PE Applied Biosystems Research Articles

Population data for the Identifier™ 15 STR loci in Hong Kong Chinese

Allele frequencies for 15 STR loci, namely D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, which are tested for by PE Applied Biosystem's Identifier kit, were obtained from a sample of 325 unrelated Chinese in Hong Kong.

The Venezuelan Type of Nondeletional Hereditary Persistence of Fetal Hemoglobin Is Due C →T Mutation at Nucleotide -211 of the Aγ Globin Gene.

Hereditary Persistence of Fetal Hemoglobin (HPFH) is a clinically benign condition characterized by continuous synthesis of fetal hemoglobin (Hb F) in adult life without major related hematological changes. The nondeletional HPFH is characterized by hemoglobin F level between 5% and 30%, in heterozygotes state. This condition has been observed among Blacks, Italians, Greeks, English and Chinese. Two different forms are known, characterized by the type of chain involve, either Gγ or Aγ. Several point mutations in the promoter region of the Aγ globin gene have been identified in ndHPFH: −202, −198, −196, −195, −117, −114, deletion 4pb (−222 −225) and −158.The propositus is a 36 year old black female with a moderate increased level of Hb F, also her sister and her sibling had the same condition. She was discovered during studies due to the presence of splenomegaly and gastroesophageal reflux. The Hbs A, A2 and F were eluted and quantified by cation exchange high-performance liquid chromatography (HPLC-CE), using Beta Tal short program, Variant BioRad (Hercules, California, USA). DNA was isolated from peripheral blood leukocytes by a salting-out extraction procedure. The Aγ and Gγ promoters globins gene were amplified independently by PCR and the presence of the HPFH mutation was determined by denaturing gradient gel electrophoresis technique (DGGE) as previously described Gottardi et al., (1992). The Aγ promoter globin gene was sequenced in three members of the family using the BigDyeTM Terminator Cycler Sequencing Kit and the ABI PRISMTM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CQ, USA).Her hematological studies revealed Hb: 14.2 g/dl, Hcto: 41.1 %, MCV: 94.4 fl, MCHC: 34.5 fl, Hb A: 81.9 %, Hb A2: 2.1 % and Hb F: 6.3 %. The sibling hematological finding was Hb: 13.1 g/dl, Hcto: 41.8 %, MCV: 83.7 fl, MCHC: 31.3 fl, Hb A: 83.2 %, Hb A2: 3.2 % and Hb F: 5.1. The sister was Hb: 14.3 g/dl, Hcto: 46.4 %, MCV: 105.9 fl, MCHC: 30.8 fl, Hb A: 83.7 %, Hb A2: 3.3 % and Hb F: 2.7 %. No spleen enlargement was seen in the other two affected members of the family. The DGGE results in the Aγ promoter, suggested the presence of a non-deletion form of HPFH in heterozygotes state. The Aγ promoter region revealed the presence of a substitution C→T at position −211. This mutation does not modify any restriction site.To our knowledge the −211 (C→T) mutation in the Aγ promoter have not been reported previously. Since modulation of g-globin gene expression in adults could provide a therapeutic intervention in hemoglobinopathy, the study of the molecular mechanisms generating HPFH is medically relevant.

Demonstration That Mast Cells, T Cells, and B Cells Bearing the Activating Kit Mutation D816V Occur in Clusters within the Marrow of Patients with Mastocytosis

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

Utility of fast mycobacterial interspersed repetitive unit-variable number tandem repeat genotyping in clinical mycobacteriological analysis.

Analysis of variable numbers of tandem repeats (VNTR) of genetic elements called mycobacterial interspersed repetitive units (MIRUs) is a recently described, polymerase chain reaction (PCR)-based method used to genotype Mycobacterium tuberculosis. It is much faster, requires a smaller amount of DNA, and has approximately the same discriminatory power as the standard IS6110 restriction fragment-length polymorphism (RFLP) method. We report the adaptation and optimization of MIRU-VNTR genotyping on a capillary electrophoresis system. We describe its application to 3 typical clinical situations encountered in our laboratory (Institut Pasteur de Bruxelles, Laboratoire Tuberculose et Mycobacteries; Brussels, Belgium). MIRU-VNTR genotyping was performed on heat-inactivated M. tuberculosis cultures obtained from clinical specimens on Lowenstein solid medium or in mycobacteria growth indicator liquid tubes (Becton Dickinson). After amplification of 12 genomic loci using 4 different multiplex PCRs, DNA fragments were separated by capillary electrophoresis using the ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems). Sizing of the PCR fragments and assignment of the various MIRU-VNTR alleles were done using the GeneScan and customized Genotyper software packages (PE Applied Biosystem). Clustering on the basis of IS6110 fingerprinting of isolates from 3 different patients attending the same hospital was confirmed by MIRU-VNTR typing. This concordance between 2 independent, highly discriminatory techniques was decisive in triggering an epidemiological inquiry that led to identification of a bronchoscopy-related tuberculosis nosocomial infection. A mixed tuberculosis infection in a patient whose infection was initially suspected as a result of the IS6110 RFLP method was clearly identified by MIRU-VNTR typing. Finally, automated MIRU-VNTR analysis permitted the identification of laboratory contamination in 6 liquid cultures of M. tuberculosis within several hours. These examples illustrate the utility of this genotyping technique for quick and accurate resolution of problems commonly encountered in clinical mycobacteriology.

Methylation in the Core-promoter Region of the Chondromodulin-I Gene Determines the Cell-specific Expression by Regulating the Binding of Transcriptional Activator Sp3

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

Increased Susceptibility of Mice Lacking Clara Cell 10-kDa Protein to Lung Tumorigenesis by 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a Potent Carcinogen in Cigarette Smoke

Ninety percent of all human lung cancers are related to cigarette smoking. Both tobacco smoke and lung tumorigenesis are associated with drastically reduced levels of Clara cell 10-kDa protein (CC10), a multifunctional secreted protein, naturally produced by the airway epithelia of virtually all mammals. We previously reported that the expression of CC10 is markedly reduced in animals exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK, a potent carcinogen in tobacco smoke. Furthermore, it has been reported that CC10 expression, induced in certain tumor cells, reverses the transformed phenotype. We demonstrate here that NNK exposure of CC10-knock-out (CC10-KO) mice causes a significantly higher incidence of airway epithelial hyperplasia and lung adenomas compared with wild type (WT) littermates (30% CC10-KO versus 5% WT, p = 0.041). We also found that compared with NNK-treated WT mice, CC10-KO mice manifest increased frequency of K-ras mutation, elevated level of Fas ligand (FasL) expression, and increased MAPK/Erk phosphorylation, all of which are considered predisposing events in NNK-induced lung tumorigenesis. We propose that CC10 has a protective role against NNK-induced lung tumorigenesis mediated via down-regulation of the above-mentioned predisposing events.

Human Constitutive Androstane Receptor Mediates Induction of CYP2B6 Gene Expression by Phenytoin

Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.

Analysis of 12 STR loci in Antioquia (Colombia) population sample

In this study, the validation of 12 short tandem repeated (STRs) systems is presented in a sample of the population from Antioquia, central region of Colombia. The DNA for the analyses of PCR was obtained of 244 to 546 unrelated individuals. PCR amplification was performed according to the manufacturer's recommendations, using PowerPlex 1.2 System and FFFL Multiplex (Promega). Amplificated fragments were separated by capillary electrophoresis using the ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). Allele frequencies, Hardy-Weinberg equilibrium, observed and expected heterozygosity and genetic disequilibrium were calculated using GENEPOP ver. 3.3 and GDA ver. 1.1 software. Forensic parameters were calculated using the PowerStat ver. 1.2 software (Promega). The 12 of the loci met Hardy-Weinberg expectations and they reached a combined power discrimination higher than 0.999999999999 and a combined power exclusion similar to 0.9999, showing to be a powerful tool for paternity testing, individual identification and forensic applications in Antioquia population.

Allele frequencies for CODIS loci in a Sicilian population sample

Data from an island population can be useful for understanding effects of forensic evaluations of DNA evidence. In this study, allele frequencies for the thirteen CODIS loci were generated from 220 unrelated volunteer donors from a Sicilian (Italy) population sample, as well as from casework using both the AmpFlSTR™ Profiler and Co-Filer ™ Amplification Kits (PE Applied Biosystems, Foster City, CA). All loci met Hardy–Weinberg expectations. The data were compared with population studies from individuals residing in other Italian geographic locations and found not be substantially different. The combined power of discrimination is high and comparable to other European population groups.

Association Among an Ornithine Decarboxylase Polymorphism, Androgen Receptor Gene (CAG) Repeat Length and Prostate Cancer Risk

A single nucleotide substitution of guanine to adenine (A) at base +316 in the ornithine decarboxylase (ODC) gene may be associated with greater ODC expression and increased tumor growth. ODC is induced by androgens in human prostatic epithelial cells, presumably via transcriptional activation of androgen receptor (AR) and also by nicotine. A nested case-control study was done to examine the association between this ODC genotype and prostate cancer risk, and whether it varies by AR gene CAG repeat length and smoking. A total of 164 cases were matched to 2 controls each from a community based cohort. ODC and AR genotyping was performed using a TaqMan (PE Applied Biosystems, Foster City, California) based assay and automated fragment analysis, respectively. Conditional logistic regression was used to estimate the OR and 95% CI. The presence of the ODC A allele was not significantly associated with an increased risk of prostate cancer (OR 1.33, 95% CI 0.90 to 1.96). However, men who inherited at least 1 ODC A alleles and less than 22 AR CAG repeats were at twice the risk of prostate cancer compared with those with 2 guanine alleles and 22 or greater AR CAG repeats (OR 2.09, 95% CI 1.23 to 3.57). Smoking was associated with prostate cancer only in men carrying a least 1 ODC A allele (p interaction = 0.02). The ODC A allele was not associated with a statistically significant increased risk of prostate cancer. However, this association may vary according to the number of CAG repeats in the AR receptor and smoking status.

Genetic diversity and background linkage disequilibrium in the North American Holstein cattle population.

The objectives of this study were to 1) identify highly heterozygous Holstein bulls that are as unrelated as possible and widely used in the US dairy industry; 2) quantify the level of genetic diversity in US Holsteins; and 3) determine the extent of background linkage disequilibrium (BLD) and disease trait associated linkage disequilibrium (DLD) in the US Holstein population. Twenty-three Holstein bulls that are not closely related but were widely used in the US dairy industry were genotyped for 54 microsatellite loci. The genotyping was performed on automated DNA sequencers (PE Applied Biosystems, CA), following polymerase chain reaction amplification with fluorescent dye-labeled primers. The heterozygosity for the sampled population ranged from 0.43 to 0.80. This wide range of heterozygosity allows selection of the most heterozygous bulls to develop informative families for gene mapping studies. The degree of genetic diversity in this population is significant and allows selection for traits of economic importance. As expected, there is extensive linkage disequilibrium (LD) in the US Holstein population. About half of the syntenic marker pairs presented a typical pattern of LD produced by DLD. Most of the nonsyntenic marker pairs had a typical pattern of LD arising from BLD. These results suggest that the observed LD is not purely due to genetic drift and migration and that a portion might be due to DLD. This raises our hopes of successful fine-localization of genes for complex traits using LD mapping.

Population data for 11 STR loci in northeast China Han

Allele frequencies for the nine tetrameric short tandem repeats (STR) loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820 (AmpFℓSTR ® Profiler Plus™ PCR amplification kit, PE Applied Biosystems) and two pentameric STR loci Penta D and Penta E (PowerPlex ® 16 system™, Promega Corporation) were determined in a population sample of unrelated China Han.

Allele Frequencies of Y-Chromosome STR Loci DYS385 and DYS392 in Three Eastern Spanish Populations

Abstract Blood samples were collected from healthy autochthonous unre- 2. lated males from Valencia and the islands of Majorca and Minorca (Spain). DNA was extracted by means of a standard phenol/ cloroform DNA protocol (1). Coamplification of the loci DYS385 and DYS392 was performed in a duplex reaction, using approximately 20 ng of genomic DNA in a total reaction volume of 25 µL. The primers used are described in Kayser et al. (2) and de Knijff et al. (3). PCR cycling conditions were as described in Furedi et al. (4), with minor modifications. A GeneAmp PCR System 2400 (PE Applied Biosystems) was used for amplification. Detection of the amplified products was carried out using an ABI 310 automatic sequencer (Perkin-Elmer) along with the GENESCAN 2.1 Analysis software. Our own allele ladders were used for allele designations. Allele nomenclature was as proposed by Kayser et al. (2) and de Knijff et 8. al. (3). Gene diversity was estimated according to Nei (5). Allele frequencies, as well as pairwise analysis, were calculated using the ARLEQUIN package (6). Data on Iberian populations were used for comparisons (7-11). DYS392 gene diversities (0.448-0.640) were within the range of those observed in the other Y-chromosome STRs studied in these samples (12), whereas DYS385 had the highest gene diversities (0.615-0.755). Comparisons between all of the three populations studied showed no significant differences. When comparisons were carried out with other Iberian populations, significant differences were observed at DYS385. With respect to Catalonia and Andalusia, we found differences at DYS392.

Synergistic Antiviral Activity of Human Interferon Combinations in the Hepatitis C Virus Replicon System

The use of type I interferon (IFN), in combination with ribvirin, to treat chronic hepatitis C virus (HCV) infection has many drawbacks that prevent widespread application, ultimately leading to a significant unmet clinical need. Potential improvements in IFN therapy through targeted delivery, molecular alteration, and combination with other agents are ongoing in an attempt to decrease adverse effects and increase efficacy. In this report, the HCV replicon cell culture system was used to assess potential synergistic antiviral effects of multiple IFN species when administered in combination. Quantitative analysis of HCV replicon RNA by TaqMan (PE Applied Biosystems, Foster City, CA) and qualitative analysis of HCV protein expression were used to measure the antiviral efficacy of individual and combination IFN treatments, and synergistic responses of IFN combinations were determined through statistical analysis of the TaqMan results. We found that when administered simultaneously, type I/II IFN combinations (IFN-alpha2b + IFN-gamma or IFN-beta + IFN-gamma) resulted in dramatic antiviral synergy, whereas a type I/I combination (IFN-alpha2b + IFN-beta) demonstrated a slightly antagonistic profile. The synergistic effect is likely due to differential cell surface receptors and signaling pathways employed by types I and II IFNs. Conversely, all type I IFN species bind the same receptor and signal through similar pathways, possibly accounting for the nearly additive response observed. In support of this hypothesis, IFN treatment resulted in differential induction of Stat1 phosphorylation at Tyr 701. In conclusion, simultaneous type I/II IFN combination treatment may allow an overall decreased effective IFN dose, which may reduce the side effect profiles that hinder current therapy.

Performance Evaluation of Two Multiplexes Used in Fluorescent Short Tandem Repeat DNA Analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.

Allele frequencies for the 13 CODIS STR loci in Peru

Population: Unrelated Peruvian donors from Lima, Huancayo, Iquitos, Piura and Huaraz cities. N: 100. Extraction: Samples were placed on FTA paper (Whatman BioScience) and the immobilized DNA washed according to the manufacturer’s recommendations. PCR: The complete PCR amplification mix is added directly to the tube containing one punch dried of diameter 2.0 mm of target DNA following manufacturer’s instructions (AmpF‘STR Profiler Plus and AmpF‘STR COfiler PCR amplification kits—PE Applied Biosystems). Typing: ABI310 and reference sequenced ladders (PE Applied Biosystem). Analysis of data: PowerStats [1] and GDA [2]. Access to the data: adndircri@pnp.gob.pe. Other remarks: Linkage disequilibrium has been tested using shuffling test for all possible combinations between loci obtaining an exact probability upper than 0.05 indicating independence of loci in all cases. This paper follows the guidelines for publication of population data requested by the journal [3]. Forensic Science International 132 (2003) 164–165

Autosomal Microsatellite Profile of Three Socially Diverse Ethnic Tamil Populations of India

Abstract Blood samples were obtained by venipuncture from random, consenting individuals of the three populations: Kongu Vellala Gounder (56), Irular (54), and Chakkiliyar (49). DNA was isolated using standard phenol/chloroform procedure (1). Fifteen autosomal microsatellite loci were amplified using the PowerPlex® 16 system (Promega Corporation, Madison) according to manufacturer's recommendations (2). The amplified products were separated by denaturing gel electrophoresis using the ABI Prism™ 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). Alleles were determined by comparison with the allelic ladder included in the kit.

Ancient DNA analysis of the delta F508 mutation.

When working with highly degraded DNA, validating the results of a slightly polymorphic system always complicates the analysis because of the difficulties in recognizing contamination and artifacts. Recognition can be greatly simplified by employing a multiplex reaction that coamplifies the fragments together with several highly polymorphic markers, for instance, short tandem repeats. In this work, we successfully included newly designed oligonucleotide primers for the detection of delta F508, the most frequent mutation causing cystic fibrosis, in the commercial AmpFlSTR Profiler Plus PCR Amplification Kit (PE Applied Biosystems). This coamplification enabled us to test the hypothesis of a heterozygote advantage associated with cystic fibrosis-specifically, higher resistance to toxin-mediated diarrheas--in a Sicilian skeletal sample of individuals who died in a cholera epidemic in 1837. The proposed method should also be suitable for the genetic characterization of other slightly polymorphic loci tested on human and animal ancient DNA; it should permit simple authentication of results by comparing the fingerprints obtained from independent amplifications repeated several times.

Autosomal Microsatellite Profile of Three Socially Diverse Ethnic Tamil Populations of India

Abstract Blood samples were obtained by venipuncture from random, consenting individuals of the three populations: Kongu Vellala Gounder (56), Irular (54), and Chakkiliyar (49). DNA was isolated using standard phenol/chloroform procedure (1). Fifteen autosomal microsatellite loci were amplified using the PowerPlex® 16 system (Promega Corporation, Madison) according to manufacturer's recommendations (2). The amplified products were separated by denaturing gel electrophoresis using the ABI Prism™ 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). Alleles were determined by comparison with the allelic ladder included in the kit.

PCR in a Rayleigh-Bénard convection cell.

PCR in a Rayleigh-Bénard convection cell.