RESUMO
Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.
Assuntos
Transtorno do Espectro Autista , Síndrome do Cromossomo X Frágil , Doenças Mitocondriais , Humanos , Proteína do X Frágil da Deficiência Intelectual/genética , Transtorno do Espectro Autista/metabolismo , Neurônios/metabolismo , Neurogênese , Doenças Mitocondriais/metabolismo , Receptores de Quinase C Ativada/genética , Receptores de Quinase C Ativada/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.
Assuntos
Esmalte Dentário , Odontogênese , Dente , Humanos , Ameloblastos/metabolismo , Amelogênese/genéticaRESUMO
The dorsolateral prefrontal cortex (dlPFC) is an evolutionarily derived cortical region in primates critical for high-level cognitive functions and implicated in various neuropsychiatric disorders. The cells that compose the dlPFC, especially excitatory and inhibitory neurons, undergo extensive maturation throughout midfetal and late-fetal development, during which critical neurodevelopmental events, such as circuit assembly and electrophysiological maturation of neurons, occur. Despite the relevance of neuronal maturation in several neurodevelopmental and psychiatric disorders, the molecular mechanisms underlying this process remain largely unknown. Here, we performed an integrated Patch-seq and single-nucleus multiomic analysis of the rhesus macaque dlPFC to identify genes governing neuronal maturation from midfetal to late-fetal development. Our multimodal analysis identified gene pathways and regulatory networks important for the maturation of distinct neuronal populations, including upper-layer intratelencephalicprojecting neurons. We identified genes underlying the maturation of specific electrophysiological properties of these neurons. Furthermore, gene knockdown in organotypic slices revealed that RAPGEF4 regulates the maturation of resting membrane potential and inward sodium current. Using this strategy, we also found that the knockdown of CHD8, a high-confidence autism spectrum disorder risk gene, in human slices led to deficits in neuronal maturation, via the downstream downregulation of several key genes, including RAPGEF4. Our study revealed novel regulators of neuronal maturation during a critical period of prefrontal development in primates and implicated such regulators in molecular processes underlying autism.
RESUMO
Debates about the ethics of human brain organoids have proceeded without the input of individuals whose brains are being modeled. Interviews with donors of biospecimens for brain organoid research revealed overall enthusiasm for brain organoids as a tool for biomedical discovery, alongside a desire for ongoing engagement with research teams to learn the results of the research, to allow transfer of decision-making authority over time, and to ensure ethical boundaries are not crossed. Future work is needed to determine the most feasible and resource-efficient way to longitudinally engage donors participating in brain organoid research.
Assuntos
Bancos de Espécimes Biológicos , Pesquisa Biomédica , Humanos , Doadores de Tecidos , Encéfalo , Organoides , Consentimento Livre e EsclarecidoRESUMO
BACKGROUND: Joubert syndrome (JS) is a neurodevelopmental ciliopathy characterised by a distinctive mid-hindbrain malformation, the 'molar tooth sign'. Over 40 JS-associated genes are known, accounting for two-thirds of cases. METHODS: While most variants are novel or extremely rare, we report on 11 recurring variants in seven genes, including three known 'founder variants' in the Ashkenazi Jewish, Hutterite and Finnish populations. We evaluated variant frequencies in ~550 European patients with JS and compared them with controls (>15 000 Italian plus gnomAD), and with an independent cohort of ~600 JS probands from the USA. RESULTS: All variants were markedly enriched in the European JS cohort compared with controls. When comparing allele frequencies in the two JS cohorts, the Ashkenazim founder variant (TMEM216 c.218G>T) was significantly enriched in American compared with European patients with JS, while MKS1 c.1476T>G was about 10 times more frequent among European JS. Frequencies of other variants were comparable in the two cohorts. Genotyping of several markers identified four novel European founder haplotypes.Two recurrent variants (MKS1 c.1476T>G and KIAA0586 c.428delG), have been detected in homozygosity in unaffected individuals, suggesting they could act as hypomorphic variants. However, while fibroblasts from a MKS1 c.1476T>G healthy homozygote showed impaired ability to form primary cilia and mildly reduced ciliary length, ciliary parameters were normal in cells from a KIAA0586 c.428delG healthy homozygote. CONCLUSION: This study contributes to understand the complex genetic landscape of JS, explain its variable prevalence in distinct geographical areas and characterise two recurrent hypomorphic variants.
Assuntos
Anormalidades Múltiplas , Anormalidades do Olho , Doenças Renais Císticas , Humanos , Cerebelo/anormalidades , Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Doenças Renais Císticas/genética , Retina/anormalidadesRESUMO
This is the first case report of sleep-disordered breathing in patients with pontine tegmental cap dysplasia, a very rare neurological disorder characterized by an anatomic malformation in the pons. Patients present with hypotonia, cognitive dysfunction, and cranial nerve palsies (eg, hearing loss, trigeminal anesthesia, and swallow dysfunction). Extensive studies have demonstrated the relevance of different pontine neuronal nuclei in breathing regulation, which are structurally abnormal in pontine tegmental cap dysplasia. We present detailed polysomnography data for 3 patients aged 41 years, 20 years, and 1.5 years revealing significant central sleep apnea. We discuss our experience with managing their sleep-disordered breathing in the setting of multiple cranial nerve palsies and corneal anesthesia, and its relative contraindication of noninvasive positive pressure ventilation treatment. CITATION: Ju Wang J-D, Doherty D, Ramirez J-M, Chen M. Central sleep apnea in patients with pontine tegmental cap dysplasia treated with supplemental oxygen: a case report. J Clin Sleep Med. 2023;19(4):843-849.
Assuntos
Anormalidades Múltiplas , Doenças dos Nervos Cranianos , Apneia do Sono Tipo Central , Humanos , Apneia do Sono Tipo Central/complicações , Apneia do Sono Tipo Central/terapia , Respiração com Pressão Positiva , OxigênioRESUMO
Dysfunction of the primary cilium, a microtubule-based signaling organelle, leads to genetic conditions called ciliopathies. Hedgehog (Hh) signaling is mediated by the primary cilium in vertebrates and is therefore implicated in ciliopathies; however, it is not clear which immortal cell lines are the most appropriate for modeling pathway response in human disease; therefore, we systematically evaluated Hh in five commercially available, immortal mammalian cell lines: ARPE-19, HEK293T, hTERT RPE-1, NIH/3T3, and SH-SY5Y. Under proper conditions, all of the cell lines ciliated adequately for our subsequent experiments, except for SH-SY5Y which were excluded from further analysis. hTERT RPE-1 and NIH/3T3 cells relocalized Hh pathway components Smoothened (SMO) and GPR161 and upregulated Hh target genes in response to pathway stimulation. In contrast, pathway stimulation did not induce target gene expression in ARPE-19 and HEK293T cells, despite SMO and GPR161 relocalization. These data indicate that human hTERT RPE-1 cells and murine NIH/3T3 cells, but not ARPE-19 and HEK293T cells, are suitable for modeling the role of Hh signaling in ciliopathies.
Assuntos
Proteínas Hedgehog , Neuroblastoma , Humanos , Animais , Camundongos , Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Cílios/metabolismo , Células HEK293 , Neuroblastoma/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Mamíferos/metabolismoRESUMO
The Joubert syndrome (JS), Meckel syndrome (MKS), and nephronophthisis (NPH) ciliopathy spectrum could be the poster child for advances and challenges in Mendelian human genetics over the past half century. Progress in understanding these conditions illustrates many core concepts of human genetics. The JS phenotype alone is caused by pathogenic variants in more than 40 genes; remarkably, all of the associated proteins function in and around the primary cilium. Primary cilia are near-ubiquitous, microtubule-based organelles that play crucial roles in development and homeostasis. Protruding from the cell, these cellular antennae sense diverse signals and mediate Hedgehog and other critical signaling pathways. Ciliary dysfunction causes many human conditions termed ciliopathies, which range from multiple congenital malformations to adult-onset single-organ failure. Research on the genetics of the JS-MKS-NPH spectrum has spurred extensive functional work exploring the broadly important role of primary cilia in health and disease. This functional work promises to illuminate the mechanisms underlying JS-MKS-NPH in humans, identify therapeutic targets across genetic causes, and generate future precision treatments.
Assuntos
Anormalidades Múltiplas , Ciliopatias , Anormalidades do Olho , Doenças Renais Policísticas , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Cerebelo/anormalidades , Cerebelo/metabolismo , Cerebelo/patologia , Criança , Cílios/genética , Cílios/metabolismo , Cílios/patologia , Transtornos da Motilidade Ciliar , Ciliopatias/genética , Ciliopatias/metabolismo , Ciliopatias/patologia , Encefalocele , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Anormalidades do Olho/patologia , Proteínas Hedgehog/metabolismo , Humanos , Doenças Renais Císticas , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Retina/anormalidades , Retina/metabolismo , Retina/patologia , Retinose PigmentarRESUMO
Joubert syndrome (JS), a well-established ciliopathy, is characterized by the distinctive molar tooth sign on brain MRI, ataxia, and neurodevelopmental features. Other manifestations can include polydactyly, accessory frenula, renal, or liver disease. Here, we report individuals meeting criteria for JS with de novo heterozygous variants in SLC30A7 (Chr1p21.2). The first individual is a female with history of unilateral postaxial polydactyly, classic molar tooth sign on MRI, macrocephaly, ataxia, ocular motor apraxia, neurodevelopmental delay, and precocious puberty. Exome sequencing detected a de novo heterozygous missense variant in SLC30A7: NM_133496.5: c.407 T > C, (p.Val136Ala). The second individual had bilateral postaxial polydactyly, molar tooth sign, macrocephaly, developmental delay, and an extra oral frenulum. A de novo deletion-insertion variant in SLC30A7, c.490_491delinsAG (p.His164Ser) was found. Both de novo variants affect highly conserved residues. Variants were not identified in known Joubert genes for either case. SLC30A7 has not yet been associated with a human phenotype. The SLC30 family of zinc transporters, like SLC30A7, permit cellular efflux of zinc, and although it is expressed in the brain its functions remain unknown. Published data from proteomic studies support SLC30A7 interaction with TCTN3, another protein associated with JS. The potential involvement of such genes in primary cilia suggest a role in Sonic Hedgehog signaling. SLC30A7 is a candidate JS-associated gene. Future work could be directed toward further characterization of SLC30A7 variants and understanding its function.
Assuntos
Anormalidades Múltiplas , Proteínas de Transporte de Cátions/genética , Anormalidades do Olho , Doenças Renais Císticas , Megalencefalia , Polidactilia , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Ataxia , Cerebelo/anormalidades , Cerebelo/diagnóstico por imagem , Anormalidades do Olho/diagnóstico , Anormalidades do Olho/genética , Feminino , Proteínas Hedgehog , Humanos , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Proteômica , Retina/anormalidades , ZincoRESUMO
PURPOSE: Genome-wide sequencing is increasingly being performed during pregnancy to identify the genetic cause of congenital anomalies. The interpretation of prenatally identified variants can be challenging and is hampered by our often limited knowledge of prenatal phenotypes. To better delineate the prenatal phenotype of Coffin-Siris syndrome (CSS), we collected clinical data from patients with a prenatal phenotype and a pathogenic variant in one of the CSS-associated genes. METHODS: Clinical data was collected through an extensive web-based survey. RESULTS: We included 44 patients with a variant in a CSS-associated gene and a prenatal phenotype; 9 of these patients have been reported before. Prenatal anomalies that were frequently observed in our cohort include hydrocephalus, agenesis of the corpus callosum, hypoplastic left heart syndrome, persistent left vena cava, diaphragmatic hernia, renal agenesis, and intrauterine growth restriction. Anal anomalies were frequently identified after birth in patients with ARID1A variants (6/14, 43%). Interestingly, pathogenic ARID1A variants were much more frequently identified in the current prenatal cohort (16/44, 36%) than in postnatal CSS cohorts (5%-9%). CONCLUSION: Our data shed new light on the prenatal phenotype of patients with pathogenic variants in CSS genes.
Assuntos
Deformidades Congênitas da Mão , Deficiência Intelectual , Micrognatismo , Anormalidades Múltiplas , Proteínas Cromossômicas não Histona/genética , Face/anormalidades , Estudos de Associação Genética , Deformidades Congênitas da Mão/genética , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Micrognatismo/genética , Pescoço/anormalidades , FenótipoRESUMO
AIM: To clarify the extent to which medical comorbidities and goals-of-care decisions influence death among individuals with childhood-onset hydrocephalus. METHOD: This was a retrospective cohort study of 1705 individuals (759 males, 946 females, mean age 11y 5mo, SD 6y 6mo, range 0-37y 7mo at last follow-up) with childhood-onset hydrocephalus, of whom 88 (5.2%) were deceased. Existing medical records, death records, and publicly available internet sources were analyzed. We estimated hazard ratios for putative risk factors through Cox regression based upon 10 529 person-years of data and quantitatively and qualitatively analyzed the circumstances surrounding each death. RESULTS: Mortality did not differ statistically by demographic factors, although higher proportions of non-White and Hispanic individuals were deceased. Most deaths were related to medical comorbidities rather than hydrocephalus itself. Of the 14 deaths directly related to hydrocephalus, seven were caused by shunt complications and four occurred after decisions to forgo treatment, apparently in response to poor outcomes predicted by the medical team. Half the deaths were preceded by shifts to comfort-based care; however, these decisions appeared to substantially change the patient's clinical trajectory only half the time. INTERPRETATION: Children are more likely to die with, rather than from, hydrocephalus. Our results emphasize the complexities of medical decision-making and the influence of clinicians in guiding these choices.
Assuntos
Hidrocefalia/mortalidade , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%.In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. METHODS: We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. RESULTS: Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. CONCLUSION: Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk.
Assuntos
Anormalidades Múltiplas , Ataxia Cerebelar , Anormalidades do Olho , Deficiência Intelectual , Doenças Renais Císticas , Anormalidades Múltiplas/genética , Ataxia Cerebelar/genética , Cerebelo/anormalidades , Cerebelo/diagnóstico por imagem , Anormalidades do Olho/genética , Haploinsuficiência/genética , Humanos , Deficiência Intelectual/genética , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Masculino , Fenótipo , Proteínas Repressoras/genética , Retina/anormalidadesRESUMO
Primary ciliopathies are heterogenous disorders resulting from perturbations in primary cilia form and/or function. Primary cilia are cellular organelles which mediate key signaling pathways during development, such as the sonic hedgehog (SHH) pathway which is required for neuroepithelium and central nervous system development. Joubert syndrome is a primary ciliopathy characterized by cerebellar/brain stem malformation, hypotonia, and developmental delays. At least 35 genes are associated with Joubert syndrome, including the gene KIAA0753, which is part of a complex required for primary ciliogenesis. The phenotypic spectrum associated with biallelic pathogenic variants in KIAA0753 is broad and not well-characterized. We describe four individuals with biallelic pathogenic KIAA0753 variants, including five novel variants. We report in vitro results assessing the function of each variant indicating that mutant proteins are not fully competent to promote primary ciliogenesis. Ablation of KIAA0753 in vitro blocks primary ciliogenesis and SHH pathway activity. Correspondingly, KIAA0753 patient fibroblasts have a deficit in primary ciliation and improper SHH and WNT signaling, with a particularly blunted response to SHH pathway stimulation. Our work expands the phenotypic spectrum of KIAA0753 ciliopathies and demonstrates the utility of patient-focused functional assays for proving causality of genetic variants.
Assuntos
Anormalidades Múltiplas , Ciliopatias , Anormalidades do Olho , Doenças Renais Císticas , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Cerebelo/anormalidades , Cílios/genética , Cílios/patologia , Ciliopatias/genética , Ciliopatias/patologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Proteínas Associadas aos Microtúbulos , Retina/anormalidadesRESUMO
Research using human fetal tissue has saved millions of lives through vaccines and other advances, but was markedly restricted by federal regulations in 2019. Although the restrictions were partially reversed in 2021, additional regulatory changes are needed to prevent further damage to essential research programs while preserving protection for human subjects.
Assuntos
Pesquisa Fetal/legislação & jurisprudência , Controle Social Formal , Adulto , Idoso , Feminino , Pesquisa Fetal/ética , Governo , Humanos , Masculino , Pessoa de Meia-Idade , National Institutes of Health (U.S.) , Apoio à Pesquisa como Assunto/economia , Autorrelato , Estados UnidosRESUMO
ARID1B is one of the most frequently mutated genes in intellectual disability (~1%). Most variants are readily classified, since they are de novo and are predicted to lead to loss of function, and therefore classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guidelines for the interpretation of sequence variants. However, familial loss-of-function variants can also occur and can be challenging to interpret. Such variants may be pathogenic with variable expression, causing only a mild phenotype in a parent. Alternatively, since some regions of the ARID1B gene seem to be lacking pathogenic variants, loss-of-function variants in those regions may not lead to ARID1B haploinsufficiency and may therefore be benign. We describe 12 families with potential loss-of-function variants, which were either familial or with unknown inheritance and were in regions where pathogenic variants have not been described or are otherwise challenging to interpret. We performed detailed clinical and DNA methylation studies, which allowed us to confidently classify most variants. In five families we observed transmission of pathogenic variants, confirming their highly variable expression. Our findings provide further evidence for an alternative translational start site and we suggest updates for the ACMG guidelines for the interpretation of sequence variants to incorporate DNA methylation studies and facial analyses.
Assuntos
Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Deficiência Intelectual/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/epidemiologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/fisiopatologia , Adolescente , Adulto , Criança , Face/anormalidades , Feminino , Regulação da Expressão Gênica/genética , Deformidades Congênitas da Mão/epidemiologia , Deformidades Congênitas da Mão/genética , Deformidades Congênitas da Mão/fisiopatologia , Humanos , Deficiência Intelectual/epidemiologia , Deficiência Intelectual/fisiopatologia , Mutação com Perda de Função/genética , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
Despite widespread clinical genetic testing, many individuals with suspected genetic conditions lack a precise diagnosis, limiting their opportunity to take advantage of state-of-the-art treatments. In some cases, testing reveals difficult-to-evaluate structural differences, candidate variants that do not fully explain the phenotype, single pathogenic variants in recessive disorders, or no variants in genes of interest. Thus, there is a need for better tools to identify a precise genetic diagnosis in individuals when conventional testing approaches have been exhausted. We performed targeted long-read sequencing (T-LRS) using adaptive sampling on the Oxford Nanopore platform on 40 individuals, 10 of whom lacked a complete molecular diagnosis. We computationally targeted up to 151 Mbp of sequence per individual and searched for pathogenic substitutions, structural variants, and methylation differences using a single data source. We detected all genomic aberrations-including single-nucleotide variants, copy number changes, repeat expansions, and methylation differences-identified by prior clinical testing. In 8/8 individuals with complex structural rearrangements, T-LRS enabled more precise resolution of the mutation, leading to changes in clinical management in one case. In ten individuals with suspected Mendelian conditions lacking a precise genetic diagnosis, T-LRS identified pathogenic or likely pathogenic variants in six and variants of uncertain significance in two others. T-LRS accurately identifies pathogenic structural variants, resolves complex rearrangements, and identifies Mendelian variants not detected by other technologies. T-LRS represents an efficient and cost-effective strategy to evaluate high-priority genes and regions or complex clinical testing results.
Assuntos
Aberrações Cromossômicas , Análise Citogenética/métodos , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Genoma Humano , Mutação , Variações do Número de Cópias de DNA , Feminino , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Cariotipagem , Masculino , Análise de Sequência de DNARESUMO
The human neonatal cerebellum is one-fourth of its adult size yet contains the blueprint required to integrate environmental cues with developing motor, cognitive and emotional skills into adulthood. Although mature cerebellar neuroanatomy is well studied, understanding of its developmental origins is limited. In this study, we systematically mapped the molecular, cellular and spatial composition of human fetal cerebellum by combining laser capture microscopy and SPLiT-seq single-nucleus transcriptomics. We profiled functionally distinct regions and gene expression dynamics within cell types and across development. The resulting cell atlas demonstrates that the molecular organization of the cerebellar anlage recapitulates cytoarchitecturally distinct regions and developmentally transient cell types that are distinct from the mouse cerebellum. By mapping genes dominant for pediatric and adult neurological disorders onto our dataset, we identify relevant cell types underlying disease mechanisms. These data provide a resource for probing the cellular basis of human cerebellar development and disease.
Assuntos
Cerebelo/embriologia , Neurogênese , Feto , Humanos , Microdissecção e Captura a Laser , Análise de Célula Única , TranscriptomaRESUMO
The Joubert-Meckel syndrome spectrum is a continuum of recessive ciliopathy conditions caused by primary cilium dysfunction. The primary cilium is a microtubule-based, antenna-like organelle that projects from the surface of most human cell types, allowing them to respond to extracellular signals. The cilium is partitioned from the cell body by the transition zone, a known hotspot for ciliopathy-related proteins. Despite years of Joubert syndrome (JBTS) gene discovery, the genetic cause cannot be identified in up to 30% of individuals with JBTS, depending on the cohort, sequencing method, and criteria for pathogenic variants. Using exome and targeted sequencing of 655 families with JBTS, we identified three individuals from two families harboring biallelic, rare, predicted-deleterious missense TMEM218 variants. Via MatchMaker Exchange, we identified biallelic TMEM218 variants in four additional families with ciliopathy phenotypes. Of note, four of the six families carry missense variants affecting the same highly conserved amino acid position 115. Clinical features included the molar tooth sign (N = 2), occipital encephalocele (N = 5, all fetuses), retinal dystrophy (N = 4, all living individuals), polycystic kidneys (N = 2), and polydactyly (N = 2), without liver involvement. Combined with existing functional data linking TMEM218 to ciliary transition zone function, our human genetic data make a strong case for TMEM218 dysfunction as a cause of ciliopathy phenotypes including JBTS with retinal dystrophy and Meckel syndrome. Identifying all genetic causes of the Joubert-Meckel spectrum enables diagnostic testing, prognostic and recurrence risk counseling, and medical monitoring, as well as work to delineate the underlying biological mechanisms and identify targets for future therapies.