Publications by authors named "Ernesto Guzmán"

8 Publications

  • Page 1 of 1

Profiling of Short-Tandem-Repeat Disease Alleles in 12,632 Human Whole Genomes.

Am J Hum Genet 2017 Nov;101(5):700-715

Human Longevity, San Diego, CA 92121, USA. Electronic address:

Short tandem repeats (STRs) are hyper-mutable sequences in the human genome. They are often used in forensics and population genetics and are also the underlying cause of many genetic diseases. There are challenges associated with accurately determining the length polymorphism of STR loci in the genome by next-generation sequencing (NGS). In particular, accurate detection of pathological STR expansion is limited by the sequence read length during whole-genome analysis. We developed TREDPARSE, a software package that incorporates various cues from read alignment and paired-end distance distribution, as well as a sequence stutter model, in a probabilistic framework to infer repeat sizes for genetic loci, and we used this software to infer repeat sizes for 30 known disease loci. Using simulated data, we show that TREDPARSE outperforms other available software. We sampled the full genome sequences of 12,632 individuals to an average read depth of approximately 30× to 40× with Illumina HiSeq X. We identified 138 individuals with risk alleles at 15 STR disease loci. We validated a representative subset of the samples (n = 19) by Sanger and by Oxford Nanopore sequencing. Additionally, we validated the STR calls against known allele sizes in a set of GeT-RM reference cell-line materials (n = 6). Several STR loci that are entirely guanine or cytosines (G or C) have insufficient read evidence for inference and therefore could not be assayed precisely by TREDPARSE. TREDPARSE extends the limit of STR size detection beyond the physical sequence read length. This extension is critical because many of the disease risk cutoffs are close to or beyond the short sequence read length of 100 to 150 bases.
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http://dx.doi.org/10.1016/j.ajhg.2017.09.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673627PMC
November 2017

Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.

Forensic Sci Int Genet 2017 05 27;28:52-70. Epub 2017 Jan 27.

Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. Electronic address:

Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities.
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http://dx.doi.org/10.1016/j.fsigen.2017.01.011DOI Listing
May 2017

Glutamine rich and basic region/leucine zipper (bZIP) domains stabilize cAMP-response element-binding protein (CREB) binding to chromatin.

J Biol Chem 2005 Apr 9;280(15):15103-10. Epub 2005 Feb 9.

Peptide Biology Laboratories, Salk Institute for Biological Studies, La Jolla, California 92037, USA.

We have examined the dynamics of cAMP-response element-binding protein (CREB) binding to chromatin in live cells using fluorescence recovery after photobleaching (FRAP). CREB was found to bind to target sites with a residence time of 100 s, and exposure to a cAMP agonist had no effect on these kinetics. In addition to the basic region/leucine zipper (bZIP) domain, a glutamine-rich trans-activation domain in CREB called Q2 also appeared to be critical for promoter occupancy. Indeed, mutations in Q2 that reduced residence time by FRAP assay disrupted target gene activation via CREB in cells exposed to a cAMP agonist. Notably, insertion of the glutamine-rich B trans-activation domain of SP1 into a mutant CREB polypeptide lacking Q2 stabilized CREB occupancy and rescued target gene activation. These results suggest a novel mechanism by which the family of glutamine-rich activators promotes cellular gene expression.
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http://dx.doi.org/10.1074/jbc.M414144200DOI Listing
April 2005

The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector.

Cell 2004 Oct;119(1):61-74

Peptide Biology Laboratories, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.
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http://dx.doi.org/10.1016/j.cell.2004.09.015DOI Listing
October 2004

Functional characterisation of the CRE/TATA box unit of type 2 deiodinase gene promoter in a human choriocarcinoma cell line.

J Mol Endocrinol 2004 Aug;33(1):51-8

Laboratory of Molecular and Endocrine Physiopathology, Department of Experimental Medicine and Pathology, University 'La Sapienza' of Rome, Rome, Italy.

The regulation of expression of type II deiodinase (D2) is a critical mechanism to maintain appropriate intracellular concentrations of tri-iodothyronine in selected tissues. One of the major regulators of D2 concentrations is cAMP, which potently increases human type II deiodinase (hD2) gene transcription in some tissues via a conserved cAMP response element (CRE) located in the promoter region. In addition, the regulatory region of the hD2 gene contains several TATA box/transcription start site (TSS) units, suggesting the presence of different transcripts that might be characterised by different biological properties. However, it is still unclear whether one ore more TATA box/TSS units are needed in response to cAMP or to other signals able to modulate hD2 transcription. In this study we have analysed the ability of cAMP to regulate hD2 in JEG3 cells, a human choriocarcinoma cell line highly responsive to cAMP. Transient transfection assays of different hD2 gene promoter constructs revealed that cAMP induces transcription starting from the most 5' TSS, located about 80 nucleotides from the CRE. RT-PCR studies have revealed that cAMP activates the expression of a long-lived transcript in JEG3 cells. Site-directed mutagenesis and deletion analysis of promoter constructs have shown that a single CRE/TATA box/TSS unit is needed to confer responsiveness to cAMP. By using chromatin immunoprecipitation studies, we have also demonstrated that the response to cAMP involves the binding of transcription factor CRE binding protein (CREB) to the CRE located in the hD2 promoter. In summary, in JEG3 cells cAMP induces transcription of a long-lived hD2 RNA via CREB and a single CRE/TATA box/TSS unit. This study provides new insights to the regulation of expression of hD2 in placenta.
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http://dx.doi.org/10.1677/jme.0.0330051DOI Listing
August 2004

TORCs: transducers of regulated CREB activity.

Mol Cell 2003 Aug;12(2):413-23

The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

The cAMP responsive factor CREB stimulates gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator CBP. In certain cell types, CREB also functions as a constitutive activator, although the underlying mechanisms are not understood. Here, we characterize a conserved family of coactivators, designated TORCs, for Transducers of Regulated CREB activity, that enhances CRE-dependent transcription via a phosphorylation-independent interaction with the bZIP DNA binding/dimerization domain of CREB. TORC recruitment does not appear to modulate CREB DNA binding activity, but rather enhances the interaction of CREB with the TAF(II)130 component of TFIID following its recruitment to the promoter. Remarkably, in certain mucoepidermoid carcinomas, a chromosomal translocation fuses the CREB binding domain of TORC1 to the Notch coactivator Mastermind (MAML2). As expression of the TORC1-MAML2 chimera strongly induced target gene expression via CREB, our results reveal a mechanism by which CREB stimulates transcription in normal and transformed cells.
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http://dx.doi.org/10.1016/j.molcel.2003.08.013DOI Listing
August 2003

Genome-wide analysis of CREB target genes reveals a core promoter requirement for cAMP responsiveness.

Mol Cell 2003 Apr;11(4):1101-8

Salk Institute for Biological Studies, La Jolla, California 92037, USA.

We have employed a hidden Markov model (HMM) based on known cAMP responsive elements to search for putative CREB target genes. The best scoring sites were positionally conserved between mouse and human orthologs, suggesting that this parameter can be used to enrich for true CREB targets. Target validation experiments revealed a core promoter requirement for transcriptional induction via CREB; TATA-less promoters were unresponsive to cAMP compared to TATA-containing genes, despite comparable binding of CREB to both sets of genes in vivo. Indeed, insertion of a TATA box motif rescued cAMP responsiveness on a TATA-less promoter. These results illustrate a mechanism by which subsets of target genes for a transcription factor are differentially regulated depending on core promoter configuration.
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http://dx.doi.org/10.1016/s1097-2765(03)00134-5DOI Listing
April 2003

Attenuation of a phosphorylation-dependent activator by an HDAC-PP1 complex.

Nat Struct Biol 2003 Mar;10(3):175-81

Peptide Biology Laboratories, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.

The second messenger cAMP stimulates transcription with burst-attenuation kinetics that mirror the PKA-dependent phosphorylation and subsequent protein phosphatase 1 (PP1)-mediated dephosphorylation of the cAMP responsive element binding protein (CREB) at Ser133. Phosphorylation of Ser133 promotes recruitment of the co-activator histone acetylase (HAT) paralogs CBP and P300, which in turn stimulate acetylation of promoter-bound histones during the burst phase. Remarkably, histone deacetylase (HDAC) inhibitors seem to potentiate CREB activity by prolonging Ser133 phosphorylation in response to cAMP stimulus, suggesting a potential role for HDAC complexes in silencing CREB activity. Here we show that HDAC1 associates with and blocks Ser133 phosphorylation of CREB during pre-stimulus and attenuation phases of the cAMP response. HDAC1 promotes Ser133 dephosphorylation via a stable interaction with PP1, which we detected in co-immunoprecipitation and co-purification studies. These results illustrate a novel mechanism by which signaling and chromatin-modifying activities act coordinately to repress the activity of a phosphorylation-dependent activator.
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http://dx.doi.org/10.1038/nsb895DOI Listing
March 2003
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