizaron que los síndromes de NA podían estar infradiagnosticados y proponían varias señales de alerta que podían aumentar la sospecha de un síndrome de NA en pacientes que presentaban trastorno del movimiento, siendo la neuropatía periférica y la hiperCKemia alguno de ellos 98. La arreflexia y la neuropatía axonal ha sido descrita en más de la mitad de los casos de pacientes con ChAc publicados 81,93,97,98,103,104. En la mayoría de ellos, la poineuropatía es leve o no bien definida. Por otra parte, los niveles elevados de creatinquinasa (CK) en suero es un hallazgo común en las CA 93,98,104,105, incluso se ha descrito como el único y primer signo de enfermedad 106,107. Sin embargo, a pesar de los niveles de CK que pueden exceder los 1,000 UI/l, escasos estudios incluyen biopsias musculares 93,103,104. El estudio más extenso fue aportado por Vita et al. en 1989, en el que estudian la neuropatía periférica en un grupo de seis pacientes con ChAc. De manera que describen los resultados electrodiagnósticos, histológicos y utrastructurales en las biopsias de músculo y de nervio, concluyendo que la mayoría de pacientes tenían una polineuropatía sensitivomotora axonal 103. Los hallazgos neurógenos que se destacan en las biopsias musculares publicadas, no es suficiente para explicar los niveles altos de CK que se observan en algunos pacientes con ChAc. Mientras que la afectación muscular específica en las ChAc no ha sido ampliamente explorada, en el SML, que frecuentemente se asocia con CK elevada en sangre, su fenotipo de afectación neuromuscular y la histología muscular ha sido ampliamente descrita 108–110. OBJETIVOS OBJETIVOS OBJETIVO GENERAL Analizar el valor de las pruebas complementarias como herramientas de cribado en enfermedades musculares hereditarias concretas, y de las técnicas de laboratorio en el análisis de la patogenicidad de mutaciones nuevas. OBJETIVOS ESPECÍFICOS A . Caracterización de la miopatía de Laing asociada a una nueva mutación c.4679G>C /p.R1560P en el gen MYH7 en dos familias no relacionadas de Andalucía. A .1 . Describir el fenotipo clínico, radiológico y patológico del músculo esquelético A .2. Determinación del carácter fundador de la nueva mutación p.R1560P en el gen MYH7 A .3 Análisis de la patogenicidad de la mutación p.R1560P en el gen MYH7 mediante estudio in vivo en C . elegans B . Detectar los parámetros con mayor utilidad diagnóstica en el estudio de pacientes con HypoPP familiar debida a mutación p.R528H en el gen CACNA1S . B .1 . Analizar las características clínicas, radiológicas y la respuesta al test de ejercicio electrofisiológico en HypoPP. B .2 . Determinar el valor de estas pruebas como herramientas de cribado en portadores asintomáticos. C . Identificar el fenotipo completo de afectación neuromuscular en una serie de pacientes españoles con ChAc genéticamente confirmada. C .2 . Caracterizar y definir los hallazgos de la biopsia muscular. C .1 . Analizar los hallazgos clínicos y de laboratorio, electrofisiológicos y radiológicos, e identificar su utilidad como pruebas de detección precoz en esta enfermedad. CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN 64 MUSCLE BIOPSY Muscle samples were obtained by open biopsy. We processed the muscle following the standard procedures. Routine stains included hematoxylin & eosin (HE), modified Gomori trichrome, Oil Red O, periodic acid-Schiff (PAS), nicotinamide adenosine dinucleotide dehydrogenase (NADH), succinate dehydrogenase (SDH), cytochrome oxidase (COX), and combined COX/SDH, and myofibrillar adenosine triphosphatase (ATPase) after preincubation at pH 9.4, 4.5 and 4.3. Immunohistochemistry for fast and slow myosin was performed in one case. MUSCLE MRI Muscle MRI was performed at the level of the thighs and lower legs. We used a 1.5 Tesla scanner (MRI Intera, Philips Medical Systems). Axial MRI sections were obtained including a T1-weighted spin-echo sequence (TR 600-700 ms, TE 30 ms) and a short-time inversion recovery (STIR) sequence (TR 2500-3500 ms, TE 60 ms, TI 150 ms), in 10 mm slices. Fatty degeneration of muscles was identified according to the increased signal in T1 sequences and quantified according to a subjective scale 111, and STIR sequences were used to evaluate the presence of edema. MYH7 GENETIC STUDY Genomic DNA was extracted as per standard protocols from peripheral blood (Chemagic DNA Blood 5K Kit, Perkin Elmer) or saliva samples (Oragene DNA Self-Collection Kit, DNA Genotek) from patients and the rest of family members. In the index patient II.6/Family A, the coding regions of MYH7 gene were Sanger-sequenced at the Neuromuscular Unit, Hospital U. Virgen del Rocío, Sevilla (NMU-HUVR). DNA from index patient II.3/Family B was sent to the Lotterywest State Biomedical Facility Genomics (Perth, Australia) for neurogenetic sub-exomic supercapture and next generation sequencing (Proton™, Life Technologies) of 336 genes, which included 254 disease genes listed in the Neuromuscular Disorders Gene Table ( www.musclegenetable.fr ). DNA samples from all recruited relatives were PCR-am- plified using specific primers (5´-CCATGATTAGTGAGCAGGCC-3´, 5´-TCAGAGGTCCCTGCAG- TAAC-3´) and directly sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific). HAPLOTYPE ANALYSIS Microsatellite and SNP markers covering the MYH7 gene were selected based on their genomic location and heterozygosity (UCSC Genome Browser, Human GRCh37/hg19; http://genome. ucsc.edu/ ) 112. They spanned a genomic region of 2,92 Mb. Microsatellite markers were amplified by PCR using fluorescently-labelled primers and resolved in an ABI 3500 Genetic Analyzer 65 CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN (Applied Biosystems). SNP loci were PCR-amplified and genotypes were determined by visual inspection of the sequence chromatograms. Haplotypes were built assuming the minimum number of recombinations. IN VIVO FUNCTIONAL STUDY The C . elegans strains used in this study were N2, wild type Bristol and CB190, unc- 54(e190) I. Nematode strains were maintained as described 113. The plasmid that generates an UNC-54::GFP fusion protein 114 was a kind gift from Prof. Andras Malnasi-Csizmadia. This plasmid was used as bait to generate the mutant variant identified in this study with the following mutagenic primers 5´-gagcaaggttctccccgcccaggttgaag-3´ (forward) and 5´ -cttcaacctgggcggggagaaccttgctc-3´ (reverse). Mutagenesis was performed using the Quick- Change II XL Site Directed Mutagenesis Kit (Stratagene) following manufacturer instructions. Transgenic worm lines expressing wild type and mutated UNC-54::GFP proteins were generated at a concentration of 20 ng/l. GFP expression in worm muscle cells was scored on an Olympus BX61 fluorescence microscope. To assay the motility, well-fed L4 transgenic animals were transferred to fresh seeded plates and incubated further during 24 h at 20ºC prior to assaying their motility parameters. Then, worms were gently placed on seeded 60mm plates (solid medium motility) or in a drop of M9 buffer on a glass slide (liquid medium motility) and, after 1 min acclimation, the number of bends (solid) or thrashes (liquid) were recorded during the following two minutes in a Leica M165FC stereoscope equipped with a CCD camera (Greenlight Solutions). 2. RESULTS CLINICAL PHENOTYPE We performed the interview and physical examination in 23 individuals from the two families, nine women and 14 men. The age at the time of the examination ranged from eight to 79 years. The symptoms appeared between four and 30 years of age. Most carriers recalled abnormal gait and falls during infancy but did not complain of weakness until adulthood. Twelve carriers of the p.R1560P mutation (age range 8-79 years) showed a pathological physical exam. Three of them (age range 21-42 years) declared themselves to be asymptomatic and weakness restricted to big toe extension was the only sign found in the exam (III.4/Family A, II.5/Family B, III.2/Family B). Two of them presented transient cervical weakness at eight years old but not later (III-8/Family A, III-15/Family A). Three out of the 16 carriers were asymptomatic with normal physical exam (age range 11-38 years). One carrier was not examined (I.2/Family B). No patient referred respiratory or cardiac symptoms, and none of them referred a familial history of sudden death, arrhythmia or myocardiopathy. Echocardiography and respiratory functional studies were performed in some CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN 66 affected patients and all the studies were normal.CK was in normal range in all the carriers. Clinical findings are summarized in Table 2 and Figure 12. Table 2 . Clinical features in the carriers of the p.R1560P mutation in MYH7 gene. ManualmuscletestingaccordingtotheMRCscale Carriers Age at exam Onset Neck flexor Finger ext Knee ext Knee flexor Hanging big toe Toes ext Ankle dorsiflexor Pes cavus FVC/ Echocardiogram Pheno type A I-1 79 y 13 y 2/5 2/5 2/5 2/5 Yes 0/5 0/5 Yes np/np Sev II-1 59 y 8 y 3/5 4/5 4/5 5/5 Yes 2/5 0/5 Yes N/np Sev II-5 54 y 6 y 3/5 2/5 4/5 5/5 Yes 0/5 0/5 Yes N/N Sev II-6 52 y 6 y 3/5 3/5 3/5 5/5 Yes 3/5 0/5 Yes N/N Sev II-8 49 y 5 y 4/5 4/5 5/5 5/5 Yes 4/5 2/5 Yes np/np Mod II-14 39 y 30 y 5/5 5/5 5/5 5/5 Yes 4/5 2/5 Yes N/N Mod III-2 38 y - 5/5 5/5 5/5 5/5 No 5/5 5/5 Yes np/np A III-4 23 y 6 y 5/5 5/5 5/5 5/5 Yes 5/5 5/5 Yes np/np Mild III-8 15 y 8y 4/5 (T) 5/5 5/5 5/5 No 5/5 5/5 No np/np Mild III-14 11 y - 5/5 5/5 5/5 5/5 No 5/5 5/5 No np/np A III-15 8 y 4y 4/5 (T) 5/5 5/5 5/5 No 5/5 5/5 No np/np Mild B II-3 54 y 6 y 4/5 5/5 5/5 5/5 Yes 2/5 0/5 No N/N Mod II-5 42 y 7 y 5/5 5/5 5/5 5/5 Yes 5/5 5/5 No np/np Mild III-1 28 y - 5/5 5/5 5/5 5/5 No 5/5 5/5 No np/np A III-2 21 y 17 y 5/5 5/5 5/5 5/5 Yes 5/5 5/5 No np/np Mild y: Years; T: Transient; Ext: Extensor; CK: Serum creatin kinase; Numbers indicate muscle strength according to MRC scale; FVC: Forced vital capacity; N: Normal; np: not performed; A: Asymptomatic; Mild: Mild phenotype defined by hanging big toe sign; Mod: Moderate phenotype defined by weakness of toes extension and ankle dorsiflexion ± finger extension; Sev: Severe phenotype defined by weakness of toes extension, ankle dorsiflexion and knee extension ± finger extension. Bold letters denote mildly affected patients who only self-reported frequent falls during infancy. 67 CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN Figure12.ClinicalfeaturesinthepatientscarryingtheMYH7 p.R1560Pmutation. The most severely affected patients showed distal weakness involving finger extension (A) with mild contractures (B), and quadriceps weakness with atrophy only in the most advanced stages (C).Neck flexor weakness was present in patients with moderate and severe phenotype (D). Intriguingly, two infantile carriers presented isolated neck flexor weakness in the physical exam only during childhood (E), and disappeared in the course of the disease (F). The hanging big toe sign was present in early stage as first clinical sign of the disease, even in mildly affected patients who self-reported no symptoms (G, III.2/Family B; H, II.5/Family B; I, III.4/Family A), in some cases very subtle (G). No clear correlation was observed between the overall stage of clinical phenotype and the severity of the hanging big toe (i.e. compare H [II.5/Family B] to K [II.6/Family A],and I [III.4/Family A] to J [II.3/Family B]). CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN 68 MUSCLE HISTOLOGY Muscle biopsies from four patients (II.5/Family A, II.6/Family A, II.14/Family A, II.3/Family B), two tibialis anterior and two gastrocnemius muscles, were available for review. Myopathic pattern with increased variability in fiber size and internal nuclei were present in all biopsies. More severe changes with prominent atrophy and increased endomysial connective tissue were observed in both tibialis anterior muscles (Fig 13A and 13B). Small group of angulated fibers and nuclear clumps suggesting neurogenic features were seen in three of the four samples (Fig 13A and 13F). Oxidative enzyme stains (NADH, SDH and COX) revealed prominent internal sarcoplasmic disorganization with core, pseudocore and minicore lesions in all biopsies (Fig 13C and 13H). We observed type I fiber predominance in three cases (Fig 13D) and scattered rimmed vacuoles in one case. No hypotrophy of type I fibers was observed. Figure13.MusclehistologyinthepatientscarryingtheMYH7 p.R1560Pmutation. Muscle biopsies from patient II.14/Family A, tibialis anterior (A-D) and patient II.3/Family B, gastrocnemius (E-H) were stained with haematoxylin and eosin (A, B, E and F), succinate dehydrogenase (SDH) (C and G) and ATPase pH 4.5 (D and H). Atrophy, variability in fibre size, increased endomysial connective tissue, and necrotic fibers (A and D), and groups of small angulated atrophic fibers with nucler clumps (B and F) were present in both cases. SDH staining shows prominent sarcoplasmic disorganization including core, pseudocore and multiminicore lesions (C and G). Type I predominance was observed (D). Scale bar is 50 μm in all panels (A, B, C, E, F and G x 200 magnification; D and H x 100 magnification). 69 CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN IMAGING STUDY Muscle MRI was performed in 11 patients (Family A: II.5, II.6, II.14, III.2, III.4, III.14, III.15; Family B: II.3, II.5, III.1, III.2). All the affected carriers with moderate or severe phenotype (mean age 54.1±12.2 years old [range 39 to 79 years], disease duration 44.6±17.2 years [range 9 to 66 years]) showed fatty degeneration of lower limb muscles. The tibialis anterior, gluteus major and sartorius muscles were involved in all of them, although with different degrees of degeneration. Degeneration was severe in the tibialis anterior and mild in the gluteus and sartorius muscles. The other affected muscles in the MRI were the gracilis (75%), the quadriceps (50%) and the gastrocnemius (25%). Two slightly affected patients (III.4/ Family A, II.5/Family B) and two asymptomatic carriers (III.2/Family A, III.1/Family B) presented mild fatty infiltration only in the sartorius muscle. Two mildly affected patients (III.15/Family A, III.2/Family B) and one asymptomatic carrier (III.14/Family A) showed normal MRI. The sartorius muscle was the only one involved in 50% of slightly affected patients and in 66’6% of asymptomatic carriers. MRI findings are summarized in Table 3 and Figure 14. Table 3 . Muscle MRI findings in the carriers of the p.R1560P mutation in MYH7 gene. Patient/ Family Gluteus major Quadriceps Sartorius Gracilis Tibialis anterior Gastrocnemius Affected patients I.1/A np np np np np Np II.1/A np np np np np Np II.5/A + + ++ ++++ N II.6/A + ++ (asymmetric) + + +++ N II.8/A np np np np np Np II.14/A + N + + ++ N II.3/B + N + N +++ + Mildaffected patients III.4/A N N + N N N III.8/A np np np np np Np III.15/A N N N N N N II.5/B N N + N N N III.2/B N N N N N N Asymptomatic carriers III.2/A N N + + N N III.14/A N N N N N N III.1/B N N + + N N N: Normal; +: Mild fatty infiltration; ++: Moderate fatty infiltration; +++: Severe fatty infiltration; np: not performed CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN 70 Figure14.MuscleMRIinthepatientscarryingtheMYH7 p.R1560Pmutation. Muscle MRI findings of patients displaying severe (A, II.6/ Family A), moderate (B, II.14/ Family A) and mild (C, II.5/ Family B) clinical phenotype. Asymptomatic carriers can show mild sartorius muscle infiltration (D, III.1/ Family B) and mild affected patients normal MRI (E, III.2/ Family B). The vastus intermedius and lateralis muscles are involved only in severe stages (A). The tibialis anterior was the most severely degenerated muscle in symptomatic patients (A-C), and usually the only affected muscle in the lower legs. The most striking finding in our study was that the sartorius muscle (arrowhead) was the earliest involved, even in asymptomatic carriers (D),although it does not show evidence of significant progression (A-C) . 71 CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN MYH7 MUTATION SCREENING The novel heterozygous mutation c.4679G>C (NG_007884.1) p.R1560P in exon 34 of MYH7 was identified in both index patients (Fig. 11). The variant is absent from databases of controls ( http://gnomad.broadinstitute.org/ ; last accessed in November 2017) and is predicted to introduce a proline in the slow myosin tail. Next generation sequencing in the index patient II.3/B excluded pathogenic mutations in other genes related to neuromuscular disorders. The novel variant was subsequently analyzed by Sanger sequencing in all the recruited family members. In total, 26 subjects from the two families were recruited (17 from Family A and nine from Family B), comprising twelve clinically affected patients and 14 asymptomatic individuals. Segregation analysis showed that 16 individuals were heterozygous for variant p.R1560P in the MYH7 gene, including twelve symptomatic individuals. HAPLOTYPE ANALYSIS Given the identification of the same novel mutation in two families from different cities in Andalucía, Southern Spain, we studied the possibility of them being related. Haplotype analysis revealed a shared genomic region of 306 Kb in all carriers of p.R1560P mutation, restricted to four SNP markers (Fig 15). CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN 72 Figure15.HaplotypeanalysisinthetwofamiliescarryingtheMYH7 p.R1560Pmutation. Genomic position for genotyped microsatellite and SNP markers relative to the MYH7 gene, according to UCSC Genome Browser, Human GRCh37/hg19 (A).Haplotypes for families A and B (B,C). Haplotype analysis using microsatellite markers D14S50, D14S283, D14S990, D14S972, D14S64 and D14S264 in all available family members revealed a different haplotype cosegregating with the mutation in each family. To narrow down the studied genomic interval, we genotyped six SNP markers, rs365990, rs434273, rs2331979, rs2277475, rs7157716 and rs11621360 close to or within the MYH7 gene. The analysis of SNP genotypes revealed a shared haplotype for markers rs365990, rs434273, rs2331979 and rs2277475 in all carriers of the p.R1560P mutation that defined a common region of 306 Kb. The shared common haplotype is boxed. Genotype for mutation p.R1560P is denoted by a + (wild-type) and M (mutant) sign. A question mark indicates unknown genotype. Solid black symbols denote carriers of the MYH7 p.R1560P mutation. 73 CAPÍTULO 1 A NOVEL MYH7 FOUNDER MUTATION CAUSING LAING DISTAL MYOPATHY IN SOUTHERN SPAIN IN VIVO FUNCTIONAL ANALYSIS The C . elegans orthologue of human MYH7 is unc-54, one of the best studied genes in this nematode, initially identified by the motility defects shown by diverse unc-54 mutants 76,115,116. MYH7 and UNC-54 proteins are highly conserved through evolution and the MYH7 arginine residue at position 1560, mutated to proline in the families analysed in this study, is conserved in worm UNC-54, located at position 1569 (Fig 16A). As the human mutation has an autosomal dominant inheritance pattern, we generated Punc-54::unc-54(mut)::gfp wormsthat express the fusion protein UNC-54(mut)::GFP in a wild type genetic background. Transgenic worms expressing UNC-54(wt)::GFP fusion protein in the same wild type background served as controls (a kind gift of Prof. Shu G. Chen). As shown in Figure 16B, the mutated variant did not affect the morphology of the muscle cell or the longitudinal distribution of the myofilaments. To determine whether the mutation would cause any muscle-associated phenotypes, we performed motility assays in both liquid medium and on seeded plates. The first is carried out by placing worms in a 5µl drop of M9 buffer and counts the number of lateral swimming movements (thrashes) 116, while the second quantifies the locomotion rate of worms by counting the body bends as the animals moves on the bacteria lawn 56. As shown in Figure 16C and 16D, we did not observe significant differences in solid or liquid medium motility between the transgenic worms expressing UNC-54(wt)::GFP or UNC-54(mut)::GFP. However, in the thrashing assay, it was evident that transgenic animals either expressing UNC-54(wt)::GFP or UNC-54(mut)::GFP had a more erratic and irregular thrashing pattern compared to wild type or GFP controls. This behaviour explains why transgenic animals do not reach the numbers of thrashes of controls (Fig. 12D) and therefore calls for caution when interpreting rescuing data using UNC-54::GFP transgenic worms. 81 CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT *Publicación en preparación 1. METHODS PATIENTS AND STUDY PROTOCOL HUVR ethics committee approved this study and all participants provided informed consent. We selected HypoPP patients heterozygous for the common p.R528H mutation (NM_000069.2:c.1583G>A) in CACNA1S gene, from our Neuromuscular Unit. Regardless of symptoms, we contacted as many family members as possible and asked them to participate in the study. The same protocol was applied to patients and relatives: 1. Collection of clinical data through review of the charts, interviews, and physical examinations (the collected clinical data are detailed in Table 4). 2. Classification of subjects as symptomatic or asymptomatic based on the occurrence of well characterized transient paralysis attacks (at least one) or of permanent weakness during the inter-critical periods. 3. Blood collection for DNA isolation and determination of creatine kinase, ion levels, thyroid hormones, and renal function. 4. Standardized electrophysiological test during an inter-attack period, as described by Fournier et al17 and performed by the same examiner and with the same equipment (TECATM Sinergy T-EP) CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT 82 5. MRI of lower limb muscles. All subjects, except for the index patients, were studied in a blind manner because sequencing of the entire coding region and of the exon/intron boundaries of the CACNA1S gene was performed last. In retrospect, we considered non-carrier subjects as controls for exercise test and muscle MRI. MUSCLE MRI PROTOCOL Muscle MRI was performed at the level of the hips, thighs and lower legs. We used a 1.5 Tesla scanner (MRI Intera, Philips Medical Systems). MRI sections were obtained from axial planes including a T1-weighted spin-echo sequence (TR 600-700 ms, TE 30 ms) and a short-time inversion recovery (STIR) sequence (TR 2500-3500 ms, TE 60 ms, TI 150 ms), in 10 mm slices. Fatty degeneration of muscles was identified according to the increased signal in T1 sequences and quantified according to a subjective scale124to quantify increased signal (0= normal, 1= mild increased signal <30%, 2 = moderate increased signal 30-60%, 3=severe increased signal > 60%), and STIR sequences were used to evaluate the presence of edema. 2. RESULTS CLINICAL PHENOTYPE We recruited 20 subjects belonging to four unrelated Caucasian families from southern Spain (Fig. 17). We detected the p.R528H mutation in 11 patients, eight of whom were symptomatic (sHypoPP) and three asymptomatic (aHypoPP). 83 CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT Figure17.PedigreeofthefourindependentSpanishfamiliescarryingtheCACNA1S p.R528Hmutation. The symptomatic carriers are represented as solid black symbols and the asymptomatic carrier as solid grey symbols. The nine remaining subjects (five males and four females) made up the control group. In the sHypoPP group, the mean age of onset was 23±11 years (range: 15 to 42 years), the age at recruitment was 39.3±17 (range: 19 to 63 years), and the duration of the disease was 16.3±15.2 years (range: 2 and 41 years). Interestingly, all sHypoPP were male and all aHypoPP were female, the age at recruitment was 42 ± 16 years (range: 20 to 58 years). All sHypoPP patients but two had experienced transient paralysis attacks during the previous year, and we were able to identify clear triggers in six patients. Six out of eight sHypoPP were receiving treatment, either acetazolamide or oral potassium. All patients had full muscle strength at physical examination, except one, who had the first episode of paralysis five years before recruitment and showed inter-critical permanent weakness involving proximal lower limb muscles (2-3/5 according to the Medical Research Council scale for manual muscle testing, 3/6 according to the Modified Rankin Scale). The muscle biopsy in this patient displayed striking myopathic features with abundant subsarcolemmal vacuoles. All patients’ data are summarized in Table 4 and Table 5. CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT 84 Table 4 . ClinicaldatainthepatientscarryingtheCACNA1S p.R528Hmutation . p .R528H symptomatic carriers (n=8) Attacks during thelast year Attacks duration (hours) Time to last attack (months) Focal weakness during attacks Generalized weaknessduring attacks Triggers Treatment/ daily dose Family A I.1 12 4 2 Upper limbs Only the first attack None Potassium/ 4.0 gr II.4 6 6 1 Lower limbs Only the first attack Exercise Carbohydrate Potassium/ 7.5 gr II. 9 1 4 4 Upper limbs Only the first attack Exercise Potassium/ 2.5 gr Family B I.1 1 48 12 No Yes None Potassium/ 0.6 gr II.2 2 9 1 No Yes Exercise Carbohydrate Potassium/ 1.2 gr Family C I.1 0 3 24 Upper limbs Only the first attack Exercise No II.4 2 2 8 Upper-lower limbs No Exercise No Family D II.2 15 2 1 Lower limbs Only the first attack Exercise Alcohol ACZ/ 375 mg ACZ = acetazolamide Table 5 . Demographic,electrophysiologicalandradiologicaldatainthepatientscarryingthe CACNA1S p.R528Hmutation . p .R528H carriers (n=11) Sex Age at onset (years) Age at recruitment (years) Blood testa Physicalexam (inter-critical) Long exercise EMG test Muscle MRI Family A Symptomatic I.1 M 42 63 Normal Normal DD Mod II.4 M 17 36 Normal Normal DD Mild II. 9 M 17 19 Normal Normal DD Normal Asymptomatic I.4 F -58 Normal Normal DD Mild I.8 F -50 Normal Normal DD Mild Family B Symptomatic I.1 M 15 56 Normal Normal DD Mild II.2 M 18 23 Normal Normal DD Normal Asymptomatic II.3 F -20 Normal Normal DD Mild Family C Symptomatic I.1 M 17 52 Normal Normal DD Moderate II.4 M 18 21 Normal Normal DD Normal Family D Symptomatic II.2 M 40 45 ↑ CK (445 UI/l) Proximal weakness DD Severe a Blood analysis include potassium, sodium, renal function, thyroid hormone and creatine kinase levels; M = male; F = female; Age R = age at recruitment; Age O = Age at onset; DD = delayed decrement; CK = creatine kinase 85 CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT MUSCLE MRI Muscle MRI on T1w-sequences showed different degree of fatty infiltration in all the three aHypoPP carriers and in five of the eight sHypoPP patients. The distribution pattern was consistent with fatty infiltration and atrophy in the muscles of the posterior compartment of the thigh, being the adductor magnus and semimembranosus the first affected muscles, and sparing the biceps femori, sartorius and gracilis muscles. At the hip level, mild fatty degeneration also involved the gluteus maior muscle. At the lower leg level, the gastrocnemius medialis and soleus muscles were affected only in the patient with permanent weakness. When we ranked the MRI studies according to a subjective scale124, three aHypoPP and two sHypoPP patients showed mild involvement and two sHypoPP patients showed moderate involvement, whereas severe fatty replacement was seen only in one study, in the sHypoPP patient with permanent weakness, who also showed fatty degeneration of the quadriceps (Fig. 18A to C and Fig. 19). There was a direct linear association between severity of fatty infiltration and age (r = 0.62, p = 0.003) , but there was no statistical association with any other clinical parameter. STIR sequences showed increase signal only in the sHypoPP patient with permanent weakness while revealing edema in some thigh muscles with fatty replacement (vastus lateralis, adductor magnus and semimembranosus) and in muscles of the lower legs (gastrocnemius and soleus) (Fig. 18D to E). Muscle MRI studies were normal in controls. Figure18.MuscleMRIoflowerlimbsinsHypoPPpatients. Muscle imaging in sHypoPP patients showing different degrees of fatty degeneration in transverse T1-weighted images. Patient without permanent weakness (A and B) showed a moth-eaten appearance with scattered small areas of increased signal in the gluteus maior muscle and in the thigh posterior compartment muscles (especially the adductor magnus and semimembranosus muscles), while an incipient involvement of the soleus muscle was observed only in B. A patient with permanent weakness (C) showed an end-stage fatty degeneration in the gluteus maior and thigh muscles, sparing the biceps femori, semitendonosus, sartorius and gracilis muscles (asterisks). In the lower legs, the soleus and the gastrocnemius medialis showed pronounced degeneration. STIR sequences show hyperintensity only in muscles with permanent weakness (F), but are normal in patients with recurrent crisis but not permanent weakness (D, E). CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT 86 Figure19.MuscleMRIoflowerlimbsinaHypoPPpatients. Muscle imaging in the three asymptomatic carriers Muscle imaging in the three asymptomatic carriers (A, B and C), show atrophy and very mild fatty degeneration of the adductor magnus and semitendinosus (arrows) in a lesser degree at the posterior compartment of the thighs, with atrophy of the gluteus maior muscle. This pattern is similar to the initial one in sHypoPP. MRI in controls from the same families were all normal (D, E). 87 CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT EXERCISE TESTING We evaluated the electrophysiological response after short and long exercise tests in the total group of patients (sHypoPP and aHypoPP) and controls. Post-exercise compound muscular action potential (CMAP) amplitude and area (baseline to negative peak) were expressed as percentages of pre-exercise values, as previously described17. After short-exercise (three trials), we observed a slight and transient increase of CMAP amplitude and area within the first post-exercise seconds (11 ±7%) , which was not significantly different from controls (4 ±6% ) (Fig 20A and 20B). After long-exercise, we detected a delayed and progressive decrease in CMAP amplitude, which was statistically significant from 20 minutes post-exercise onwards ( -21.45 % ± 11% ; p< 0.005). The decline was up to 67% at the end of the recording (Fig 19D). We consider this an appropriate cut-off based on our non-carrier control group amplitude, which decreased only slightly (-10.8% ± 4.8%). The CMAP amplitude was significantly different between patients and controls from 10 minutes (95% confidence interval, 2.2-18.4; p<0.05) until 40 minutes post-exercise (95% confidence interval, 12.2-36.9; p<0.05). A transient increment in CMAP area was slightly but not significantly higher in patients immediately after long-ex- ercise completion (Fig 20C). This electrophysiological pattern was observed in all the mutation carriers, regardless of the symptoms (Fig. 21). CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT 88 Figure20.Shortandlongexercisetestsinthep.R528HHypoPPpatientsandcontrols. (A, B) Short exercise of the abductor digiti minimi (ADM) muscle was repeated three times successively at 1-minute intervals. The post-exercise values of CMAP area (A) and amplitude (B) were not significantly different from that observed in controls. (C, D) Long exercise test in the ADM showed a delayed decrease of CMAP area (C) and amplitude (D), while an early and transient increase was only observed in CMAP area. The amplitude and area of the CMAP, expressed as a percentage of its value before the trials, were recorded at different times after the exercise (arrows). This data represent the mean value of all patients in any group (patients and controls). We analysed separately the results in sHypoPP and aHypoPP (Fig. 21). The response of the exercise test in the aHypoPP patients group (42,6 ± 16,3 years old; range: 20 to 58 years) not showed any significant CMAP decline or increase after the short exercise neither in the successive trials. In the long exercise, no significant CMAP changes were observed during the first second after excercise, but between 10 and 35 minutes after the exercise a significant and prolonged decrease of CMAP amplitude appeared at rest in all aHypoPP patients (-42’47% ± 27’22%). The maximum decline was -67,14% (range: -24% and -67,14%). In the sHypoPP patients group (39,37±17,14 years old; range 19 to 63 years) no significant CMAP change was observed in the short exercise test. However, a significant decrease in CMAP amplitude was observed between 10-35 minutes after long exercise (-31,79%±10,76%), being the maximum decrease -52,72% (range: -20,93% and -52,72%). Only in one sHypoPP patient we observed a progressive decrease of CMAP amplitude since the first minute after long exercise. 89 CAPÍTULO 2 REDEFINING PERIODIC PARALYSIS WITH MUTATION IN CACNA1S GENE IN A SPANISH COHORT Figure21.ShortandlongexercisetestsintheasymptomaticandsymptomaticHypoPPpatients. The response of CMAP area (A, C) and CMAP amplitude (B, D) in the short (A, B) and long exercise test (C, D) were not significantly different in aHypoPP and sHypoPP. Short exercise test was repeated three times successively at 1-minute intervals. The amplitude (B, D) and area (A, C) of the CMAP, expressed as a percentage of its value before the trials, were recorded at different times after the exercise (arrows). This data represent the mean value of all patients in any group (aHypoPP and sHypoPP). Six sHypoPP, who had presented attacks during the previous year, showed the same pattern of CMAP amplitude decrement in the tests as the two sHypoPP patients with no recent attacks. CMAP amplitude and area showed concordant results both in short- and long-exercise tests (Table 6). CMAP amplitude decrement was not influenced by age, sex, disease duration, and we did not observe differences between treated or untreated patients. In our hands, this pattern for the HypoPP patients with p.R528H mutation in CACNA1S gene had 63.6% sensitivity and 100% specificity, considering the values 20 minutes post-exer- cise. However, after 35 minutes post-exercise test showed 100% sensitivity but specificity was reduced to 88%. 97 CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES *Publicación en preparación 1. METHODS We recruited all the patients carrying recessive mutations in the VPS13A gene from three centers (Hospital U. Virgen del Rocío at Sevilla, Hospital U. 12 Octubre at Madrid and Hospital U. Santa Creu i Sant Pau at Barcelona). All of them were contacted and offered the possibility of participating in a study to define the involvement of peripheral nervous system in patients with ChAc. The participants were examined using a standardized protocol which included anamnesis, physical exam, electroneurography, muscle MRI, brain MRI, muscle biopsy, serum CK level, acanthocytes in peripheral blood smear and expression of chorein in skeletal muscle and/or peripheral red cells. In the only deceased case the information was obtained from the clinical chart. This study received approval from the Hospital U. Virgen del Rocío local Ethics Committee and all participants provided informed consent. CLINICAL PHENOTYPE Based on the clinical manifestations, we considered four patterns of clinical severity: 0 = asymptomatic; 1(mild) = one symptom; 2 (moderate) = more than one symptom but independent; 3 (severe) = no walking or dependent. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 98 ELECTRONEUROGRAPHY Standardized electrophysiological protocol was performed by the same examiner and with the same equipment (TECATM Sinergy T-EP). The sensory nerve action potentials (SNAP) were elicited after surface stimulation of the median, ulnar and sural nerves. The compound motor action potentials (CMAP) were measured in the median, ulnar, common peroneal and posterior tibial nerves. The needle electromyography study was performed in proximal and distal muscles of the upper and lower limbs. MUSCLE MRI Muscle MRI was performed at the level of the thighs and lower legs. We used a 1.5 Tesla scanner (MRI Intera, Philips Medical Systems). Axial MRI sections were obtained including a T1-weighted spin-echo sequence (TR 600-700 ms, TE 30 ms) and a short-time inversion recovery (STIR) sequence (TR 2500-3500 ms, TE 60 ms, TI 150 ms), in 10 mm slices. Fatty degeneration of muscles was identified according to the increased signal in T1 sequences and STIR sequences were used to evaluate the presence of edema. In one patient was performed muscle computed tomography (CT) of lower legs because he did not tolerate MRI study. MUSCLE BIOPSY The fresh muscle samples were obtained by open biopsy in the gastrocnemius medialis. Additionally, we already had frozen muscle biopsies from some patients, previously obtained from the biceps brachii or the tibialis anterior muscles for diagnostic purposes. Routine stains included hematoxylin & eosin (HE), modified Gomori trichrome, Oil Red O, periodic acid-Schiff (PAS), nicotinamide adenine dinucleotide dehydrogenase (NADH), succinate dehydrogenase (SDH), cytochrome oxidase (COX), combined COX/SDH, and myofibrillar adenosine triphosphatase (ATPase) after preincubation at pH 9.4, 4.5 and 4.3. CHOREIN EXPRESSION IN MUSCLE We studied the chorein expression in muscle. For Wb 25μg of muscle lysate were loaded per lane on 3-8% Tris-Acetate gels for detection of chorein with SIGMA HPA021662 antibody. For detection of putative reactive bands of low molecular weight, a 4-12% Bis-Tris gel was also used and checked with HPA021662 antibody. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 99 VPS13A GENETIC STUDY For all probands, genomic DNA was extracted from peripheral blood. Eight patients were studied by Sanger sequencing as previously described 135. PCR amplification was carried out with standard reaction. Amplification products from proband patients were combined in a 3 : 1 ratio with the appropriate wild-type homozygous amplicon. This mixture was then annealed and analysed by denaturing high performance liquid chromatography (DHPLC), as described previously 136. PCR products showing a variant DHPLC pattern were purified using a QIAquick PCR purification kit (QIAGEN, Surrey, UK). For 11 exons (exons 2, 16, 29, 33, 35, 48, 49, 50, 66, 67 and 72), analysis by DHPLC was not appropriate, these were therefore sequenced directly as above. Two patients were diagnosed by NGS technics with a panel that included genes involved in the most frequent neurogenetic conditions related with hyperCKemia. Point variants and copy number variants (CNV) were included in the analysis. In one patient that presented only one mutation in heterozygous in VPS13A gene, was completed the genetic analysis by an exome including other neuromuscular diseases. 2. RESULTS CLINICAL AND LABORATORY FINDINGS We studied and obtained full data from ten patients belonging to eight unrelated families (Figure 22). Although we identified twelve cases, the patient C/II.1 was not included in the study because we did not have the clinical information, and the patient H/II.2 refused to be examined but have mild symptoms and he is carrier of the same mutations of his brother. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 100 Figure22.PedigreesoffivefamiliescarryingVPS13Amutations. Circles denote female members, squares male members, solid black symbols affected patients and solid grey symbol heterozygous carrier of only one mutation in VPS13A. In the text the patients will be referred Family/Patient (example: C/II.1) The age at the time of the examination ranged from 16 to 47 years (33.63 ± 10.71). The first manifestation (clinical symptom or hyperCKemia) appeared between 14 and 43 years of age (26.27± 8.33). There was a mean diagnostic delay of 6.4 ± 4.4 years (range between 2 and 17 years), from the first manifestation to the diagnosis. The first symptom was epilepsy in four patients (36.3%), movement disorder in two patients (18.1%) and psychiatric disorder (schizophrenia like) in one patient (9%). Interestingly, asymptomatic hyperCKemia was the first manifestation in four patients (36.3%) (at 14, 17, 19 and 43 years old respectively), but one of them associated a unique epileptic seizure eight years before. All patients but one presented elevated CK level since the moment at diagnosis, ranged from 182 UI/l to 4,096 UI/l (1849.1±1183.3 UI/L), and they were 34.5±10.8 years old (range 16-47 years). Strikingly, six patients (54.5%) were recruited from a Unit of Neuromuscular Disorders because a muscle disorder was suspected: two siblings referred because seizures and high CK level in order to investigate a probable mitochondrial disease, and three patients because asymptomatic hyperCKemia. Other five patients (45.4%) were identified in Units of Movement Disorders. Chorea was the first manifestation only in one patient, but it appeared in 63.6% during the CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 101 course of the disease, becoming a generalized and severe chorea causing loss of ambulance in 71.4% of these patients. Seizures appeared in 54.5%of the patients, being the most frequent clinical presentation among the symptomatic patients, and 63.6% of the patients associated psychiatric disorder. Generalized areflexia was observed in 18% of the patients and areflexia limited to the lower limbs in 27.2% of the patients. No patient presented weakness or sensory symptoms. Cardiopathy (atrioventricular node ablation) was detected only one patient (9%). At least one patient from each family, except the family H, showed varying degree of acanthocytes in peripheral blood smear by light microscopy (10±9.7% ranging from 2.2% to 30%). Atrophy of the caudate nucleus and putamen by neuroimaging (MRI or CT) was observed in all the studies performed except in one at 20 years of age with manifesting as asymptomatic hyperCKemia (II-1/G). Clinical findings are summarized in Table 7. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 102 Table 7 . ClinicalfeaturesinthepatientscarryingVPS13Amutations . A B C D E F G H Patients II-1 II-1 II-2 II-3 II-1 II-1 II-2 II-1 II-1 II-2(*) II-1 Onset(years) 32 20 43 28 37 26 25 14 19 17 28 Ageatexam(years) 43 38 45 45 47 28 35 16 19 21 33 Age at diagnosis (years) 40 -45 45 45 28 35 16 25 21 33 First symptom Chorea Seizures HyperCKemia Seizures Seizures Seizures Psychiatric disorder HyperCKemia HyperCKemia HyperCKemia Oropharyngeal tics and depression Weakness No No No No No No No No No No No Amiotrophy No Unknown No Gastrocnemius muscles No No No No No No No Sensitivity Normal Normal Normal Normal Normal Normal Normal No No No Normal Upperlimbsarreflexia No No No No No Yes No No No No Yes Lowerlimbsarreflexia No Unknown No Yes Yes Yes Yes No No No Yes Cardiopathy No Yes No No No No No No No No No Dysphagia Yes Unknown Yes Yes Yes Yes Yes No No No Yes Psychiatricdisorder No Yes Yes Yes Yes Yes Yes No No No Yes Cognitiveimpairment Yes Yes No No No No No No No No Yes Dependence Yes Yes No Yes Yes No Yes No No No Yes Exitus No Yes (41 yrs) No No No No No No No No No CKmax(UI/l) 765 3757 1200 1084 4096 1920 182 2000 2700 1787 850 Acanthocytes 7% np 2.2% np 2.2% 30% 18.4% 2.5% 8.3% No No ChoreinWB Negative np Negative np Negative np Negative np np np Negative Caudate/putamen atrophy(MRI/CT) Yes np Yes Yes (CT) Yes Yes Yes np No (CT) np Yes CK: creatine kinase; max: maximum; WB: Western Blot; MRI: magnetic resonance image; CT: computed tomography; np: not performed. (*) Carrier in heterozygous of only one mutation in VPS13A gene CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 103 Table 7 . ClinicalfeaturesinthepatientscarryingVPS13Amutations . A B C D E F G H Patients II-1 II-1 II-2 II-3 II-1 II-1 II-2 II-1 II-1 II-2(*) II-1 Onset(years) 32 20 43 28 37 26 25 14 19 17 28 Ageatexam(years) 43 38 45 45 47 28 35 16 19 21 33 Age at diagnosis (years) 40 -45 45 45 28 35 16 25 21 33 First symptom Chorea Seizures HyperCKemia Seizures Seizures Seizures Psychiatric disorder HyperCKemia HyperCKemia HyperCKemia Oropharyngeal tics and depression Weakness No No No No No No No No No No No Amiotrophy No Unknown No Gastrocnemius muscles No No No No No No No Sensitivity Normal Normal Normal Normal Normal Normal Normal No No No Normal Upperlimbsarreflexia No No No No No Yes No No No No Yes Lowerlimbsarreflexia No Unknown No Yes Yes Yes Yes No No No Yes Cardiopathy No Yes No No No No No No No No No Dysphagia Yes Unknown Yes Yes Yes Yes Yes No No No Yes Psychiatricdisorder No Yes Yes Yes Yes Yes Yes No No No Yes Cognitiveimpairment Yes Yes No No No No No No No No Yes Dependence Yes Yes No Yes Yes No Yes No No No Yes Exitus No Yes (41 yrs) No No No No No No No No No CKmax(UI/l) 765 3757 1200 1084 4096 1920 182 2000 2700 1787 850 Acanthocytes 7% np 2.2% np 2.2% 30% 18.4% 2.5% 8.3% No No ChoreinWB Negative np Negative np Negative np Negative np np np Negative Caudate/putamen atrophy(MRI/CT) Yes np Yes Yes (CT) Yes Yes Yes np No (CT) np Yes CK: creatine kinase; max: maximum; WB: Western Blot; MRI: magnetic resonance image; CT: computed tomography; np: not performed. (*) Carrier in heterozygous of only one mutation in VPS13A gene CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 104 ELECTRONEUROGRAPHY The EMG was performed in eight out of eleven patients. Sensory nerve conduction studies showed absent of sensory nerve action potential (SNAP) in sural nerve in lower limbs in four patients (50%), mild decreased amplitude in two patients (25%) and normal values in two patients (25%). In upper limbs sensory nerve conductions SNAPs were in normal range in all patients Motor nerve conduction studies showed normal CMAPs in all the nerves except in the common peroneal that showed mild reduced amplitude in three patients). Altogether we detected a pure sensory axonal polyneuropathy limited to lower limbs in four patients (50%) and a mild sensorymotor axonal polyneuropathy in two patients (25%). The study was normal in two patients (25%) with clinical pattern 0 and 2 respectively. We did not find any correlation between the result of the conduction studies and the clinical presentation or duration of the disease. MUSCLE MRI Muscle MRI or CT was performed in nine patients (mean age 34.7±10.7 years old) (Fig. 23). Five patients with moderate or severe clinical phenotype (age 41.5 ± 6.2 years old; range of disease duration between 1-17 years), displayed fatty degeneration of the gastrocnemius muscle, being the gastrocnemius medialis the first involved and the most severely affected (Fig. 23B-E). Notice that the patient B/II-2 showed severe fatty infiltration of the gastrocnemius muscle only one year after the disease onset with hyperCKemia and psychiatric manifestation (Fig. 23B). Besides the gastrocnemius involvement, mild atrophy and fatty infiltration in the adductor major muscle at thigh level was observed in one patient with 17 years of disease duration (B/II-3) (Fig. 23C). Three patients (A/II-1, E/II-2, H/II-1) aged 43, 35 and 35 years old (11, 10 and 7 years of disease duration respectively) showed normal MRI in spite of the presence of pathologic alterations in muscle biopsy and severe clinical phenotype in all of them (Fig. 23A). Two patients with asymptomatic hyperCKemia (F/II-1 and G/II-1) showed normal MRI (T1 sequence) at age of 15 and 19 years old respectively, but in G/II-1 observed a subtle hiperintensity in both medial gastrocnemius (right predominantly) only in STIR sequence after seven years (26 years old) in a control MRI (Fig. 23F). The patient G/II.2, carrier of one mutation in heterozygous and hyperCKemia presented normal muscle MRI. We did not find association between the presence of muscle involvement in imaging studies and the duration or severity of the disease at the moment of the imaging study. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 105 Figure23.LowerlegsmuscleimaginginthepatientscarryingVPS13Amutationswithdifferent stagesofthedisease. Muscle MRI was performed in five patients (A, B, D, E and F) and muscle TC in one patient (C). Normal MRI was observed although 11 years of disease and severe clinical phenotype (A) or asymptomatic patient (F). The gastrocnemius (asterisk) was the most severely degenerated muscle, the medial portion specially. It show severe degeneration (B, C) although one year or 17 years of evolution, respectively. Moderate affectation (D, E) although 10 years and 5 years of evolution of the disease, respectively. Adductor muscles of the hip(arrow) were mild involved only in one patient in severe stage (C). Mild asymmetry was observed in some patients (B, D). In one asymptomatic patient with normal MRI in T1 sequence (F) observed mild asymmetric hyperintensity in medial gastrocnemius in STIR sequence (arrowhead). Patterns of clinical severity: 0 = asymptomatic; 1= mild; 2 = moderate; 3= severe MUSCLE BIOPSY We obtained muscle biopsy from the gastrocnemius muscle in six patients. Four more muscle samples were already available from the biceps brachii and tibialis anterior muscles. The patient D/II-1 refused the muscle biopsy. In total, we had two biopsies from proximal muscles and eight biopsies from distal muscles. All patients showed alterations in the muscle biopsy. When proximal muscles (biceps brachii) were studied (II.1/B and II.1/C), we observed minimum changes, such as mild fiber size variability and scattered internal nuclei. The type 1 fibres showed irregular internal pattern with NADH, with no fibre grouping (Fig. 24A-C). When we studied the distal muscles (gastrocnemius or tibialis anterior), the biopsies revealed neurogenic atrophy with groups of atrophic fibres, fibre type grouping of variable degree and occasional angulated and target fibres (Fig. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 112 Figure28.ChoreinexpressioninmusclebyWesternblotting. To detect chorein, we used SIGMA anti-VPS13A antibodyPanels on the left show the Ponceau staining (A) of the membranes, then used for WB (B). Specific chorein band of 360 kDa is shown in control muscle samples (B, C), as well as some strong unspecific bands of about 30kDa (B), that showed similar loading for all the samples. WB showed absence or marked reduction of chorein in ChAc patients (panel B lanes 4a, 6a, 8a; panel C lanes P1, P2, P3) compared to healthy controls (panel B lanes C1, C2; panel C lanes Ctrl1, Ctrl 2) and disease control (McLeod syndrome patient,panel B, lane 5a). GENETIC ANALYSIS Genetic study confirmed recessive mutations in VPS13A in all the patients (Table 9). All of them resulted in a truncated protein and have been previously described gene. We did not find any genotype-phenotype correlation between the clinical, muscle imaging or neuropathological features. Table 9 . VPS13AvariantsdetectedinthestudiedChAcfamilies Family(Pat.) Genot .aDNAchangebEffect Notes A (II.1) Homo c.556-290_697-483delcp.T186_L232del Exons 8-9, deletion B (II.2, II.3) Het c.4864-581A>G p.V1622Tfs*21 Intron 39, splicing c.7581_7652+1028del p.D2474Ffs*2 Exon 54, deletion C (II.1) Homo c.4864-581A>G p.V1622Tfs*21 Intron 39, splicing D (II.1) Het c.3235+2T>C p.? Exon 30, splicing c.6880-1G>C p.? Exon 50, splicing E (II.2) Homo 7581_7652+1028del p.D2474Ffs*2 Exon 54, deletion F (II.1) Homo c.556-290_697-483deldp.T186_L232del Exons 8-9, deletion G (II.1) Homo c.9275+1G>A p.? Exon 69, splicing (II.2) Het c.9275+1G>A H (II.1, II.2) Het c.145-1G>T p.? Exon 3, splicing c.4411C>T p.R1471* Exon 37, nonsense a: Patient’s genotype: Homo, homozygous; Het, heterozygous. b: Description using main transcript (variant A, 72 exons) as reference (NM_033305.2). c: Same variant characterised in a German ChAc patient 137 CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 113 3. DISCUSSION The neuromuscular involvement in ChAc has been classically focused on the sensorymotor axonal polyneuropathy reported in many studies, usually asymptomatic and supported by subtle nerve conduction findings 97,98,101,103,104. However, a high CK level is a constant finding in ChAc patients, difficult to be explained by a mild polyneuropathy. The persistent movement due to the chorea could explain a mild increase of CK but not levels above 1000 UI/l, detected in many cases. In addition, here we report two young patients with asymptomatic hyper- CKemia who turned out to be carriers of homozygous VPS13A gene mutations, like previously reported in several presymtomatic ChAc cases 106,107. The high level and temporal profile of this hyperCKemia supports a sarcolemmal alteration causing a significant myofiber degeneration, that probably preceded the rest of clinical manifestations. This suggests a primary muscle involvement as part of the physiopathology of ChAc. Expression analysis revealed a higher amount of CHAC RNA and chorein protein in brain and skeletal muscle than in other tissues 94,138, that would be in line with a main role of chorein in muscle. Most of VPS13A gene mutations lead to truncated proteins that are expected to be degraded 93,94,135. We studied the expression of chorein by Wb in muscle, showing absence of protein in all of them. These findings are foreseeable with mutations that produce a dashed protein, such as in all our cases. Nevertheless, a previous report demonstrated absence or markedly reduction of chorein expression in erythrocyte membrane, skin fibroblasts and lymphoblastoid cell line from genetically confirmed ChAc patients carrying missense mutations, compared to normal controls and other NA 105. Thus, the study of chorein expression could be useful to support the diagnoses of ChAc in cases with high clinical suspicion but only one mutation detected, novel mutations or variants of uncertain significance. The molecular pathways in which chorein is postulated to be involved would be critically impaired as a consequence of a loss of function due to recessive CHAC mutations93. Considering the high protein expression in normal muscle, the absence of chorein would yield to specific primary myopathic manifestations in ChAc. Overall, the muscle biopsies in our cohort displayed the neurogenic and myopathic features already described in ChAc as variable fiber diameter, groups of atrophic fibers, increased central nuclei and fiber splitting 99,103,104,139–142. But additionally, in our cohort we described a more specific myopathic alterations such as abundant sarcoplasmic inclusions with myofibrillar proteins acumulation. Moreover, we could observed important morphological differences between proximal and distal muscles, even in different samples from the same patient. As expected, distal muscles showed a more severe neurogenic pattern. The myopathic features were also found in distal muscles, identifying the sarcoplasmic inclusions exclusively in biopsies from distal locations. Interestingly, a previous work demonstrated higher tTGase levels in muscles from ChAc patients that could cause sarcolemmal distortion in myofibers with increased release of CK 143. The same authors described protein aggregates in proximal CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 114 muscles by electron microscopy possibly related to the tTGase excess production. These findings strongly suggest that the sarcoplasmic inclusions that we observed in the distal muscles by histochemical staining have the same origin. In this line, nemaline rods have been reported in a biopsy from a distal muscle of a ChAc case, which could also be the result of the same protein aggregates, anyway sustaining an underlying myopathic process 140. The muscle imaging in our series seem to uncover a consistent pattern in ChAc patients defined by an early fatty degeneration of the gastrocnemius medialis muscle in lower legs and atrophy of the adductor major muscle in thigh. In line with our results, a previous publication reported two ChAc cases with similar muscle imaging findings. 144. The selectivity of involved muscles with striking contrast between involved and spared muscle in muscle imaging is a typical finding in myopathies. The main differential diagnosis of ChAc has to be established with some distal myopathies, that could also present asymtomatic hyperCKemia in preclinical stages. Dysferlin myopathies are characterized by involvement of the gastrocnemius medialis and adductor major muscles in the first stages of the disease, 20, together with very high serum CK level. In this case, absence of dysferlin expression in muscle biopsy is the clue. Similar muscle MRI and clinical features are associated to distal muscular dystrophies with anoctamin-5 mutations 19,145, but this entity do not show specific alterations in muscle biopsy, so the genetic study is mandatory for diagnostic confirmation. Among hereditary neuropathies, selective involvement of the posterior compartment of lower legs has been reported in some axonal types of Charcot Marie Tooth (CMT), but, in contrast with our study, the soleus muscle was affected earlier and more severely than gastrocnemius 146,147. In one patient with CMT2F have been reported slightly reduced CMAPs in the lower limbs and mild fatty infiltration of the medial gastrocnemius, very similar to the pattern we described in ChAc, however in CMT2F the soleus muscle was affected at the same time 148. ChAc presents a wide variability in the type of presenting symptoms and their severity, causing considerable proportion of misdiagnosis and diagnosis delay 98,100, which hampers the genetic counselling. In our series we observed the diagnostic delay was 6.4 ± 4.4 years, from the first manifestation to the definite diagnosis. The movement disorders and neuropsychiatric alterations are the most frequent and disabling symptoms in ChAc 96,100,105, but rarely present at the onset of the disease. 98,149 and the diagnosis is usually unsuspected at early stages when initial symptoms are other manifestations such as epilepsy o neuromuscular disorders. Epilepsy is observed in up to half of the reported ChAc, being the most frequent initial symptom in our series (40%)98,150–153. The cardiopathy is not considered a common finding in ChAc 97,100, however there are a few cases reported whose associate variate types heart involvement of unknown cause 106,154,155. In our case, we think the heart involvement is a causal finding. In fact, the two affected sibling don´t associate cardiopathy. CAPÍTULO 3 NEUROMUSCULAR INVOLVEMENT IN CHOREOACANTHOCYTOSIS: A SPANISH SERIES 115 Areflexia and elevated CK levels are described in almost all ChAc cases 93,97,98,105,106 and probably these findings are present before other symptoms but are unnoticed. Peikert et al., considered the hiperCKemia is the most consistent paraclinical finding in ChAc 96. Three patients of our series presented asymptomatic hiperCKemia as the first sign, as well as other reports 106,107. Moreover, one patient (G/II.1), genetically confirmed of ChAc, presented an isolated epileptic seizure and after some years was studied because his brother (G/II.2) presented asymptomatic hyperCKemia showing high level of CK also. Probably the hyperCKemia was present in the moment of the epileptic episode but it has not been tested. The patient G/II.2 was studied for asymptomatic hyperCKemia and presented only one mutation (c.9275+1G>A) in heterozygous in VPS13A gene, then we consider that the carrier condition could associate high levels of CK. We want to underline this finding of hyperCKemia in heterozygous carriers of mutations in VPS13A gene, because it has not been previously reported and it is very important to be taken into account in the study of patients with asymptomatic hyperCKemia. Mention to this should be consider to be included in diagnostic guidelines in the field of muscle disorders156. In the light of the above, we consider that the isolated hyperCKemia is an early and common sign in ChAc patients. In these cases, the findings of the muscle MRI (selective fatty infiltration of medial gastrocnemius) could be useful. Then, we would recommend investigate the acanthocytes in blood, furthermore there are cases reported of ChAc symptoms without or with a low percentage of blood acanthocytosis 96,102. Moreover, we recommend complete the study including the CNV in the gene VPS13A within the next generation sequencing on patients with hyperCKemia. In fact, we confirmed two patients carrying recessive mutations in the VPS13A gene studying when we analyzed the CNV after sequencing a panel including genes involved in different neurodegenerative disorders, once we excluded mutations in genes associated to hyperCKemia. To conclude, our findings describe the neuromuscular phenotype in a cohort of ChAc genetically confirmed and definitely support a primary muscle involvement in this disease. We consider that the asymptomatic hyperCKemia is an early and common sign in ChAc patients and recommend to include the study of the VPS13A gene in the genetic study of patients with hyperCKemia. We described focal structural alterations and sarcoplasmic inclusions in the biopsy of distal muscles. Gastrocnemius muscle show early and significant degeneration in MRI showing relevant alterations in the biopsy. The expresion of chorein in muscle tissue is a useful method to support the diagnosis, specially in cases with novel mutations or variants of uncertain significance. All these findings are important to improve the diagnosis of the ChAc and recognize this disease in cases of asymptomatic hyperCKemia. DISCUSIÓN FINAL DISCUSIÓN FINAL La heterogenenidad clínica y genética de las ENM junto con su baja prevalencia hace que el diagnóstico definitivo se retrase. Al ser enfermedades incapacitantes y que en la mayoría de los casos requiere un consejo genético, resulta de especial interés el hallazgo de pruebas que sirvan de cribado para estas patologías. En este trabajo hemos estudiado detalladamente el fenotipo clínico, electrofisiológico, radiológico, anatomo-patológico y genético en distintas patologías de origen genético con afectación neuromuscular, como son la MDL, la HypoPP y la ChAc, centrándonos en aquellos hallazgos o signos que puedan ser útiles como herramientas de cribado y apoyo al diagnóstico. Respecto a los hallazgos clínicos, el primer dedo del pie caído, que es un hallazgo muy típico en la MDL, supone con frecuencia el primer signo visible sin que los pacientes refieran síntomas o hayan apreciado este hallazgo, que en ocasiones puede ser muy sutil. Además de forma novedosa, nosotros objetivamos en dos pacientes infantiles una debilidad cervical transitoria en la primera década de la vida y la mayoría de pacientes referían torpeza en la infancia aunque no apreciaron debilidad hasta después de muchos años. Por tanto, la debilidad cervical y la torpeza motora transitoria en la infancia junto con el primer dedo caído pueden ser considerados como signo de detección precoz en la MDL. La hiperCKemia asintomática puede ser la manifestación inicial en las ChAc como ya se ha referido en otros trabajos, y probablemente sea un signo precoz en muchos pacientes. Por este motivo, hemos incluido el gen VPS13A en el panel de genes para hiperCKemias de nuestro hospital y nos ha permitido diagnosticar a dos pacientes de esta serie que fueron valorados en la consulta de Enfermedades Neuromusculares por hiperCKemia. Además, hemos detectado que la condición de portador en heterocigosis de una mutación en el gen VPS13A también puede asociarse con hiperCKemia. Por ello, consideramos que la inclusión del gen VPS13A en los estudios NGS para hiperCKemias debería hacerse extensivo a todos los laboratorios genéticos. El test de ejercicio electrofisiológico en canalopatías musculares nos ha permitido comprobar que es una prueba muy sensible para detectar portadores de la mutación p.R528H en HypoPP asociada al gen CACNA1S, pero lo más relevante y de nueva aportación respecto a otros estudios es que es una herramienta de cribado útil también en portadores asintomáticos. La RMm puede ser utilizada como prueba de screening en las tres patologías estudiadas (MDL, HypoPP y ChAc), incluso en pacientes asintomáticos pueden aportar información. El músculo sartorio se afecta de forma leve y precoz en la MDL, incluso en asintomáticos, manteniéndose estable a lo largo de la evolución de la enfermedad. La infiltración grasa del músculo adductor magnus y el semimembranoso en RMm, pueden ser un signo de detección precoz para portadores asintomáticos de la mutación p.R528H en el gen CACNA1S. El gastrocnemio medial en las ChAc se afecta precozmente y puede orientarnos al diagnóstico incluso en asintomáticos, además podría ser el músculo de elección para realizar una biopsia muscular pues muestra hallazgos relevantes. El estudio morfológico detallado en la biopsia muscular nos ha permitido describir nuevos hallazgos miopáticos en las ChAc y así poder justificar la hiperCKemia tan elevada a veces en estos pacientes. Por tanto, la biopsia muscular junto con los hallazgos de RMm y la hiperCKemia aportan objetividad diagnóstica y pueden ser pruebas útiles para mejorar el diagnóstico precoz en las ChAc. Los estudios de NGS han supuesto un gran avance en el diagnóstico genético definitivo, pero resulta primordial una orientación clínica adecuada, integrando los hallazgos clínicos, electrofisiológicos, radiológicos y patológicos oportunos. El apoyo de los laboratorios de investigación a la clínica resulta de gran ayuda para suplir las limitaciones de los estudios de secuenciación masiva. En nuestro caso, el estudio de haplotipos y los estudios funcionales in vivo han sido útiles para describir la nueva variante p.R1560P en el gen MYH7 como fundadora en Andalucía y la sugerimos como primera opción a estudiar en los pacientes originarios del sur de España con sospecha de MDL. Por otra parte, advertimos que C . Elegans no es el modelo de elección para recapitular in vivo mutaciones en el gen MYH7 humano. CONCLUSIONES/ CONCLUSIONS APÉNDICE 129 Available online at www.sciencedirect.com Neuromuscular Disorders 28 (2018) 828–836 www.elsevier.com/locate/nmd A novel MYH7 founder mutation causing Laing distal myopathy in Southern Spain P. Carbonell-Corvillo a , E. Tristán-Clavijo b , M. Cabrera-Serrano a , c , E. Servián-Morilla a , G. García-Martín d , L. Villarreal-Pérez a , E. Rivas-Infante e , E. Area-Gómez f , M.I. Chamorro-Muñoz d , A. Gil-Gálvez g , A. Miranda-Vizuete g , A. Martinez-Mir b , N. Laing c , C. Paradas a , ∗ a Neuromuscular Unit, Department of Neurology, Hospital U. Virgen del Rocío/ Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain b Instituto de Biomedicina de Sevilla (IBIS)/Hospital Virgen del Rocío/CSIC/Universidad de Sevilla, Spain c Centre for Medical Research, University of Western Australia, Harry Perkins Institute of Medical Research, Perth, Australia d Department of Neurology, Hospital Universitario Virgen de la Victoria, Málaga, Spain e Department of Neuropathology, Hospital U. Virgen del Rocío/ Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain f Department of Neurology, Columbia University Medical Center, New York, NY, USA g Redox Homeostasis Group, Instituto de Biomedicina de Sevilla (IBIS)/Hospital Virgen del Rocío/CSIC/Universidad de Sevilla, Spain Received 29 May 2018; received in revised form 12 July 2018; accepted 19 July 2018 Abstract MYH7 gene mutations are associated with wide clinical and genetic heterogeneity. We report a novel founder mutation in MYH7 in Southern Spain (Andalucía). We studied two index patients and 24 family members from two apparently independent families by physical examination, serum creatine-kinase, muscle MRI, sequencing studies and genetic linkage analysis. Sixteen individuals were heterozygous for a (p.R1560P) variant in the MYH7 gene. Haplotype was consistent with a common ancestor for the two families. The patients displayed the classic Laing distal myopathy phenotype, with hanging first toe as the initial presentation, even in mildly affected patients who declared themselves asymptomatic, although neck flexor weakness was revealed as an early sign in some cases. MRI showed that the sartorius was the first muscle involved, even in two out of three asymptomatic carriers. Our findings support the novel variant p.R1560P in MYH7 as a founder mutation in Andalucía. The early involvement of the sartorius muscle in MRI may be useful as an indicator of affection status. © 2018 Elsevier B.V. All rights reserved. Keywords: MYH7 ; Founder mutation; Distal myopathy; Muscle MRI; Genetic linkage analysis. 1. Introduction Laing distal myopathy is an autosomal dominant disease, commonly characterized by slowly progressive distal weakness, involving the anterior compartment of the legs, thus causing the typical “hanging big toe” sign [1] . Nevertheless, highly variable clinical and histological phenotypes have been widely reported in Laing distal myopathy [2–4] , and the same mutation in the MYH7 gene can cause a different clinical picture regarding age of onset, weakness pattern or severity ∗Corresponding author. E-mail address: [email protected] (C. Paradas). and variable pathologic profiles, even within the same family [3] . Concerning muscle MRI in the Laing distal myopathy, the tibialis anterior is the earliest and more severely affected muscle in all the reported cases. To date, several case reports [3,5,6] and one study with detailed analysis on radiological pattern in a cohort of Laing distal myopathy patients [7] have been published, reporting quite variable and not always consistent conclusions regarding the muscle imaging findings other than the tibialis anterior early involvement. The largest series of distal myopathy patients with mutations in MYH7 reported a high rate of de novo mutations. This high frequency of sporadic mutations suggests that there https://doi.org/10.1016/j.nmd.2018.07.006 0960-8966/© 2018 Elsevier B.V. All rights reserved. PRUEBAS COMPLEMENTARIAS Y TÉCNICAS DE LABORATORIO: IMPORTANCIA PARA EL CRIBADO Y DIAGNÓSTICO DE ENFERMEDADES NEUROMUSCULARES RARAS DE ORIGEN GENÉTICO Y UTILIDAD EN EL APOYO A LA CLÍNICA 130 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 829 Table 1 Clinical features. Manual muscle testing according to the MRC scale Carriers Age at exam Onset Neck flexor Finger ext Knee ext Knee flexor Hanging big toe Toes ext Ankle dorsiflexor Pes cavus FVC/ Echocardiogram Pheno type A I-1 79 y 13 y 2/5 2/5 2/5 2/5 Yes 0/5 0/5 Yes np/np Sev II-1 59 y 8 y 3/5 4/5 4/5 5/5 Yes 2/5 0/5 Yes N/np Sev II-5 54 y 6 y 3/5 2/5 4/5 5/5 Yes 0/5 0/5 Yes N/N Sev II-6 52 y 6 y 3/5 3/5 3/5 5/5 Yes 3/5 0/5 Yes N/N Sev II-8 49 y 5 y 4/5 4/5 5/5 5/5 Yes 4/5 2/5 Yes np/np Mod II-14 39 y 30 y 5/5 5/5 5/5 5/5 Yes 4/5 2/5 Yes N/N Mod III-2 38 y – 5/5 5/5 5/5 5/5 No 5/5 5/5 Yes np/np A III-4 23 y 6 y 5/5 5/5 5/5 5/5 Yes 5/5 5/5 Yes np/np Mild III-8 15 y 8y 4/5 (T) 5/5 5/5 5/5 No 5/5 5/5 No np/np Mild III-14 11 y – 5/5 5/5 5/5 5/5 No 5/5 5/5 No np/np A III-15 8 y 4y 4/5 (T) 5/5 5/5 5/5 No 5/5 5/5 No np/np Mild B II-3 54 y 6 y 4/5 5/5 5/5 5/5 Yes 2/5 0/5 No N/N Mod II-5 42 y 7 y 5/5 5/5 5/5 5/5 Yes 5/5 5/5 No np/np Mild III-1 28 y – 5/5 5/5 5/5 5/5 No 5/5 5/5 No np/np A III-2 21 y 17 y 5/5 5/5 5/5 5/5 Yes 5/5 5/5 No np/np Mild y: Years; T: Transient; Ext: Extensor; CK: Serum creatin kinase; Numbers indicate muscle strength according to MRC scale; FVC: Forced vital capacity; N: Normal; np: not performed; A: Asymptomatic; Mild: Mild phenotype defined by hanging big toe sign or transient cervical weakness Mod: Moderate phenotype defined by weakness of toes extension and ankle dorsiflexion ±finger extension; Sev: Severe phenotype defined by weakness of toes extension, ankle dorsiflexion and knee extension ±finger extension. Bold letters denote mildly affected patients who only self-reported frequent falls during infancy. should be a wide geographical distribution [7–9] . Exceptionally, a founder mutation in the MYH7 gene, p.K1729del has been found to cause the majority of Laing distal myopathy cases in the Safor region, Spain [10] . Most mutations are associated with the classic phenotype of Laing distal myopathy. However, in the series reported by Fiorillo et al. [7] some atypical clinical features were reported which delayed the diagnosis. Lamont et al reported eight out of 15 families with missense mutations introducing a proline in MYH7 associated with distal myopathy [9] . These mutations involve aminoacid residues at all positions of the heptad repeat of the protein which would induce significant modifications in the protein structure, which suggests that proline substitutions at any of these positions has a pathogenic effect [11,12] . Here, we report a novel mutation in the MYH7 gene with a founder effect in Andalucía (South of Spain). The description of founder mutations and the analysis of a potential genotypephenotype correlation are important from a diagnostic point of view, especially when variable phenotypical expression is associated with the disease. 2. Methods 2.1. Patients and study protocol We initially identified two apparently unrelated patients with a distal anterior myopathy, both from Andalucía ( Fig. 1 ). Patient II.6/Family A reported several affected relatives including his father and at least four siblings. Patient II.3/Family B was a sporadic case. Affected and unaffected relatives from both families were contacted and offered the possibility of participating in the study. The participants were examined using a standardized protocol which included interview, physical exam, muscle MRI, serum CK and blood sample to obtain DNA. The first step of the clinical protocol was the interview of the recruited family members to collect the clinical data and classify them as symptomatic or asymptomatic, followed by a complete general and neurological examination, including manual muscle testing, and the rest of the tests. This study received approval from the HUVR local Ethics Committee and all participants provided informed consent. 2.2. Muscle biopsy Muscle samples were obtained by open biopsy. We processed the muscle following the standard procedures. Routine stains included hematoxylin & eosin (HE), modified Gomori trichrome, Oil Red O, periodic acid-Schiff (PAS), nicotinamide adenosine dinucleotide dehydrogenase (NADH) nicotinamide adenine dinucleotide (NADH), succinate dehydrogenase (SDH), cytochrome oxidase (COX), and combined COX/SDH, and myofibrillar adenosine triphosphatase (AT- Pase) after preincubation at pH 9.4, 4.5 and 4.3. Immunohistochemistry for fast and slow myosin was performed in one case. 2.3. Muscle MRI Muscle MRI was performed at the level of the thighs and lower legs. We used a 1.5 Tesla scanner (MRI Intera, Philips Medical Systems). Axial MRI sections were obtained including a T1-weighted spin-echo sequence (TR 600–700 ms, TE 30 ms) and a short-time inversion recovery (STIR) sequence (TR 2500–3500 ms, TE 60 ms, TI 150 ms), in 10 mm slices. Fatty degeneration of muscles was identified according to the increased signal in T1 sequences and quantified according to APÉNDICE 131 830 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 Fig. 1. Pedigrees of the two families with Laing distal myopathy. Clinical examination and genetic study were performed in all available family members. Circles denote female members, squares male members, solid black symbols affected patients, solid dark grey symbols mildly affected patients, solid light grey symbols asymptomatic carriers, striped symbols not-carriers, squared symbols not-examined carrier, and white symbols not-studied family members. a subjective scale [13] , and STIR sequences were used to evaluate the presence of edema. 2.4. MYH7 genetic study Genomic DNA was extracted as per standard protocols from peripheral blood (Chemagic DNA Blood 5 K Kit, Perkin Elmer) or saliva samples (Oragene DNA Self-Collection Kit, DNA Genotek) from patients and the rest of family members. In the index patient II.6/Family A, the coding regions of MYH7 gene were Sanger-sequenced at the Neuromuscular Unit, Hospital U. Virgen del Rocío, Sevilla (NMU-HUVR). DNA from index patient II.3/Family B was sent to the Lotterywest State Biomedical Facility Genomics (Perth, Australia) for neurogenetic sub-exomic supercapture and next generation sequencing (Proton TM , Life Technologies) of 336 genes, which included 254 disease genes listed in the Neuromuscular Disorders Gene Table ( www.musclegenetable.fr). DNA samples from all recruited relatives were PCR-amplified using specific primers (5  -CCATGATTAGTGAGCAGGCC- 3  , 5  -TCAGAGGTCCCTGCAGTAAC-3  ) and directly sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific). 2.5. Haplotype analysis Microsatellite and SNP markers covering the MYH7 gene were selected based on their genomic location and heterozygosity (UCSC Genome Browser, Human GRCh37/hg19; http: // genome.ucsc.edu/ ) [14] . They spanned a genomic region of 2,92 Mb. Microsatellite markers were amplified by PCR using fluorescently-labeled primers and resolved in an ABI 3500 Genetic Analyzer (Applied Biosystems). SNP loci were PCR- amplified and genotypes were determined by visual inspection of the sequence chromatograms. Haplotypes were built assuming the minimum number of recombinations. 3. Results 3.1. Clinical phenotype We performed the interview and physical examination in 23 individuals from the two families, nine women and 14 men. The age at the time of the examination ranged from eight to 79 years. The symptoms appeared between four and 30 years of age. Most carriers recalled abnormal gait and falls during infancy but did not complain of weakness until adulthood. Twelve carriers of the p.R1560P mutation PRUEBAS COMPLEMENTARIAS Y TÉCNICAS DE LABORATORIO: IMPORTANCIA PARA EL CRIBADO Y DIAGNÓSTICO DE ENFERMEDADES NEUROMUSCULARES RARAS DE ORIGEN GENÉTICO Y UTILIDAD EN EL APOYO A LA CLÍNICA 132 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 831 Fig. 2. Clinical features. The most severely affected patients showed distal weakness involving finger extension (A) with mild contractures (B) , and quadriceps weakness with atrophy only in the most advanced stages (C). Neck flexor weakness was present in patients with moderate and severe phenotype (D) . Intriguingly, two infantile carriers presented isolated neck flexor weakness in the physical exam only during childhood (E) , and disappeared in the course of the disease (F) . The hanging big toe sign was present in early stage as first clinical sign of the disease, even in mildly affected patients who self-reported no symptoms (G, III.2/Family B; H, II.5/Family B; I , III.4/Family A), in some cases very subtle (G). No clear correlation was observed between the overall stage of clinical phenotype and the severity of the hanging big toe (i.e. compare H [II.5/Family B] to K [II.6/Family A], and I [III.4/Family A] to J [II.3/Family B]). (age range 8–79 years) showed a pathological physical exam. Three of them (age range 21–42 years) declared themselves to be asymptomatic and weakness restricted to big toe extension was the only sign found in the exam (III.4/Family A, II.5/Family B, III.2/Family B). Two of them presented transient cervical weakness at eight years old but not later (III-8/Family A, III-15/Family A). Three out of the 16 carriers were asymptomatic with normal physical exam (age range 11–38 years). One carrier was not examined (I.2/Family B). No patient referred respiratory or cardiac symptoms, and none of them referred a familial history of sudden death, arrhythmia or myocardiopathy. Echocardiography and respiratory functional studies were performed in some affected patients and all the studies were normal. CK was in normal range in all the carriers. Clinical findings are summarized in Table 1 and Fig. 2 . 3.2. Muscle histology Muscle biopsies from four patients (II.5/Family A, II.6/Family A, II.14/Family A, II.3/Family B), two tibialis anterior and two gastrocnemius muscles, were available for review. Myopathic pattern with increased variability in fiber size and internal nuclei were present in all biopsies. More severe changes with prominent atrophy and increased endomysial connective tissue were observed in both tibialis anterior muscles ( Fig. 3 A and B). Small group of angulated fibers and nuclear clumps suggesting neurogenic features were seen in three of the four samples ( Fig. 3 B and F). Oxidative enzyme stains (NADH, SDH and COX) revealed prominent internal sarcoplasmic disorganization with core, core with poor defined bordersand minicore lesions in all biopsies ( Fig. 3 C and G). We observed type I fiber predominance in three cases APÉNDICE 133 832 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 Fig. 3. Muscle histology. Muscle biopsies from patient II.14/Family A, tibialis anterior ( A–D ) and patient II.3/Family B, gastrocnemius ( E–H ) were stained with haematoxylin and eosin ( A, B, E and F ), succinate dehydrogenase (SDH) ( C and G ) and ATPase pH 4.5 ( D and H ). Atrophy, variability in fibre size, increased endomysial connective tissue, and necrotic fibers (arrow) ( A and E ), and groups of small angulated atrophic fibers with nuclear clumps (arrowheads) ( B and F ) were present in both cases. SDH staining shows prominent sarcoplasmic disorganization including core (black arrowhead), core with poor defined borders (open arrow), and multiminicore (black arrow) lesions ( C and G ). Type I predominance was observed ( D ). Scale bar is 50 μm in all panels ( A , B , C , E , F and G x 200 magnification; D and H x 100 magnification). ( Fig. 3 D) and scattered rimmed vacuoles in one case. No hypotrophy of type I fibers was observed. 3.3. Imaging study Muscle MRI was performed in 11 patients (Family A: II.5, II.6, II.14, III.2, III.4, III.14, III.15; Family B: II.3, II.5, III.1, III.2). All the affected carriers with moderate or severe phenotype (mean age 54.1 ±12.2 years old [range 39 to 79 years], disease duration 44.6 ±17.2 years [range 9–66 years]) showed fatty degeneration of lower limb muscles. The tibialis anterior, gluteus major and sartorius muscles were involved in all of them, although with different degrees of degeneration. Degeneration was severe in the tibialis anterior and mild in the gluteus and sartorius muscles. The other affected muscles in the MRI were the gracilis (75%), the quadriceps (50%) and the gastrocnemius (25%). Two slightly affected patients (III.4/ Family A, II.5/Family B) and two asymptomatic carriers (III.2/Family A, III.1/Family B) presented mild fatty infiltration only in the sartorius muscle. Two mildly affected patients (III.15/Family A, III.2/Family B) and one asymptomatic carrier (III.14/Family A) showed normal MRI. The sartorius muscle was the only one involved in 50% of slightly affected patients and in 66’6% of asymptomatic carriers. MRI findings are summarized in Table 2 and Fig. 4 . 3.4. MYH7 mutation screening The novel heterozygous mutation c.4679G > C (NG_007884.1) p.R1560P in exon 34 of MYH7 was identified in both index patients ( Fig. 1 ). The variant is absent from databases of controls ( http:// gnomad.broadinstitute.org/ ; last accessed in November 2017) and is predicted to introduce a proline in the slow myosin tail. Next generation sequencing in the index patient II.3/B excluded pathogenic mutations in other genes related to neuromuscular disorders. The novel variant was subsequently analyzed by Sanger sequencing in all the recruited family members. In total, 26 subjects from the two families were recruited (17 from Family A and nine from Family B), comprising twelve clinically affected patients and 14 asymptomatic individuals. Segregation analysis showed that 16 individuals were heterozygous for variant p.R1560P in the MYH7 gene, including twelve symptomatic individuals. 3.5. Haplotype analysis Given the identification of the same novel mutation in two families from different cities in Andalucía, Southern Spain, we studied the possibility of them being related. Haplotype analysis revealed a shared genomic region of 306 Kb in all carriers of p.R1560P mutation, restricted to four SNP markers ( Fig. 5 ). 4. Discussion We report a novel founder mutation in MYH7 causing Laing distal myopathy in Andalucía. The shared haplotype identified in our study spans a short genomic region (306 Kb) defined by SNP markers, however, the data are in accordance with the findings by Muelas et al. [10] , that show a common SNP-defined genomic region of 41,2–71,2 Kb. In our Neuromuscular Unit at HUVR, which is a national reference unit, only patients with ancestors from Southern Spain carry the novel c.4679G > C (p.R1560P) mutation in MYH7 , while in patients from other geographic regions of Spain the disease PRUEBAS COMPLEMENTARIAS Y TÉCNICAS DE LABORATORIO: IMPORTANCIA PARA EL CRIBADO Y DIAGNÓSTICO DE ENFERMEDADES NEUROMUSCULARES RARAS DE ORIGEN GENÉTICO Y UTILIDAD EN EL APOYO A LA CLÍNICA 134 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 833 Table 2 Muscle MRI findings. Patient/Family Gluteus major Quadriceps Sartorius Gracilis Tibialis anterior Gastrocnemius Affected patients I.1/A np np np np np np II.1/A np np np np np np II.5/A + + ++ + +++ N II.6/A + ++ (asymmetric) + + +++ N II.8/A np np np np np np II.14/A + N + + ++ N II.3/B + N + N +++ + Mild affected patients III.4/A N N + N N N III.8/A np np np np np np III.15/A N N N N N N II.5/B N N + N N N III.2/B N N N N N N Asymptomatic carriers III.2/A N N + + N N III.14/A N N N N N N III.1/B N N + + N N N: Normal; + : Mild fatty infiltration; ++ : Moderate fatty infiltration; +++ : Severe fatty infiltration; np: not performed. Fig. 4. Muscle MRI. Muscle MRI findings of patients displaying severe ( A, II.6/ Family A), moderate ( B, II.14/ Family A) and mild ( C, II.5/ Family B) clinical phenotype. Asymptomatic carriers can show mild sartorius muscle infiltration ( D, III.1/ Family B) and mild affected patients normal MRI ( E, III.2/ Family B). The vastus intermedius and lateralis muscles are involved only in severe stages (A) . The tibialis anterior was the most severely degenerated muscle in symptomatic patients (A–C) , and usually the only affected muscle in the lower legs. The most striking finding in our study was that the sartorius muscle (arrowhead) was the earliest involved, even in asymptomatic carriers (D), although it does not show evidence of significant progression (A–C) . APÉNDICE 135 834 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 Fig. 5. Haplotype analysis in the two families carrying the MYH7 p.R1560P mutation. Genomic position for genotyped microsatellite and SNP markers relative to the MYH7 gene, according to UCSC Genome Browser, Human GRCh37/hg19 (A). Haplotypes for families A and B (B, C) . Haplotype analysis using microsatellite markers D14S50 , D14S283 , D14S990 , D14S972 , D14S64 and D14S264 in all available family members revealed a different haplotype cosegregating with the mutation in each family. To narrow down the studied genomic interval, we genotyped six SNP markers, rs365990 , rs434273 , rs2331979 , rs2277475 , rs7157716 and rs11621360 close to or within the MYH7 gene. The analysis of SNP genotypes revealed a shared haplotype for markers rs365990 , rs434273 , rs2331979 and rs2277475 in all carriers of the p.R1560P mutation that defined a common region of 306 Kb. The shared common haplotype is boxed. Genotype for mutation p.R1560P is denoted by a + (wild-type) and M (mutant) sign. A question mark indicates unknown genotype. Solid black symbols denote carriers of the MYH7 p.R1560P mutation. PRUEBAS COMPLEMENTARIAS Y TÉCNICAS DE LABORATORIO: IMPORTANCIA PARA EL CRIBADO Y DIAGNÓSTICO DE ENFERMEDADES NEUROMUSCULARES RARAS DE ORIGEN GENÉTICO Y UTILIDAD EN EL APOYO A LA CLÍNICA 136 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 835 is due to different mutations, all of them already described. Thus, this mutation should be the first mutation to study in patients with this disease phenotype from Southern Spain. Proline residues are widely known to have a pathogenic effect on the assembly of the protein [15,16] . Moreover, most mutations in MYH7 associated with distal myopathy are located in the mid-rod region of MYH7 protein encoded by exons 32 to 36 [17,18] . All together, these findings support the pathogenic role of this novel mutation. The high rate of de novo mutations demonstrated in MYH7 together with the long lifespan of patients with Laing distal myopathy could explain the high prevalence of cases, regardless of geographic or ethnic distribution [7,9] . Hence, the identification of founder mutations in specific geographic areas facilitates the molecular diagnosis of patients. A noticeable clinical and genetic heterogeneity is associated with the MYH7 -related myopathies [3,4,9] . Until now, only one founder mutation in MYH7 has been reported, and it was also associated with a wide range of clinical and morphologic features among the carriers [3,10] . In this study, we identified the clinical variability previously reported in other series, finding patients severely or mildly affected at a similar age, in the same family. Four previously reported large series of patients showed that age at onset varied between 0 and 50 years [3,7,9,19] and in our series the onset was between four and 30 years of age (none of the patients with congenital onset), and the asymptomatic ones ranged from 11 to 38 years old. Fiorillo et al. described a series of 21 patients identifying 12 heterozygous mutations in MYH7 affecting the C-terminal rod domain, and the patients with congenital or early onset presented predominant distal weakness, while patients with later onset presented prevalent proximal weakness [7] . In contrast, in our series with a novel mutation affecting the rod domain we found no differences in the distribution of the weakness related to the age of onset. In our cohort, we found three asymptomatic carriers (age ranged 11–38 years old) and five slightly affected patients (age ranged 8–42 years old). Tasca et al. reported one family carrying a novel mutation in the myosin tail, and the age of onset of all three affected members was not prior the fifth decade and shared mild weakness limited to long toe and foot extensors [4] . Feinstein-Linial et al., who reported two novel heterozygous proline substitutions located in exon 31 of MYH7, described also one asymptomatic carrier aged 40 [5] . This confirms that more benign forms than the classic phenotype exist, probably associated with better prognosis regarding the progression of the disease. We uncovered interesting manifestations after a thorough anamnesis and clinical exam. The majority of carriers of the mutation reported abnormal gait with increasing tendency to stumble and fall in infancy or childhood, although most of them did not complain of motility or muscle problems until the third or fourth decade, a finding also described by Muelas et al. [3] . A review of 34 patients with Laing distal myopathy by Lamont et al. revealed that initial weakness of great toe/ankle dorsiflexion and later development of neck flexor weakness were consistent findings [9] . Interestingly, two infantile carriers in our series presented neck flexor weakness only during childhood. Recently, one atypical case with dropped head at the age of four has been reported, but authors do not refer if it disappeared later and revealed also calf hypertrophy and cardiopathy at the age of eight [7] . Thus, we must be attentive to abnormal gait or cervical weakness during infancy because it can be the first transient symptom or sign of the disease. Hanging big toe is the typical sign of this disease, it is frequently the first obvious sign [4] , even we uncovered hanging big toe in three mildly affected patients who self-reported no symptoms, as already reported by Muelas et al. in two patients [3] . However, we observed no correlation between the severity of the toe extensor weakness and the severity of other manifestations. Thus, weakness of the big toe extension may represent the most consistent clinical sign in every stage of the disease, although neck flexor weakness and abnormal gait during infancy could represent the first manifestation in some cases. Calf hypertrophy and pes cavus are findings reported in other families [3,4] , and we found these symptoms only in family A, so they do not seem to be specific to the novel mutation c.4679G > C. Regarding the muscle biopsy, our series also showed some of the variable morphological findings previously reported: hypotrophy of type I fibers, fiber type disproportion, areas devoid of oxidative activity forming cores/pseudocores, angulated fibers, nuclear clumps, and rimmed vacuoles [3,9,19] . However, which is the most prominent morphological feature varies between series, thus atrophic type I fiber was found as the most common abnormality in some previous series but it was not observed in our study, while cores/pseudocores were highly frequent in most of the series [3,9] and ours but were not described by Lamont et al. [19] . We have reviewed the MRI descriptions reported until now, demonstrating variability in radiological findings [3,4,6,7] . As previously described, in our series the tibialis anterior muscle becomes the most severely degenerated muscle in early stages of the disease, and the gastrocnemius medialis muscle was simultaneously affected in one patient which is in accordance with recent reports showing involvement of the gastrocnemius medialis in about half of the patients [5–8] . However, some authors described that the gastrocnemius together with the gracilis and the sartorius muscles are usually preserved even in severely affected patients [3,20] , and our data contradict this assertion. We observed that the involvement of the sartorius muscle is a very early radiological finding in our patients, even in presymptomatic stages. This is quite surprising, since the sartorius muscle is usually preserved until late stages in many other neuromuscular disorders, except for some myofibrillar myopathies where it seems to be a hallmark [21] . The involvement of the adductor magnus and the sartorius muscles has only been mentioned in few reports of Laing distal myopathy, with no further analysis regarding their evolution [5,7] . We observed that the degree of involvement of the sartorius is always mild at early stages, showing no further progression of the degeneration during the course of the disease. Although this has not been described as a typical radiological sign in Laing distal myopathy, our results support that the APÉNDICE 137 836 P. Carbonell-Corvillo et al. / Neuromuscular Disorders 28 (2018) 828–836 mild involvement of the sartorius muscle is a common and early radiological sign in this myopathy, prior to the involvement of the tibialis anterior muscle, even in asymptomatic carriers, so it could be a useful sign for screening. To conclude, our findings support the novel variant p.R1560P in MYH7 as a founder mutation in Andalucía, and it should be considered the first mutation to study in patients from Southern Spain. These large pedigrees allowed us to better describe the progression and diagnostic clues in very early stages of the disease, such as neck flexor weakness, hanging big toe and the sartorius muscle degeneration in muscle MRI, which could be useful for detecting asymptomatic carriers of the MYH7 mutation. All these findings are important to improve the diagnosis and the genetic counseling of Laing distal myopathy. Author contributions C.P. and P.C designed the study. E.T.C and A.M.M performed the haplotype analysis. E.S., C.P., P.C., L.V., M.C.S., E.A-G, G.G., A.G-G., and M-I.C. handled patients and collected biological samples. M.C.S. and N.L. performed next generation sequencing and analyzed the results. E.R. processed and studied muscle biopsy. C.P. and P.C analyzed the radiological findings. C.P., A.M.M. and A.M-V. supervised and provided critical discussion on the research. C.P. coordinated all the study. P.C wrote the initial manuscript. All authors contributed to the final version of the manuscript. Conflict of interest Professor Laing reports grants from Australian National Health and Medical Research Council, during the conduct of the study. The rest of authors have declared that no conflict of interest exists. Acknowledgments This work was supported in part by grants from the Health Institute Carlos III and FEDER ( FIS PI16-01843 to Paradas, JR15/00042 to Cabrera-Serrano), the Consejería de Salud ( PI- 0085-2016 to Paradas), the Spanish Ministry of Economy and Competitiveness ( BFU2015-71464-R to Martinez-Mir, BFU2015-64408-P to Miranda-Vizuete), the Alfonso Martín Escudero Foundation ( AME-2012 ) (to Cabrera-Serrano), and the NHMRC Principal Research Fellowship ( APP1117510 to Laing). References [1] Voit T , Kutz P , Leube B , Neuen-Jacobc E , Schröder JM , Cavallottiet D , et al. Autosomal dominant distal myopathy: further evidence of a chromosome 14 locus. Neuromuscul Disord 2001;11:11–19 . [2] Tajsharghi H , Thornell LE , Lindberg C , Lindvall B , Henriksson KG , Oldfors A . Myosin storage myopathy associated with a heterozygous missense mutation in MYH7 . Ann Neurol 2003;54:494–500 . [3] Muelas N , Hackman P , Luque H , Garcés-Sánchez M , Azorín I , Suominen T , et al. 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