RESUMEN
BACKGROUND: The diagnostic yield of next-generation sequencing (NGS) technologies in the diagnosis of monogenic inborn errors of immunity (IEI) remains limited, rarely exceeding 30%. Monoallelic pathogenic germline variants in cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) result in variable immunodeficiency and immune dysregulation. The genetic diagnosis of CTLA-4 insufficiency can affect follow-up procedures and may lead to consideration of treatment with CTLA-4-Ig. OBJECTIVES: The aim of the study was to identify the genetic cause of familial immunodeficiency and immune dysregulation in cases where single nucleotide variant analysis of short-read NGS data yielded no diagnostic result. METHODS: Analysis of copy number variants (CNVs) was applied on short-read NGS data. RESULTS: We identified a novel monoallelic deletion-insertion variant in CTLA-4 (c.445_568-544delinsTTTGCGATTG) resulting in familial autoimmunity. This is the second larger scale variant in CTLA-4, which despite consistently reduced expression of CTLA-4 displayed variable expressivity, ranging from typical juvenile idiopathic arthritis to common variable immunodeficiency-like immunodeficiency. CONCLUSIONS: Our report suggests the significance of integration of CNV analysis in routine evaluation of NGS, which may increase its diagnostic yield in IEI.
Asunto(s)
Inmunodeficiencia Variable Común , Síndromes de Inmunodeficiencia , Humanos , Pruebas Genéticas/métodos , Antígeno CTLA-4/genética , Variaciones en el Número de Copia de ADN , Abatacept/genética , Síndromes de Inmunodeficiencia/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inmunodeficiencia Variable Común/genéticaRESUMEN
Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood and adolescence. In more than 60% of cases of this heterogeneous disease, a genetic marker is identified via cytogenetic or molecular analyses. TCF3 gene fusions occur in 5%-11% of ALL patients. In < 1%, the TCF3 alteration in ALL leads to a TCF3-HLF fusion gene. Even though this is a very rare event, the detection of a TCF3-HLF fusion gene is associated with a very poor prognosis with incurable relapses in almost all patients. The frequent TCF3-PBX1 fusion gene, which is detectable in 5%-10% of childhood B-cell precursor ALLs and ~3.8% of adult B-cell precursor ALLs, is associated with a rather good prognosis, that is, an observed event-free 5-year survival of approximately 85%. Thus, the distinction of the different partner genes fused to TCF3 is essential for risk assessment. To verify RNA sequencing as a tool for detection of known and unknown fusion genes, we screened 200 cases of pediatric B-cell precursor ALL with "targeted" RNA sequencing in a pilot project in comparison to classical cytogenetic analyses (chromosome R-banding analysis), fluorescence in situ hybridization, and PCR. We observed a TCF3 fusion gene in 6.5% (13/200) of the patients. Ten (5%) patients displayed a TCF3-PBX1 fusion gene, two (1%) patients a TCF3-FLI1 fusion gene, and one (0.5%) patient a TCF3-HLF fusion gene. For the TCF3 fusions, we obtained discrepant results with the different methods, which are described in the article. Taken together, translocations leading to TCF3 fusion genes might appear cryptic and may remain undetected by a single method.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Análisis de Secuencia de ARN , Niño , Bandeo Cromosómico , Humanos , Hibridación Fluorescente in Situ , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Pronóstico , Proteína Proto-Oncogénica c-fli-1/genética , Translocación GenéticaRESUMEN
Inborn errors of immunity (IEI) are genetically driven disorders. With the advancement of sequencing technologies, a rapidly increasing number of gene defects has been identified, thereby mirroring the high heterogeneity in immunological and clinical presentations observed in patients. However, for a large majority of patients, no causative single nucleotide variant (SNV) or small indel can be identified using next-generation sequencing. First studies have shown that also copy number variants (CNVs) can cause IEI. Unfortunately, CNVs are not well examined in many routine diagnostic settings and the aim of this study was to assess the number of clinically relevant chromosomal losses and gains in a large cohort. We identified a total of 20 CNVs using whole exome sequencing data of a cohort of 191 patients with a suspected IEI. A definite molecular diagnosis could be made in five patients (2.6%), including pathogenic deletions affecting ICOS, TNFAIP3, and 22q11.2. CNVs of uncertain significance were observed in fifteen patients (7.9%), including deletions of 11q22.1q22.3 and 16p11.2 but also duplications affecting entire or parts of genes previously associated with IEI. Importantly, five patients carrying a CNV of uncertain significance also carried pathogenic or likely pathogenic SNVs (PIK3R1, NFKB1, NLRC4, DOCK2), or SNVs of unknown significance (NFKB2). This cooccurrence of SNVs and CNVs suggests modifying effects in some patients, and functional follow-up is warranted now in order to better understand phenotypic heterogeneity. In summary, the diagnostic yield of IEI can be increased substantially by evaluating CNVs, which allows an improved therapeutic management in those patients.
Asunto(s)
Aberraciones Cromosómicas , Variaciones en el Número de Copia de ADN , Enfermedades del Sistema Inmune , Estudios de Cohortes , Enfermedades Genéticas Congénitas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Enfermedades del Sistema Inmune/genética , Secuenciación del ExomaRESUMEN
Perturbed vitamin-A metabolism is associated with type 2 diabetes and mitochondrial dysfunction that are pathophysiologically linked to the development of diabetic cardiomyopathy (DCM). However, the mechanism, by which vitamin A might regulate mitochondrial energetics in DCM has previously not been explored. To test the hypothesis that vitamin-A deficiency accelerates the onset of cardiomyopathy in diet-induced obesity (DIO), we subjected mice with lecithin retinol acyltransferase (Lrat) germline deletion, which exhibit impaired vitamin-A stores, to vitamin A-deficient high-fat diet (HFD) feeding. Wild-type mice fed with a vitamin A-sufficient HFD served as controls. Cardiac structure, contractile function, and mitochondrial respiratory capacity were preserved despite vitamin-A deficiency following 20 wk of HFD feeding. Gene profiling by RNA sequencing revealed that vitamin A is required for the expression of genes involved in cardiac fatty acid oxidation, glycolysis, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation in DIO as expression of these genes was relatively preserved under vitamin A-sufficient HFD conditions. Together, these data identify a transcriptional program, by which vitamin A preserves cardiac energetic gene expression in DIO that might attenuate subsequent onset of mitochondrial and contractile dysfunction.NEW & NOTEWORTHY The relationship between vitamin-A status and the pathogenesis of diabetic cardiomyopathy has not been studied in detail. We assessed cardiac mitochondrial respiratory capacity, contractile function, and gene expression by RNA sequencing in a murine model of combined vitamin-A deficiency and diet-induced obesity. Our study identifies a role for vitamin A in preserving cardiac energetic gene expression that might attenuate subsequent development of mitochondrial and contractile dysfunction in diet-induced obesity.
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Diabetes Mellitus Tipo 2 , Cardiomiopatías Diabéticas , Ratones , Animales , Vitamina A , Modelos Animales de Enfermedad , Dieta , Obesidad/genética , Expresión Génica , VitaminasRESUMEN
Hereditary mutations in polynucleotide kinase-phosphatase (PNKP) result in a spectrum of neurological pathologies ranging from neurodevelopmental dysfunction in microcephaly with early onset seizures (MCSZ) to neurodegeneration in ataxia oculomotor apraxia-4 (AOA4) and Charcot-Marie-Tooth disease (CMT2B2). Consistent with this, PNKP is implicated in the repair of both DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs); lesions that can trigger neurodegeneration and neurodevelopmental dysfunction, respectively. Surprisingly, however, we did not detect a significant defect in DSB repair (DSBR) in primary fibroblasts from PNKP patients spanning the spectrum of PNKP-mutated pathologies. In contrast, the rate of SSB repair (SSBR) is markedly reduced. Moreover, we show that the restoration of SSBR in patient fibroblasts collectively requires both the DNA kinase and DNA phosphatase activities of PNKP, and the fork-head associated (FHA) domain that interacts with the SSBR protein, XRCC1. Notably, however, the two enzymatic activities of PNKP appear to affect different aspects of disease pathology, with reduced DNA phosphatase activity correlating with neurodevelopmental dysfunction and reduced DNA kinase activity correlating with neurodegeneration. In summary, these data implicate reduced rates of SSBR, not DSBR, as the source of both neurodevelopmental and neurodegenerative pathology in PNKP-mutated disease, and the extent and nature of this reduction as the primary determinant of disease severity.
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Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Enzimas Reparadoras del ADN/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Apraxias/genética , Apraxias/patología , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Reparación del ADN/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Microcefalia/genética , Microcefalia/patología , Mutación/genética , Convulsiones/genética , Convulsiones/patologíaRESUMEN
PAX5 is a member of the paired box (PAX) family of transcription factors involved in B-cell development. PAX5P80R has recently been described as a distinct genetic B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) subtype with a favorable prognosis in adults. In contrast, an unfavorable outcome has been observed in children. Our aim was to determine the frequency of PAX5P80R in childhood BCP-ALL treated according to the Associazione Italiana Ematologia ed Oncologia Pediatrica-Berlin-Frankfurt-Muenster (AIEOP-BFM) ALL 2000 protocol and to evaluate its clinical significance within this study cohort. The analyses included 1237 patients with ALL treated in the AIEOP-BFM ALL 2000 trial with complete information for copy number variations (CNVs) of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, and ERG. A customized TaqMan genotyping assay was used to screen for PAX5P80R . Sanger sequencing was used to confirm PAX5P80R -positive results as well as to screen for second variants in PAX5. Agilent CGH + SNP arrays (e-Array design 85 320; Agilent Technologies) were performed in PAX5P80R -positive patients to verify additional CNVs. Almost 2% (20/1028) of our BCP-ALL cohort were PAX5P80R -positive. White blood cell counts higher than 50 000/µl as well as male sex were significantly (P < .05) associated with PAX5P80R . Most of the PAX5P80R -positive cases were 10 years of age or older. PAX5P80R -positive samples were enriched for deletions affecting PAX5, IKZF1, CDKN2A, and CDKN2B. Compared to PAX5P80R -wildtype BCP-ALL, PAX5P80R -positive patients showed a significantly reduced 5-year overall survival (P = .042). Further studies should evaluate the interaction of PAX5P80R with other genetic aberrations to further stratify intermediate risk pediatric BCP-ALL.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Asparaginasa/uso terapéutico , Ciclofosfamida/uso terapéutico , Citarabina/uso terapéutico , Daunorrubicina/uso terapéutico , Frecuencia de los Genes , Humanos , Mercaptopurina/uso terapéutico , Metotrexato/uso terapéutico , Mutación Missense , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Prednisona/uso terapéutico , Vincristina/uso terapéuticoRESUMEN
We present two independent cases of syndromic thrombocytopenia with multiple malformations, microcephaly, learning difficulties, dysmorphism and other features. Exome sequencing identified two novel de novo heterozygous variants in these patients, c.35G>T p.(Gly12Val) and c.178G>C p.(Gly60Arg), in the RAP1B gene (NM_001010942.2). These variants have not been described previously as germline variants, however functional studies in literature strongly suggest a clinical implication of these two activating hot spot positions. We hypothesize that pathogenic missense variants in the RAP1B gene cause congenital syndromic thrombocytopenia with a spectrum of associated malformations and dysmorphism, possibly through a gain of function mechanism.
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Discapacidad Intelectual/genética , Microcefalia/genética , Trombocitopenia/genética , Proteínas de Unión al GTP rap/genética , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Adolescente , Adulto , Niño , Preescolar , Exoma/genética , Femenino , Heterocigoto , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/patología , Masculino , Microcefalia/diagnóstico , Microcefalia/patología , Mutación Missense/genética , Linaje , Fenotipo , Trombocitopenia/diagnóstico , Trombocitopenia/patología , Secuenciación del ExomaRESUMEN
Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.
Asunto(s)
Hibridación Genómica Comparativa , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , ARN Mensajero/análisis , ARN Neoplásico/análisis , Análisis de Secuencia de ARN , Cariotipo Anormal , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Asparaginasa/administración & dosificación , Ciclofosfamida/administración & dosificación , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Genes Relacionados con las Neoplasias , Humanos , Factor de Transcripción Ikaros/genética , Hibridación Fluorescente in Situ , Mercaptopurina/administración & dosificación , Metotrexato/administración & dosificación , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prednisona/administración & dosificación , Estudios Prospectivos , Factores de Riesgo , Transcriptoma , Vincristina/administración & dosificación , Flujo de TrabajoRESUMEN
NGS-based multiple gene panel resequencing in combination with a high resolution CGH-array was used to identify genetic risk factors for hereditary breast and/or ovarian cancer in 237 high risk patients who were previously tested negative for pathogenic BRCA1/2 variants. All patients were screened for pathogenic variants in 94 different cancer predisposing genes. We identified 32 pathogenic variants in 14 different genes (ATM, BLM, BRCA1, CDH1, CHEK2, FANCG, FANCM, FH, HRAS, PALB2, PMS2, PTEN, RAD51C and NBN) in 30 patients (12.7%). Two pathogenic BRCA1 variants that were previously undetected due to less comprehensive and sensitive methods were found. Five pathogenic variants are novel, three of which occur in genes yet unrelated to hereditary breast and/or ovarian cancer (FANCG, FH and HRAS). In our cohort we discovered a remarkably high frequency of truncating variants in FANCM (2.1%), which has recently been suggested as a susceptibility gene for hereditary breast cancer. Two patients of our cohort carried two different pathogenic variants each and 10 other patients in whom a pathogenic variant was confirmed also harbored a variant of unknown significance in a breast and ovarian cancer susceptibility gene. We were able to identify pathogenic variants predisposing for tumor formation in 12.3% of BRCA1/2 negative breast and/or ovarian cancer patients.
Asunto(s)
Neoplasias de la Mama Masculina/genética , Neoplasias de la Mama/genética , ADN Helicasas/genética , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Neoplasias Ováricas/genética , Adolescente , Adulto , Anciano , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/patología , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas , Humanos , Masculino , Anamnesis , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Breast cancer is the most prevalent tumor entity in Li-Fraumeni syndrome. Up to 80% of individuals with a Li-Fraumeni-like phenotype do not harbor detectable causative germline TP53 variants. Yet, no systematic panel analyses for a wide range of cancer predisposition genes have been conducted on cohorts of women with breast cancer fulfilling Li-Fraumeni(-like) clinical diagnostic criteria. METHODS: To specifically help explain the diagnostic gap of TP53 wild-type Li-Fraumeni(-like) breast cancer cases, we performed array-based CGH (comparative genomic hybridization) and panel-based sequencing of 94 cancer predisposition genes on 83 breast cancer patients suggestive of Li-Fraumeni syndrome who had previously had negative test results for causative BRCA1, BRCA2, and TP53 germline variants. RESULTS: We identified 13 pathogenic or likely pathogenic germline variants in ten patients and in nine genes, including four copy number aberrations and nine single-nucleotide variants or small indels. Three patients presented as double-mutation carriers involving two different genes each. In five patients (5 of 83; 6% of cohort), we detected causative pathogenic variants in established hereditary breast cancer susceptibility genes (i.e., PALB2, CHEK2, ATM). Five further patients (5 of 83; 6% of cohort) were found to harbor pathogenic variants in genes lacking a firm association with breast cancer susceptibility to date (i.e., Fanconi pathway genes, RECQ family genes, CDKN2A/p14ARF, and RUNX1). CONCLUSIONS: Our study details the mutational spectrum in breast cancer patients suggestive of Li-Fraumeni syndrome and indicates the need for intensified research on monoallelic variants in Fanconi pathway and RECQ family genes. Notably, this study further reveals a large portion of still unexplained Li-Fraumeni(-like) cases, warranting comprehensive investigation of recently described candidate genes as well as noncoding regions of the TP53 gene in patients with Li-Fraumeni(-like) syndrome lacking TP53 variants in coding regions.
Asunto(s)
Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , Síndrome de Li-Fraumeni/genética , Adulto , Estudios de Cohortes , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN/métodos , Femenino , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Proteína p53 Supresora de Tumor/genética , Adulto JovenRESUMEN
Myelodysplastic syndrome (MDS) can easily transform into acute myeloid leukemia (AML), a process which is often associated with clonal evolution and development of complex karyotypes. Deletion of 5q (del(5q)) is the most frequent aberration in complex karyotypes. This prompted us to analyze clonal evolution in MDS patients with del(5q). There were 1684 patients with low and intermediate-risk MDS and del(5q) with or without one additional cytogenetic abnormality, who were investigated cytogenetically in our department, involving standard karyotyping, fluorescence in situ hybridization (FISH) and multicolor FISH. We identified 134 patients (8%) with aspects of clonal evolution. There are two main routes of cytogenetic clonal evolution: a stepwise accumulation of cytogenetic events over time and a catastrophic event, which we defined as the occurrence of two or more aberrations present at the same time, leading to a sudden development of highly complex clones. Of the 134 patients, 61% underwent a stepwise accumulation of events whereas 39% displayed a catastrophic event. Patients with isolated del(5q) showed significantly more often a stepwise accumulation of events rather than a catastrophic event. The most frequent aberrations in the group of stepwise accumulation were trisomy 8 and trisomy 21 which were significantly more frequent in this group compared to the catastrophic event group. In the group with catastrophic events, del(7q)/-7 and del(17p)/-17 were the most common aberrations. A loss of 17p, containing the tumor suppressor gene TP53, was found significantly more frequent in this group compared to the group of stepwise accumulation. This leads to the assumption that the loss of TP53 is the driving force in patients with del(5q) who undergo a sudden catastrophic event and evolve into complex karyotypes.
Asunto(s)
Anemia Macrocítica/genética , Evolución Clonal , Cariotipo , Células Cultivadas , Deleción Cromosómica , Cromosomas Humanos Par 5/genética , HumanosRESUMEN
Complex karyotypes are associated with a poor prognosis in chronic lymphocytic leukemia (CLL). Using mFISH, iFISH, and T/C-FISH, we thoroughly characterized 59 CLL patients regarding parameters known to be involved in chromosomal instability: status of the genes ATM and TP53 and telomere length. Interestingly, a deletion of the ATM locus in 11q, independent of the cytogenetic context, was associated with significantly diminished risk (p<0.05) of carrying a mutation in TP53. In patients with loss or mutation of TP53, chromosomal breakage occurred more frequently (p<0.01) in (near-) heterochromatic regions. Median telomere length in patients with complex karyotypes was significantly shorter than that of healthy controls and shorter than in all other cytogenetic cohorts. Furthermore, the median telomere length of patients carrying a TP53 mutation was significantly shorter than without mutation. We conclude that telomere shortening in combination with loss of TP53 induces increased chromosomal instability with preferential involvement of (near-) heterochromatic regions.
Asunto(s)
Secuencia de Bases , Inestabilidad Cromosómica , Heterocromatina/genética , Leucemia Linfocítica Crónica de Células B/genética , Eliminación de Secuencia , Homeostasis del Telómero/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Heterocromatina/metabolismo , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Persona de Mediana Edad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Acute myeloid leukemia with complex karyotype (CK-AML) is a distinct biological entity associated with a very poor outcome. Since complex karyotypes frequently contain deletions of the chromosomal region 12p13 encompassing the tumor suppressor genes ETV6 and CDKN1B, we aimed to unravel their modes of inactivation in CK-AML. RESULTS: To decipher deletions, mutations and methylation of ETV6 and CDKN1B, arrayCGH, SNP arrays, direct sequencing of all coding exons and pyrosequencing of the 5'UTR CpG islands of ETV6 and CDKN1B were performed. In total, 39 of 79 patients (49%) showed monoallelic deletions of 12p13 according to karyotypic data and 20 of 43 patients (47%) according to genomic profiling. Genomic profiling led to the minimal deleted region covering the 3'-UTR of ETV6 and CDKN1B. Direct sequencing revealed one novel monoallelic frameshift mutation in ETV6 while no mutations in CDKN1B were identified. Furthermore, methylation levels of ETV6 and CDKN1B did not indicate transcriptional silencing of any of these genes. ETV6 and CDKN1B had reduced expression levels in CK-AML patients with deletion in 12p13 as compared to CK-AML without deletion in 12p13, while the other genes (BCL2L14, LRP6, DUSP16 and GPRC5D) located within the minimal deleted region in 12p13 had very low or missing expression in CK-AML irrespective of their copy number status. CONCLUSIONS: ETV6 and CDKN1B are mainly affected by small monoallelic deletions, whereas mutations and hypermethylation play a minor role in CK-AML. Reduced gene dosage led to reduced gene expression levels, pointing to haploinsufficiency as the relevant mechanism of inactivation of ETV6 and CDKN1B in CK-AML.
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Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Haploinsuficiencia , Cariotipo , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Represoras/genética , Puntos de Rotura del Cromosoma , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 12 , Metilación de ADN , Análisis Mutacional de ADN , Dosificación de Gen , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Genómica , Humanos , Mutación , Proteína p53 Supresora de Tumor/genética , Proteína ETS de Variante de Translocación 6RESUMEN
Suv39h1 mediates heterochromatin formation in pericentric and telomeric regions by trimethylation of lysine 9 of histone 3 (H3K9me3). Yet, its role in the induction of chromosomal instability is poorly understood. We established a leukemia model by retrovirally expressing Myc in wild-type and histone methyltransferase Suv39h1-deficient hematopoietic cells and characterized the resulting leukemias for chromosomal instability. All mice that received cells overexpressing Myc developed myeloid leukemia with a median survival of 44 days posttransplantation. Myc-overexpressing wild-type leukemias demonstrated clones with numerical chromosomal aberrations (5/16). In secondary transplantations of these leukemic cells, structural changes, mostly end-to-end fusions of chromosomes, appeared (10/12). In contrast, leukemic cells overexpressing Myc with reduced or no Suv39h1 expression had a normal karyotype in primary, secondary, and tertiary transplantations (16/16). Myc-transduced Suv39h1-deficient cells showed less critically short telomeres (P < 0.05) compared with Myc-transduced wild-type bone marrow cells. Gene expression analysis showed upregulation of genes involved in the alternative lengthening of telomeres (ALT) mechanism. Thus, we hypothesize that loss of Suv39h1 implies activation of the ALT mechanism, in turn ensuring telomere length and stability. Our data show for the first time that Suv39h1 deficiency may prevent chromosomal instability by more efficient telomere stabilization in hematopoietic bone marrow cells overexpressing Myc.
Asunto(s)
Inestabilidad Cromosómica , Leucemia Mieloide/genética , Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Represoras/genética , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hibridación Fluorescente in Situ , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Masculino , Metiltransferasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cariotipificación Espectral , Telómero/genética , Homeostasis del Telómero/genética , Acortamiento del Telómero/genéticaRESUMEN
BACKGROUND: Alveolar hypoxia is protective in the context of cardiovascular and ischemic heart disease; however, the underlying mechanisms are incompletely understood. The present study sought to test the hypothesis that hypoxia is cardioprotective in left ventricular pressure overload (LVPO)-induced heart failure. We furthermore aimed to test that overlapping mechanisms promote cardiac recovery in heart failure patients following left ventricular assist device-mediated mechanical unloading and circulatory support. METHODS AND RESULTS: We established a novel murine model of combined chronic alveolar hypoxia and LVPO following transverse aortic constriction (HxTAC). The HxTAC model is resistant to cardiac hypertrophy and the development of heart failure. The cardioprotective mechanisms identified in our HxTAC model include increased activation of HIF (hypoxia-inducible factor)-1α-mediated angiogenesis, attenuated induction of genes associated with pathological remodeling, and preserved metabolic gene expression as identified by RNA sequencing. Furthermore, LVPO decreased Tbx5 and increased Hsd11b1 mRNA expression under normoxic conditions, which was attenuated under hypoxic conditions and may induce additional hypoxia-mediated cardioprotective effects. Analysis of samples from patients with advanced heart failure that demonstrated left ventricular assist device-mediated myocardial recovery revealed a similar expression pattern for TBX5 and HSD11B1 as observed in HxTAC hearts. CONCLUSIONS: Hypoxia attenuates LVPO-induced heart failure. Cardioprotective pathways identified in the HxTAC model might also contribute to cardiac recovery following left ventricular assist device support. These data highlight the potential of our novel HxTAC model to identify hypoxia-mediated cardioprotective mechanisms and therapeutic targets that attenuate LVPO-induced heart failure and mediate cardiac recovery following mechanical circulatory support.
Asunto(s)
Estenosis de la Válvula Aórtica , Insuficiencia Cardíaca , Humanos , Ratones , Animales , Insuficiencia Cardíaca/etiología , Cardiomegalia/metabolismo , Miocardio/metabolismo , Hipoxia/complicaciones , Remodelación Ventricular , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: The increasing volume and intricacy of sequencing data, along with other clinical and diagnostic data, like drug responses and measurable residual disease, creates challenges for efficient clinical comprehension and interpretation. Using paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) as a use case, we present an artificial intelligence (AI)-assisted clinical framework clinALL that integrates genomic and clinical data into a user-friendly interface to support routine diagnostics and reveal translational insights for hematologic neoplasia. METHODS: We performed targeted RNA sequencing in 1365 cases with haematological neoplasms, primarily paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) from the AIEOP-BFM ALL study. We carried out fluorescence in situ hybridization (FISH), karyotyping and arrayCGH as part of the routine diagnostics. The analysis results of these assays as well as additional clinical information were integrated into an interactive web interface using Bokeh, where the main graph is based on Uniform Manifold Approximation and Projection (UMAP) analysis of the gene expression data. At the backend of the clinALL, we built both shallow machine learning models and a deep neural network using Scikit-learn and PyTorch respectively. FINDINGS: By applying clinALL, 78% of undetermined patients under the current diagnostic protocol were stratified, and ambiguous cases were investigated. Translational insights were discovered, including IKZF1plus status dependent subpopulations of BCR::ABL1 positive patients, and a subpopulation within ETV6::RUNX1 positive patients that has a high relapse frequency. Our best machine learning models, LDA and PASNET-like neural network models, achieve F1 scores above 97% in predicting patients' subgroups. INTERPRETATION: An AI-assisted clinical framework that integrates both genomic and clinical data can take full advantage of the available data, improve point-of-care decision-making and reveal clinically relevant insights promptly. Such a lightweight and easily transferable framework works for both whole transcriptome data as well as the cost-effective targeted RNA-seq, enabling efficient and equitable delivery of personalized medicine in small clinics in developing countries. FUNDING: German Ministry of Education and Research (BMBF), German Research Foundation (DFG) and Foundation for Polish Science.
Asunto(s)
Inteligencia Artificial , Investigación Biomédica Traslacional , Humanos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Biología Computacional/métodos , Niño , Hibridación Fluorescente in Situ/métodos , Femenino , Masculino , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica/métodosRESUMEN
Monogenic autoinflammatory diseases (AID) encompass a growing group of inborn errors of the innate immune system causing unprovoked or exaggerated systemic inflammation. Diagnosis of monogenic AID requires an accurate description of the patients' phenotype, and the identification of highly penetrant genetic variants in single genes is pivotal. We performed whole exome sequencing (WES) of 125 pediatric patients with suspected monogenic AID in a routine genetic diagnostic setting. Datasets were analyzed in a step-wise approach to identify the most feasible diagnostic strategy. First, we analyzed a virtual gene panel including 13 genes associated with known AID and, if no genetic diagnosis was established, we then analyzed a virtual panel including 542 genes published by the International Union of Immunological Societies associated including all known inborn error of immunity (IEI). Subsequently, WES data was analyzed without pre-filtering for known AID/IEI genes. Analyzing 13 genes yielded a definite diagnosis in 16.0% (n = 20). The diagnostic yield was increased by analyzing 542 genes to 20.8% (n = 26). Importantly, expanding the analysis to WES data did not increase the diagnostic yield in our cohort, neither in single WES analysis, nor in trio-WES analysis. The study highlights that the cost- and time-saving analysis of virtual gene panels is sufficient to rapidly confirm the differential diagnosis in pediatric patients with AID. WES data or trio-WES data analysis as a first-tier diagnostic analysis in patients with suspected monogenic AID is of limited benefit.
RESUMEN
Inborn errors of immunity are known to influence susceptibility to mycobacterial infections. The aim of this study was to characterize the genetic profile of nine patients with mycobacterial infections (eight with BCGitis and one with disseminated tuberculosis) from the Republic of Moldova using whole-exome sequencing. In total, 12 variants in eight genes known to be associated with Mendelian Susceptibility to Mycobacterial Disease (MSMD) were detected in six out of nine patients examined. In particular, a novel splice site mutation c.373-2A>C in STAT1 gene was found and functionally confirmed in a patient with disseminated tuberculosis. Trio analysis was possible for seven out of nine patients, and resulted in 23 candidate variants in 15 novel genes. Four of these genes - GBP2, HEATR3, PPP1R9B and KDM6A were further prioritized, considering their elevated expression in immune-related tissues. Compound heterozygosity was found in GBP2 in a single patient, comprising a maternally inherited missense variant c.412G>A/p.(Ala138Thr) predicted to be deleterious and a paternally inherited intronic mutation c.1149+14T>C. Functional studies demonstrated that the intronic mutation affects splicing and the level of transcript. Finally, we analyzed pathogenicity of variant combinations in gene pairs and identified five patients with putative oligogenic inheritance. In summary, our study expands the spectrum of genetic variation contributing to susceptibility to mycobacterial infections in children and provides insight into the complex/oligogenic disease-causing mode.