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PURPOSE: To summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS. METHODS: Phenotypic characterisation was performed through an international and multicentre collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG. RESULTS: Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurological, immunological and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis was not consistently abnormal. CONCLUSION: The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry can be used as a reliable biochemical test for screening and confirmation of disease.
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WAC-related intellectual disability (ID) is a rare genetic condition characterized by a spectrum of neurodevelopmental disorders of varying severity, including global developmental delay (GDD), ID, and autism spectrum disorder. Here, we describe five affected individuals, age range 9-20 years, and provide proof of pathogenicity of a novel splicing variant. All individuals presented with GDD, some degree of ID, and variable dysmorphism. Except for feeding difficulties, all patients were healthy without major congenital malformations or medical comorbidities. All individuals were heterozygous for de novo, previously unreported, loss of function variants in WAC. Three unrelated patients from different ethnic backgrounds shared the intronic variant c.381+4_381+7delAGTA, which was predicted to alter splicing and was initially classified as a variant of uncertain significance. Reverse transcription-polymerase chain reaction analysis from one patient's cells confirmed aberrant splicing of the WAC transcript resulting in premature termination and a truncated protein p.(Gly92Alafs*2). These functional studies and the identification of several nonrelated individuals provide sufficient evidence to classify this variant as pathogenic. The clinical description of these five individuals and the three novel variants expand the genotypic and phenotypic spectrum of this ultrarare disease.
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Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Adulto , Trastorno del Espectro Autista/diagnóstico , Trastorno del Espectro Autista/genética , Niño , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Mutación , Adulto JovenRESUMEN
Smithies et al. (1985) and Jasin and colleagues (1994) provided proof of concept that homologous recombination (HR) could be applied to the treatment of human disease and that its efficiency could be improved by the induction of double-strand breaks (DSBs). A key advance was the discovery of engineered nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs), that can generate site-specific DSBs. The democratization and widespread use of genome editing was enabled by the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease system. While genome editing using ZFNs and TALENs has already reached clinical trials, the pace at which genome editing enters human trials is bound to accelerate in the next several years with multiple promising preclinical studies heralding cures for monogenic diseases that are currently difficult to manage or even incurable. Here we review recent advances and current limitations and discuss the path forward using genome editing to understand, treat, and cure genetic diseases.
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Sistemas CRISPR-Cas , Edición Génica , Recombinación Homóloga , Animales , Roturas del ADN , Reparación del ADN , Edición Génica/historia , Edición Génica/métodos , Marcación de Gen , Ingeniería Genética , Terapia Genética , Genómica/métodos , Historia del Siglo XX , Historia del Siglo XXI , Humanos , ARN Guía de Kinetoplastida/genéticaRESUMEN
ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD+ to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals' fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.
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Ataxia Cerebelosa/genética , Discapacidades del Desarrollo/genética , Glicósido Hidrolasas/genética , Mutación/genética , Enfermedades Neurodegenerativas/genética , ADP-Ribosilación/genética , Adenosina Difosfato Ribosa/genética , Adolescente , Alelos , Niño , Preescolar , Exoma/genética , Femenino , Humanos , Lactante , Masculino , Malformaciones del Sistema Nervioso/genética , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
The signals regulating stem cell activation during tissue regeneration remain poorly understood. We investigated the baldness associated with mutations in the voltage-gated calcium channel (VGCC) Cav1.2 underlying Timothy syndrome (TS). While hair follicle stem cells express Cav1.2, they lack detectable voltage-dependent calcium currents. Cav1.2(TS) acts in a dominant-negative manner to markedly delay anagen, while L-type channel blockers act through Cav1.2 to induce anagen and overcome the TS phenotype. Cav1.2 regulates production of the bulge-derived BMP inhibitor follistatin-like1 (Fstl1), derepressing stem cell quiescence. Our findings show how channels act in nonexcitable tissues to regulate stem cells and may lead to novel therapeutics for tissue regeneration.
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Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Calcio/metabolismo , Folículo Piloso/citología , Células Madre/citología , Células Madre/metabolismo , Animales , Trastorno Autístico , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Señalización del Calcio/efectos de los fármacos , Proteínas Relacionadas con la Folistatina/biosíntesis , Proteínas Relacionadas con la Folistatina/metabolismo , Síndrome de QT Prolongado/metabolismo , Ratones , Células Madre/efectos de los fármacos , Sindactilia/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a fatal disorder characterized by progressive gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, skeletal myopathy, ophthalmoparesis, and ptosis. MNGIE stems from deficient thymidine phosphorylase activity (TP) leading to toxic elevations of plasma thymidine. Hematopoietic stem cell transplant (HSCT) restores TP activity and halts disease progression but has high transplant-related morbidity and mortality. Liver transplant (LT) was reported to restore TP activity in two adult MNGIE patients. We report successful LT in four additional MNGIE patients, including a pediatric patient. Our patients were diagnosed between ages 14 months and 36 years with elevated thymidine levels and biallelic pathogenic variants in TYMP. Two patients presented with progressive gastrointestinal dysmotility, and three demonstrated progressive peripheral neuropathy with two suffering limitations in ambulation. Two patients, including the child, had liver dysfunction and cirrhosis. Following LT, thymidine levels nearly normalized in all four patients and remained low for the duration of follow-up. Disease symptoms stabilized in all patients, with some manifesting improvements, including intestinal function. No patient died, and LT appeared to have a more favorable safety profile than HSCT, especially when liver disease is present. Follow-up studies will need to document the long-term impact of this new approach on disease outcome. Take Home Message: Liver transplantation is effective in stabilizing symptoms and nearly normalizing thymidine levels in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and may have an improved safety profile over hematopoietic stem cell transplant.
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Trasplante de Hígado/métodos , Mitocondrias/metabolismo , Encefalomiopatías Mitocondriales/terapia , Timidina Fosforilasa/genética , Adolescente , Adulto , Trastornos de la Motilidad Esofágica/genética , Femenino , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Lactante , Trasplante de Hígado/mortalidad , Imagen por Resonancia Magnética , Masculino , Mitocondrias/enzimología , Mitocondrias/patología , Encefalomiopatías Mitocondriales/diagnóstico por imagen , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/fisiopatología , Enfermedades del Sistema Nervioso Periférico/genética , Timidina/sangre , Secuenciación del ExomaRESUMEN
Genome editing holds the promise of one-off and potentially curative therapies for many patients with genetic diseases. This is especially true for patients affected by mucopolysaccharidoses as the disease pathophysiology is amenable to correction using multiple approaches. Ex vivo and in vivo genome editing platforms have been tested primarily on MSPI and MPSII, with in vivo approaches having reached clinical testing in both diseases. Though we still await proof of efficacy in humans, the therapeutic tools established for these two diseases should pave the way for other mucopolysaccharidoses. Herein, we review the current preclinical and clinical development studies, using genome editing as a therapeutic approach for these diseases. The development of new genome editing platforms and the variety of genetic modifications possible with each tool provide potential applications of genome editing for mucopolysaccharidoses, which vastly exceed the potential of current approaches. We expect that in a not-so-distant future, more genome editing-based strategies will be established, and individual diseases will be treated through multiple approaches.
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Edición Génica , Mucopolisacaridosis II/genética , Mucopolisacaridosis II/terapia , Mucopolisacaridosis I/genética , Mucopolisacaridosis I/terapia , HumanosRESUMEN
Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2ß, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification.
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Adenosina Trifosfato/metabolismo , Autoantígenos/genética , Ensamble y Desensamble de Cromatina/genética , Discapacidad Intelectual/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Mutación Missense/genética , Anomalías Múltiples/genética , Adolescente , Animales , Núcleo Celular/metabolismo , Niño , Preescolar , ADN Helicasas/genética , Discapacidades del Desarrollo/genética , Exoma/genética , Cara/anomalías , Femenino , Deformidades Congénitas de la Mano/genética , Pérdida Auditiva/genética , Histona Desacetilasa 1/metabolismo , Humanos , Masculino , Megalencefalia/genética , Ratones , Micrognatismo/genética , Cuello/anomalías , Proteínas Nucleares/genética , Síndrome , Factores de Transcripción/genéticaRESUMEN
PURPOSE: Haploinsufficiency of DYRK1A causes a recognizable clinical syndrome. The goal of this paper is to investigate congenital anomalies of the kidney and urinary tract (CAKUT) and genital defects (GD) in patients with DYRK1A variants. METHODS: A large database of clinical exome sequencing (ES) was queried for de novo DYRK1A variants and CAKUT/GD phenotypes were characterized. Xenopus laevis (frog) was chosen as a model organism to assess Dyrk1a's role in renal development. RESULTS: Phenotypic details and variants of 19 patients were compiled after an initial observation that one patient with a de novo pathogenic variant in DYRK1A had GD. CAKUT/GD data were available from 15 patients, 11 of whom presented with CAKUT/GD. Studies in Xenopus embryos demonstrated that knockdown of Dyrk1a, which is expressed in forming nephrons, disrupts the development of segments of embryonic nephrons, which ultimately give rise to the entire genitourinary (GU) tract. These defects could be rescued by coinjecting wild-type human DYRK1A RNA, but not with DYRK1AR205* or DYRK1AL245R RNA. CONCLUSION: Evidence supports routine GU screening of all individuals with de novo DYRK1A pathogenic variants to ensure optimized clinical management. Collectively, the reported clinical data and loss-of-function studies in Xenopus substantiate a novel role for DYRK1A in GU development.
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Discapacidad Intelectual/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Anomalías Urogenitales/genética , Adolescente , Adulto , Animales , Niño , Preescolar , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Exoma/genética , Femenino , Haploinsuficiencia/genética , Humanos , Discapacidad Intelectual/complicaciones , Riñón/anomalías , Riñón/embriología , Masculino , Nefronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Sistema Urinario/embriología , Sistema Urinario/metabolismo , Secuenciación del Exoma/métodos , Xenopus laevis/genética , Xenopus laevis/metabolismo , Adulto Joven , Quinasas DyrKRESUMEN
Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.
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Discapacidades del Desarrollo/genética , Megalencefalia/genética , Mutación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Encéfalo/diagnóstico por imagen , Niño , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/patología , Femenino , Estudios de Asociación Genética , Células HEK293 , Humanos , Inmunoprecipitación , Imagen por Resonancia Magnética , Masculino , Megalencefalia/diagnóstico por imagen , Megalencefalia/patología , Mutagénesis Sitio-Dirigida/métodos , Fosfatidilinositoles/metabolismo , TransfecciónRESUMEN
F-box and leucine-rich repeat protein 4 (FBXL4) is a mitochondrial protein whose exact function is not yet known. However, cellular studies have suggested that it plays significant roles in mitochondrial bioenergetics, mitochondrial DNA (mtDNA) maintenance, and mitochondrial dynamics. Biallelic pathogenic variants in FBXL4 are associated with an encephalopathic mtDNA maintenance defect syndrome that is a multisystem disease characterized by lactic acidemia, developmental delay, and hypotonia. Other features are feeding difficulties, growth failure, microcephaly, hyperammonemia, seizures, hypertrophic cardiomyopathy, elevated liver transaminases, recurrent infections, variable distinctive facial features, white matter abnormalities and cerebral atrophy found in neuroimaging, combined deficiencies of multiple electron transport complexes, and mtDNA depletion. Since its initial description in 2013, 36 different pathogenic variants in FBXL4 were reported in 50 affected individuals. In this report, we present 37 additional affected individuals and 11 previously unreported pathogenic variants. We summarize the clinical features of all 87 individuals with FBXL4-related mtDNA maintenance defect, review FBXL4 structure and function, map the 47 pathogenic variants onto the gene structure to assess the variants distribution, and investigate the genotype-phenotype correlation. Finally, we provide future directions to understand the disease mechanism and identify treatment strategies.
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ADN Mitocondrial/genética , Proteínas F-Box/genética , Estudios de Asociación Genética , Encefalomiopatías Mitocondriales/genética , Ubiquitina-Proteína Ligasas/genética , Acidosis Láctica/genética , Cardiomiopatía Hipertrófica/genética , Predisposición Genética a la Enfermedad , Humanos , Estimación de Kaplan-Meier , Mitocondrias/genética , Encefalomiopatías Mitocondriales/epidemiología , Encefalomiopatías Mitocondriales/patología , Proteínas Mitocondriales/genética , Hipotonía Muscular/genética , Mutación , Fosforilación Oxidativa , Proteoma/genéticaRESUMEN
Mutations in GLI3, which encodes a transcription factor of the Hedgehog signaling pathway, cause several developmental anomalies linked to inappropriate tissue patterning. Here, we report a novel missense variant in the fifth zinc finger domain of GLI3 (c.1826G>A; p.(Cys609Tyr)) initially identified in a proband with preaxial polydactyly type IV, developmental delay, sensorineural hearing loss, skeletal, and genitourinary anomalies. Additional family members exhibited various digital anomalies such as preaxial polydactyly, syndactyly, and postaxial polydactyly either in isolation or combined. Functional studies of Cys609Tyr GLI3 in cultured cells showed abnormal GLI3 processing leading to decreased GLI3 repressor production, increased basal transcriptional activity, and submaximal GLI reporter activity with Hedgehog pathway activation, thus demonstrating an intriguing molecular mechanism for this GLI3-related phenotype. Given the complexity of GLI3 post-translational processing and opposing biological functions as a transcriptional activator and repressor, our findings highlight the importance of performing functional studies of presumed GLI3 variants. This family also demonstrates how GLI3 variants are variably expressed.
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Acrocefalosindactilia/genética , Dedos/anomalías , Proteínas del Tejido Nervioso/genética , Polidactilia/genética , Pulgar/anomalías , Dedos del Pie/anomalías , Proteína Gli3 con Dedos de Zinc/genética , Acrocefalosindactilia/diagnóstico , Secuencia de Aminoácidos , Animales , Preescolar , Femenino , Fibroblastos , Genes Reporteros , Técnicas de Genotipaje , Humanos , Ratones , Mutación Missense , Linaje , Fenotipo , Polidactilia/diagnóstico , Proteínas Represoras/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Transducción de Señal , Dedos de ZincRESUMEN
Pierre Robin sequence (PRS) is an important craniofacial anomaly that can be seen as an isolated finding or manifestation of multiple syndromes. 22q11.2 deletion and Stickler syndrome are cited as the two most common conditions associated with PRS, but their frequencies are debated. We performed a retrospective study of 66 patients with PRS and reviewed their genetic testing, diagnoses, and clinical findings. The case series is complemented by a comprehensive literature review of the nature and frequency of genetic diagnosis in PRS. In our cohort 65% of patients had associated anomalies; of these, a genetic diagnosis was established in 56%. Stickler syndrome was the most common diagnosis, comprising approximately 11% of all cases, followed by Treacher Collins syndrome (9%). The frequency of 22q11.2 deletion was 1.5%. Chromosome arrays, performed for 72% of idiopathic PRS with associated anomalies, revealed two cases of 18q22âqter deletion, a region not previously reported in association with PRS. A review of the cytogenetic anomalies identified in this population supports an association between the 4q33-qter, 17q24.3, 2q33.1, and 11q23 chromosomal loci and PRS. We found a low frequency of 22q11.2 deletion in PRS, suggesting it is less commonly implicated in this malformation. Our data also indicate a higher frequency of cytogenetic anomalies in PRS patients with associated anomalies, and a potential new link with the 18q22âqter locus. The present findings underscore the utility of chromosomal microarrays in cases of PRS with associated anomalies and suggest that delaying testing for apparently isolated cases should be considered.
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Síndrome de Pierre Robin/diagnóstico , Síndrome de Pierre Robin/genética , Aberraciones Cromosómicas , Deleción Cromosómica , Cromosomas Humanos Par 22 , Comorbilidad , Femenino , Pruebas Genéticas , Humanos , Masculino , Fenotipo , Síndrome de Pierre Robin/cirugíaRESUMEN
Costello syndrome (CS) is a multisystem disorder caused by heterozygous germline mutations in the HRAS proto-oncogene. Respiratory system complications have been reported in individuals with CS, but a comprehensive description of the full spectrum and incidence of respiratory symptoms in these patients is not available. Here, we report the clinical course of four CS patients with respiratory complications as a major cause of morbidity. Review of the literature identified 56 CS patients with descriptions of their neonatal course and 17 patients in childhood/adulthood. We found that in the neonatal period, respiratory complications are seen in approximately 78% of patients with transient respiratory distress reported in 45% of neonates. Other more specific respiratory diagnoses were reported in 62% of patients, the majority of which comprised disorders of the upper and lower respiratory tract. Symptoms of upper airway obstruction were reported in CS neonates but were more commonly diagnosed in childhood/adulthood (71%). Analysis of HRAS mutations and their respiratory phenotype revealed that the common p.Gly12Ser mutation is more often associated with transient respiratory distress and other respiratory diagnoses. Respiratory failure and dependence on mechanical ventilation occurs almost exclusively with rare mutations. In cases of prenatally diagnosed CS, the high incidence of respiratory complications in the neonatal period should prompt anticipatory guidance and development of a postnatal management plan. This may be important in cases involving rarer mutations. Furthermore, the high frequency of airway obstruction in CS patients suggests that otorhinolaryngological evaluation and sleep studies should be considered. © 2016 Wiley Periodicals, Inc.
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Síndrome de Costello/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Anomalías del Sistema Respiratorio/genética , Síndrome de Costello/complicaciones , Síndrome de Costello/diagnóstico , Síndrome de Costello/fisiopatología , Femenino , Mutación de Línea Germinal , Humanos , Recién Nacido , Masculino , Fenotipo , Embarazo , Proto-Oncogenes Mas , Anomalías del Sistema Respiratorio/complicaciones , Anomalías del Sistema Respiratorio/diagnóstico , Anomalías del Sistema Respiratorio/fisiopatologíaRESUMEN
Hawkinsinuria is a rare disorder of tyrosine metabolism that can manifest with metabolic acidosis and growth arrest around the time of weaning off breast milk, typically followed by spontaneous resolution of symptoms around 1 year of age. The urinary metabolites hawkinsin, quinolacetic acid, and pyroglutamic acid can aid in identifying this condition, although their relationship to the clinical manifestations is not known. Herein we describe clinical and laboratory findings in two fraternal twins with hawkinsinuria who presented with failure to thrive and metabolic acidosis. Close clinical follow-up and laboratory testing revealed previously unrecognized hypoglycemia, hypophosphatemia, combined hyperlipidemia, and anemia, along with the characteristic urinary metabolites, including massive pyroglutamic aciduria. Treatment with N-acetyl-L-cysteine (NAC) restored normal growth and normalized or improved most biochemical parameters. The dramatic response to NAC therapy supports the idea that glutathione depletion plays a key role in the pathogenesis of hawkinsinuria.
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Acetilcisteína/uso terapéutico , Oxigenasas de Función Mixta/deficiencia , Tirosinemias/tratamiento farmacológico , Acidosis/patología , Errores Innatos del Metabolismo de los Aminoácidos/tratamiento farmacológico , Errores Innatos del Metabolismo de los Aminoácidos/patología , Femenino , Glutatión Sintasa/deficiencia , Humanos , Recién Nacido , Masculino , Fenotipo , Gemelos , Tirosinemias/patologíaRESUMEN
Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.
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Busulfano , Microglía , Progranulinas , Animales , Microglía/metabolismo , Microglía/efectos de los fármacos , Progranulinas/metabolismo , Progranulinas/genética , Ratones , Busulfano/farmacología , Trasplante de Células Madre Hematopoyéticas , Aminopiridinas/farmacología , Encéfalo/metabolismo , Pirroles/farmacología , Ratones Endogámicos C57BL , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Trasplante de Médula Ósea , Masculino , Sistema Nervioso Central/metabolismo , Ratones Noqueados , Acondicionamiento Pretrasplante/métodos , Análisis de la Célula Individual , Citocinas/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidoresRESUMEN
Myhre syndrome (MS, MIM 139210) is a rare multisystemic disorder caused by recurrent pathogenic missense variants in SMAD4. The clinical features have been mainly documented in childhood and comprise variable neurocognitive development, recognizable craniofacial features, a short stature with a pseudo-muscular build, hearing loss, thickened skin, joint limitations, diverse cardiovascular and airway manifestations, and increased fibrosis often following trauma or surgery. In contrast, adults with MS are underreported obscuring potential clinical variability. Here, we describe 24 adults with MS, including 17 diagnosed after the age of 18 years old, and we review the literature on adults with MS. Overall, our cohort shows a milder phenotype as well as lower mortality rates compared to what has been published in literature. Individuals with a codon 500 variant in SMAD4 present with a more pronounced neurodevelopmental and systemic phenotype. However, in contrast to the literature, we observe cardiovascular abnormalities in individuals with the p.(Arg496Cys) variant. In addition, we describe scoliosis as a new manifestation and we report fertility in two additional males with the p.(Arg496Cys). In conclusion, our study contributes novel insights into the clinical variability of MS and underscores the importance of variant-specific considerations, and we provide recommendations for the management of MS in adulthood.
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Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn-/- mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.
RESUMEN
Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.