RESUMEN
Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and ß2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date. Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014-19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1-56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0-49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%. Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected. This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects.
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Síndromes Miasténicos Congénitos , Masculino , Femenino , Humanos , Niño , Adolescente , Adulto Joven , Adulto , Síndromes Miasténicos Congénitos/diagnóstico , Acetilcolinesterasa , Diagnóstico Tardío , Unión Neuromuscular/genética , Pruebas Genéticas , Mutación/genéticaRESUMEN
PURPOSE: Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the "ClinVar low-hanging fruit" reanalysis, reasons for the failure of previous analyses, and lessons learned. METHODS: Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. RESULTS: We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). CONCLUSION: The "ClinVar low-hanging fruit" analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock.
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Discapacidad Intelectual , Humanos , Secuenciación del Exoma , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Alelos , GenotipoRESUMEN
Consanguineous marriages have a prevalence rate of 24% in Turkey. These carry an increased risk of autosomal recessive genetic conditions, leading to severe disability or premature death, with a significant health and economic burden. A definitive molecular diagnosis could not be achieved in these children previously, as infrastructures and access to sophisticated diagnostic options were limited. We studied the cause of neurogenetic disease in 246 children from 190 consanguineous families recruited in three Turkish hospitals between 2016 and 2020. All patients underwent deep phenotyping and trio whole exome sequencing, and data were integrated in advanced international bioinformatics platforms. We detected causative variants in 119 known disease genes in 72% of families. Due to overlapping phenotypes 52% of the confirmed genetic diagnoses would have been missed on targeted diagnostic gene panels. Likely pathogenic variants in 27 novel genes in 14% of the families increased the diagnostic yield to 86%. Eighty-two per cent of causative variants (141/172) were homozygous, 11 of which were detected in genes previously only associated with autosomal dominant inheritance. Eight families carried two pathogenic variants in different disease genes. De novo (9.3%), X-linked recessive (5.2%) and compound heterozygous (3.5%) variants were less frequent compared to non-consanguineous populations. This cohort provided a unique opportunity to better understand the genetic characteristics of neurogenetic diseases in a consanguineous population. Contrary to what may be expected, causative variants were often not on the longest run of homozygosity and the diagnostic yield was lower in families with the highest degree of consanguinity, due to the high number of homozygous variants in these patients. Pathway analysis highlighted that protein synthesis/degradation defects and metabolic diseases are the most common pathways underlying paediatric neurogenetic disease. In our cohort 164 families (86%) received a diagnosis, enabling prevention of transmission and targeted treatments in 24 patients (10%). We generated an important body of genomic data with lasting impacts on the health and wellbeing of consanguineous families and economic benefit for the healthcare system in Turkey and elsewhere. We demonstrate that an untargeted next generation sequencing approach is far superior to a more targeted gene panel approach, and can be performed without specialized bioinformatics knowledge by clinicians using established pipelines in populations with high rates of consanguinity.
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Exoma , Consanguinidad , Exoma/genética , Homocigoto , Humanos , Mutación , Linaje , Fenotipo , Secuenciación del ExomaRESUMEN
Several clinical trials are working on drug development for Duchenne and Becker muscular dystrophy (DMD and BMD) treatment, and, since the expected increase in dystrophin is relatively subtle, high-sensitivity quantification methods are necessary. There is also a need to quantify dystrophin to reach a definitive diagnosis in individuals with mild BMD, and in female carriers. We developed a method for the quantification of dystrophin in DMD and BMD patients using spectral confocal microscopy. It offers the possibility to capture the whole emission spectrum for any antibody, ensuring the selection of the emission peak and allowing the detection of fluorescent emissions of very low intensities. Fluorescence was evaluated first on manually selected regions of interest (ROIs), proving the usefulness of the methodology. Later, ROI selection was automated to make it operator-independent. The proposed methodology correctly classified patients according to their diagnosis, detected even minimal traces of dystrophin, and the results obtained automatically were statistically comparable to the manual ones. Thus, spectral imaging could be implemented to measure dystrophin expression and it could pave the way for detailed analysis of how its expression relates to the clinical course. Studies could be further expanded to better understand the expression of dystrophin-associated protein complexes (DAPCs).
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Distrofina , Distrofia Muscular de Duchenne , Humanos , Femenino , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/diagnóstico por imagen , Distrofia Muscular de Duchenne/metabolismoRESUMEN
Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.
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Genómica , Enfermedades Raras , Exoma , Estudios de Asociación Genética , Genómica/métodos , Humanos , Fenotipo , Enfermedades Raras/diagnóstico , Enfermedades Raras/genéticaRESUMEN
PURPOSE: The chaperone protein BiP is the master regulator of the unfolded protein response in the endoplasmic reticulum. BiP chaperone activity is regulated by the post-translational modification AMPylation, exclusively provided by FICD. We investigated whether FICD variants identified in patients with motor neuron disease could interfere with BiP activity regulation. METHODS: Exome sequencing was performed to identify causative pathogenic variants associated with motor neuron diseases. Functional studies were conducted on fibroblasts from patients to explore the molecular mechanism of the disease. RESULTS: We identified biallelic variants in FICD causing a neurodegenerative disease of upper and lower motor neurons. Affected individuals harbor a specific missense variant, Arg374His, positioned in the catalytic motif of the enzyme and important for adenosine triphosphate binding. The mutated residue abolishes intramolecular interaction with the regulatory residue Glu234, essential to inhibit AMPylation and to promote de-AMPylation by FICD. Consequently, fibroblasts from patients with FICD variants have abnormally increased levels of AMPylated and thus inactivated BiP. CONCLUSION: Loss of BiP chaperone activity in patients likely results in a chronic impairment of the protein quality control system in the endoplasmic reticulum. These findings will guide the development of therapeutic strategies for motoneuron and related diseases linked to proteotoxic stress.
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Enfermedad de la Neurona Motora , Enfermedades Neurodegenerativas , Humanos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Enfermedad de la Neurona Motora/genética , Enfermedad de la Neurona Motora/metabolismoRESUMEN
We present a Turkish family with two cousins (OC15 and OC15b) affected with syndromic developmental delay, microcephaly, and trigonocephaly but with some phenotypic traits distinct between them. OC15 showed asymmetrical skeletal defects and syndactyly, while OC15b presented with a more severe microcephaly and semilobal holoprosencephaly. All four progenitors were related and OC15 parents were consanguineous. Whole Exome Sequencing (WES) analysis was performed on patient OC15 as a singleton and on the OC15b trio. Selected variants were validated by Sanger sequencing. We did not identify any shared variant that could be associated with the disease. Instead, each patient presented a de novo heterozygous variant in a different gene. OC15 carried a nonsense mutation (p.Arg95*) in PORCN, which is a gene responsible for Goltz-Gorlin syndrome, while OC15b carried an indel mutation in ZIC2 leading to the substitution of three residues by a proline (p.His404_Ser406delinsPro). Autosomal dominant mutations in ZIC2 have been associated with holoprosencephaly 5. Both variants are absent in the general population and are predicted to be pathogenic. These two de novo heterozygous variants identified in the two patients seem to explain the major phenotypic alterations of each particular case, instead of a homozygous variant that would be expected by the underlying consanguinity.
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Aciltransferasas/genética , Consanguinidad , Proteínas de la Membrana/genética , Mutación , Proteínas Nucleares/genética , Factores de Transcripción/genética , Niño , Preescolar , Análisis Mutacional de ADN , Facies , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Linaje , Fenotipo , Radiografía , TurquíaRESUMEN
Alazami syndrome (MIM#615071) is a rare developmental disorder caused by biallelic variants in the LARP7 gene. Hallmark features include short stature, global developmental delay, and distinctive facial features. To date, 23 patients from 11 families have been reported in the literature. Here we describe a 19-year-old man who, in association with the typical features of Alazami syndrome, was diagnosed at the age of 14 years with papillary thyroid carcinoma, harboring the somatic BRAF V600E mutation. Whole exome sequencing revealed two novel LARP7 variants in compound heterozygosity, whereas only common variants were detected in genes associated with familial nonmedullary thyroid cancer (MIM#188550). LARP7 acts as a tumor suppressor in breast and gastric cancer, and possibly, according to recent studies, in thyroid tumors. Since thyroid cancer is rare among children and adolescents, we hypothesize that the LARP7 variants identified in our patient are responsible for both Alazami syndrome and tumor susceptibility. We also provide an overview of the clinical findings in all Alazami syndrome patients reported to date and discuss the possible pathogenetic mechanism that may underlie this condition, including the role of LARP7 in tumor susceptibility.
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Discapacidades del Desarrollo/genética , Enanismo/genética , Discapacidad Intelectual/genética , Proteínas Proto-Oncogénicas B-raf/genética , Ribonucleoproteínas/genética , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genética , Mutación del Sistema de Lectura , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Italia , Masculino , Fenotipo , Cáncer Papilar Tiroideo/diagnóstico por imagen , Cáncer Papilar Tiroideo/patología , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/patología , Secuenciación del Exoma , Adulto JovenRESUMEN
3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.
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Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Biomarcadores , Transporte de Electrón , Metabolismo Energético , Fibroblastos/metabolismo , Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Fenotipo , Transporte de Proteínas , Espasmos Infantiles/diagnóstico , Espasmos Infantiles/genética , Secuenciación del ExomaRESUMEN
Congenital disorders of glycosylation (CDG) are a heterogeneous and rapidly growing group of diseases caused by abnormal glycosylation of proteins and/or lipids. Mutations in genes involved in the homeostasis of the endoplasmic reticulum (ER), the Golgi apparatus (GA), and the vesicular trafficking from the ER to the ER-Golgi intermediate compartment (ERGIC) have been found to be associated with CDG. Here, we report a patient with defects in both N- and O-glycosylation combined with a delayed vesicular transport in the GA due to mutations in TRAPPC11, a subunit of the TRAPPIII complex. TRAPPIII is implicated in the anterograde transport from the ER to the ERGIC as well as in the vesicle export from the GA. This report expands the spectrum of genetic alterations associated with CDG, providing new insights for the diagnosis and the understanding of the physiopathological mechanisms underlying glycosylation disorders.
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Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/genética , Mutación , Proteínas de Transporte Vesicular/genética , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Alelos , Sustitución de Aminoácidos , Encéfalo/anomalías , Encéfalo/diagnóstico por imagen , Genotipo , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Fenotipo , Secuenciación Completa del GenomaRESUMEN
Most inborn errors of metabolism (IEM) remain without effective treatment mainly due to the incapacity of conventional therapeutic approaches to target the neurological symptomatology and to ameliorate the multisystemic involvement frequently observed in these patients. However, in recent years, the therapeutic use of small molecules has emerged as a promising approach for treating this heterogeneous group of disorders. In this review, we focus on the use of therapeutically active small molecules to treat IEM, including readthrough agents, pharmacological chaperones, proteostasis regulators, substrate inhibitors, and autophagy inducers. The small molecules reviewed herein act at different cellular levels, and this knowledge provides new tools to set up innovative treatment approaches for particular IEM. We review the molecular mechanism underlying therapeutic properties of small molecules, methodologies used to screen for these compounds, and their applicability in preclinical and clinical practice.
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Errores Innatos del Metabolismo/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Animales , Humanos , Recién Nacido , Tamizaje Neonatal/métodosRESUMEN
Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.
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Aciltransferasas/genética , Lipoilación/genética , Oxo-Ácido-Liasas/genética , Acidosis Láctica/genética , Acidosis Láctica/mortalidad , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Metabolismo Energético/genética , Femenino , Humanos , Recién Nacido , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación Missense , Complejo Piruvato Deshidrogenasa/genética , Ácido Tióctico/metabolismoRESUMEN
Gaucher's disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-ß-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-ß-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-ß-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.
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1-Desoxinojirimicina/análogos & derivados , Adamantano/análogos & derivados , Enfermedad de Gaucher/tratamiento farmacológico , Células Madre Pluripotentes Inducidas/efectos de los fármacos , 1-Desoxinojirimicina/farmacología , Adamantano/farmacología , Antígenos de Diferenciación/metabolismo , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , Análisis Mutacional de ADN , Neuronas Dopaminérgicas/enzimología , Evaluación Preclínica de Medicamentos , Estabilidad de Enzimas/efectos de los fármacos , Enfermedad de Gaucher/patología , Expresión Génica , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/fisiología , Factor 4 Similar a Kruppel , Lisosomas/enzimología , Macrófagos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transporte de Proteínas , Bibliotecas de Moléculas Pequeñas , TranscriptomaRESUMEN
Coenzyme Q10 (CoQ10) plays a key role in the exchange of electrons in lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of intra-lysosomal medium, which is essential for the proteolytic function of hydrolases responsible -when deficient- of a wide range of inherited lysosomal diseases such as Sanfilippo syndromes. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail (α-tocopherol, N-acetylcysteine and α-lipoic acid) were able to ameliorate the biochemical phenotype in cultured fibroblasts of Sanfilippo patients. Basal CoQ10 was analyzed in fibroblasts and Sanfilippo A patients showed decreased basal levels. However, no dysfunction in the CoQ10 biosynthesis pathways was found, revealing for the first time a secondary CoQ10 deficiency in Sanfilippo A fibroblasts. Cultured fibroblasts from five patients affected by Sanfilippo A and B diseases were treated with CoQ10 and an antioxidant cocktail. Upon CoQ10 treatment, none of the Sanfilippo A fibroblasts increased their residual enzymatic activity, but the two Sanfilippo B cell lines showed a statistically significant increase of their residual activity. The antioxidant treatment had no effect on the residual activity in all tested cell lines. Moreover, one Sanfilippo A and two Sanfilippo B fibroblasts showed a statistically significant reduction of glycosaminoglycans accumulation both, after 50 µmol/L CoQ10 and antioxidant treatment. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results are encouraging as some cellular alterations observed in Sanfilippo syndrome can be partially restored by CoQ10 or other antioxidant treatment in some patients.
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Antioxidantes/uso terapéutico , Fibroblastos/efectos de los fármacos , Mucopolisacaridosis III/tratamiento farmacológico , Ubiquinona/análogos & derivados , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Exocitosis , Glicosaminoglicanos/metabolismo , Humanos , Lisosomas/metabolismo , Mucopolisacaridosis III/metabolismo , Mucopolisacaridosis III/patología , Ubiquinona/uso terapéuticoRESUMEN
Background: The genetic diagnosis of mitochondrial disorders is complicated by its genetic and phenotypic complexity. Next generation sequencing techniques have much improved the diagnostic yield for these conditions. A cohort of individuals with multiple respiratory chain deficiencies, reported in the literature 10 years ago, had a diagnostic rate of 60% by whole exome sequencing (WES) but 40% remained undiagnosed. Objective: We aimed to identify a genetic diagnosis by reanalysis of the WES data for the undiagnosed arm of this 10-year-old cohort of patients with suspected mitochondrial disorders. Methods: The WES data was transferred and processed by the RD-Connect Genome-Phenome Analysis Platform (GPAP) using their standardized pipeline. Variant prioritisation was carried out on the RD-Connect GPAP. Results: Singleton WES data from 14 individuals was reanalysed. We identified a possible or likely genetic diagnosis in 8 patients (8/14, 57%). The variants identified were in a combination of mitochondrial DNA (nâ=â1, MT-TN), nuclear encoded mitochondrial genes (nâ=â2, PDHA1, and SUCLA2) and nuclear genes associated with nonmitochondrial disorders (nâ=â5, PNPLA2, CDC40, NBAS and SLC7A7). Variants in both the NBAS and CDC40 genes were established as disease causing after the original cohort was published. We increased the diagnostic yield for the original cohort by 15% without generating any further genomic data. Conclusions: In the era of multiomics we highlight that reanalysis of existing WES data is a valid tool for generating additional diagnosis in patients with suspected mitochondrial disease, particularly when more time has passed to allow for new bioinformatic pipelines to emerge, for the development of new tools in variant interpretation aiding in reclassification of variants and the expansion of scientific knowledge on additional genes.
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Secuenciación del Exoma , Enfermedades Mitocondriales , Humanos , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/diagnóstico , Secuenciación del Exoma/métodos , Niño , Masculino , Femenino , Estudios de Cohortes , ADN Mitocondrial/genéticaRESUMEN
Rare diseases (RD) have a prevalence of not more than 1/2000 persons in the European population, and are characterised by the difficulty experienced in obtaining a correct and timely diagnosis. According to Orphanet, 72.5% of RD have a genetic origin although 35% of them do not yet have an identified causative gene. A significant proportion of patients suspected to have a genetic RD receive an inconclusive exome/genome sequencing. Working towards the International Rare Diseases Research Consortium (IRDiRC)'s goal for 2027 to ensure that all people living with a RD receive a diagnosis within one year of coming to medical attention, the Solve-RD project aims to identify the molecular causes underlying undiagnosed RD. As part of this strategy, we developed a phenotypic similarity-based variant prioritization methodology comparing submitted cases with other submitted cases and with known RD in Orphanet. Three complementary approaches based on phenotypic similarity calculations using the Human Phenotype Ontology (HPO), the Orphanet Rare Diseases Ontology (ORDO) and the HPO-ORDO Ontological Module (HOOM) were developed; genomic data reanalysis was performed by the RD-Connect Genome-Phenome Analysis Platform (GPAP). The methodology was tested in 4 exemplary cases discussed with experts from European Reference Networks. Variants of interest (pathogenic or likely pathogenic) were detected in 8.8% of the 725 cases clustered by similarity calculations. Diagnostic hypotheses were validated in 42.1% of them and needed further exploration in another 10.9%. Based on the promising results, we are devising an automated standardized phenotypic-based re-analysis pipeline to be applied to the entire unsolved cases cohort.
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Genómica , Enfermedades Raras , Humanos , Enfermedades Raras/diagnóstico , Enfermedades Raras/epidemiología , Enfermedades Raras/genética , Fenotipo , Mapeo CromosómicoRESUMEN
Congenital myopathies (CMs) are rare genetic disorders for which the diagnostic yield does not typically exceed 60% . We performed deep phenotyping, histopathological studies, clinical exome and trio genome sequencing and a phenotype-driven analysis of the genomic data, that led to the molecular diagnosis in a child with CM. We identified a heterozygous variant in RYR1 in the affected child, inherited from her asymptomatic mother. Given the alignment of the clinical and histopathological phenotype with RYR1-CM, we considered the potential existence of a missing second variant in trans in the proband, but also hypothesized that the variant might be mosaic in the mother, as subsequently demonstrated. Our study is an example of how heterozygous variants inherited from asymptomatic parents are frequently dismissed. When the genotype-phenotype correlation is strong, it is recommended to consider a parental mosaicism.
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Mosaicismo , Fenotipo , Canal Liberador de Calcio Receptor de Rianodina , Humanos , Estudios de Asociación Genética , Miotonía Congénita/genética , Miotonía Congénita/diagnóstico , Canal Liberador de Calcio Receptor de Rianodina/genética , Masculino , PreescolarRESUMEN
Recessive desminopathies are rare and often present as severe early-onset myopathy. Here we report a milder phenotype in three unrelated patients from southern India (2 M, 1F) aged 16, 21, and 22 years, who presented with childhood-onset, gradually progressive, fatigable limb-girdle weakness, ptosis, speech and swallowing difficulties, without cardiac involvement. Serum creatine kinase was elevated, and repetitive nerve stimulation showed decrement in all. Clinical improvement was noted with pyridostigmine and salbutamol in two patients. All three patients had a homozygous substitution in intron 5: DES(NM_001927.4):c.1023+5G>A, predicted to cause a donor splice site defect. Muscle biopsy with ultrastructural analysis suggested myopathy with myofibrillar disarray, and immunohistochemistry showed partial loss of desmin with some residual staining, while western blot analysis showed reduced desmin. RT-PCR of patient muscle RNA revealed two transcripts: a reduced normal desmin transcript and a larger abnormal transcript suggesting leaky splicing at the intron 5 donor site. Sequencing of the PCR products confirmed the inclusion of intron 5 in the longer transcript, predicted to cause a premature stop codon. Thus, we provide evidence for a leaky splice site causing partial loss of desmin associated with a unique phenotypic presentation of a milder form of desmin-related recessive myopathy overlapping with congenital myasthenic syndrome.
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Desmina , Humanos , Masculino , Desmina/genética , Desmina/metabolismo , Femenino , Adulto Joven , Adolescente , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Músculo Esquelético/metabolismo , Sitios de Empalme de ARN/genética , Transmisión Sináptica , Fenotipo , MutaciónRESUMEN
BACKGROUND: Congenital myasthenic syndromes (CMS) are a group of inherited neuromuscular junction (NMJ) disorders arising from gene variants encoding diverse NMJ proteins. Recently, the VAMP1 gene, responsible for encoding the vesicle-associated membrane protein 1 (VAMP1), has been associated with CMS. METHODS: This study presents a characterization of five new individuals with VAMP1-related CMS, providing insights into the phenotype. RESULTS: The individuals with VAMP1-related CMS exhibited early disease onset, presenting symptoms prenatally or during the neonatal period, alongside severe respiratory involvement and feeding difficulties. Generalized weakness at birth was a common feature, and none of the individuals achieved independent walking ability. Notably, all cases exhibited scoliosis. The clinical course remained stable, without typical exacerbations seen in other CMS types. The response to anticholinesterase inhibitors and salbutamol was only partial, but the addition of 3,4-diaminopyridine (3,4-DAP) led to significant and substantial improvements, suggesting therapeutic benefits of 3,4-DAP for managing VAMP1-related CMS symptoms. Noteworthy is the identification of the VAMP1 (NM_014231.5): c.340delA; p.Ile114SerfsTer72 as a founder variant in the Iberian Peninsula and Latin America. CONCLUSIONS: This study contributes valuable insights into VAMP1-related CMS, emphasizing their early onset, arthrogryposis, facial and generalized weakness, respiratory involvement, and feeding difficulties. Furthermore, the potential efficacy of 3,4-DAP as a useful therapeutic option warrants further exploration. The findings have implications for clinical management and genetic counseling in affected individuals. Additional research is necessary to elucidate the long-term outcomes of VAMP1-related CMS.
Asunto(s)
Amifampridina , Síndromes Miasténicos Congénitos , Fenotipo , Proteína 1 de Membrana Asociada a Vesículas , Humanos , Síndromes Miasténicos Congénitos/tratamiento farmacológico , Síndromes Miasténicos Congénitos/genética , Síndromes Miasténicos Congénitos/fisiopatología , Femenino , Masculino , Amifampridina/farmacología , Proteína 1 de Membrana Asociada a Vesículas/genética , Niño , Adolescente , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacología , 4-Aminopiridina/uso terapéutico , Preescolar , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/uso terapéutico , LactanteRESUMEN
Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.