Charcot-Marie-Tooth type 4B2 demyelinating neuropathy in miniature Schnauzer dogs caused by a novel splicing SBF2 (MTMR13) genetic variant: a new spontaneous clinical model

This study reports the first genetic variant in Miniature Schnauzer dogs responsible for the occurrence of a demyelinating peripheral neuropathy with abnormally folded myelin. This discovery establishes a genotype/phenotype correlation in affected Miniature Schnauzers that can be used for the diagnosis of these dogs. It further supports the dog as a natural model of a human disease; in this instance, Charcot-Marie-Tooth disease. It opens avenues to search the biological mechanisms responsible for the disease and to test new therapies in a non-rodent large animal model. In particular, recent gene editing methods that led to the restoration of dystrophin expression in a canine model of muscular dystrophy could be applied to other canine models such as this before translation to humans

The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms

7 Souhrn V rámci disertační práce "Objasňování příčin neurogenetických onemocnění ana- lýzou dat z MPS pomocí moderních algoritmů" jsme zpracovávali MPS data sek- venovaná pomocí panelu genů, celoexomového (WES) a celogenomového (WGS) sekvenování. Sekvenování panelem genů Při sekvenování pomocí panelu genů jsme využívali na našem pracovišti navržený panel genů, které jsou asociovány s onemocněním. Obecnou podmínkou pro zařazení genu do panelu jsou minimálně dvě nezávislé publikace asociující gen s onemocněním a nebo alespoň jedna publikace popisu- jící kauzální varianty v genu ve dvou nebo více nepříbuzných pacientech. Těmto kritériím v případě panelu pro epileptickou encefalopatii (EE) vyhovovalo t.č. 112 genů. Sekvenování pomocí panelu genů bylo provedeno u 257 pacientů s epileptickou encefalopatií. Patogenní či pravděpodobně patogenní variantu jsme nalezli u 28 % případů (72 z 257 pacientů). U patogenních a pravděpodobně patogenních 76 variant jsme provedli další ana- lýzu variant - rozdělili jsme varianty dle genů do skupin dle dědičnosti a dle původu varianty na de novo, zděděné a s neznámým původem. Ze 112 genů v panelu jsme nalezli patogenní nebo pravděpodobně patogenní variantu ve 33 genech, z nich se nejčastěji jednalo o geny s autozomálně dominantní dědičností (50 variant ve 22 genech). Dle...8 Summary The thesis "The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms" is focused on processing the massively parallel sequencing (MPS) data from a gene panel, whole-exome sequencing (WES) and whole-genome sequencing (WGS). The aim of the study was to develop a suitable pipeline to evaluate at least 250 MPS gene panel data, 150 WES data and 20 WGS data in order to improve molecular genetic testing of rare neurogenetic disorders. Associated data management and database implementation is also described. Targeted gene panel sequencing A custom-designed gene panel consisting of ge- nes previously associated with the disease was used. In the Epileptic Encephalopathy (EE) panel, two prerequisites need to be met for inclusion into the panel: the gene has to have been published in at least two independent publications OR at least in one publication but in multiple independent families. In the case of the EE panel, 112 genes were included. The targeted gene panel sequencing was then performed on 257 patients with EE. Pathogenic or likely pathogenic (according to ACMG criteria) variants have been found in 28% of patients (72 out of 257). Further analysis of the pathogenic or likely pathogenic variants was performed (76 in total); the variants were grouped by...Department of Paediatric NeurologyKlinika dětské neurologie2. lékařská fakultaSecond Faculty of Medicin

Genetic Investigations of Sporadic Inclusion Body Myositis and Myopathies with Structural Abnormalities and Protein Aggregates in Muscle

The application of whole-exome sequencing (WES) has not only dramatically accelerated the discovery of pathogenic genes of Mendelian diseases, but has also shown promising findings in complex diseases. This thesis focuses on exploring genetic risk factors for a large series of sporadic inclusion body myositis (sIBM) cases, and identifying disease-causing genes for several groups of patients with abnormal structure and/or protein aggregates in muscle. Both conventional and advanced techniques were applied. Based on the International IBM Genetics Consortium (IIBMGC), the largest sIBM cohort of blood and muscle tissue for DNA analysis was collected as the initial part of this thesis. Candidate gene studies were carried out and revealed a disease modifying effect of an intronic polymorphism in TOMM40, enhanced by the APOE ε3/ε3 genotype. Rare variants in SQSTM1 and VCP genes were identified in seven of 181 patients, indicating a mutational overlap with neurodegenerative diseases. Subsequently, a first whole-exome association study was performed on 181 sIBM patients and 510 controls. This reported statistical significance of several common variants located on chromosome 6p21, a region encompassing genes related to inflammation/infection. WES was performed on a group of 35 cases with tubular aggregates/cylindrical spirals, and detected rare variants in known/candidate genes. Disease-causing genes were identified in four families with protein aggregates in muscle also by WES. In one family identified with a novel homozygous deletion in SBF1 with a rare autosomal-recessive neuromuscular condition, functional analysis was carried out indicating a loss-of-function mechanism underlying the pathogenesis of the disease. The collection of a large series of sIBM patients through the IIBMGC has been shown here to reveal important genetic findings and will be a valuable resource for the future. WES proved to be important in sIBM and also to be an efficient method to investigate the genetics basis of rare complex muscle disorders

Two newly defined inherited disorders due to cholinergic transporter dysfunction with distinct clinical outcomes, disease mechanisms and modes of inheritance.

Neurodegenerative diseases are becoming increasingly prevalent due to the ageing population, and are among the major contributors to disability and disease worldwide. The identification of the gene defects responsible for many of these conditions has played a major role in our understanding of the pathogenic processes involved, and provided opportunity to develop targeted treatment strategies. Cholinergic neurotransmission supports a wide range of physiological and behavioural processes and its dysfunction of cholinergic signalling has been associated with a number of disorders, including myasthenias, cardiovascular disease(1), attention-deficit hyperactivity disorder (ADHD) (2), Alzheimer’s disease (ADi), schizophrenia, addiction(3), and depression(4). SLC5A7 encodes the Na+/Cl- dependent, high-affinity choline transporter (CHT) which represents the rate limiting step in acetylcholine (Ach) synthesis and is critical for normal cholinergic signalling. The work in this thesis details two new inherited disorders, caused by distinct pathogenic disease mechanisms, associated with novel SLC5A7 mutations. Chapter three documents the discovery of two autosomal-dominantly acting SLC5A7/CHT mutations associated with adult onset motor neurone disorders. Initially we identified a frameshift mutation that results in premature truncation of the transporter protein in a large Welsh kindred affected with distal hereditary motor neuropathy type VII (dHMN-VII), in which neurodegeneration and muscle paresis is largely restricted to the distal limb muscles and vocal cords. The mutation responsible results in the dominant-negative interference of the mutant molecule with function of the wild type choline transporter, resulting in significantly reduced (although not completely abolished) transporter activity. This finding is further evidenced by the discovery of a second dHMN family associated with a distinct frameshift SLC5A7 mutation indicative of a similar dominant-negative disease mechanism. Together these findings corroborate a dominant-negative disease mechanism arising from C-terminal truncating SLC5A7 mutations associated with dHMN, and provide further insight into the role of aberrant choline transporter function in neurological disease. Chapter four describes N-terminal missense mutations located in the transmembrane spanning region of SLC5A7/CHT, associated with a severe infantile neuromuscular disorder characterised by predominantly central hypotonia and developmental delay. The phenotypic effects of these mutations are likely to result from the near abolition of CHT-mediated choline transport in homozygous individuals, and are in keeping with those observed in CHT knock-out mouse models(5). The development of a mouse model of the human motor neurone disease arising from SLC5A7 frameshift mutations should allow for further investigation of the mechanism by which truncated CHT leads to the dHMN phenotype. Chapter 5 details treatment hypotheses for dHMN, as well as the generation of a patient-specific knock-in mouse model carrying an Slc5a7 mutation orthologous to that identified in dHMN-VII families in chapter 3, and results from preliminary neurological phenotyping of the mouse model. This model will be crucially important for the exploration of treatment options in dHMN-VII motor neurone disease as a prelude to clinical trials in humans.Neurosciences Research FoundationJohn McDowall Bursary (for MND research

Análisis de la marcha en pacientes con enfermedad de Charcot Marie Tooth 1A

[Resumen] INTRODUCCIÓN. La enfermedad de Charcot Marie Tooth, también conocida como neuropatía motora y sensorial hereditaria, se compone de un grupo de neuropatías heterogéneas y progresivas que representan la enfermedad neuromuscular genética más común en Europa. Se divide principalmente en dos formas, siendo la 1A la más común, con un total de casos entre el 60-80%. Su presentación clínica incluye deformidades musculoesqueléticas, síntomas motores y sensitivos, y una reducción o ausencia de reflejos profundos en el tendón. La pérdida de propiocepción y la ataxia sensitiva contribuyen a cambios característicos en el patrón y en la mecánica de la marcha; produciendo dificultades para correr o saltar, tropiezos y esguinces de tobillo repetitivos. OBJETIVO. El objetivo de esta revisión sistemática es conocer en profundidad los cambios que se producen en la marcha de los pacientes con Charcot Marie Tooth 1A, con el fin de llevar a cabo una detección precoz de los mecanismos de compensación que estos pacientes utilizan al caminar. Lo cual, facilitará su correcto diagnóstico y tratamiento, repercutiendo así en la mejora su la calidad asistencial. METODOLOGÍA. Se realizó una búsqueda bibliográfica en las bases de datos Pubmed, CINHAL, Scopus y Web of Science en el mes de diciembre de 2020. RESULTADOS. Tras la búsqueda se obtuvieron un total de 323 estudios de los cuales, tras aplicar los criterios de inclusión y exclusión, fueron seleccionados 8 documentos con el fin de responder a la pregunta del estudio. CONCLUSIONES. Debido a la lenta progresión de la enfermedad, el análisis de la marcha es una herramienta poderosa que permite cuantificar las características de pacientes con alteraciones de la marcha, por lo que es importante adecuar las tareas motrices a la capacidad funcional del paciente. La debilidad muscular de los miembros inferiores altera los rangos de movimiento del tobillo. Al subir escaleras hay una disminución de la flexión dorsal y mala posición del pie, produciendo una cadena de compensaciones que permitirán el avance de la pierna contraria. En general, la rodilla sufre un exceso de flexión para compensar la caída del pie típica de la enfermedad.[Resumo] INTRODUCIÓN. A enfermidade de Charcot Marie Tooth, tamén coñecida como neuropatía motora e sensorial hereditaria, componse dun grupo de neuropatías heteroxéneas e progresivas que representan a enfermidade neuromuscular xenética máis común en Europa. Divídese principalmente en dúas formas, sendo a 1A a máis común, cun total de casos entre o 60-80% A súa presentación clínica inclúe deformidades musculoesqueléticas, síntomas motores e sensitivos, e una redución ou ausencia de reflexos profundos no tendón. A perda de propiocepción e a ataxia sensitiva contribúen a cambios característicos no patrón e na mecánica da marcha; producindo dificultades para correr ou saltar, tropezos e torceduras de nocello repetitivos. OBXECTIVO. O obxectivo desta revisión sistemática é coñecer en profundidade os cambios producidos na marcha dos pacientes con Charcot Marie Tooth 1A, co fin de levar a cabo unha detección precoz dos mecanismos de compensación que estes pacientes utilizan ao camiñar. O cal facilitará o seu correcto diagnóstico e tratamento, repercutindo desta forma na mellora da súa calidade asistencial. METODOLOXÍA. Realizouse una busca bibliográfica nas bases de datos Pubmed, CINHAL, Scopus e Web of Science no mes de decembro do 2020. RESULTADOS. Trala busca obtivéronse un total de 323 estudos dos cales, tras aplicar os criterios de inclusión e exclusión, seleccionáronse 8 documentos co fin de responder á pregunta de estudo. CONCLUSIÓNS. Debido á lenta progresión da enfermidade, o análise da marcha é una poderosa ferramenta que permite cuantificar as características dos pacientes con alteracións da marcha, polo que é importante adecuar as tarefas motrices á capacidade funcional do paciente. A debilidade muscular dos membros inferiores altera os rangos de movemento do nocello. Ao subir escaleiras hai unha diminución da flexión dorsal e un mal posicionamento do pé, producindo unha cadea de compensacións que permitirán o avance da perna contraria. En xeral, o xeonllo sofre un exceso de flexión para compensar á caída do pé típica da enfermidade.[Abstract] INTRODUCTION. Charcot Marie Tooth disease, also known as hereditary sensory and motor neuropathy, is made up of a group of heterogeneous and progressive neuropathies that represent the most common genetic neuromuscular disease in Europe. It is mainly divided into two forms, 1A being the most common, with a total of cases between 60-80%. Its clinical presentation includes musculoskeletal deformities, motor and sensory symptoms, and a reduction or absence of deep reflexes in the tendon. Loss of proprioception and sensory ataxia contribute to characteristic changes in gait pattern and mechanics; causing difficulties in running or jumping, stumbling, and repetitive ankle sprains. OBJECTIVE. This systematic review aims to know in depth the changes that occur in the gait of patients with Charcot Marie Tooth 1A, to carry out early detection of the compensation mechanisms that these patients use when they walk. Which will facilitate its correct diagnosis and treatment, thus having an impact on improving the quality of care. METHODOLOGY. A bibliographic search was carried out in the Pubmed, CINHAL, Scopus, and Web of Science databases in December 2020. RESULTS. After the search, a total of 323 studies were obtained, of which, after applying the inclusion and exclusion criteria, 8 documents were selected to answer the study question. CONCLUSIONS. Due to the slow progression of the disease, gait analysis is a powerful tool that allows quantifying the characteristics of patients with gait disturbances, so it is important to adapt motor tasks to the functional capacity of the patient. Muscle weakness in the lower limbs alters the ankle's range of motion. When climbing stairs there is a decrease in dorsiflexion and poor foot position, producing a chain of compensations that will allow the opposite leg to advance. In general, the knee is excessively flexed to compensate for the foot drop typical of the disease.Traballo fin de mestrado (UDC.FCS). Asistencia e investigación sanitaria. Especialidade en Reeducación funcional, Auitonomía personal y calidad de vida

Caracterización fenotípica y molecular de las neuropatías hereditarias en la infancia y la adolescencia

ESTADO ACTUAL DEL TEMA: La mayoría de las polineuropatías en la infancia son genéticamente determinadas, estimándose alrededor del 70-90% del total de neuropatías. Este grupo de neuropatías hereditarias (NH) comprende las NH tipo Charcot-Marie-Tooth y los síndromes hereditarios complejos bien sean enfermedades neurodegenerativas o errores innatos del metabolismo en los que la polineuropatía es una de sus características. La literatura médica es escasa en estudios de neuropatías hereditarias en población pediátrica, lo que puede ser atribuible a la escasez de programas nacionales e internacionales de bases de datos y escalas uniformes de evaluación pediátricas de aplicación generalizada. OBJETIVOS: El objetivo principal es la caracterización genética y fenotípica de una serie de pacientes menores de 20 años con NH incluyendo la búsqueda de nuevos genes causantes. Los objetivos secundarios son: determinar la variabilidad de la gravedad de los distintos tipos de NH y estimar la sensibilidad de la escala CMTPedS como medida de progresión en las distintas NH. METODOLOGÍA: Se trata de un estudio descriptivo longitudinal de 3 años de duración de una serie hospitalaria reclutados y valorados de forma prospectiva entre septiembre 2017 y septiembre de 2020 en la Unidad de Enfermedades Neuromusculares y Neuropediatría del Hospital Universitario y Politécnico La Fe (Valencia, España). Se incluyeron solo los pacientes que tenían menos de 20 años en el momento del inicio del estudio y contaban con un diagnóstico definitivo de neuropatía periférica de origen genético, aunque no se conociese el defecto genético causal tanto los casos índices como los secundarios (algún progenitor o hermano/a afectos). Los pacientes fueron estudiados desde el punto de vista clínico, neurofisiológico, de neuroimagen, de discapacidad (con la escala CMTPedS) y genético siguiendo los protocolos reflejados en la presente tesis. Para el estudio centrado en la utilidad de la escala CMTPedS en las formas motoras puras, se contó además con la aportación de pacientes por parte del grupo de investigación en neuropatías hereditarias en edades pediátricas de The Children’s Hospital at Westmead (NSW, Australia). Los criterios de inclusión y exclusión que debían cumplir los pacientes procedentes de estos dos centros eran los mismos. RESULTADOS: Se estudió un total de 110 pacientes con NH que en el momento del inicio tenían 20 años o menos. La mayoría pertenecen a la cohorte de pacientes remitidos al Hospital Universitario y Politécnico La Fe mientras que 8 de ellos fueron aportados por The Children’s Hospital at Westmead. De los 102 pacientes procedentes del Hospital Universitario y Politécnico La Fe, tres presentaron un fenotipo tan único y compartían el mismo genotipo TRMT5 que fueron descritos en un extenso trabajo aparte (Argente-Escrig et al., 2022). Así pues, 99 pacientes fueron los reflejados en el trabajo Argente-Escrig et al., 2021a. De estos 99 (59 hombres), 14 debutaron con neuropatía motora hereditaria distal (NHMd) y 85 debutaron con una forma sensitivomotora (NHSM) con 2/3 de subtipo desmielinizantes (Argente-Escrig et al., 2021a). El diagnóstico genético se logró en el 79,5% de las familias, con una tasa de detección de mutaciones en las formas desmielinizantes (88,7%) y axonales (89,5%), significativamente mayor que en las familias NHMd (27,3%). CMT1A fue el subtipo más frecuente (n = 37), seguido de los que tienen mutaciones heterocigóticas en los genes GDAP1 (n = 9) o GJB1 (n = 8). Se identificaron mutaciones en otros 15 genes, incluyendo una nueva variante patogénica en el gen ATP1A (Argente-Escrig et al., 2021a). La cohorte de 22 pacientes con NHMd (13 mujeres) de 19 familias procedió de los dos centros universitarios (Argente-Escrig et al., 2021b). 14 personas fueron sintomáticas en el primer año de vida. La discapacidad intelectual estuvo presente en 6 individuos y se observaron signos de neurona motora superior en 8. Se encontraron variantes patogénicas en 9 familias, más frecuentemente en BICD2 (BICD2-4, DYNC1H1-2, MFN2-2, GARS1-1). Se identificó una nueva variante patogénica en el gen GARS1 (Argente-Escrig et al., 2021b). La CMTpedS detectó una progresión significativa de la enfermedad en todos los subtipos genéticos de NHSM (Argente-Escrig et al., 2021a), a un ritmo de 1,84 (±3,7) durante 1 año (p < 0,0005, n = 62) y una tasa de 3,6 (± 4,4: p < 0,0005, n = 45) a los 2 años. También se detectó un empeoramiento significativo para CMT1A al año (1,7 ± 3,6, p < 0,05) y a los 2 años (4,2 ± 4,3, p < 0,0005). En las NHMd, la puntuación total de la CMTPedS a lo largo de 1 año se deterioró, en promedio, 1,5 puntos (DE 3,7) o 9% (n = 12), con variabilidad significativa en la tasa de progresión dentro de la cohorte (Argente-Escrig et al., 2021b). Finalmente, se fenotipa en profundidad las neuropatías asociadas a dos genes distintos: FGD4 y TRMT5. Los dos pacientes hermanos portadores de las variantes patogénicas c.514delG (p.Ala172Glnfs*28) y c.2211dupA (Ala738Serfs*5) en el gen FGD4 debutaron en la adolescencia y mostraron un fenotipo muy leve a diferencia de lo publicado previamente (Argente-Escrig et al., 2019). La proteína truncada p.Ala738Serfs*5 puede haber conservado parcialmente la actividad de FGD4 ya que se conservan los principales dominios funcionales. Las mutaciones recesivas en el gen TRMT5 en tres pacientes procedentes de tres familias distintas se asociaron con un fenotipo no descrito previamente (Argente-Escrig et al., 2022). Se presentaron con retraso global del desarrollo, neuropatía desmielinizante de predominio sensitivo de inicio congénito o infantil, signos piramidales, ataxia cerebelosa leve y ausencia de un perfil bioquímico compatible con una deficiencia de OXPHOS. El análisis patológico rutinaria de músculo y nervio en estos pacientes resultó aparentemente normal mientras que el estudio ultraestructural estaba claramente alterado. CONCLUSIONES: Nuestra serie pediátrica presenta características diferenciadoras con otras cohortes descritas por la mayor proporción de portadores heterocigotos en el gen GDAP1. La afectación del tracto piramidal y la cognitiva son frecuentes en las formas motoras puras en la edad pediátrica. Se confirmó la progresión de las NHSM a los dos años de seguimiento medida con la CMTPedS, y como novedad se detectó que la CMTPedS es sensible al cambio también en el primer año de seguimiento de las NHSM. Los presentes datos apoyan a la escala CMTPedS como una medida de discapacidad sensible a la progresión al año también en las formas motoras y nuestro estudio apunta a que la CMTpedS se puede adaptar a los pacientes con formas motoras. El fenotipado en profundidad y el exhaustivo análisis genético realizado nos ayudan a comprender los mecanismos patogénicos asociados a las diferentes variantes y su influencia en el fenotipo final. Las muestras ultraestructurales de músculos y nervios pueden indicar una etiología mitocondrial en casos en que las imágenes histopatológicas de rutina parecen normales.BACKGROUND: Most polyneuropathies in childhood are genetically determined, with an estimated 70-90% of all neuropathies. Inherited peripheral neuropathies (IPN) include Charcot-Marie-Tooth disease and complex hereditary syndromes, whether they are neurodegenerative diseases or inborn errors of metabolism in which polyneuropathy is one of their characteristics. Medical literature is scarce on studies of inherited peripheral neuropathies in the pediatric population, which may be attributable to the short supply of national and international database programs and uniform pediatric evaluation scales for general application. OBJECTIVES: The main objective is the genetic and phenotypic characterization of a series of patients under 20 years of age with IPN, including the search for new causative genes. The secondary objectives are the following: to determine the variability of the severity of the different types of IPN and to estimate the sensitivity of the CMTPedS scale as a measure of progression in the different IPN subtypes. METHODOLOGY: This is a 3-year longitudinal descriptive study of a hospital-based series prospectively recruited and assessed between September 2017 and September 2020 in the Neuromuscular Diseases and Child Neurology Unit of the Hospital Universitario y Politécnico La Fe (Valencia, Spain). Only patients who were under 20 years of age at the start of the study and had a definitive diagnosis of peripheral neuropathy of genetic origin were included, even if the causal genetic defect was not known, both in index cases and in secondary cases (either parent or affected sibling). Patients were studied from the clinical, neurophysiological, neuroimaging, disability (with the CMTPedS scale) and genetic perspective following the protocols reflected in this thesis. The research group on inherited neuropathies at The Children's Hospital at Westmead (NSW, Australia) contributed with patients with pure motor IPN for the study on the usefulness of the CMTPedS scale in pure motor forms. The inclusion and exclusion criteria that patients from these two centers had to meet were the same. RESULTS: A total of 110 patients with IPN who were 20 years old or younger at onset were studied. Most belonged to the cohort of patients referred to Hospital Universitario y Politécnico La Fe, while 8 of them were contributed by The Children's Hospital at Westmead. Of the 102 patients from the Hospital Universitario y Politécnico La Fe, three presented with such a unique phenotype and shared the same TRMT5 genotype that they were described in an extensive separate study (Argente-Escrig et al., 2022). Thus, 99 patients were reflected in the study Argente-Escrig et al., 2021a. Of these 99 (59 men), 14 presented with distal hereditary motor neuropathy (dHMN) and 85 had a sensorimotor form with 2/3 of the demyelinating subtype (Argente-Escrig et al., 2021a). Genetic diagnosis was achieved in 79.5% of families, with a detection rate of mutations in demyelinating forms (88.7%) and axonal forms (89.5%), significantly higher than in dHMN families (27.3%). CMT1A was the most frequent subtype (n = 37), followed by those with heterozygous mutations in the GDAP1 (n = 9) or GJB1 (n = 8) genes. Mutations in another 15 genes were identified, including a new pathogenic variant in the ATP1A gene (Argente-Escrig et al., 2021a). The cohort of 22 dHMN patients (13 women) from 19 families came from the two university centers (Argente-Escrig et al., 2021b). 14 people were symptomatic in the first year of life. Intellectual disability was present in 6 individuals and upper motor neuron signs were seen in 8. Pathogenic variants were found in 9 families, most frequently in BICD2 (BICD2-4, DYNC1H1-2, MFN2-2, GARS1-1). A new pathogenic variant in the GARS1 gene was identified (Argente-Escrig et al., 2021b). The CMTpedS detected significant disease progression in all sensorimotor genetic subtypes (Argente-Escrig et al., 2021a), at a rate of 1.84 (±3.7) over 1 year (p < 0.0005, n = 62) and a rate of 3.6 (± 4.4: p < 0.0005, n = 45) at 2 years. Significant worsening was also detected for CMT1A at 1 year (1.7 ± 3.6, p < 0.05) and at 2 years (4.2 ± 4.3, p < 0.0005). In the dHMN, the CMTPedS total score over 1 year deteriorated, on average, 1.5 points (SD 3.7) or 9% (n = 12), with significant variability in the rate of progression within 1 year of follow-up (Argente-Escrig et al., 2021b). Finally, neuropathies associated with two different genes are phenotyped in depth: FGD4 and TRMT5. The two sibling patients who were carriers of the pathogenic variants c.514delG (p.Ala172Glnfs*28) and c.2211dupA (Ala738Serfs*5) in the FGD4 gene had their onset in adolescence and showed a very mild phenotype in contrast to what was previously published (Argente -Escrig et al., 2019). The p.Ala738Serfs*5 truncated protein may have partially conserved FGD4 activity since the main functional domains are preserved. Recessive mutations in the TRMT5 gene in three patients from three different families were associated with a phenotype not previously described (Argente-Escrig et al., 2022). They presented with global developmental delay, predominantly sensory demyelinating neuropathy of congenital or infantile onset, pyramidal signs, mild cerebellar ataxia, and no biochemical profile consistent with OXPHOS deficiency. The routine pathological analysis of muscle and nerve in these patients was apparently normal while the ultrastructural study was clearly abnormal. CONCLUSIONS: Our pediatric series presents differentiating characteristics with other cohorts described by the higher proportion of heterozygous carriers in the GDAP1 gene. The involvement of the pyramidal tract and the cognitive are frequent in the pure motor forms in the pediatric age. The progression of the sensorimotor forms was confirmed at two years of follow-up measured with the CMTPedS, and as a novelty it was detected that the CMTPedS is sensitive to change also in the first year of follow-up of the sensorimotor neuropathies. The present data support the CMTPedS scale as a measure of disability sensitive to progression at one year also in motor forms, and our study suggests that the CMTpedS can be adapted to patients with motor forms. The in-depth phenotyping and exhaustive genetic analysis carried out help us to understand the pathogenic mechanisms associated with the different variants and their influence on the final phenotype. Ultrastructural muscle and nerve specimens may indicate a mitochondrial etiology in cases where routine histopathologic imaging appears normal

Clinical, laboratory, and genetic studies of families with Charcot-Marie-Tooth type 2C disease

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. We studied five unrelated families with CMT2C, of which two showed significant linkage to chromosome 12q24.11. Linkage analysis excluded this locus in one of the remaining families, suggesting genetic heterogeneity within this CMT subtype. SNP genotyping of samples from affected individuals in the three linked families did not reveal any deletion or copy number variation. All genes in this region were sequenced and two heterozygous missense mutations were identified in the transient receptor potential, subfamily V, member 4 (TRPV4), at positions c.805C>T and c.806G>A in two families, predicting the amino acid substitutions R269C and R269H, respectively. Two other mutations (R186Q, R232C) and one change of uncertain significance (D584N) were subsequently identified. The R269C and R269H variants were not present in more than 200 controls. TRPV4 is a well known member of the TRP superfamily of cation channels. We confirmed that it is expressed in motor and sensory neurons. The R186, R232, and R269 residues are located in the intracellular N-terminal portion of the TRPV4 protein in the ankyrin repeat domain (ARD); functional domains known to mediate protein-protein interactions. When expressed in HEK293 cells and Xenopus oocytes, R269C and R269H TRPV4 trafficked normally to the cell surface, but caused increased cell toxicity and increased constitutive and activated channel currents compared to wild-type TRPV4. Other mutations in TRPV4 have been previously associated with inherited forms of skeletal dysplasia. When mapped onto the crystal structure of the TRPV4 ARD, the mutations found in bone diseases lie on the opposite face of the domain compared to those causing neuropathy. Our findings indicate that TRPV4 mutations cause a degenerative disorder of peripheral nerves. Thus, there is striking phenotypic variability with different mutations in this important channel protein

Distribución mutacional y correlación genotipo-fenotipo del síndrome de Charcot-Marie-Tooth en la Comunidad Valenciana

i. Objetivo general: Describir la distribución mutacional de pacientes con CMT evaluados en el Hospital Universitari i Politècnic La Fe durante el periodo de 1990-2012 y compararla con otras poblaciones El análisis de la distribución mutacional de pacientes con CMT seguidos en el Hospital Universitari i Politècnic La Fe (centro de referencia para esta patología en la Comunidad Valenciana) confirma que la misma tiene características diferenciadoras con otras series publicadas. Entre ellas destaca la elevada frecuencia de formas con herencia autosómica recesiva y de mutaciones causales en el gen GDAP1. La frecuencia de pacientes con herencia recesiva es explicable en parte por la existencia de un grupo de pacientes con mutaciones recesivas en el gen GDAP1 y principalmente por una numerosa población de etnia gitana, que presentan mutaciones fundadoras causales de CMT4. La elevada frecuencia de mutaciones en el gen GDAP1 en los pacientes con un subtipo axonal probablemente esté en relación con un presumible efecto fundador de la mutación p.R120W en el oeste de Europa. ii. Objetivo específico: Profundizar en la caracterización del fenotipo de pacientes con mutaciones que aporten rasgos diferenciadores con respecto a otras series. Para ello se trató de ampliar el conocimiento fenotípico de los pacientes con mutaciones dominantes en GDAP1, recesivas en SH3TC2, así como de las mutaciones noveles que se detectaron. La elevada frecuencia de pacientes con la mutación p.R120W en GDAP1 en nuestra serie clínica motivó que se tratase de definir con exactitud el fenotipo de dichos pacientes. Se observó un cuadro leve-moderado con amplia variabilidad clínica, una afectación 152 equivalente de la flexión y extensión del tobillo y un patrón de RM muscular de pantorrillas característico. Las mutaciones recesivas en SH3TC2 se estudiaron mediante la realización de un estudio vestibular, observando una disfunción vestibular objetivable en todos los pacientes y muy marcada en el 70% de los mismos. Dicha vestibulopatía probablemente influye deletéreamente en la estabilidad del paciente, y puede modificar las estrategias rehabilitadoras empleadas. Aparte, se aporta información fenotípica detallada de las 13 mutaciones que no habían sido descritas previamente. Entre ellas destaca uno de los pocos casos descritos de una neuropatía hipomielinizante congénita debida a una mutación novel en el gen MPZ. iii. Objetivo específico: Proponer algoritmos diagnósticos para poder orientar el cribaje mutacional de pacientes afectos de enfermedad de Charcot-Marie-Tooth en la Comunidad Valenciana.. Gracias a la distribución mutacional encontrada y a los estudios fenotípicos realizados, y teniendo en cuenta el conocimiento previo acumulado, se proponen una serie de algoritmos diagnósticos para orientar el cribaje mutacional de futuros pacientes con CMT en la Comunidad Valenciana. Hasta qué punto dichos algoritmos son extrapolables a otras poblaciones cercanas debe ser valorado en años venideros, mediante la realización de cribajes mutacionales comparables en otras áreas de España o de países mediterráneos

CLINICAL DIAGNOSTIC WHOLE EXOME SEQUENCING FOR INFANTS IN INTENSIVE CARE SETTINGS: OUTCOMES ANALYSIS AND ECONOMIC EVALUATION

Whole exome sequencing (ES) is an extensive form of genetic testing and increasingly used as a diagnostic tool. Clinical uptake of genome-scale sequencing occurred without clear guidelines for application or robust information regarding potential impact on patient health outcomes or cost of care. For infants in intensive care with suspected genetic conditions, ES can be especially powerful to identify a specific diagnosis and inform crucial decisions about medical care. However, little is known about the cost-effectiveness of ES compared to other diagnostic strategies. This project first assessed the literature on pediatric clinical ES. Then, using electronic medical record, diagnostic laboratory, and hospital cost data, we analyzed and compared outcomes and costs of care for patients with suspected genetic etiologies admitted to intensive care within the first year of life in two patient cohorts: those who had ES (ES, n=368) and did not have ES (No-ES, n=368) as part of a diagnostic workup at a large children’s hospital. Molecular diagnostic yield (25.8% No-ES, 27.7% ES; p=0.56) and 1-year survival (84.8% No-ES, 80.2% ES; p=0.10) were similar between cohorts, while ES patients had higher total cost, diagnostic investigation cost, and genetic test cost during the index admission and for the year after the date of first inpatient genetics consultation (all p\u3c0.01). ES demonstrated important diagnostic utility for patients with monogenic disease, yet other genetic tests, especially chromosomal microarray, remain important given the burden of chromosomal abnormalities in this population. As clinically applied over the first 5 years, ES does not appear to be a cost-effective diagnostic tool for the broad population of newborns and infants with suspected genetic disease compared to standard diagnostic tests such as chromosomal microarray analysis and panel/single gene testing. Further work is needed to develop outcome measures to capture utility of ES results – both diagnostic and non-diagnostic – for clinicians, patients, and patients’ families, and to specify clinical guidelines for appropriate ES application