RESUMO
INTRODUCTION: Mutations in the thyroid hormone receptor alpha (THRA) gene are a rare cause of thyroid hormone resistance, which leads to a pleomorphic phenotypic spectrum. Hormonal profiles are variable and subtle, making laboratory diagnoses challenging. Genetic evaluation can be a helpful tool in diagnosing these cases. CASE PRESENTATION: Three patients (P1, P2, and P3) from unrelated families presented to their endocrinologists with short stature and abnormalities in thyroid function results. P1 showed hypoactivity and mild thyroid-stimulating hormone (TSH) elevation. P2 presented with a mild developmental delay and a hormonal profile initially interpreted as central hypothyroidism. Patient P3 had severe symptoms, including hypotonia, developmental delay, normal TSH, hypercholesterolemia, severe hypertriglyceridemia, high amylase levels, and mild pericardial effusion. All the patients had low free thyroxine (FT4) levels, mild constipation, and short stature. The patients underwent exome sequencing analysis that identified three different heterozygous variants in the THRA gene (P1 and P2 had missense variants, and P3 had a stop codon variant). All patients were treated with levothyroxine replacement, improving their clinical symptoms, such as constipation, and neurological symptoms. P1 and P2 were also treated with the recombinant human growth hormone (rhGH). The improvements in growth velocity and height standard deviation scores (SDS) were remarkable. Notably, P1 had a total height gain of 2.5 SDS, reaching an adult height within the normal range. CONCLUSION: THRA gene defects can lead to growth disorders with different phenotypes. Children with THRA mutations can benefit from adequate treatment with levothyroxine and may respond well to rhGH treatment.
RESUMO
Collapsing glomerulopathy (CG) is most often associated with fast progression to kidney failure with an incidence apparently higher in Brazil than in other countries. However, the reason for this occurrence is unknown. To better understand this, we performed an integrated analysis of clinical, histological, therapeutic, causative genetic and genetic ancestry data in a highly genetically admixed cohort of 70 children and adult patients with idiopathic CG (ICG). The disease onset occurred at 23 (interquartile range: 17-31) years and approximately half of patients progressed to chronic kidney disease requiring kidney replacement therapy (CKD-KRT) 36 months after diagnosis. Causative genetic bases, assessed by targeted-gene panel or whole-exome sequencing, were identified in 58.6% of patients. Among these cases, 80.5% harbored APOL1 high-risk genotypes (HRG) and 19.5% causative Mendelian variants (MV). Self-reported non-White patients more frequently had HRG. MV was an independent risk factor for progression to CKD-KRT by 36 months and the end of follow-up, while remission was an independent protective factor. All patients with HRG manifested CG at 9-44 years of age, whereas in those with APOL1 low-risk genotype, the disease arose throughout life. HRGs were associated with higher proportion of African genetic ancestry. Novel causative MVs were identified in COL4A5, COQ2 and PLCE1 and previously described causative MVs were identified in MYH9, TRPC6, COQ2, COL4A3 and TTC21B. Three patients displayed HRG combined with a variant of uncertain significance (ITGB4, LAMA5 or PTPRO). MVs were associated with worse kidney prognosis. Thus, our data reveal that the genetic status plays a major role in ICG pathogenesis, accounting for more than half of cases in a highly admixed Brazilian population.
Assuntos
Apolipoproteína L1 , Insuficiência Renal Crônica , Adulto , Criança , Humanos , Apolipoproteína L1/genética , Genótipo , Rim/patologia , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Fatores de Risco , Adolescente , Adulto JovemRESUMO
OBJECTIVE: Our study aimed to assess the impact of genetic modifiers on the significant variation in phenotype that is observed in individuals with SHOX deficiency, which is the most prevalent monogenic cause of short stature. DESIGN AND METHODS: We performed a genetic analysis in 98 individuals from 48 families with SHOX deficiency with a target panel designed to capture the entire SHOX genomic region and 114 other genes that modulate growth and/or SHOX action. We prioritized rare potentially deleterious variants. RESULTS: We did not identify potential deleterious variants in the promoter or intronic regions of the SHOX genomic locus. In contrast, we found eight heterozygous variants in 11 individuals from nine families in genes with a potential role as genetic modifiers. In addition to a previously described likely pathogenic (LP) variant in CYP26C1 observed in two families, we identified LP variants in PTHLH and ACAN, and variants of uncertain significance in NPR2, RUNX2, and TP53 in more affected individuals from families with SHOX deficiency. Families with a SHOX alteration restricted to the regulatory region had a higher prevalence of a second likely pathogenic variant (27%) than families with an alteration compromising the SHOX coding region (2.9%, P = .04). CONCLUSION: In conclusion, variants in genes related to the growth plate have a potential role as genetic modifiers of the phenotype in individuals with SHOX deficiency. In individuals with SHOX alterations restricted to the regulatory region, a second alteration could be critical to determine the penetrance and expression of the phenotype.
Assuntos
Nanismo , Humanos , Íntrons , Genômica , Lâmina de Crescimento , Fenótipo , Doenças Raras , Proteína de Homoeobox de Baixa Estatura/genéticaRESUMO
OBJECTIVES: To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. MATERIAL AND METHODS: Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. RESULTS: Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. CONCLUSION: The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Estudos Retrospectivos , Centros de Atenção Terciária , Mutação/genética , FenótipoRESUMO
Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.
Assuntos
Astrócitos , Glioblastoma , Astrócitos/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transição Epitelial-Mesenquimal , Glioblastoma/patologia , Humanos , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Hypospadias is a common congenital disorder of male genital formation. Children born small for gestational age (SGA) present a high frequency of hypospadias of undetermined etiology. No previous study investigated the molecular etiology of hypospadias in boys born SGA using massively parallel sequencing. Our objective is to report the genetic findings of a cohort of patients born SGA with medium or proximal hypospadias. We identified 46 individuals with this phenotype from a large cohort of 46,XY DSD patients, including 5 individuals with syndromic features. DNA samples from subjects were studied by either whole exome sequencing or target gene panel approach. Three of the syndromic patients have 5 main clinical features of Silver-Russell syndrome (SRS) and were first studied by MLPA. Among the syndromic patients, loss of DNA methylation at the imprinting control region H19/IGF2 was identified in 2 individuals with SRS clinical diagnosis. Two novel pathogenic variants in compound heterozygous state were identified in the CUL7 gene establishing the diagnosis of 3M syndrome in one patient, and a novel homozygous variant in TRIM37 was identified in another boy with Mulibrey nanism phenotype. Among the non-syndromic subjects, 7 rare heterozygous variants were identified in 6 DSD-related genes. However, none of the variants found can explain the phenotype by themselves. In conclusion, a genetic defect that clarifies the etiology of hypospadias was not found in most of the non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition.
Assuntos
Transtorno 46,XY do Desenvolvimento Sexual , Hipospadia , Metilação de DNA/genética , Transtorno 46,XY do Desenvolvimento Sexual/genética , Idade Gestacional , Humanos , Hipospadia/complicações , Hipospadia/genética , Recém-Nascido , Recém-Nascido Pequeno para a Idade Gestacional , Masculino , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Abstract Objectives To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.
RESUMO
Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350.
Assuntos
Neoplasias Encefálicas , Matriz Extracelular , Glioblastoma , Meduloblastoma , Neoplasias Encefálicas/genética , Matriz Extracelular/patologia , Glioblastoma/genética , Humanos , Meduloblastoma/genética , Proteoma/genética , Proteômica , Microambiente TumoralRESUMO
Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.
Assuntos
Aminoácido Oxirredutases/metabolismo , Adesão Celular , Regulação Neoplásica da Expressão Gênica , Glioblastoma/enzimologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Simulação por Computador , Citoesqueleto/metabolismo , Endocitose , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/fisiopatologia , Humanos , Lisossomos/fisiologia , Invasividade NeoplásicaRESUMO
Objective: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. Design: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods: We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.