Palmitate-Induced Insulin Hypersecretion and Later Secretory Decline Associated with Changes in Protein Expression Patterns in Human Pancreatic Islets.

In obese children with high circulating concentrations of free fatty acid palmitate, we have observed that insulin levels at fasting and in response to a glucose challenge were several times higher than in obese children with low concentrations of the fatty acid as well as in lean controls. Declining and even insufficient insulin levels were observed in obese adolescents with high levels of the fatty acid. In isolated human islets exposed to palmitate we have observed insulin hypersecretion after 2 days exposure. In contrast, insulin secretion from the islets was reduced after 7 days culture in the presence of the fatty acid. This study aims at identifying islet-related biological events potentially linked with the observed insulin hypersecretion and later secretory decline in these obese children and adolescents using the islet model. We analyzed protein expression data obtained from human islets exposed to elevated palmitate levels for 2 and 7 days by an improved methodology for statistical analysis of differentially expressed proteins. Protein profiling of islet samples by liquid chromatography-tandem mass spectrometry identified 115 differentially expressed proteins (DEPs). Several DEPs including sorcin were associated with increased glucose-stimulated insulin secretion in islets after 2 days of exposure to palmitate. Similarly, several metabolic pathways including altered protein degradation, increased autophagy, altered redox condition, and hampered insulin processing were coupled to the functional impairment of islets after 7 days of culture in the presence of palmitate. Such biological events, once validated in the islets, may give rise to novel treatment strategies aiming at normalizing insulin levels in obese children with high palmitate levels, which may reduce or even prevent obesity-related type 2 diabetes mellitus.

Sensitive plasma protein analysis by microparticle-based proximity ligation assays.

Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.

Multiplexed genotyping of methicillin-resistant Staphylococcus aureus isolates by use of padlock probes and tag microarrays.

We developed and tested a ligase-based assay for simultaneous probing of core genome diversity and typing of methicillin resistance determinants in Staphylococcus aureus isolates. This assay uses oligonucleotide padlock probes whose two ends are joined through ligation when they hybridize to matching target DNA. Circularized probes are subsequently amplified by PCR with common primers and analyzed by using a microarray equipped with universal tag probes. Our set of padlock probes includes oligonucleotides targeting diagnostic regions in the mecA, ccrB, and ccrC genes of the SCCmec cassette in methicillin-resistant S. aureus (MRSA). These probes determine the presence and type of SCCmec cassettes (i.e., SCCmec types I to VI). Additional oligonucleotides interrogate a number of highly informative single nucleotide polymorphisms retrieved from a multilocus sequence typing (MLST) database. These latter probes enable the exploration of isolates' phylogenetic affiliation with clonal lineages of MRSA as revealed by MLST. The described assay enables multiplexed genotyping of MRSA based on a single-tube reaction. With a set of clinical isolates of MRSA and methicillin-susceptible S. aureus (n=66), 100% typeability and 100% accuracy were achieved. The assay described here provides valuable genotypic information that may usefully complement existing genotyping procedures. Moreover, the assay is easily extendable by incorporating additional padlock probes and will be valuable for the quick and cost-effective probing of large numbers of polymorphisms at different genomic locations, such as those ascertained through currently ongoing mutation discovery and genome resequencing projects.

Pyrosequencing analysis identifies discrete populations of Haemonchus contortus from small ruminants.

The genus Haemonchus consists of blood-sucking parasitic nematodes in the abomasum of ruminants. Members of this genus are responsible for extensive production losses, particularly of small ruminants in the tropics but are also found in temperate regions. In this study, we examined the internal transcribed spacers-1 and -2 of rRNA in Haemonchus spp. The rRNA region spanning the internal transcribed spacers-1, -2 and the 5.8S rRNA gene was amplified by PCR from each of 10 worms from Swedish sheep, a Swedish goat and Kenyan sheep. The fragments were sequenced and examined with respect to genetic differences fixed among the three isolates. These and additional worms were further analysed with Pyrosequencing technology. This technique allowed us to rapidly analyse 110 individuals in three putative polymorphic nucleotide positions that were initially identified with dideoxy sequencing. The geographical isolates could to a large extent be genetically distinguished, but none of the polymorphic positions were consistent among all individuals within each isolate. Furthermore an alignment of our sequences and a consensus sequence published for Haemonchus contortus revealed two differences in positions 123 and 196 in internal transcribed spacers-2. Although these positions were previously reported as heterogenic, no polymorphism was detected among the 30 worms sequenced in the present study. Modelling of the internal transcribed spacers-2 secondary structure based on our data also identified a new putative long-range interaction. The isolates are best described as populations. In conclusion, consistent differences were not identified and the studied isolates are therefore best described as discrete populations. This study also reveals for the first time the potential of Pyrosequencing technology as a tool in the analysis of nematode population genetics.

Cytochrome p450 genotyping by multiplexed real-time dna sequencing with pyrosequencing technology.

Individual differences in xenobiotic metabolism influence the therapeutic value of many drugs and are of major concern during the development of new drug candidates. A number of polymorphic cytochrome p450 enzymes account for a significant part of this variation. A better understanding of these genetic factors would be of value for drug development, as well as clinical practice. To fulfill the goal of a personalized medicine, methods for simple and accurate assessment of cytochrome p450 genes are required. We report on the development of multiplex assays for genotyping of the cytochrome p450 drug-metabolizing enzymes CYP2D6, CYP2C9, and CYP2C19 with Pyrosequencing technology. Eleven variable positions, representing 12 of the most frequent alleles, were scored: CYP2D6 alleles *2, *3, *4, *6, *7, *8, and *14, CYP2C19 alleles *2, *3, and *4, and CYP2C9 alleles *2 and *3. Four multiplex Pyrosequencing reactions per patient sample were performed to cover these positions, using either simplex or multiplex PCR for amplification of target DNA sequences. Unequivocal genotypes were obtained for all patient samples, and the results were validated by comparing with results obtained using PCR-RFLP. For positions addressed with both methods, the results were in complete agreement. Pyrosequencing technology offers a highly automated, rapid, and accurate method for identification of cytochrome p450 alleles, which is suitable for pharmacogenomic research, as well as for routine assessment of patient genotypes.

Pyrosequencing technology and the need for versatile solutions in molecular clinical research.

An increasing amount of genetic information is rapidly becoming available to the practitioners of medicine and pharmacology. This knowledge promises to revolutionize the determination of diagnoses and prognoses for genetically-based disorders as well as infectious diseases and to enable tailoring of treatment to suit the individual patient. As genomics becomes ripe for clinical implementation, versatile technologies that can handle all the relevant types of analyses will be requested by many clinicians. The recently established Pyrosequencing technology for rapid determination of short DNA sequences has gained widespread acceptance and is being used in a broad range of applications. It can provide a solution for many emerging issues in molecular clinical research and applications, owing to its reliability and high flexibility together with its user-friendly attributes.

Method for one-step preparation of double-stranded DNA template applicable for use with Pyrosequencing technology.

A new one-step method for fast and efficient preparation of double-stranded DNA template, suitable for use with Pyrosequencing technology, has been developed. In the new method, two different types of oligonucleotides were used to prevent reannealing of remaining PCR primers to the template: oligonucleotides complementary to the PCR primers and 3'-end modified oligonucleotides with the same sequence as the PCR primers. Advantages with the new strategy are: (i) faster and simpler template preparation procedure (one-step); (ii) no need for exonuclease I treatment; and (iii) less problem with unspecific priming from loop structures and dimers. By careful oligonucleotide design, and/or by addition of single-stranded DNA-binding protein, problems with unspecific sequence signals due to mispriming can be reduced. The new method was used for analysis of genotype variations within the renin-angiotensin-aldosterone system.

Rapid detection of rifampin resistance in Mycobacterium tuberculosis by Pyrosequencing technology.

We developed an assay for rapid detection of rifampin resistance in Mycobacterium tuberculosis based on Pyrosequencing technology, involving a technique for real-time sequencing. A 180-bp region of the rpoB gene was amplified in clinical isolates of both rifampin-resistant and -susceptible M. tuberculosis. The PCR products were subjected to Pyrosequencing analysis using four different sequencing primers in four overlapping reactions. These four sequencing reactions covered the 81-bp region where > 96% of the mutations associated with rifampin resistance are located. The results were compared to those obtained with two other molecular methods, the line probe assay and cycle sequencing, and the phenotypic BACTEC method. The genotypic determination methods all detected the mutations that previously have been correlated with rifampin resistance. In addition, Pyrosequencing analysis and the two other molecular methods found additional mutations within the rpoB gene in phenotypically susceptible strains. We found that Pyrosequencing technology, in particular, offers high accuracy, short turnaround time, and a potentially high throughput in detection of rifampin resistance in M. tuberculosis.

Determination of CYP2D6 gene copy number by pyrosequencing.

BACKGROUND: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. METHODS: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. RESULTS: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0-4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. CONCLUSIONS: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

Molecular haplotype determination using allele-specific PCR and pyrosequencing technology.

Haplotyping of single-nucleotide polymorphisms (SNPs) is usually performed statistically by computational analysis or by time-consuming cloning techniques. Here we present a simple molecular approach for reliable haplotype determination on individual samples. The procedure is based on allele-specific PCR (AS-PCR) in combination with Pyrosequencing analysis. AS-PCR primers for each allelic variant of the investigated SNPs were used. A mismatch introduced at the second base from the 3' end dramatically improved allele specificity. Analysis of multiple SNPs on amplified fragments using Pyrosequencing technology allowed determination of haplotypes. Genotyping of heterozygote samples after AS-PCR gave a typical monoallelic pattern at each SNP, in which the identity of the present allele depended on the allele-specific initial amplification. Haplotype determination by the described procedure proved to be highly reliable. The results obtained by Pyrosequencing technology have the benefit of being truly quantitative, enabling detection of any nonspecific allele amplification.

Rapid combined characterization of microorganism and host genotypes using a single technology.

BACKGROUND: Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrosequencing trade mark technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection. MATERIALS AND METHODS: DNA from 87 clinical isolates of H. pylori, 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples. RESULTS: All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called. CONCLUSIONS: We conclude that genetic analysis using Pyrosequencing trade mark technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markers.