RESUMEN
Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.
Asunto(s)
Enfermedad/genética , Drosophila melanogaster/genética , Pruebas Genéticas , Patrón de Herencia , Interferencia de ARN , Animales , Modelos Animales de Enfermedad , Humanos , Cromosoma XRESUMEN
BACKGROUND: Clubfoot, presenting as a rigid inward and downward turning of the foot, is one of the most common congenital musculoskeletal anomalies. The aetiology of clubfoot is poorly understood and variants in known clubfoot disease genes account for only a small portion of the heritability. METHODS: Exome sequence data were generated from 1190 non-syndromic clubfoot cases and their family members from multiple ethnicities. Ultra-rare variant burden analysis was performed comparing 857 unrelated clubfoot cases with European ancestry with two independent ethnicity-matched control groups (1043 in-house and 56 885 gnomAD controls). Additional variants in prioritised genes were identified in a larger cohort, including probands with non-European ancestry. Segregation analysis was performed in multiplex families when available. RESULTS: Rare variants in 29 genes were enriched in clubfoot cases, including PITX1 (a known clubfoot disease gene), HOXD12, COL12A1, COL9A3 and LMX1B. In addition, rare variants in posterior HOX genes (HOX9-13) were enriched overall in clubfoot cases. In total, variants in these genes were present in 8.4% (100/1190) of clubfoot cases with both European and non-European ancestry. Among these, 3 are de novo and 22 show variable penetrance, including 4 HOXD12 variants that segregate with clubfoot. CONCLUSION: We report HOXD12 as a novel clubfoot disease gene and demonstrate a phenotypic expansion of known disease genes (myopathy gene COL12A1, Ehlers-Danlos syndrome gene COL9A3 and nail-patella syndrome gene LMX1B) to include isolated clubfoot.
Asunto(s)
Pie Equinovaro , Secuenciación del Exoma , Proteínas de Homeodominio , Femenino , Humanos , Masculino , Pie Equinovaro/genética , Pie Equinovaro/patología , Exoma/genética , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio/genética , Linaje , Factores de Transcripción/genéticaRESUMEN
Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.
Asunto(s)
Discapacidades del Desarrollo/genética , Proteínas de Drosophila/genética , Enfermedades Hereditarias del Ojo/genética , Discapacidad Intelectual/genética , Carioferinas/genética , Anomalías Musculoesqueléticas/genética , beta Carioferinas/genética , Proteína de Unión al GTP ran/genética , Alelos , Secuencia de Aminoácidos , Animales , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Hereditarias del Ojo/patología , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Genoma Humano , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Masculino , Anomalías Musculoesqueléticas/metabolismo , Anomalías Musculoesqueléticas/patología , Mutación , Neuronas/metabolismo , Neuronas/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Secuenciación Completa del Genoma , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.
Asunto(s)
Axonema/metabolismo , Trastornos de la Motilidad Ciliar/genética , Codón sin Sentido , Dineínas/metabolismo , Proteínas/genética , Células 3T3 , Adulto , Animales , Axonema/fisiología , Células Cultivadas , Chlamydomonas reinhardtii , Cilios/metabolismo , Cilios/fisiología , Trastornos de la Motilidad Ciliar/patología , Secuencia Conservada , Humanos , Masculino , Ratones , Proteínas Asociadas a Microtúbulos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas/química , Proteínas/metabolismo , Mucosa Respiratoria/metabolismoRESUMEN
BACKGROUND: Clubfoot, a congenital deformity that presents as a rigid, inward turning of the foot, affects approximately 1 in 1000 infants and occurs as an isolated birth defect in 80% of patients. Despite its high level of heritability, few causative genes have been identified, and mutations in known genes are only responsible for a small portion of clubfoot heritability. QUESTIONS/PURPOSES: (1) Are any rare gene variants enriched (that is, shared) in unrelated patients with isolated clubfoot? (2) Are there other rare variants in the identified gene (Filamin B) in these patients with clubfoot? METHODS: Whole-exome sequence data were generated from a discovery cohort of 183 unrelated probands with clubfoot and 2492 controls. Variants were filtered with minor allele frequency < 0.02 to identify rare variants as well as small insertions and deletions (indels) resulting in missense variants, nonsense or premature truncation, or in-frame deletions. A candidate deletion was then genotyped in another cohort of 974 unrelated patients with clubfoot (a replication cohort). Other rare variants in the candidate gene were also investigated. A segregation analysis was performed in multigenerational families of individuals with clubfoot to see if the genotypes segregate with phenotypes. Single-variant association analysis was performed using the Fisher two-tailed exact test (exact p values are presented to give an indication of the magnitude of the association). RESULTS: There were no recurrent variants in the known genes causing clubfoot in this study. A three-base pair in-frame codon deletion of Filamin B (FLNB) (p.E1792del, rs1470699812) was identified in 1.6% (3 of 183) of probands with clubfoot in the discovery cohort compared with 0% of controls (0 of 2492) (odds ratio infinity (inf) [95% CI 5.64 to inf]; p = 3.18 x 10-5) and 0.0016% of gnomAD controls (2 of 125,709) (OR 1.01 x 103 [95% CI 117.42 to 1.64 x 104]; p = 3.13 x 10-8). By screening a replication cohort (n = 974 patients), we found two probands with the identical FLNB deletion. In total, the deletion was identified in 0.43% (5 of 1157) of probands with clubfoot compared with 0% of controls and 0.0016% of gnomAD controls (OR 268.5 [95% CI 43.68 to 2.88 x 103]; p = 1.43 x 10-9). The recurrent FLNB p.E1792del variant segregated with clubfoot, with incomplete penetrance in two families. Affected individuals were more likely to be male and have bilateral clubfoot. Although most patients had isolated clubfoot, features consistent with Larsen syndrome, including upper extremity abnormalities such as elbow and thumb hypermobility and wide, flat thumbs, were noted in affected members of one family. We identified 19 additional rare FLNB missense variants located throughout the gene in patients with clubfoot. One of these missense variants, FLNB p.G2397D, exhibited incomplete penetrance in one family. CONCLUSION: A recurrent FLNB E1792 deletion was identified in 0.43% of 1157 isolated patients with clubfoot. Given the absence of any recurrent variants in our discovery phase (n = 183) for any of the known genes causing clubfoot, our findings support that novel and rare missense variants in FLNB in patients with clubfoot, although rare, may be among the most commonly known genetic causes of clubfoot. Patients with FLNB variants often have isolated clubfoot, but they and their family members may be at an increased risk of having additional clinical features consistent with Larsen syndrome. CLINICAL RELEVANCE: Identification of FLNB variants may be useful for determining clubfoot recurrence risk and comorbidities.
Asunto(s)
Pie Equinovaro/genética , Secuenciación del Exoma , Filaminas/genética , Adolescente , Adulto , Anciano , Niño , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Adulto JovenRESUMEN
From a GeneMatcher-enabled international collaboration, we identified ten individuals affected by intellectual disability, speech delay, ataxia, and facial dysmorphism and carrying a deleterious EBF3 variant detected by whole-exome sequencing. One 9-bp duplication and one splice-site, five missense, and two nonsense variants in EBF3 were found; the mutations occurred de novo in eight individuals, and the missense variant c.625C>T (p.Arg209Trp) was inherited by two affected siblings from their healthy mother, who is mosaic. EBF3 belongs to the early B cell factor family (also known as Olf, COE, or O/E) and is a transcription factor involved in neuronal differentiation and maturation. Structural assessment predicted that the five amino acid substitutions have damaging effects on DNA binding of EBF3. Transient expression of EBF3 mutant proteins in HEK293T cells revealed mislocalization of all but one mutant in the cytoplasm, as well as nuclear localization. By transactivation assays, all EBF3 mutants showed significantly reduced or no ability to activate transcription of the reporter gene CDKN1A, and in situ subcellular fractionation experiments demonstrated that EBF3 mutant proteins were less tightly associated with chromatin. Finally, in RNA-seq and ChIP-seq experiments, EBF3 acted as a transcriptional regulator, and mutant EBF3 had reduced genome-wide DNA binding and gene-regulatory activity. Our findings demonstrate that variants disrupting EBF3-mediated transcriptional regulation cause intellectual disability and developmental delay and are present in â¼0.1% of individuals with unexplained neurodevelopmental disorders.
Asunto(s)
Ataxia/genética , Cara/anomalías , Discapacidad Intelectual/genética , Trastornos del Desarrollo del Lenguaje/genética , Mutación , Trastornos del Neurodesarrollo/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Adolescente , Adulto , Sustitución de Aminoácidos , Niño , Preescolar , Cromatina/genética , Cromatina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Discapacidades del Desarrollo/genética , Exoma/genética , Femenino , Regulación de la Expresión Génica/genética , Genes Reporteros , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mosaicismo , Transporte de Proteínas/genética , Síndrome , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Primary ciliary dyskinesia (PCD) is a rare disorder that affects the biogenesis or function of motile cilia resulting in chronic airway disease. PCD is genetically and phenotypically heterogeneous, with causative mutations identified in over 40 genes; however, the genetic basis of many cases is unknown. Using whole-exome sequencing, we identified three affected siblings with clinical symptoms of PCD but normal ciliary structure, carrying compound heterozygous loss-of-function variants in CFAP221. Computational analysis suggests that these variants are the most damaging alleles shared by all three siblings. Nasal epithelial cells from one of the subjects demonstrated slightly reduced beat frequency (16.5 Hz vs 17.7 Hz, p = 0.16); however, waveform analysis revealed that the CFAP221 defective cilia beat in an aberrant circular pattern. These results show that genetic variants in CFAP221 cause PCD and that CFAP221 should be considered a candidate gene in cases where PCD is suspected but cilia structure and beat frequency appear normal.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Variación Genética , Proteínas/genética , Proteínas/metabolismo , Alelos , Proteínas de Unión a Calmodulina , Cilios/genética , Trastornos de la Motilidad Ciliar/diagnóstico por imagen , Células Epiteliales , Exones/genética , Humanos , Mutación , Secuenciación del ExomaRESUMEN
BACKGROUND: Wolff-Parkinson-White (WPW) syndrome is a relatively common arrhythmia affecting ~1-3/1,000 individuals. Mutations in PRKAG2 have been described in rare patients in association with cardiomyopathy. However, the genetic basis of WPW in individuals with a structurally normal heart remains poorly understood. Sudden death due to atrial fibrillation (AF) can also occur in these individuals. Several studies have indicated that despite ablation of an accessory pathway, the risk of AF remains high in patients compared to general population. METHODS: We applied exome sequencing in 305 subjects, including 65 trios, 80 singletons, and 6 multiple affected families. We used de novo analysis, candidate gene approach, and burden testing to explore the genetic contributions to WPW. RESULTS: A heterozygous deleterious variant in PRKAG2 was identified in one subject, accounting for 0.6% (1/151) of the genetic basis of WPW in this study. Another individual with WPW and left ventricular hypertrophy carried a known pathogenic variant in MYH7. We found rare de novo variants in genes associated with arrhythmia and cardiomyopathy (ANK2, NEBL, PITX2, and PRDM16) in this cohort. There was an increased burden of rare deleterious variants (MAF ≤ 0.005) with CADD score ≥ 25 in genes linked to AF in cases compared to controls (P = .0023). CONCLUSIONS: Our findings show an increased burden of rare deleterious variants in genes linked to AF in WPW syndrome, suggesting that genetic factors that determine the development of accessory pathways may be linked to an increased susceptibility of atrial muscle to AF in a subset of patients.
Asunto(s)
Proteínas Quinasas Activadas por AMP/genética , Fibrilación Atrial/genética , Predisposición Genética a la Enfermedad , Síndrome de Wolff-Parkinson-White/genética , Adolescente , Adulto , Ancirinas/genética , Fibrilación Atrial/patología , Proteínas Portadoras/genética , Niño , Estudios de Cohortes , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Femenino , Estudios de Asociación Genética , Atrios Cardíacos/patología , Proteínas de Homeodominio/genética , Humanos , Proteínas con Dominio LIM/genética , Masculino , Mutación/genética , Factores de Transcripción/genética , Secuenciación del Exoma , Síndrome de Wolff-Parkinson-White/patología , Adulto Joven , Proteína del Homeodomínio PITX2RESUMEN
The paradigm of a single gene associated with one specific phenotype and mode of inheritance has been repeatedly challenged. Genotype-phenotype correlations can often be traced to different mutation types, localization of the variants in distinct protein domains, or the trigger of or escape from nonsense-mediated decay. Using whole-exome sequencing, we identified homozygous variants in EMC1 that segregated with a phenotype of developmental delay, hypotonia, scoliosis, and cerebellar atrophy in three families. In addition, a de novo heterozygous EMC1 variant was seen in an individual with a similar clinical and MRI imaging phenotype. EMC1 encodes a member of the endoplasmic reticulum (ER)-membrane protein complex (EMC), an evolutionarily conserved complex that has been proposed to have multiple roles in ER-associated degradation, ER-mitochondria tethering, and proper assembly of multi-pass transmembrane proteins. Perturbations of protein folding and organelle crosstalk have been implicated in neurodegenerative processes including cerebellar atrophy. We propose EMC1 as a gene in which either biallelic or monoallelic variants might lead to a syndrome including intellectual disability and preferential degeneration of the cerebellum.
Asunto(s)
Atrofia/genética , Discapacidades del Desarrollo/genética , Variación Genética , Hipotonía Muscular/genética , Proteínas/genética , Escoliosis/genética , Adolescente , Alelos , Secuencia de Aminoácidos , Atrofia/diagnóstico , Cerebelo/patología , Niño , Preescolar , Discapacidades del Desarrollo/diagnóstico , Degradación Asociada con el Retículo Endoplásmico , Femenino , Estudios de Asociación Genética , Heterocigoto , Humanos , Imagen por Resonancia Magnética , Masculino , Proteínas de la Membrana , Datos de Secuencia Molecular , Hipotonía Muscular/diagnóstico , Mutación , Linaje , Pliegue de Proteína , Proteínas/metabolismo , Escoliosis/diagnósticoRESUMEN
We developed an algorithm, HMZDelFinder, that uses whole exome sequencing (WES) data to identify rare and intragenic homozygous and hemizygous (HMZ) deletions that may represent complete loss-of-function of the indicated gene. HMZDelFinder was applied to 4866 samples in the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) cohort and detected 773 HMZ deletion calls (567 homozygous or 206 hemizygous) with an estimated sensitivity of 86.5% (82% for single-exonic and 88% for multi-exonic calls) and precision of 78% (53% single-exonic and 96% for multi-exonic calls). Out of 773 HMZDelFinder-detected deletion calls, 82 were subjected to array comparative genomic hybridization (aCGH) and/or breakpoint PCR and 64 were confirmed. These include 18 single-exon deletions out of which 8 were exclusively detected by HMZDelFinder and not by any of seven other CNV detection tools examined. Further investigation of the 64 validated deletion calls revealed at least 15 pathogenic HMZ deletions. Of those, 7 accounted for 17-50% of pathogenic CNVs in different disease cohorts where 7.1-11% of the molecular diagnosis solved rate was attributed to CNVs. In summary, we present an algorithm to detect rare, intragenic, single-exon deletion CNVs using WES data; this tool can be useful for disease gene discovery efforts and clinical WES analyses.
Asunto(s)
Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Exoma , Enfermedades Genéticas Congénitas/genética , Hemicigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Algoritmos , Empalme Alternativo , Estudios de Cohortes , Consanguinidad , Conjuntos de Datos como Asunto , Enfermedades Genéticas Congénitas/diagnóstico , Humanos , Patrón de Herencia , Modelos Genéticos , Linaje , Reproducibilidad de los Resultados , Eliminación de Secuencia , Flujo de TrabajoRESUMEN
Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5' end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1(st) coding exon), c.16A>T (p.Lys6(∗)) and c.35_38delTCAA (p.Ile12Lysfs(∗)4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5' end of the geminin protein. All three GMNN mutations identified alter sites 5' to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS.
Asunto(s)
Microtia Congénita/genética , Enanismo/genética , Geminina/genética , Trastornos del Crecimiento/genética , Micrognatismo/genética , Mutación , Rótula/anomalías , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular/genética , Preescolar , Microtia Congénita/metabolismo , Enanismo/metabolismo , Enanismo/patología , Exones , Femenino , Geminina/metabolismo , Expresión Génica , Genes Dominantes , Trastornos del Crecimiento/metabolismo , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Patrón de Herencia , Masculino , Micrognatismo/metabolismo , Datos de Secuencia Molecular , Rótula/metabolismo , Linaje , Estabilidad Proteica , Proteolisis , Empalme del ARN , Alineación de SecuenciaRESUMEN
[This corrects the article DOI: 10.1371/journal.pbio.1001847.].
RESUMEN
Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of -3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (-70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes.
Asunto(s)
Mapeo Cromosómico/métodos , Drosophila melanogaster/genética , Mutagénesis , Animales , Metanosulfonato de Etilo , Femenino , Genes Esenciales , Genes de Insecto , Masculino , Datos de Secuencia Molecular , Mutágenos , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Cromosoma XRESUMEN
Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/metabolismo , Proteínas de Transporte Vesicular/genética , Animales , Proteínas de Drosophila/genética , Endocitosis/fisiología , Luz , Lisosomas/metabolismo , Mutación , Células Fotorreceptoras de Invertebrados/citología , Transporte de Proteínas , Degeneración Retiniana/fisiopatología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Vesicular trafficking plays a key role in tuning the activity of Notch signaling. Here, we describe a novel and conserved Rab geranylgeranyltransferase (RabGGT)-α-like subunit that is required for Notch signaling-mediated lateral inhibition and cell fate determination of external sensory organs. This protein is encoded by tempura, and its loss affects the secretion of Scabrous and Delta, two proteins required for proper Notch signaling. We show that Tempura forms a heretofore uncharacterized RabGGT complex that geranylgeranylates Rab1 and Rab11. This geranylgeranylation is required for their proper subcellular localization. A partial dysfunction of Rab1 affects Scabrous and Delta in the secretory pathway. In addition, a partial loss Rab11 affects trafficking of Delta. In summary, Tempura functions as a new geranylgeranyltransferase that regulates the subcellular localization of Rab1 and Rab11, which in turn regulate trafficking of Scabrous and Delta, thereby affecting Notch signaling.
Asunto(s)
Dimetilaliltranstransferasa/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Procesamiento Proteico-Postraduccional , Subunidades de Proteína/genética , Receptores Notch/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab1/genética , Animales , Dimetilaliltranstransferasa/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Prenilación de Proteína , Subunidades de Proteína/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Rodopsina/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Envejecimiento/metabolismo , Animales , Citoplasma/metabolismo , Citoplasma/efectos de la radiación , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Electrorretinografía , Femenino , Técnicas de Silenciamiento del Gen , Genes de Insecto/genética , Luz , Masculino , Mutación/genética , Células Fotorreceptoras de Invertebrados/patología , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Células Fotorreceptoras de Invertebrados/ultraestructura , Unión Proteica/efectos de la radiación , Transporte de Proteínas/efectos de la radiación , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatologíaRESUMEN
BACKGROUND: Congenital vertical talus (CVT), also known as "rocker-bottom foot", is a rare foot deformity associated with a dislocation of the talonavicular joint. Although genetic causes of CVT have been described in single isolated and syndromic families, whole-exome sequencing (WES) of large cohorts have not yet been reported. METHODS: In this study, 62 probands with CVT were evaluated for likely causative single nucleotide variants (SNVs) and copy number variants (CNVs) using WES. Segregation of variants within families was determined by Sanger sequencing. RESULTS: In this cohort, CVT occurred as an isolated anomaly in 75.8% (47/62) and was familial in 19.3% (12/62) of cases. Analysis of WES data led to the identification of likely causative variants in known disease genes in 30.6% (19/62) of all CVT probands. More than one proband had likely causative SNVs in TSHZ1, GDF5, and LMX1B. Only two probands had likely causative CNVs: a chromosome 12q13.13 deletion of the 5' HOXC gene cluster, and a chromosome 18q22.3q23 deletion involving TSHZ1. Familial CVT was strongly predictive of identifying a molecular diagnosis [75% (9/12) of familial cases compared to 20% (10/50) of non-familial cases (Chi-square test, P-value = 0.0002)]. There was no difference in the solved rate based on isolated or syndromic presentation, unilateral or bilateral affectation, or sex. CONCLUSIONS: CVT is genetically heterogeneous and more often caused by SNVs than CNVs. There is a high yield of WES in familial CVT cases (â¼75%). Additional research is needed to identify the causes of sporadic CVT, which had much lower solved rates.
Asunto(s)
Pie Plano , Deleción Cromosómica , Variaciones en el Número de Copia de ADN , Exoma/genética , Humanos , Linaje , Secuenciación del ExomaRESUMEN
Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification.
Asunto(s)
Azoospermia , Infertilidad Masculina , Humanos , Masculino , Animales , Ratones , Azoospermia/genética , Azoospermia/patología , Testículo/patología , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Espermatogénesis/genéticaRESUMEN
ABL1 is a proto-oncogene well known as part of the fusion gene BCR-ABL1 in the Philadelphia chromosome of leukemia cancer cells. Inherited germline ABL1 changes have not been associated with genetic disorders. Here we report ABL1 germline variants cosegregating with an autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. The variant c.734A>G (p.Tyr245Cys) was found to occur de novo or cosegregate with disease in five individuals (families 1-3). Additionally, a de novo c.1066G>A (p.Ala356Thr) variant was identified in a sixth individual (family 4). We overexpressed the mutant constructs in HEK 293T cells and observed increased tyrosine phosphorylation, suggesting increased ABL1 kinase activities associated with both the p.Tyr245Cys and p.Ala356Thr substitutions. Our clinical and experimental findings, together with previously reported teratogenic effects of selective BCR-ABL inhibitors in humans and developmental defects in Abl1 knockout mice, suggest that ABL1 has an important role during organismal development.
Asunto(s)
Anomalías Múltiples/genética , Enfermedades del Desarrollo Óseo/genética , Trastornos de los Cromosomas/genética , Anomalías Craneofaciales/genética , Trastornos de Alimentación y de la Ingestión de Alimentos/genética , Proteínas de Fusión bcr-abl/genética , Mutación de Línea Germinal/genética , Cardiopatías Congénitas/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Deformidades Congénitas de las Extremidades/genética , Animales , Línea Celular , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Cromosoma Filadelfia/efectos de los fármacos , Fosforilación/genética , Proto-Oncogenes Mas , Transducción de Señal/genéticaRESUMEN
The ability to sense energy status is crucial in the regulation of metabolism via the mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTT-Pontin/Reptin complex is responsive to changes in energy status. Under energy-sufficient conditions, the TTT-Pontin/Reptin complex promotes mTORC1 dimerization and mTORC1-Rag interaction, which are critical for mTORC1 activation. We show that WAC is a regulator of energy-mediated mTORC1 activity. In a Drosophila screen designed to isolate mutations that cause neuronal dysfunction, we identified wacky, the homolog of WAC. Loss of Wacky leads to neurodegeneration, defective mTOR activity, and increased autophagy. Wacky and WAC have conserved physical interactions with mTOR and its regulators, including Pontin and Reptin, which bind to the TTT complex to regulate energy-dependent activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating mTORC1 dimerization and mTORC1-Rag interaction.