RESUMEN
Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function (LoF) variation in ZFHX3 as a cause for syndromic intellectual disability (ID). ZFHX3 is a zinc-finger homeodomain transcription factor involved in various biological processes, including cell differentiation and tumorigenesis. We describe 42 individuals with protein-truncating variants (PTVs) or (partial) deletions of ZFHX3, exhibiting variable intellectual disability and autism spectrum disorder, recurrent facial features, relative short stature, brachydactyly, and, rarely, cleft palate. ZFHX3 LoF associates with a specific methylation profile in whole blood extracted DNA. Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation. ZFHX3 was found to interact with the chromatin remodeling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex, suggesting a function in chromatin remodeling and mRNA processing. Furthermore, ChIP-seq for ZFHX3 revealed that it predominantly binds promoters of genes involved in nervous system development. We conclude that loss-of-function variants in ZFHX3 are a cause of syndromic ID associating with a specific DNA methylation profile.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/complicaciones , Haploinsuficiencia/genética , Trastornos del Neurodesarrollo/genética , Encéfalo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismoRESUMEN
OBJECTIVE: Hereditary spastic paraplegia (HSP) is a highly heterogeneous neurologic disorder characterized by lower-extremity spasticity. Here, we set out to determine the genetic basis of an autosomal dominant, pure, and infantile-onset form of HSP in a cohort of 8 patients with a uniform clinical presentation. METHODS: Trio whole-exome sequencing was used in 5 index patients with infantile-onset pure HSP to determine the genetic cause of disease. The functional impact of identified genetic variants was verified using bioinformatics and complementary cellular and biochemical assays. RESULTS: Distinct heterozygous KPNA3 missense variants were found to segregate with the clinical phenotype in 8 patients; in 4 of them KPNA3 variants had occurred de novo. Mutant karyopherin-α3 proteins exhibited a variable pattern of altered expression level, subcellular distribution, and protein interaction. INTERPRETATION: Our genetic findings implicate heterozygous variants in KPNA3 as a novel cause for autosomal dominant, early-onset, and pure HSP. Mutant karyopherin-α3 proteins display varying deficits in molecular and cellular functions, thus, for the first time, implicating dysfunctional nucleocytoplasmic shuttling as a novel pathomechanism causing HSP. ANN NEUROL 2021;90:738-750.
Asunto(s)
Mutación/genética , Paraplejía Espástica Hereditaria/genética , alfa Carioferinas/genética , Adulto , Preescolar , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Secuenciación del Exoma/métodos , Adulto JovenRESUMEN
Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. The genome editing process relies on creating a site-specific DNA double-strand break (DSB) by engineered nucleases and then allowing the cell's repair machinery to repair the break such that precise changes are made to the DNA sequence. The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing accelerates the progress towards using genome editing as a new approach to human therapeutics. Here we review how genome editing using engineered nucleases works and how using different genome editing outcomes can be used as a tool set for treating human diseases. We then review the major challenges of therapeutic genome editing and we discuss how its potential enhancement through CRISPR guide RNA and Cas9 protein modifications could resolve some of these challenges.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Edición Génica/métodos , Terapia Genética/métodos , ARN Guía de Kinetoplastida/uso terapéutico , Animales , Sistemas CRISPR-Cas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Genes Transgénicos Suicidas , Ingeniería Genética/métodos , HumanosRESUMEN
Chromosomal microarray analysis (CMA) is a technology used for the detection of clinically-significant microdeietions or duplications, with a high sensitivity for submicroscopic aberrations. It is able to detect changes as small as 5-10Kb in size - a resolution up to 1000 times higher than that of conventional karyotyping. CMA is used for uncovering copy number variants (CNVs) thought to play an important role in the pathogenesis of a variety of disorders, primarily neurodevelopmental disorders and congenital anomalies. CMA may be applied in the prenatal or postnatal setting, with unique benefits and limitations in each setting. The growing use of CMA makes it essential for practicing physicians to understand the principles of this technology and be aware of its powers and limitations.
Asunto(s)
Cromosomas/genética , Análisis por Micromatrices/métodos , Diagnóstico Prenatal/métodos , Adulto , Femenino , Pruebas Genéticas , Humanos , Recién Nacido , Cariotipificación , Análisis por Micromatrices/instrumentación , EmbarazoRESUMEN
Importance: National implementation of rapid trio genome sequencing (rtGS) in a clinical acute setting is essential to ensure advanced and equitable care for ill neonates. Objective: To evaluate the feasibility, diagnostic efficacy, and clinical utility of rtGS in neonatal intensive care units (NICUs) throughout Israel. Design, Setting, and Participants: This prospective, public health care-based, multicenter cohort study was conducted from October 2021 to December 2022 with the Community Genetics Department of the Israeli Ministry of Health and all Israeli medical genetics institutes (n = 18) and NICUs (n = 25). Critically ill neonates suspected of having a genetic etiology were offered rtGS. All sequencing, analysis, and interpretation of data were performed in a central genomics center at Tel-Aviv Sourasky Medical Center. Rapid results were expected within 10 days. A secondary analysis report, issued within 60 days, focused mainly on cases with negative rapid results and actionable secondary findings. Pathogenic, likely pathogenic, and highly suspected variants of unknown significance (VUS) were reported. Main Outcomes and Measures: Diagnostic rate, including highly suspected disease-causing VUS, and turnaround time for rapid results. Clinical utility was assessed via questionnaires circulated to treating neonatologists. Results: A total of 130 neonates across Israel (70 [54%] male; 60 [46%] female) met inclusion criteria and were recruited. Mean (SD) age at enrollment was 12 (13) days. Mean (SD) turnaround time for rapid report was 7 (3) days. Diagnostic efficacy was 50% (65 of 130) for disease-causing variants, 11% (14 of 130) for VUS suspected to be causative, and 1 novel gene candidate (1%). Disease-causing variants included 12 chromosomal and 52 monogenic disorders as well as 1 neonate with uniparental disomy. Overall, the response rate for clinical utility questionnaires was 82% (107 of 130). Among respondents, genomic testing led to a change in medical management for 24 neonates (22%). Results led to immediate precision medicine for 6 of 65 diagnosed infants (9%), an additional 2 (3%) received palliative care, and 2 (3%) were transferred to nursing homes. Conclusions and Relevance: In this national cohort study, rtGS in critically ill neonates was feasible and diagnostically beneficial in a public health care setting. This study is a prerequisite for implementation of rtGS for ill neonates into routine care and may aid in design of similar studies in other public health care systems.
Asunto(s)
Enfermedad Crítica , Cuidado Intensivo Neonatal , Lactante , Recién Nacido , Femenino , Masculino , Humanos , Estudios de Cohortes , Estudios Prospectivos , Unidades de Cuidado Intensivo NeonatalRESUMEN
Neurodevelopmental disorders (NDDs) result from impaired development and functioning of the brain. Here, we identify loss-of-function variation in ZFHX3 as a novel cause for syndromic intellectual disability (ID). ZFHX3, previously known as ATBF1, is a zinc-finger homeodomain transcription factor involved in multiple biological processes including cell differentiation and tumorigenesis. Through international collaboration, we collected clinical and morphometric data (Face2Gene) of 41 individuals with protein truncating variants (PTVs) or (partial) deletions of ZFHX3 . We used data mining, RNA and protein analysis to identify the subcellular localization and spatiotemporal expression of ZFHX3 in multiple in vitro models. We identified the DNA targets of ZFHX3 using ChIP seq. Immunoprecipitation followed by mass spectrometry indicated potential binding partners of endogenous ZFHX3 in neural stem cells that were subsequently confirmed by reversed co-immunoprecipitation and western blot. We evaluated a DNA methylation profile associated with ZFHX3 haploinsufficiency using DNA methylation analysis on whole blood extracted DNA of six individuals with ZFHX3 PTVs and four with a (partial) deletion of ZFHX3 . A reversed genetic approach characterized the ZFHX3 orthologue in Drosophila melanogaster . Loss-of-function variation of ZFHX3 consistently associates with (mild) ID and/or behavioural problems, postnatal growth retardation, feeding difficulties, and recognizable facial characteristics, including the rare occurrence of cleft palate. Nuclear abundance of ZFHX3 increases during human brain development and neuronal differentiation in neural stem cells and SH-SY5Y cells, ZFHX3 interacts with the chromatin remodelling BRG1/Brm-associated factor complex and the cleavage and polyadenylation complex. In line with a role for chromatin remodelling, ZFHX3 haploinsufficiency associates with a specific DNA methylation profile in leukocyte-derived DNA. The target genes of ZFHX3 are implicated in neuron and axon development. In Drosophila melanogaster , z fh2, considered to be the ZFHX3 orthologue, is expressed in the third instar larval brain. Ubiquitous and neuron-specific knockdown of zfh2 results in adult lethality underscoring a key role for zfh2 in development and neurodevelopment. Interestingly, ectopic expression of zfh2 as well as ZFHX3 in the developing wing disc results in a thoracic cleft phenotype. Collectively, our data shows that loss-of-function variants in ZFHX3 are a cause of syndromic ID, that associates with a specific DNA methylation profile. Furthermore, we show that ZFHX3 participates in chromatin remodelling and mRNA processing.