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

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.

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

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.

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

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.

Edulcorantes no nutritivos e ingesta diaria admisible en adultos y niños de peso normal y obesos de tres niveles socioeconómicos, y un grupo de diabéticos de la Región Metropolitana / Non-caloric sweeteners and daily acceptable intake in adults and children with normal weight and obesity from three different socioeconomic levels, and a diabetic group from Metropolitan Region

Introduction: There is wide availability of products containing sweeteners but there is no regulation on its consumption. Objective: To establish if adults and children with normal weight or obesity from three socioeconomic levels, and a group of adults and children with diabetes; do not exceed ADI levels for some sweeteners. Methods: Group 1 (477 adults, Group 2 (516 children) from socioeconomic levels: ABC1, C2 and C3, normal nutritional status and obesity, and group 3 (218) adults and children with diabetes. The daily intake of sweeteners was recorded, including: aspartame (ASP), acesulfame K (AK), cyclamate (CICL), saccharin (SAC), sucralose (SUC) and stevia (STV). Results: 85% adults and 75%% of children consumed products with sweeteners, and of these 50% were instant powdered beverages, soft drinks or diet yogurts. When comparing the consumption between groups 1 and 2, group 1 consumed a larger amount of sweeteners (p<0.05). Group 1 ABC1 ate more AK, ASP and SUC than C2 and C3 (p<0.05). Group 3 did not surpass the acceptable daily intake of AK, ASP, SUC and STE, but 5.8% of adults and 25% of diabetic children exceeded the ADI for SAC. Conclusions: The 97.5% and the 98.8% had a safe consumption of artificial sweeteners. It should be emphasized that 5.8% of adults and 25% of diabetic children exceeded the maximum ADI for SAC, finding that suggests to be continued long-term studies to elucidate whether this has implications for health.

Introducción: Existe gran disponibilidad de productos con edulcorantes pero no existe regulación sobre su consumo. Objetivo: determinar si individuos adultos y niños con estado nutricional normal u obesidad de tres niveles socioeconómicos y un grupo de adultos y niños con diabetes, no excedían la ingesta diaria admisible de los edulcorantes permitidos. Metodología: Grupo 1 (477 adultos) y grupo 2 (516 niños) de niveles socioeconómicos (NSE): ABC1, C2 y C3, estado nutricional normal y obesos, y grupo 3 (218) adultos y niños diabéticos. Se registró la ingesta diaria de edulcorantes incluyéndose: aspartame (ASP), acesulfamo K (AK), ciclamato (CICL), sacarina (SAC), sucralosa (SUC) y estevia (STV). Resultados: El 85 % adultos y 75 % de niños consumían productos con edulcorantes y de estos el 50% eran bebidas instantáneas en polvo, bebidas gaseosas o yogurts dietéticos. Al comparar la ingesta de edulcorantes entre los grupos 1 y 2, el grupo 1 tuvo una mayor ingesta (p<0.05) que el grupo 2. El grupo 1 del NSE ABC1, consumió mas AK, ASP y SUC que NSE C2 y C3 (p<0.05). En el grupo 3, el 5.8% de adultos y el 25% de niños diabéticos sobrepasaron el IDA sólo para SAC. Conclusiones: El 97.5% adultos y el 98.8% niños tuvieron ingesta dentro del nivel seguro en cada edulcorante. Se debe enfatizar que el 5,8% de adultos y 25% de niños diabéticos excedieron el IDA máximo para SAC, hallazgo que sugiere continuar con estudios a largo plazo que permitan dilucidar si esto tiene repercusión para la salud.

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

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.

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

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.

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

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.

TORCs: transducers of regulated CREB activity.

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.

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

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.

Proteínas de lupino dulce (Lupinus luteus, var. Aurea/Weico, y Lupinus albus, var. Multolupa): I. Extracción y filtración por Sephadex / Sweet lupine (Lupinus luteus, var. Aurea/Weico and Lupinus albus, var. Multolupa) proteins: I. Extraction and filtration by Sephadex

Las investigaciones de índole nutricional realizadas en la harina de lupino en Chile, han motivado el tener un conocimiento más a fondo de las proteínas de esta leguminosa. Las especies sometidas a estudio fueron Lupinus lu teus var. Aurea/Weico y Lupinus albus var Multolupa. A estas especies se les extrajeron y fraccionaron las proteínas, determinándose la distribución porcentual de globulinas y albúminas. Las semillas descarcaradas, previo análisis proximal, se molieron, desgrasaron y extrajeron sucesivamente con agua destilada a un pH de 5.0, con solución amortiguadora de fosfato 0.05 M, pH 8.5. Las proteínas extraídas se determinaron según el método de Lowry simplificado. Las globulinas (pH 8.5) de ambos lupins se filtraron por Sephadex G-100 y las del L albus se filtraron además por Sephadex G-150; a las fracciones colectadas se les midió la absorbancia a 280 nm. Las semillas descascaradas (SD) de L. luteus y de L. albus evidenciaron un contenido de proteínas de 58.2 y 41.0%, respectivamente

Proteínas de lupino dulce (Lupinus luteus var. Aurea/Weico, y Lupinus albus var. Multolupa): II. Separación por electroforesis / Sweet lupine (Lupinus luteus, var. Aurea/Weico and Lupinus albus, var. Multolupa) proteins: II. Separation by electrophoresis

En este estudio se analizaron las albúminas y globulinas extraídas del Lupinus luteus var. Aurea/Weico y Lupinus albus var. Multolupa mediante la técnica de electroforesis en gel de poliacrilamida. El propósito fue el de determinar la distribución porcentual de albúminas y globulinas de las proteínas en los electroforetogramas, con su consiguiente caracterización, según su movilidad. El densitograma de las albúminas del L. luteus evidenció seis fracciones de las cuales las fracciones 2 y 4 contribuyeron con 58.1%. En cuanto a las globulinas, el densitograma reveló cinco fracciones de las cuales las fracciones 1, 2 y 3 contribuyeron con 90.7%. El densitograma de las globulinas eluídas en el pico I a través de Sephadex G-100 , reveló cinco fracciones que guardaron semejanza con las fracciones 1 y 2 de las globulinas totales y cuya movilidad relativa dio valores bajos comprendidos entre 0.26 y 0.39. Las albúminas del L. albus se resolvieron en cuatro fracciones, siendo la más representativa la fracción 2, con 54.3%. Respecto al fraccionamiento de las globulinas, se obtuvieron cinco fracciones que coincidieron en su distribución con las globulinas del pico I, acusando la fracción 2 la mayor contribución (48.2%). Se evidenció que el Sephadex G-100 no lograba una buena separación. En cuanto a la movilidad electroforética de estas globulinas, se destacaron las fracciones 2 y 3, con movilidades de 0.35 y 0.48. A partir de este trabajo, se puede concluir que las albúnas del L. luteus que presentan un mayor número de fracciones son de menor movilidad relativa que las albúminas del L. albus. En cuanto a las globulinas de ambas especies de lupino, éstas se caracterizan por presentar agrupaciones de baja y alta movilidad, respectivamente

Valor nutritivo de raciones entregadas a escuelas básicas en el Area Metropolitana de Santiago / Nutritive value of meals delivered to basic schools of the Metropolitan Area of Santiago

Se determinó la composición química y aporte energético de raciones de desayunos y almuerzos entregados por una industria a Escuelas Básicas del Area Metropolitana, acogida al Programa de Alimentación Escolar (PAE) de la Junta Nacional de Auxilio Escolar y Becas (JUNAEB). La muestra fue seleccionada por muestreo dirigido, aplicando estratificación simple. Esta quedó constituida por 20 escuelas para almuerzos (23%) del total) y 15 escuelas para desayuno (17% del total). El análisis químico incluyó determinaciones del humedad, cenizas totales, proteína total (Nx6,25) y extracto etéreo. El extracto no nitrogenado (ENN) se calculó por diferencia; el contenido energético se calculó utilizando los factores de Atwater. Los resultados del aporte energético y proteico de las raciones se compararon con lo estipulado por las JUNAEB. Para desayuno, se encontró que la ración aportaba en promedio 217 Kcal y que el 100% de dichas raciones entregaba menos de las 300Kcal exigidas por la JUNAEB. El valor promedio para almuerzos fue de 372 Kcal/ración y el 100% de las raciones entregaba menos de las 500 Kcal exigidas por la JUNAEB. El aporte de proteína fue 15,5 g/día en promedio, lo que superó en un 3% al mínino exigido por la JUNAEB. En una submuesrtra (N = 18) se realizó un control del número de raciones entregadas comparado con el número de raciones programadas, los resultados de este análisis señalaron que la mayoría de las escuelas cubre el 100% de las raciones entregadas respecto a las programadas

Efectos de la germinación sobre los factores antinutricionales termorresistentes y nutrientes esenciales del lupino / Effect of germination on the levels of thermoresistant antinutritional factors and essential nutrients of lupin

Se estudiaron los efectos del proceso de germinación en los contenidos de proteínas, aminoácidos, riboflavina, oligosacáridos, fitatos y alcaloides de la semilla de lupino dulce (Lupinus albus cv Multolupa), así como en la calidad biológica de la proteína de este grano. Debido a que en Chile, el lupino presenta ventajas comparativas en relación a la soya, se tomó a ésta como muestra control. Las semillas sin germinar de ambas leguminosas también se usaron para las comparaciones. Los granos se germinaron a 25-C y 85% HR durante 72 hrs., evaluándose la capacidad germinativa mediante determinaciones del largo del hipocotilo y epicotilo, cambios de peso y porcentaje de semillas germinadas. Posteriormente, se molieron y secaron por liofilización para los análisis. Se encontró que la capacidad de germinación del lupino fue inferior a la de soya, aunque los cambios bioquímicos y nutricionales buscados fueron similares en ambas leguminosas. Al término de la germinación se encontraron aumentos significativos en los contenidos de riboflavina y de aminoácidos. Este efecto explicó el mejoramiento de la calidad biológica de la proteína de ambos granos. Asimismo, se lograron reducciones significativas de los azúcares de la flatulencia (estaquiosa y rafinosa) y de los fitatos. Los cambios observados se atribuyeron a un aumento en la concentración de proteasas, alfa-galactosidasa y fitasa; así como a una acumulación de los nutrientes solubles requeridos por la nueva plántula para su nutrición

Composición química y calidad nutricional de variedades mejoradas de lentejas, lens esculenta / Chemical composition and nutritional quality of improved varieties of lentils, lens esculenta

El objetivo del presente estudio fue conocer la composición química proximal de variedades mejoradas de lentejas y evaluar la calidad de la proteína mediante estudios biológicos (PER y NPR). Los cultivares utilizados fueron proporcionados por el Programa de Leguminosas de Grano del Instituto de Investigaciones Agropecuarias (INIA) La Platina y corresponden a lenteja: tekoa, centinela INIA, y araucana empleando como control la variedad corriente. El análisis químico incluyó la determinación de humedad, cenizas, proteínas (N x 6,25), extracto etereo y fibra cruda, empleando las técnicas establecidas por la AOAC. Se determinó además el contenido de riboflavina y vitamina C; encontrándose valores de vitamina B2 entre 0,21 y 0,46 mg/100 g. y de vitamina C entre 0,05 y 4,2 mg/100 g. Los resultados del análisis proximal indican que las lentejas araucana y corriente presentan el menor contenido de proteínas, 22,9 y 23,0 g/100 g (en base húmeda) respectivamente. Los valores más altos los presentaron tekoa y araucana INIA alcanzando valores de NPR de 2,12 ñ 0,17 y 2,14 ñ 0,32 (p<0,05); éstos al ser expresados como NPR rel por ciento fueron: araucana INIA 55,3, tekoa 54,8, lentejas corriente 50,1 y centinela INIA 48,8. Los valores del PER fueron en general bajos fluctuando los NPR rel por ciento entre 16,9 para centinela y 23,9 para tekoa, la lenteja corriente dio un valor bastante bajo (2,6 por ciento). En cuanto a la digestibilidad los valores obtenidos fueron concordante con el origen de las proteínas estudiadas