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The genomics era has facilitated discovery of new genes predisposing to bone marrow failure (BMF) and hematological malignancy (HM). We report the discovery of ERG as a novel autosomal dominant BMF/HM predisposition gene. ERG is a highly constrained transcription factor critical for definitive hematopoiesis, stem cell function and platelet maintenance. ERG colocalizes with other transcription factors including RUNX1 and GATA2 on promoters/enhancers of genes orchestrating hematopoiesis. We identified a rare heterozygous ERG missense variant in 3 thrombocytopenic individuals from one family and 14 additional ERG variants in unrelated individuals with BMF/HM including 2 de novo cases and 3 truncating variants. Phenotypes associated with pathogenic germline ERG variants included cytopenias (thrombocytopenia, neutropenia, pancytopenia) and HMs (acute myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia) with onset before 40 years. Twenty ERG variants (19 missense, 1 truncating) including 3 missense population variants were functionally characterized. Thirteen potentially pathogenic ETS domain missense variants displayed loss-of-function characteristics disrupting transcriptional transactivation, DNA-binding and/or nuclear localization. Selected variants overexpressed in mouse fetal liver cells failed to drive myeloid differentiation and cytokine-independent growth in culture, and to promote acute erythroleukemia when transplanted into mice, concordant with these variants being loss-of-function. Four individuals displayed somatic genetic rescue by copy neutral loss of heterozygosity. Identification of predisposing germline ERG variants has clinical implications for patient/family diagnosis, counselling, surveillance, and treatment strategies including selection of bone marrow donors or cell/gene therapy.
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PURPOSE: Determine the variant detection rate for ENG, ACVRL1, and SMAD4 in individuals who meet consensus (Curaçao) criteria for the clinical diagnosis of hereditary hemorrhagic telangiectasia. METHODS: Review of HHT center database for individuals with three or more HHT diagnostic criteria, in whom molecular genetic analysis for ENG, ACVRL1, and SMAD4 had been performed. RESULTS: A variant known or suspected to be causal was detected in ENG in 67/152 (44.1%; 95% confidence interval [CI], 36.0-52.4%), ACVRL1 in 79/152 (52.0%; 95% CI, 43.7-60.1%), and SMAD4 in 2/152 (1.3%; 95% CI, 0.2-4.7%) family probands with definite HHT. Only 4/152 (2.6%; 95% CI, 0.7-6.6%) family probands did not have a variant in one of these genes. CONCLUSION: Previous reports of the variant detection rate for ENG and ACVRL1 in HHT patients have come from laboratories, which receive samples from clinicians with a wide range of expertise in recognizing clinical manifestations of HHT. These studies suggest a significantly lower detection rate (~75-85%) than we have found in patients who meet strictly applied consensus criteria (96.1%). Analysis of SMAD4 adds an additional detection rate of 1.3%. HHT as defined by the Curaçao criteria is highly predictive of a causative variant in either ENG or ACVRL1.
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Telangiectasia Hemorrágica Hereditaria , Receptores de Activinas Tipo II/genética , Curazao , Endoglina/genética , Humanos , Mutación , Telangiectasia Hemorrágica Hereditaria/diagnóstico , Telangiectasia Hemorrágica Hereditaria/genéticaRESUMEN
Connexin 26 (Cx26), encoded by the GJB2 gene, is a key protein involved in the formation of gap junctions in epithelial organs including the inner ear and palmoplantar epidermis. Pathogenic variants in GJB2 are responsible for approximately 50% of inherited sensorineural deafness. The majority of these variants are associated with autosomal recessive inheritance; however, rare reports of dominantly co-segregating variants have been published. Since we began offering GJB2 testing in 2003, only about 2% of detected GJB2 variants from our laboratory have been classified as dominant. Here we report three novel dominant GJB2 variants (p.Thr55Ala, p.Gln57_Pro58delinsHisSer, and p.Trp44Gly); two associated with syndromic sensorineural hearing loss and one with nonsyndromic hearing loss. In the kindred with the p.Thr55Ala variant, the proband and his father present with only leukonychia as a cutaneous finding of their syndromic hearing loss. This phenotype has been previously documented in conjunction with palmoplantar hyperkeratosis, but isolated leukonychia is a novel finding likely associated with the unique threonine to alanine change at codon 55 (other variants at this codon have been reported in cases of nonsyndromic hearing loss). This report contributes to the short list of GJB2 variants associated with autosomal dominant hearing loss, highlights the variability of skin and nail findings associated with such cases, and illustrates the occurrence of both syndromic and nonsyndromic presentations with changes in the same gene.
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Conexina 26/genética , Sordera/genética , Genes Dominantes , Estudios de Asociación Genética , Variación Genética , Niño , Preescolar , Biología Computacional/métodos , Conexina 26/química , Sordera/diagnóstico , Femenino , Humanos , Patrón de Herencia , Masculino , Modelos Moleculares , Fenotipo , Conformación Proteica , Secuencias Repetidas en TándemRESUMEN
Background: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney,caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative (DN) mode-of-action, wherein an increased level of AFF3 resulted in pathological effects. Methods: Evolutionary constraints suggest that other mode-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be deleterious variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. Results: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous LoF or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not complement. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring +/+, DN/DN, LoF/+, LoF/LoF or DN/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the DN/DN or the LoF/LoF lines. While the same pathways are affected, only about one-third of the differentially expressed genes are common to these homozygote datasets, indicating that AFF3 LoF and DN variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. Conclusions: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.
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BACKGROUND: We previously described the KINSSHIP syndrome, an autosomal dominant disorder associated with intellectual disability (ID), mesomelic dysplasia and horseshoe kidney, caused by de novo variants in the degron of AFF3. Mouse knock-ins and overexpression in zebrafish provided evidence for a dominant-negative mode of action, wherein an increased level of AFF3 resulted in pathological effects. METHODS: Evolutionary constraints suggest that other modes-of-inheritance could be at play. We challenged this hypothesis by screening ID cohorts for individuals with predicted-to-be damaging variants in AFF3. We used both animal and cellular models to assess the deleteriousness of the identified variants. RESULTS: We identified an individual with a KINSSHIP-like phenotype carrying a de novo partial duplication of AFF3 further strengthening the hypothesis that an increased level of AFF3 is pathological. We also detected seventeen individuals displaying a milder syndrome with either heterozygous Loss-of-Function (LoF) or biallelic missense variants in AFF3. Consistent with semi-dominance, we discovered three patients with homozygous LoF and one compound heterozygote for a LoF and a missense variant, who presented more severe phenotypes than their heterozygous parents. Matching zebrafish knockdowns exhibit neurological defects that could be rescued by expressing human AFF3 mRNA, confirming their association with the ablation of aff3. Conversely, some of the human AFF3 mRNAs carrying missense variants identified in affected individuals did not rescue these phenotypes. Overexpression of mutated AFF3 mRNAs in zebrafish embryos produced a significant increase of abnormal larvae compared to wild-type overexpression further demonstrating deleteriousness. To further assess the effect of AFF3 variation, we profiled the transcriptome of fibroblasts from affected individuals and engineered isogenic cells harboring + / + , KINSSHIP/KINSSHIP, LoF/ + , LoF/LoF or KINSSHIP/LoF AFF3 genotypes. The expression of more than a third of the AFF3 bound loci is modified in either the KINSSHIP/KINSSHIP or the LoF/LoF lines. While the same pathways are affected, only about one third of the differentially expressed genes are common to the homozygote datasets, indicating that AFF3 LoF and KINSSHIP variants largely modulate transcriptomes differently, e.g. the DNA repair pathway displayed opposite modulation. CONCLUSIONS: Our results and the high pleiotropy shown by variation at this locus suggest that minute changes in AFF3 function are deleterious.
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Discapacidad Intelectual , Transcriptoma , Pez Cebra , Animales , Femenino , Humanos , Masculino , Discapacidad Intelectual/genética , Mutación con Pérdida de Función , Mutación Missense , Fenotipo , Pez Cebra/genéticaRESUMEN
Per-Arnt-Sim (PAS) kinase (PASK, PASKIN, and PSK) is a member of the group of nutrient sensing protein kinases. These protein kinases sense the energy or nutrient status of the cell and regulate cellular metabolism appropriately. PAS kinase responds to glucose availability and regulates glucose homeostasis in yeast, mice, and man. Despite this pivotal role, the molecular mechanisms of PAS kinase regulation and function are largely unknown. This review focuses on what is known about PAS kinase, including its conservation from yeast to man, identified substrates, associated phenotypes and role in metabolic disease.
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Glucosa/metabolismo , Homeostasis , Proteínas Serina-Treonina Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Diabetes Mellitus/fisiopatología , Activación Enzimática , Regulación de la Expresión Génica , Humanos , Fenotipo , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
PAS kinase 1 (Psk1) is a key regulator of respiration in Saccharomyces cerevisiae Herein the molecular mechanisms of this regulation are explored through the characterization of its substrate, Centromere binding factor 1 (Cbf1). CBF1-deficient yeast displayed a significant decrease in cellular respiration, while PAS kinase-deficient yeast, or yeast harboring a Cbf1 phosphosite mutant (T211A) displayed a significant increase. Transmission electron micrographs showed an increased number of mitochondria in PAS kinase-deficient yeast consistent with the increase in respiration. Although the CBF1-deficient yeast did not appear to have an altered number of mitochondria, a mitochondrial proteomics study revealed significant differences in the mitochondrial composition of CBF1-deficient yeast including altered Atp3 levels, a subunit of the mitochondrial F1-ATP synthase complex. Both beta-galactosidase reporter assays and western blot analysis confirmed direct transcriptional control of ATP3 by Cbf1 In addition, we confirmed the regulation of yeast lipid genes LAC1 and LAG1 by Cbf1 The human homolog of Cbf1, Upstream transcription factor 1 (USF1), is also known to be involved in lipid biogenesis. Herein, we provide the first evidence for a role of USF1 in respiration since it appeared to complement Cbf1 in vivo as determined by respiration phenotypes. In addition, we confirmed USF1 as a substrate of human PAS kinase (hPASK) in vitro Combined, our data supports a model in which Cbf1/USF1 functions to partition glucose toward respiration and away from lipid biogenesis, while PAS kinase inhibits respiration in part through the inhibition of Cbf1/USF1.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Respiración de la Célula/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores Estimuladores hacia 5'/genética , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Glucosa/metabolismo , Humanos , Lipogénesis/genética , Mitocondrias/genética , Mutación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Transcripción Genética/genéticaRESUMEN
We describe the interplay between three sensory protein kinases in yeast: AMP-regulated kinase (AMPK, or SNF1 in yeast), PAS kinase 1 (Psk1 in yeast), and the target of rapamycin complex 1 (TORC1). This signaling cascade occurs through the SNF1-dependent phosphorylation and activation of Psk1, which phosphorylates and activates poly(A)- binding protein binding protein 1 (Pbp1), which then inhibits TORC1 through sequestration at stress granules. The SNF1-dependent phosphorylation of Psk1 appears to be direct, in that Snf1 is necessary and sufficient for Psk1 activation by alternate carbon sources, is required for altered Psk1 protein mobility, is able to phosphorylate Psk1 in vitro, and binds Psk1 via its substrate-targeting subunit Gal83. Evidence for the direct phosphorylation and activation of Pbp1 by Psk1 is also provided by in vitro and in vivo kinase assays, including the reduction of Pbp1 localization at distinct cytoplasmic foci and subsequent rescue of TORC1 inhibition in PAS kinase-deficient yeast. In support of this signaling cascade, Snf1-deficient cells display increased TORC1 activity, whereas cells containing hyperactive Snf1 display a PAS kinase-dependent decrease in TORC1 activity. This interplay between yeast SNF1, Psk1, and TORC1 allows for proper glucose allocation during nutrient depletion, reducing cell growth and proliferation when energy is low.
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Proteínas Portadoras/metabolismo , Glucosa/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proliferación Celular , Regulación hacia Abajo , Activación Enzimática , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Fosforilación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.