Rebecca Sanders

Evaluation of digital PCR for absolute DNA quantification.

The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.

Evaluation of Digital PCR for Absolute RNA Quantification

Gene expression measurements detailing mRNA quantities are widely employed in molecular biology and are increasingly important in diagnostic fields. Reverse transcription (RT), necessary for generating complementary DNA, can be both inefficient and imprecise, but remains a quintessential RNA analysis tool using qPCR. This study developed a Transcriptomic Calibration Material and assessed the RT reaction using digital (d)PCR for RNA measurement. While many studies characterise dPCR capabilities for DNA quantification, less work has been performed investigating similar parameters using RT-dPCR for RNA analysis. RT-dPCR measurement using three, one-step RT-qPCR kits was evaluated using single and multiplex formats when measuring endogenous and synthetic RNAs. The best performing kit was compared to UV quantification and sensitivity and technical reproducibility investigated. Our results demonstrate assay and kit dependent RT-dPCR measurements differed significantly compared to UV quantification. Different values were reported by different kits for each target, despite evaluation of identical samples using the same instrument. RT-dPCR did not display the strong inter-assay agreement previously described when analysing DNA. This study demonstrates that, as with DNA measurement, RT-dPCR is capable of accurate quantification of low copy RNA targets, but the results are both kit and target dependent supporting the need for calibration controls.

Further support for an association between a polymorphic CAG repeat in the hKCa3 gene and schizophrenia.

A recent study has suggested that a polymorphism in the hKCa3 potassium channel may be associated with raised susceptibility to schizophrenia.1 Despite its modest statistical significance, the study1 is intriguing for two reasons. First, hKCa3 contains a polymorphic CAG repeat in its coding sequence, with large repeats more common in schizophrenics compared with controls.1 This is interesting in view of several repeat expansion detection (RED) studies2 that have reported an excess of large CAG repeats in psychotic probands.3–7 Second, the hKCa3 gene is a functional candidate gene because studies of antipsychotic and psychotogenic compounds suggest that glutamatergic systems modulated by SKCa channels may be important in schizophrenia pathogenesis.1 In the light of the above, we have tested the hypothesis of an association between schizophrenia and the hKCa3 CAG repeat polymorphism using a case control study design. Under the same model of analysis as the earlier study, schizophrenic probands had a higher frequency of alleles with greater than 19 repeats than controls (χ2u2009=u20092.820, Pu2009=u20090.047, 1-tail). Our data therefore provide modest support for the hypothesis that polymorphism in the hKCa3 gene may contribute to susceptibility to schizophrenia.

Application of next generation qPCR and sequencing platforms to mRNA biomarker analysis.

Recent years have seen the emergence of new high-throughput PCR and sequencing platforms with the potential to bring analysis of transcriptional biomarkers to a broader range of clinical applications and to provide increasing depth to our understanding of the transcriptome. We present an overview of how to process clinical samples for RNA biomarker analysis in terms of RNA extraction and mRNA enrichment, and guidelines for sample analysis by RT-qPCR and digital PCR using nanofluidic real-time PCR platforms. The options for quantitative gene expression profiling and whole transcriptome sequencing by next generation sequencing are reviewed alongside the bioinformatic considerations for these approaches. Considering the diverse technologies now available for transcriptome analysis, methods for standardising measurements between platforms will be paramount if their diagnostic impact is to be maximised. Therefore, the use of RNA standards and other reference materials is also discussed.

CAG repeat length in the hKCa3 gene and symptom dimensions in schizophrenia

BACKGROUNDnLong CAG repeats in the hKCa3 potassium channel gene have been associated with schizophrenia. We sought evidence for associations between this polymorphism and aspects of the schizophrenia phenotype.nnnMETHODSnAssociations were investigated between CAG repeat length and gender, age of illness onset, and psychotic symptom dimensions in 203 unrelated individuals with DSM-IIIR schizophrenia.nnnRESULTSnNo association was found between CAG repeat length and gender or age of onset. Long CAG repeats were associated with higher negative symptom dimension scores.nnnCONCLUSIONSnThis study provides preliminary evidence that genetic liability to negative symptoms in schizophrenia may be partly mediated through the hKCa3 gene.

Linked polymorphisms upstream of exons 1 and 2 of the human cholecystokinin gene are not associated with schizophrenia or bipolar disorder

The evidence for a significant genetic contribution to the functional psychoses (schizophrenia and bipolar disorder) is now well established. However, in both cases, the non-mendelian mode of inheritance has made the identification of susceptibility loci particularly challenging.1–3 The neuropeptide cholecystokinin (CCK) is present both in the gut and the CNS. Studies of CCK-like immunoreactivity and CCK mRNA levels in human brains have revealed high concentrations in numerous loci and shown colocalisation of CCK with, for example, dopamine and tyrosine hydroxylase.4 Furthermore, antagonists of CCK-B receptors, which are found most frequently in the brain, inhibit the activity of brain dopamine neurons.5 Such findings suggest that, with respect to neuropsychiatric disorders, CCK is a suitable candidate for analysis using methods to detect gene variations which have the potential to affect protein structure or expression.6 In the present study, mutation analyses were carried out on the human CCK gene. Linked polymorphisms were found in the promoter region and in intron 1 close to the 3′ mRNA splice acceptor site. However, the allele frequencies of these polymorphisms in samples of individuals affected with either schizophrenia (nu2009=u2009117) or bipolar disorder (nu2009=u2009124) did not differ from those of control subjects (nu2009=u2009234), suggesting that these variations do not confer a predisposition to either of the functional psychoses.

Considerations for accurate gene expression measurement by reverse transcription quantitative PCR when analysing clinical samples

AbstractReverse transcription quantitative PCR is an established, simple and effective method for RNA measurement. However, technical standardisation challenges combined with frequent insufficient experimental detail render replication of many published findings challenging. Consequently, without adequate consideration of experimental standardisation, such findings may be sufficient for a given publication but cannot be translated to wider clinical application. This article builds on earlier standardisation work and the MIQE guidelines, discussing processes that need consideration for accurate, reproducible analysis when dealing with patient samples. By applying considerations common to the science of measurement (metrology), one can maximise the impact of gene expression studies, increasing the likelihood of their translation to clinical tools.n ᅟ

Sibling pairs with schizophrenia or schizoaffective disorder: associations of subtypes, symptoms and demographic variables

BACKGROUNDnAffected sibling pairs provide a valuable means of investigating the familial basis of clinical heterogeneity in schizophrenia.nnnMETHODSnAssociations of schizophrenia subtypes, psychotic symptoms (defined by SAPS/SANS and OPCRIT), affective episodes and demographic variables were studied in 109 sibling pairs with DSM-IV schizophrenia or schizoaffective disorder.nnnRESULTSnNone of the subtypes or affective episodes were significantly associated within pairs. A broad definition of positive formal thought disorder, grandiose delusions and delusions of influence (all from OPCRIT) were modestly associated. There was no excess of same-sex pairs. There were modest associations for age of illness onset, pre-morbid adjustment and illness severity. Caution is required in interpreting the results because many statistical tests were carried out.nnnCONCLUSIONSnNone of the variables appears to be closely associated with specific genetic or shared environmental factors that contribute liability to schizophrenia. They are at best only weakly associated with such factors, and/or are associated with factors unrelated to the aetiology of schizophrenia.

Mutation screening of the KCNN3 gene reveals a rare frameshift mutation

259 posing an a of 0.05 with 18 patients (homozygous insertion), a satisfactory power of 0.84 was reached. Demographic, genetic and clinical data are summarised in Table 1. We were unable to replicate the previous finding since we observed no significant differences in genotype or allelic distributions between patients and controls (both followed the Hardy–Weinberg Equilibrium). Ethnic differences or differences in patient selection between both studies might be responsible for these discrepancies. No significant differences in genotypes or allele frequencies were found with respect to treatment procedures. A divergent clinical outcome was observed in that, after 4 weeks of treatment, D-allele carriers showed significantly lower HAM-D17 scores (P , 0.0001), remitted more often (HAM-D17 ,9; D/D+I/D:I/I = 18:1; P = 0.049) and had a significantly shorter duration of hospitalisation (P = 0.020). Also, the number of treatment alterations during hospitalisation was significantly higher in I/I-genotypes. Thus, the Dallele might positively influence the onset of therapeutic efficacy. Homozygosity for the I-allele seems to be associated with delayed response. Since, in functional studies, ACE allele-D was associated with increased neuropeptide degradation, increased SP concentrations might be responsible for the delayed effect on I/I-genotypes. This, however, has not yet been proven; SP levels were not controlled in this study and the role of SP in the treatment of depression still has to be clarified. Although therapeutic outcome in different genotypes cannot yet be related to the diversity of medication, our results give first hints that variants in the ACE gene could contribute to differential drug response. Further studies will have to clarify whether this effect is related to different drugs or a common feature of antidepressant treatment. In addition to association studies, family studies might help to further clarify the relationship between treatment response as a complex phenotype and ACE gene polymorphism.

Comparative sequencing of the proneurotensin gene and association studies in schizophrenia

Neurotensin (NT) is an endogenous tridecapetide1 cleaved from a precursor proneurotensin/ proneuromedin protein. NT localises within dopaminergic neurones in the mesocortical, mesolimbic and nigrostriatal systems1, 2, 3 and it is now clear that NT can selectively modulate dopaminergic neurotransmission.2, 3, 4, 5, 6, 7, 8, 9 These anatomical and functional connections have led to the hypothesis that NT dysfunction might contribute to the pathogenesis of neuropsychiatric disorders in which disordered dopaminergic neurotransmission is suspected, particularly schizophrenia.3 The latter hypothesis has been supported circumstantially by the observation that central administration of NT produces effects similar to those produced by the peripheral administration of atypical antipsychotics,10, 11 and more directly by studies showing levels of NT in cerebral spinal fluid (CSF) is lower in schizophrenics than in controls.12, 13 To allow such hypotheses to be tested, we used denaturing high performance liquid chromatography (DHPLC)14 to identify three sequence variants in the neurotensin gene (NTS) that might alter NT structure or expression. However, using a case-control study design and a novel genotyping system based upon a primer extension protocol and HPLC detection,15 we found no evidence to support the hypothesis that variation in the proneurotensin gene contributes to susceptibility to schizophrenia.