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Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.
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Inversión Cromosómica , Enfermedades Raras , Humanos , Enfermedades Raras/genética , Masculino , Femenino , Inversión Cromosómica/genética , Linaje , Genoma Humano , Secuenciación Completa del Genoma , Proteína 2 de Unión a Metil-CpG/genética , Mutación , Proteínas de Homeodominio/genética , Persona de Mediana EdadRESUMEN
BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.
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Variación Genética , Enfermedades Raras , Humanos , Enfermedades Raras/diagnóstico , Enfermedades Raras/genética , Secuenciación Completa del Genoma , Pruebas Genéticas , Mutación , Proteínas de Ciclo CelularRESUMEN
PURPOSE: Studies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis. METHODS: Trio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. RESULTS: Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. CONCLUSION: This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.
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Craneosinostosis , Proteínas de Homeodominio , Animales , Humanos , Ratones , Secuencia de Bases , Suturas Craneales/patología , Craneosinostosis/genética , Genes Homeobox , Proteínas de Homeodominio/genética , PenetranciaRESUMEN
Craniosynostosis, the premature fusion of the cranial sutures, affects ~1 in 2000 children. Although many patients with a genetically determined cause harbor a variant in one of just seven genes or have a chromosomal abnormality, over 60 genes are known to be recurrently mutated, thus comprising a long tail of rarer diagnoses. Genome sequencing for the diagnosis of rare diseases is increasingly used in clinical settings, but analysis of the data is labor intensive and involves a trade-off between achieving high sensitivity or high precision. PanelApp, a crowd-sourced disease-focused set of gene panels, was designed to enable prioritization of variants in known disease genes for a given pathology, allowing enhanced identification of true-positives. For heterogeneous disorders like craniosynostosis, these panels must be regularly updated to ensure that diagnoses are not being missed. We provide a systematic review of genetic literature on craniosynostosis over the last 5 years, including additional results from resequencing a 42-gene panel in 617 affected individuals. We identify 16 genes (representing a 25% uplift) that should be added to the list of bona fide craniosynostosis disease genes and discuss the insights that these new genes provide into pathophysiological mechanisms of craniosynostosis.
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Craneosinostosis , Niño , Humanos , Craneosinostosis/diagnóstico , Craneosinostosis/genética , Craneosinostosis/patología , Suturas CranealesRESUMEN
INTRODUCTION: SPRY1 encodes protein sprouty homolog 1 (Spry-1), a negative regulator of receptor tyrosine kinase signalling. Null mutant mice display kidney/urinary tract abnormalities and altered size of the skull; complete loss-of-function of Spry-1 in humans has not been reported. METHODS: Analysis of whole-genome sequencing data from individuals with craniosynostosis enrolled in the 100,000 Genomes Project identified a likely pathogenic variant within SPRY1. Reverse-transcriptase PCR and western blot analysis were used to investigate the effect of the variant on SPRY1 mRNA and protein, in lymphoblastoid cell lines from the patient and both parents. RESULTS: A nonsense variant in SPRY1, encoding p.(Leu27*), was confirmed to be heterozygous in the unaffected parents and homozygous in the child. The child's phenotype, which included sagittal craniosynostosis, subcutaneous cystic lesions overlying the lambdoid sutures, hearing loss associated with bilateral cochlear and vestibular dysplasia and a unilateral renal cyst, overlapped the features reported in Spry1-/- null mice. Functional studies supported escape from nonsense-mediated decay, but western blot analysis demonstrated complete absence of full-length protein in the affected child and a marked reduction in both parents. CONCLUSION: This is the first report of complete loss of Spry-1 function in humans, associated with abnormalities of the cranial sutures, inner ear, and kidneys.
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Craneosinostosis , Oído Interno , Sistema Urinario , Ratones , Animales , Niño , Humanos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfoproteínas/genética , Ratones Noqueados , Craneosinostosis/genéticaRESUMEN
PURPOSE: The study aimed to identify novel genes for idiopathic hypogonadotropic hypogonadism (IHH). METHODS: A cohort of 1387 probands with IHH underwent exome sequencing and de novo, familial, and cohort-wide investigations. Functional studies were performed on 2 p190 Rho GTPase-activating proteins (p190 RhoGAP), ARHGAP35 and ARHGAP5, which involved in vivo modeling in larval zebrafish and an in vitro p190A-GAP activity assay. RESULTS: Rare protein-truncating variants (PTVs; n = 5) and missense variants in the RhoGAP domain (n = 7) in ARHGAP35 were identified in IHH cases (rare variant enrichment: PTV [unadjusted P = 3.1E-06] and missense [adjusted P = 4.9E-03] vs controls). Zebrafish modeling using gnrh3:egfp phenotype assessment showed that mutant larvae with deficient arhgap35a, the predominant ARHGAP35 paralog in the zebrafish brain, display decreased GnRH3-GFP+ neuronal area, a readout for IHH. In vitro GAP activity studies showed that 1 rare missense variant [ARHGAP35 p.(Arg1284Trp)] had decreased GAP activity. Rare PTVs (n = 2) also were discovered in ARHGAP5, a paralog of ARHGAP35; however, arhgap5 zebrafish mutants did not display significant GnRH3-GFP+ abnormalities. CONCLUSION: This study identified ARHGAP35 as a new autosomal dominant genetic driver for IHH and ARHGAP5 as a candidate gene for IHH. These observations suggest a novel role for the p190 RhoGAP proteins in GnRH neuronal development and integrity.
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Hipogonadismo , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Hipogonadismo/genética , Hormona Liberadora de Gonadotropina/genética , Proteínas Represoras , Factores de Intercambio de Guanina Nucleótido , Proteínas Activadoras de GTPasa/genéticaRESUMEN
Chromatin is essentially an array of nucleosomes, each of which consists of the DNA double-stranded fiber wrapped around a histone octamer. This organization supports cellular processes such as DNA replication, DNA transcription, and DNA repair in all eukaryotes. Human histone H4 is encoded by fourteen canonical histone H4 genes, all differing at the nucleotide level but encoding an invariant protein. Here, we present a cohort of 29 subjects with de novo missense variants in six H4 genes (H4C3, H4C4, H4C5, H4C6, H4C9, and H4C11) identified by whole-exome sequencing and matchmaking. All individuals present with neurodevelopmental features of intellectual disability and motor and/or gross developmental delay, while non-neurological features are more variable. Ten amino acids are affected, six recurrently, and are all located within the H4 core or C-terminal tail. These variants cluster to specific regions of the core H4 globular domain, where protein-protein interactions occur with either other histone subunits or histone chaperones. Functional consequences of the identified variants were evaluated in zebrafish embryos, which displayed abnormal general development, defective head organs, and reduced body axis length, providing compelling evidence for the causality of the reported disorder(s). While multiple developmental syndromes have been linked to chromatin-associated factors, missense-bearing histone variants (e.g., H3 oncohistones) are only recently emerging as a major cause of pathogenicity. Our findings establish a broader involvement of H4 variants in developmental syndromes.
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Histonas , Pez Cebra , Animales , Cromatina , ADN , Histonas/metabolismo , Humanos , Síndrome , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Introduction: Saethre-Chotzen syndrome, a craniosynostosis syndrome characterized by the premature closure of the coronal sutures, dysmorphic facial features and limb anomalies, is caused by haploinsufficiency of TWIST1. Although the majority of variants localize in the coding region of the gene, two variants in the 5' UTR have been recently reported to generate novel upstream initiation codons. Methods: Skeletal dysplasia Next-generation sequencing (NGS) panel was used for genetic analysis in a patient with bicoronal synostosis, facial dysmorphisms and limb anomalies. The variant pathogenicity was assessed by a luciferase reporter promoter assay. Results: Here, we describe the identification of a third ATG-creating de novo variant, c.-18C>T, in the 5' UTR of TWIST1 in the patient with a clinical diagnosis of Saethre-Chotzen syndrome. It was predicted to create an out-of-frame new upstream translation initiation codon resulting in a 40 amino acid larger functionally inactive protein. We performed luciferase reporter promoter assays to demonstrate that the variant does indeed reduce translation from the main open reading frame. Conclusion: This is the third variant identified in this region and confirms the introduction of upstream ATGs in the 5' UTR of TWIST1 as a pathogenic mechanism in Saethre-Chotzen syndrome. This case report shows the necessity for performing functional characterization of variants of unknown significance within national health services.
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BACKGROUND: Pathogenic heterozygous SIX1 variants (predominantly missense) occur in branchio-otic syndrome (BOS), but an association with craniosynostosis has not been reported. METHODS: We investigated probands with craniosynostosis of unknown cause using whole exome/genome (n=628) or RNA (n=386) sequencing, and performed targeted resequencing of SIX1 in 615 additional patients. Expression of SIX1 protein in embryonic cranial sutures was examined in the Six1nLacZ/+ reporter mouse. RESULTS: From 1629 unrelated cases with craniosynostosis we identified seven different SIX1 variants (three missense, including two de novo mutations, and four nonsense, one of which was also present in an affected twin). Compared with population data, enrichment of SIX1 loss-of-function variants was highly significant (p=0.00003). All individuals with craniosynostosis had sagittal suture fusion; additionally four had bilambdoid synostosis. Associated BOS features were often attenuated; some carrier relatives appeared non-penetrant. SIX1 is expressed in a layer basal to the calvaria, likely corresponding to the dura mater, and in the mid-sagittal mesenchyme. CONCLUSION: Craniosynostosis is associated with heterozygous SIX1 variants, with possible enrichment of loss-of-function variants compared with classical BOS. We recommend screening of SIX1 in craniosynostosis, particularly when sagittal±lambdoid synostosis and/or any BOS phenotypes are present. These findings highlight the role of SIX1 in cranial suture homeostasis.
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Craneosinostosis/genética , Proteínas de Homeodominio/genética , Animales , Preescolar , Estudios de Cohortes , Suturas Craneales/embriología , Suturas Craneales/patología , Craneosinostosis/complicaciones , Craneosinostosis/embriología , Análisis Mutacional de ADN , Estudios de Asociación Genética , Proteínas de Homeodominio/fisiología , Humanos , Lactante , Ratones , Linaje , Fenotipo , RNA-Seq , Secuenciación Completa del GenomaRESUMEN
PURPOSE: Genome sequencing (GS) for diagnosis of rare genetic disease is being introduced into the clinic, but the complexity of the data poses challenges for developing pipelines with high diagnostic sensitivity. We evaluated the performance of the Genomics England 100,000 Genomes Project (100kGP) panel-based pipelines, using craniosynostosis as a test disease. METHODS: GS data from 114 probands with craniosynostosis and their relatives (314 samples), negative on routine genetic testing, were scrutinized by a specialized research team, and diagnoses compared with those made by 100kGP. RESULTS: Sixteen likely pathogenic/pathogenic variants were identified by 100kGP. Eighteen additional likely pathogenic/pathogenic variants were identified by the research team, indicating that for craniosynostosis, 100kGP panels had a diagnostic sensitivity of only 47%. Measures that could have augmented diagnoses were improved calling of existing panel genes (+18% sensitivity), review of updated panels (+12%), comprehensive analysis of de novo small variants (+29%), and copy-number/structural variants (+9%). Recent NHS England recommendations that partially incorporate these measures should achieve 85% overall sensitivity (+38%). CONCLUSION: GS identified likely pathogenic/pathogenic variants in 29.8% of previously undiagnosed patients with craniosynostosis. This demonstrates the value of research analysis and the importance of continually improving algorithms to maximize the potential of clinical GS.
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Craneosinostosis , Pruebas Genéticas , Secuencia de Bases , Mapeo Cromosómico , Craneosinostosis/diagnóstico , Craneosinostosis/genética , Humanos , Enfermedades Raras/genéticaRESUMEN
Heterozygous intragenic loss-of-function mutations of ERF, encoding an ETS transcription factor, were previously reported to cause a novel craniosynostosis syndrome, suggesting that ERF is haploinsufficient. We describe six families harboring heterozygous deletions including, or near to, ERF, of which four were characterized by whole-genome sequencing and two by chromosomal microarray. Based on the severity of associated intellectual disability (ID), we identify three categories of ERF-associated deletions. The smallest (32 kb) and only inherited deletion included two additional centromeric genes and was not associated with ID. Three larger deletions (264-314 kb) that included at least five further centromeric genes were associated with moderate ID, suggesting that deletion of one or more of these five genes causes ID. The individual with the most severe ID had a more telomerically extending deletion, including CIC, a known ID gene. Children found to harbor ERF deletions should be referred for craniofacial assessment, to exclude occult raised intracranial pressure.
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Cromosomas Humanos Par 19 , Discapacidad Intelectual , Niño , Deleción Cromosómica , Haploinsuficiencia , Heterocigoto , Humanos , Discapacidad Intelectual/genética , Mutación , Proteínas Represoras/genéticaRESUMEN
Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.
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Discapacidad Intelectual , Trastornos del Neurodesarrollo , Aminoácidos , Animales , Humanos , Discapacidad Intelectual/genética , Sistema de Señalización de MAP Quinasas , Ratones , Hipotonía Muscular , Trastornos del Neurodesarrollo/genéticaRESUMEN
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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PURPOSE: Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype. METHODS: We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants. RESULTS: We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10-7). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype. CONCLUSION: Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.
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Craneosinostosis , Craneosinostosis/genética , Genotipo , Humanos , Mutación Missense/genética , Penetrancia , Fenotipo , Proteína smad6/genéticaRESUMEN
SOX6 belongs to a family of 20 SRY-related HMG-box-containing (SOX) genes that encode transcription factors controlling cell fate and differentiation in many developmental and adult processes. For SOX6, these processes include, but are not limited to, neurogenesis and skeletogenesis. Variants in half of the SOX genes have been shown to cause severe developmental and adult syndromes, referred to as SOXopathies. We here provide evidence that SOX6 variants also cause a SOXopathy. Using clinical and genetic data, we identify 19 individuals harboring various types of SOX6 alterations and exhibiting developmental delay and/or intellectual disability; the individuals are from 17 unrelated families. Additional, inconstant features include attention-deficit/hyperactivity disorder (ADHD), autism, mild facial dysmorphism, craniosynostosis, and multiple osteochondromas. All variants are heterozygous. Fourteen are de novo, one is inherited from a mosaic father, and four offspring from two families have a paternally inherited variant. Intragenic microdeletions, balanced structural rearrangements, frameshifts, and nonsense variants are predicted to inactivate the SOX6 variant allele. Four missense variants occur in residues and protein regions highly conserved evolutionarily. These variants are not detected in the gnomAD control cohort, and the amino acid substitutions are predicted to be damaging. Two of these variants are located in the HMG domain and abolish SOX6 transcriptional activity in vitro. No clear genotype-phenotype correlations are found. Taken together, these findings concur that SOX6 haploinsufficiency leads to a neurodevelopmental SOXopathy that often includes ADHD and abnormal skeletal and other features.
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Trastorno por Déficit de Atención con Hiperactividad/genética , Craneosinostosis/genética , Trastornos del Neurodesarrollo/genética , Osteocondroma/genética , Factores de Transcripción SOXD/genética , Transporte Activo de Núcleo Celular , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Niño , Preescolar , Simulación por Computador , Femenino , Variación Estructural del Genoma/genética , Humanos , Lactante , Masculino , Mutación Missense , Trastornos del Neurodesarrollo/diagnóstico , RNA-Seq , Factores de Transcripción SOXD/química , Factores de Transcripción SOXD/metabolismo , Síndrome , Transcripción Genética , Transcriptoma , Translocación Genética/genéticaRESUMEN
SUMMARY: amplimap is a command-line tool to automate the processing and analysis of data from targeted next-generation sequencing experiments with PCR-based amplicons or capture-based enrichment systems. From raw sequencing reads, amplimap generates output such as read alignments, annotated variant calls, target coverage statistics and variant allele counts and frequencies for each target base pair. In addition to its focus on user-friendliness and reproducibility, amplimap supports advanced features such as consensus base calling for read families based on unique molecular identifiers and filtering false positive variant calls caused by amplification of off-target loci. AVAILABILITY AND IMPLEMENTATION: amplimap is available as a free Python package under the open-source Apache 2.0 License. Documentation, source code and installation instructions are available at https://github.com/koelling/amplimap.
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Secuenciación de Nucleótidos de Alto Rendimiento , Programas Informáticos , Alelos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Human retrocopies, that is messenger RNA transcripts benefitting from the long interspersed element 1 machinery for retrotransposition, may have specific consequences for genomic testing. Next genetration sequencing (NGS) techniques allow the detection of such mobile elements but they may be misinterpreted as genomic duplications or be totally overlooked. We report eight observations of retrocopies detected during diagnostic NGS analyses of targeted gene panels, exome, or genome sequencing. For seven cases, while an exons-only copy number gain was called, read alignment inspection revealed a depth of coverage shift at every exon-intron junction where indels were also systematically called. Moreover, aberrant chimeric read pairs spanned entire introns or were paired with another locus for terminal exons. The 8th retrocopy was present in the reference genome and thus showed a normal NGS profile. We emphasize the existence of retrocopies and strategies to accurately detect them at a glance during genetic testing and discuss pitfalls for genetic testing.
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Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Retroelementos , Adolescente , Adulto , Anciano , Niño , Preescolar , Pruebas Diagnósticas de Rutina , Femenino , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.
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Ensamble y Desensamble de Cromatina , Suturas Craneales/crecimiento & desarrollo , Craneosinostosis/metabolismo , Mutación Missense , Nucleosomas/metabolismo , Osteogénesis , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Animales , Suturas Craneales/metabolismo , Craneosinostosis/genética , Craneosinostosis/fisiopatología , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Humanos , Lactante , Masculino , Ratones , Unión Proteica , Conformación Proteica , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Proteína 4 de Unión a Retinoblastoma/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiología , Población Blanca , Secuenciación Completa del GenomaRESUMEN
The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms.