Amalia Dutra - Academia.edu (original) (raw)

Papers by Amalia Dutra

Nature Communications

Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often actin... more Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10−10 per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate.

Science, 1997

Parkinson's disease (PD) is a common neurodegenerative disorder with a lifetime incidence of ... more Parkinson's disease (PD) is a common neurodegenerative disorder with a lifetime incidence of approximately 2 percent. A pattern of familial aggregation has been documented for the disorder, and it was recently reported that a PD susceptibility gene in a large Italian kindred is located on the long arm of human chromosome 4. A mutation was identified in the α-synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred and in three unrelated families of Greek origin with autosomal dominant inheritance for the PD phenotype. This finding of a specific molecular alteration associated with PD will facilitate the detailed understanding of the pathophysiology of the disorder.

ACS Pharmacology & Translational Science, 2021

Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, cha... more Charcot-Marie-Tooth 1A (CMT1A) is the most common form of hereditary peripheral neuropathies, characterized by genetic duplication of the critical myelin gene Peripheral Myelin Protein 22 (PMP22). PMP22 overexpression results in abnormal Schwann cell differentiation, leading to axonal loss and muscle wasting. Since regulation of PMP22 expression is a major target of therapeutic discovery for CMT1A, we sought to establish unbiased approaches that allow the identification of therapeutic agents for this disease. Using genome editing, we generated a coincidence reporter assay that accurately monitors Pmp22 transcript levels in the S16 rat Schwann cell line, while reducing reporter-based false positives. A quantitative high-throughput screen (qHTS) of 42 577 compounds using this assay revealed diverse novel chemical classes that reduce endogenous Pmp22 transcript levels. Moreover, some of these classes show pharmacological specificity in reducing Pmp22 over another major myelin-associated gene, Mpz (Myelin protein zero). Finally, to investigate whether compound-mediated reduction of Pmp22 transcripts translates to reduced PMP22 protein levels, we edited the S16 genome to generate a reporter assay that expresses a PMP22-HiBiT fusion protein using CRISPR/Cas9. Overall, we present a screening platform that combines genome edited cell lines encoding reporters that monitor transcriptional and posttranslational regulation of PMP22 with titration-based screening (e.g., qHTS), which could be efficiently incorporated into drug discovery campaigns for CMT1A.

SummaryResearchers have presumed that the “inactive” X chromosome (Xi) has little impact, in tran... more SummaryResearchers have presumed that the “inactive” X chromosome (Xi) has little impact, in trans, on the “active” X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xi’s. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X-chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes (≥ 121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X-chromosome genes most likely to drive sex differences in common disease, and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of cis and trans effects differs widely among genes.

Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Prote... more Determination of the chromosomal location and genomic structure of the Hedgehog-Interacting Protein gene, and analysis of its role in Holoprosencephaly Hedgehog-Interacting Protein (HIP) is a novel component of the Sonic Hedgehog (SHH) signaling pathway. Recently, defects in this pathway have been shown to cause holoprosencephaly (HPE), which is the most common birth defect of the brain and face in humans. In animal models knockout mice with homozygous null mutations for Shh displayed abnormalities consistent with HPE. Hip has been shown to function as a co-receptor and attenuate Hedgehog signaling by cell-surface binding of the Hedgehog protein and is hypothesized to act as part of a negative regulatory feedback loop. Although Hip is an important factor in the Shh signaling pathway, its potential role in HPE had not been examined in humans. Here, we report the complete gene structure of the human HIP gene and present its mutational analysis in HPE patients. No mutations were found ...

Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (... more Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phos-phatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN–/ – neural precursor cells with a retrovirus encoding EGFRvIII, which is a con-stitutively activated receptor. EGFRvIII PTEN–/ – cells formed highly mitotic tumors with nuclear pleomor-phism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radia-tion. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/ Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphor-ylated at S280 by Akt, suppressi...

Investigative Ophthalmology & Visual Science, 2013

SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environ... more SUMMARYEndoC-βH1 is emerging as a critical human beta cell model to study the genetic and environmental etiologies of beta cell function, especially in the context of diabetes. Comprehensive knowledge of its molecular landscape is lacking yet required to fully take advantage of this model. Here, we report extensive chromosomal (spectral karyotyping), genetic (genotyping), epigenetic (ChIP-seq, ATAC-seq), chromatin interaction (Hi-C, Pol2 ChIA-PET), and transcriptomic (RNA-seq, miRNA-seq) maps of this cell model. Integrated analyses of these maps define known (e.g.,PDX1, ISL1) and putative (e.g.,PCSK1, mir-375) beta cell-specific chromatin interactions and transcriptionalcis-regulatory networks, and identify allelic effects oncis-regulatory element use and expression.Importantly, comparative analyses with maps generated in primary human islets/beta cells indicate substantial preservation of chromatin looping, but also highlight chromosomal heterogeneity and fetal genomic signatures i...

Proceedings of the National Academy of Sciences, 2004

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion ... more Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11 . We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11 . The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2 , with eight and two indepen...

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most... more After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on ...

Cell Reports

Highlights d Comprehensive multiomic maps of EndoC-bH1 human b cell line and primary islets d Seq... more Highlights d Comprehensive multiomic maps of EndoC-bH1 human b cell line and primary islets d Sequence motifs enriched in EndoC-specific enhancers reflect its precursor state d Identification of regulatory hubs preserved between EndoC-bH1 and human islets d Identified SNP alleles (including T2D GWAS) altering cisregulatory signatures

Human molecular genetics, Jan 15, 2018

Peripheral nerve myelination is adversely affected in the most common form of the hereditary peri... more Peripheral nerve myelination is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot-Marie-Tooth Disease. This form, classified as CMT1A, is caused by a 1.4 Mb duplication on chromosome 17, which includes the abundantly expressed Schwann cell myelin gene, Peripheral Myelin Protein 22 (PMP22). This is one of the most common copy number variants causing neurological disease. Overexpression of Pmp22 in rodent models recapitulates several aspects of neuropathy, and reduction of Pmp22 in such models results in amelioration of the neuropathy phenotype. Recently we identified a potential super-enhancer approximately 90-130 kb upstream of the Pmp22 transcription start sites. This super-enhancer encompasses a cluster of individual enhancers that have the acetylated histone H3K27 active enhancer mark, and coincides with smaller duplications identified in patients with milder CMT1A-like symptoms, where the PMP22 coding region itself was not part of ...

BMC cancer, Feb 13, 2017

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition d... more Neurofibromatosis type 2 (NF2) is a rare autosomal dominant nervous system tumor predisposition disorder caused by constitutive inactivation of one of the two copies of NF2. Meningiomas affect about one half of NF2 patients, and are associated with a higher disease burden. Currently, the somatic mutation landscape in NF2-associated meningiomas remains largely unexamined. Here, we present an in-depth genomic study of benign and atypical meningiomas, both from a single NF2 patient. While the grade I tumor was asymptomatic, the grade II tumor exhibited an unusually high growth rate: expanding to 335 times its initial volume within one year. The genomes of both tumors were examined by whole-exome sequencing (WES) complemented with spectral karyotyping (SKY) and SNP-array copy-number analyses. To better understand the clonal composition of the atypical meningioma, the tumor was divided in four sections and each section was investigated independently. Both tumors had second copy inactivat...

The Faseb Journal, Apr 1, 2009

Chromosoma, Oct 31, 1984

Analysis of meiotic prophase in the male cricket Discophogryllus ontophagus (2n = 19, XO/XX) show... more Analysis of meiotic prophase in the male cricket Discophogryllus ontophagus (2n = 19, XO/XX) showed a diffuse stage, characterized by a high decondensation of euchromatin, a large nucleus, peculiar behaviour of the sex chromosome, and separation of homologues. Labelling with 3H-thymidine showed that it occurred during middiplotene.-Transcription of genetic information during the meiotic prophase was determined in vitro by the incorporation of tritiated uridine into the spermatocytes. During the diffuse stage only 6.5% of the scored cells showed grains in the autoradiographic analysis.-An intense transcriptional activity has been attributed to this phase, based on its morphological resemblance to the lampbrush configuration. However, the data suggest that-at least in this species-the diffuse stage is in fact a period of brief or no transcriptional activity.

J Immunother, 1997

Fas-associated death domain protein (FADD)/MORT1 is a 23-kDa cytoplasmic protein containing a C-t... more Fas-associated death domain protein (FADD)/MORT1 is a 23-kDa cytoplasmic protein containing a C-terminal death domain that interacts with the intracellular death domain of the Fas transmembrane receptor. Cross-linking of Fas mediates apoptosis in a variety of cells, primarily peripheral T lymphocytes, for which this pathway plays a major role in mature lymphocyte homeostasis. We report the characterization of the human FADD gene, which spans approximately 3.6 kb and contains two exons (286 and 341 bp) separated by a 2.0-kb intron. FADD was mapped to chromosome 11q13.3 by the independent techniques of PCR screening of somatic cell hybrid mapping panels and fluorescence in situ hybridization. In addition FADD was shown by fluorescence in situ hybridization to be amplified along with other 11q13.3 genes previously studied in the breast cancer cell line MDA-MB-134-VI, raising the possibility that overexpression of mutant FADD could contribute to poor prognosis and increased invasiveness of tumors. Its known role in apoptosis has made FADD a candidate susceptibility gene for autoimmune lymphoproliferative syndrome. Now that it has been colocalized in 11q13.3 with IDDM4, a diabetes susceptibility locus, alterations in FADD should also be considered as potential contributors to insulin-dependent familial diabetes. Elucidation of the map position and gene structure of FADD will make possible linkage and mutation analysis to study the role of this gene in human diseases.

Disease Models & Mechanisms, 2016

Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gauche... more Glucocerebrosidase is a lysosomal hydrolase involved in the breakdown of glucosylceramide. Gaucher disease, a recessive lysosomal storage disorder, is caused by mutations in the gene GBA1. Dysfunctional glucocerebrosidase leads to accumulation of glucosylceramide and glycosylsphingosine in various cell types and organs. Mutations in GBA1 are also a common genetic risk factor for Parkinson disease and related synucleinopathies. In recent years, research on the pathophysiology of Gaucher disease, the molecular link between Gaucher and Parkinson disease, and novel therapeutics, have accelerated the need for relevant cell models with GBA1 mutations. Although induced pluripotent stem cells, primary rodent neurons, and transfected neuroblastoma cell lines have been used to study the effect of glucocerebrosidase deficiency on neuronal function, these models have limitations because of challenges in culturing and propagating the cells, low yield, and the introduction of exogenous mutant GBA1. To address some of these difficulties, we established a high yield, easy-to-culture mouse neuronal cell model with nearly complete glucocerebrosidase deficiency representative of Gaucher disease. We successfully immortalized cortical neurons from embryonic null allele gba −/− mice and the control littermate (gba +/+) by infecting differentiated primary cortical neurons in culture with an EF1α-SV40T lentivirus. Immortalized gba −/− neurons lack glucocerebrosidase protein and enzyme activity, and exhibit a dramatic increase in glucosylceramide and glucosylsphingosine accumulation, enlarged lysosomes, and an impaired ATP-dependent calcium-influx response; these phenotypical characteristics were absent in gba +/+ neurons. This null allele gba −/− mouse neuronal model provides a much-needed tool to study the pathophysiology of Gaucher disease and to evaluate new therapies.

Amer J Med Genet, 2004

We report a 19-year-old, non-Amish Caucasian female patient with primary amenorrhea caused by com... more We report a 19-year-old, non-Amish Caucasian female patient with primary amenorrhea caused by complete lack of Mü llerian fusion with vaginal agenesis or Mü llerian aplasia (MA), postaxial polydactyly (PAP), and tetralogy of Fallot. The genital tract anomaly of MA with and without renal or skeletal anomalies comprises Mayer-Rokitansky-Kuster-Hauser syndrome, which has not been reported with tetralogy of Fallot. The phenotypic triad of anomalies most closely resembled McKusick-Kaufman syndrome (MKS; OMIM 236700), a rare multiple congenital anomaly syndrome comprised of hydrometrocolpos (HMC), PAP, and congenital heart malformation that is inherited in an autosomal recessive pattern. While upper reproductive tract anomalies have not been reported with MKS, they have been reported with Bardet-Biedl syndrome (BBS), a syndrome that significantly overlaps with MKS. Both MKS and BBS can be caused by mutations in the MKKS or BBS6 gene on chromosome 20p12 and BBS is also associated with mutations in other genes (BBS1, BBS2, BBS4, and BBS7). To address this heterogenity, we sequenced the causative genes in MKS and BBS but no mutations in these five genes were identified. Fluorescence in situ hybridization (FISH) excluded large deletions of chromosome 20p12 and microsatellite marker studies confirmed biparental inheritance for all of the known BBS loci. The dual midline fusion defects of tetralogy of Fallot and MA suggests that either this patient has a unique syndrome with a distinct genetic etiology or that she has a genetically heterogeneous or variant form of MKS.