Analysis of the Human T Cell Receptor α/δ Locus: New Approaches to Mapping and Sequencing

The human T cell receptor (TCR) α/δ locus has been mapped and sequenced. This region occupies roughly one megabase (Mb) of DNA or equivalent to one three thousandth of the entire human genome, the longest continuous piece of human DNA yet sequenced. The sequence has provided new insights into the complex organization, structure and evolution of two intermingled multigene families (α and δ), and will hopefully in the future help answer interesting questions concerning the complex expression patterns of TCR α and δ chains and about possible associations between specific polymorphisms in the TCR α/δ locus and susceptibility to autoimmune diseases. Comparison to cDNA data has provided information about expression of each of the TCR elements and about the striking diversification in the third hypervariable or junctional region. The sequence has contributed a glimpse of closely associated genomic DNA, in that the sequences surrounding the TCR locus, include the defender against death gene as well as five olfactory receptor genes. The sequence also harbors many other stretches of DNA, highly similar to previously identified genes, although in most cases, these have been found to be nonfunctional due to one or a few mutations. Comparison of 130 kilobases (kb) in the 3' region of the human sequence with its murine counterpart, suggests this region is highly conserved. The same 3' region has also been found to be limited in the concentration of genome wide repeats compared to the remainder of the locus. Furthermore, it contains a substantially reduced frequency of DNA variations compared to the rest of the locus. Apart from DNA variations in noncoding sequence, polymorphisms have also been identified in the coding regions of the TCR variable (V) gene segments, where, if they lead to amino acid changes, may alter the function of the TCR. During the physical clone mapping and sequencing, new strategies were tested using primarily bacterial artificial chromosome (BAC) clones. These clones proved to be much more reliable and stable than clones currently employed in the human genome project (e.g., cosmids and yeast artificial chromosomes, YACs). BAC inserts can be sequenced completely by the high redundancy shotgun approach. Their insert size, stability, and capacity to be easily sequenced suggests that BAC clones are excellent mapping and sequencing reagents. The ends of BAC clone inserts can be sequenced directly. This has led to the proposal of a new strategy for obtaining the entire DNA sequence of the human genome without physical mapping.</p

The Reproducibility of Lists of Differentially Expressed Genes in Microarray Studies

Reproducibility is a fundamental requirement in scientific experiments and clinical contexts. Recent publications raise concerns about the reliability of microarray technology because of the apparent lack of agreement between lists of differentially expressed genes (DEGs). In this study we demonstrate that (1) such discordance may stem from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion, the lists become much more reproducible, especially when fewer genes are selected; and (3) the instability of short DEG lists based on P cutoffs is an expected mathematical consequence of the high variability of the t-values. We recommend the use of FC ranking plus a non-stringent P cutoff as a baseline practice in order to generate more reproducible DEG lists. The FC criterion enhances reproducibility while the P criterion balances sensitivity and specificity

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies

<p>Abstract</p> <p>Background</p> <p>Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists.</p> <p>Results</p> <p>Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan – the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (<it>P</it>) derived from widely used simple <it>t</it>-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent <it>P</it>-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on <it>P</it>-value ranking is an expected mathematical consequence of the high variability of the <it>t</it>-values; the more stringent the <it>P</it>-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations.</p> <p>Conclusion</p> <p>We recommend the use of FC-ranking plus a non-stringent <it>P </it>cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the <it>P</it>-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and <it>P</it>-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the <it>P </it>criterion balances sensitivity and specificity.</p

Genomic Analysis of the Olfactory Receptor Region of the Mouse and Human T-Cell Receptor α/δ Loci

We have conducted a comparative genomic analysis of several olfactory receptor (OR) genes that lie immediately 5′ to the V-α gene segments at the mouse and human T-cell receptor (TCR) α/δ loci. Five OR genes are identified in the human cluster. The murine cluster has at least six OR genes; the first five are orthologous to the human genes. The sixth mouse gene has arisen since mouse-human divergence by a duplication of a ∼10-kb block. One pair of OR paralogs found at the mouse and human loci are more similar to each other than to their corresponding orthologs. This paralogous “twinning” appears to be under selection, perhaps to increase sensitivity to particular odorants or to resolve structurally-similar odorants. The promoter regions of the mouse OR genes were identified by RACE-PCR. Orthologs share extensive 5′ UTR homology, but we find no significant similarity among paralogs. These findings extend previous observations that suggest that OR genes do not share local significant regulatory homology despite having a common regulatory agenda. We also identified a diverged TCR-α gene segment that uses a divergent recombination signal sequence (RSS) to initiate recombination in T-cells from within the OR region. We explored the hypothesis that OR genes may use DNA recombination in expressing neurons, e.g., to recombine ORs into a transcriptionally active locus. We searched the mouse sequence for OR-flanking RSS motifs, but did not find evidence to suggest that these OR genes use TCR-like recombination target sequences

Accuracy and User Acceptability of 24-hour Ambulatory Blood Pressure Monitoring by a Prototype Cuffless Multi-Sensor Device Compared to a Conventional Oscillometric Device

Objective 24-hour ambulatory blood pressure monitoring (24ABPM) is state of the art in out-of-office blood pressure (BP) monitoring. Due to discomfort and technical limitations related to cuff-based 24ABPM devices, methods for non-invasive and continuous estimation of BP without the need for a cuff have gained interest. The main aims of the present study were to compare accuracy of a pulse arrival time (PAT) based BP-model and user acceptability of a prototype cuffless multi-sensor device (cuffless device), developed by Aidee Health AS, with a conventional cuff-based oscillometric device (ReferenceBP) during 24ABPM. Methods Ninety-five normotensive and hypertensive adults underwent simultaneous 24ABPM with the cuffless device on the chest and a conventional cuff-based oscillometric device on the non-dominant arm. PAT was calculated using the electrocardiogram (ECG) and photoplethysmography (PPG) sensors incorporated in the chest-worn device. The cuffless device recorded continuously, while ReferenceBP measurements were taken every 20 minutes during daytime and every 30 minutes during nighttime. Two-minute PAT-based BP predictions corresponding to the ReferenceBP measurements were compared with ReferenceBP measurements using paired t-tests, bias, and limits of agreement. Results Mean (SD) of ReferenceBP compared to PAT-based daytime and nighttime systolic BP (SBP) were 129.7 (13.8) mmHg versus 133.6 (20.9) mmHg and 113.1 (16.5) mmHg versus 131.9 (23.4) mmHg. Ninety-five % limits of agreements were [-26.7, 34.6 mmHg] and [-20.9, 58.4 mmHg] for daytime and nighttime SBP respectively. The cuffless device was reported to be significantly more comfortable and less disturbing than the ReferenceBP device during 24ABPM. Conclusions In the present study, we demonstrated that a general PAT-based BP model had unsatisfactory agreement with ambulatory BP during 24ABPM, especially during nighttime. If sufficient accuracy can be achieved, cuffless BP devices have promising potential for clinical assessment of BP due to the opportunities provided by continuous BP measurements during real-life conditions and high user acceptability