RNA isolation and purification steps greatly influence the results of gene expression profiling. There are two commercially available products for whole blood RNA collection, PAXgene™ and Tempus™ blood collection tubes, and each comes with their own RNA purification method. In both systems the blood is immediately lysed when collected into the tube and RNA stabilized using proprietary reagents. Both systems enable minimal blood handling procedures thus minimizing the risk of inducing changes in gene expression through blood handling or processing. Because the RNA purification steps could influence the total RNA pool, we examined the impact of RNA isolation, using the PAXgene™ or Tempus™ method, on gene expression profiles.Using microarrays as readout of RNA from stimulated whole blood we found a common set of expressed transcripts in RNA samples from either PAXgene™ or Tempus™. However, we also found several to be uniquely expressed depending on the type of collection tube, suggesting that RNA purification methods impact results of differential gene expression profiling. Specifically, transcripts for several known PHA-inducible genes, including IFNγ, IL13, IL2, IL3, and IL4 were found to be upregulated in stimulated vs. control samples when RNA was isolated using the ABI Tempus™ method, but not using the PAXgene™ method . Sequenom Quantiative Gene Expression (QGE)  measures confirmed IL2, IL4 and IFNγ up-regulation in Tempus™ purified RNA from PHA stimulated cells while only IL2 was up-regulated using PAXgene™ purified (p < 0.05).Here, we demonstrate that peripheral blood RNA isolation methods can critically impact differential expression results, particularly in the clinical setting where fold-change differences are typically small and there is inherent variability within biological cohorts. A modified method based upon the Tempus™ system was found to provide high yield, good post-hybridization array quality, low variability in expression measures and was shown to produce differential expression results consistent with the predicted immunologic effects of PHA stimulation. Microarrays have rapidly become the assay of choice for clinical investigators wanting to measure gene expression, owing to their high-throughput and relative ease of use. As with any assay, it is critical that experimental variance is minimized in order to permit measurement of true biological variance. In clinical microarray studies, the sources of experimental variance can be considerable. While a range of corrections exist to detect and correct for variability introduced during hybridization and due to chip quality, little attention has been paid to the impact of specimen collection, handling and processing on the resulting gene expression measures.For immunological studies, peripheral blood is commonly used in microarray experiments, as it is the most easily obtained source of lymphocytes, granulocytes, and other cells that may provide insight into immune function. Historically, gradient density-based methods have been used to purify white blood cells from peripheral blood. However, it is known that, within minutes of collection, peripheral blood gene expression profiles change significantly due to transcript induction and transcript degradation, and it To address these concerns, RNA whole blood collection tubes have been developed that have the considerable advantage of lysing whole blood at the time of collection, while simultaneously stabilizing RNA for later purification. The PAXgene™ Whole Blood RNA isolation system contains a proprietary solution that reduces RNA degradation and transcript induction upon peripheral blood collection ,3. UsingIn this report, we compare these two whole blood RNA purification methods for use in microarray experiments to measure immune response gene expression, and show that the choice of RNA purification method can have significant implications. Using phytohemagglutinin (PHA) stimulated whole blood as a test case, we found that RNA yield and hybridization quality indicators were better for RNA isolated using the Tempus™ RNA purification method. While PHA induced a set of transcript expression changes detected in both Tempus™ and PAXgene™ samples, use of the Tempus system resulted in the identification of a greater number of gene expression changes that would be expected to result from PHA stimulation.260/230ratios , a mitogen known to induce expression of immune activation transcripts including those for IL-2, IL-4, and IFNγ. However® microarray. Only two transcripts, for the B-cell translocation transcript BTG-1, were identified as down-regulated in samples collected with Li Heparin tubes, compared to those collected in Tempus tubes. Interestingly, just one transcript was up-regulated using PAXgene™ tubes, corresponding to one of the transcripts up-regulated in the Tempus™ system, BTG1. These results suggest that collection of samples into Li Heparin tubes prior to PHA stimulation had a minimal effect on differential expression and would have negligible effect on the interpretation of differences between the two tube types.To control for the effect of Li Heparin tube collection, we performed an initial comparison of 5 healthy control samples drawn directly in PAXgene™ or Tempus™ tubes vs. the same 5 healthy control samples drawn in Li Heparin tubes with no PHA stimulation. RNAs were hybridized to the HG-U133 2.0 Plus Affymetrix GeneChip® microarray.To compare PAXgene™ and Tempus™ tubes, whole blood samples collected in Li Heparin tubes from 7 healthy controls were split into PHA-stimulated or unstimulated aliquots. After 3 hrs, samples were transferred to each respective RNA isolation system and hybridized to the HG-U133 2.0 Plus Affymetrix GeneChipHierarchical clustering of absolute expression levels, irrespective of gene function, showed that the primary separation occurred between stimulated vs. unstimulated samples (as opposed to different sample collection systems). The clustering was generated on the 28 individual samples using the 1,266 differentially expressed transcripts that were either up- or down-regulated by PHA-stimulation Fig. . StimulaPHA-stimulation resulted in 538 transcripts being detected as upregulated, and 392 downregulated when the Tempus™ system was used. For the PAXgene™ system, 400 were found to be upregulated and 539 downregulated p < 0.01, FDR) by PHA stimulation Fig. . Among a, FDR by To validate differential expression measured by microarray, real time PCR was performed on four of the seven samples that had sufficient RNA remaining. Using the Sequenom Quantitative Gene Expression (QGE) platform, eighteen immune function transcripts were assessed, including IL-2, IL-4, IFNγ, controls and others not known to be regulated by PHA. Other transcripts assessed include: TGFβ, P19, Perforin, MIG, IP10, IL10, GB, FOXP3, CXCR3, CTLA4, CTGF, CD3, CD25, CD20, and CD103. Transcripts for IFNγ, IL-2, IL-4 were statistically significant for differential expression between un-stimulated and stimulated samples using the Tempus™ system (p < 0.05). Samples prepared using the PAXgene™ system exhibited greater variability in transcript levels in both unstimulated and stimulated conditions, thus trending towards upregulation for IL-2, IL-4, and IFNγ, but not statistically significant  . Both PAXgene™ and Tempus™ whole blood collection tubes were used in this step. Since cells are immediately lysed upon collection with either PAXgene™ or Tempus™, blood had to be initially drawn in Li Heparin tubes  prior to transfer into either system. A secondary comparison was made between samples drawn directly in both collection systems versus. Samples were drawn in Li Heparin and then immediately transferred into their respective tube types, to determine if Li Heparin adversely effected gene expression on its own.Our objective was to assess the effect of two available blood collection systems with regard to differential gene expression in clinical samples. The primary comparison was of unstimulated peripheral blood samples with those stimulated For these comparisons, whole blood was collected from seven healthy individuals who provided informed consent, under the approval of the Institutional Review Board of Brigham and Women's Hospital. A total of 110 mL of peripheral blood was collected from each participant using Li Heparin tubes. Whole blood from individual participants was pooled into a 200 mL plastic container and a set of seven aliquots from the pool was incubated for 3 hrs at room temperature with no stimulant added; a second set of seven aliquots was stimulated with 25 μg/mL of PHA and incubated for 3 hrs. After incubation, samples were subsequently transferred to either PAXgene™ or Tempus™ tubes. To assess whether stimulation by PHA was successful, FACS detection for IFNγ  was performed. Samples from five of the seven subjects were drawn directly into PAXgene™ and Tempus™ tubes for assessing the effect of Li Heparin alone as described above. In total, 48 samples were collected and hybridized as part of the analysis.RNA was extracted at the ITN Central Nucleic Acid Isolation Core Facility in Pittsburgh, PA according to the ITN-modified method for the Tempus™ system. The PAXgene™ Whole Blood RNA samples were processed using the PAXgene™ Blood RNA Kit based on the Qiagen method for column purification of nucleic acids . Whole blood samples collected into Tempus™ vacuette were extracted using ABI Prism™ 6100 Nucleic Acid PrepStation™ and using Tempus™ extraction reagents. Samples were frozen immediately at -70°C upon collection. Extraction steps included addition of PBS buffer to compensate for short blood sample draws, a wash step with Purification Wash Solution 1 two times at 80% vacuum for 500 seconds, followed by a single washing step using Purification Wash Solution 2 at 80% vacuum for 120 seconds. An extra vacuum step was performed to eliminate Purification Wash Solution 2 from the filter. Upon changing the reservoirs on the ABI 6100 PrepStation™, three more washing steps were performed using Purification Wash Solution 2 at 80% vacuum for 120 seconds. At the elution step, adapter plates were changed one extra time to ensure the purity of the eluted RNA. Eluted RNA was then concentrated using the Microcon YM-100 . RNA purity and yield were assessed prior to hybridization using the Agilent 2100 Bioanalyzer  [The ITN Central Microarray Facility  performed all hybridizations with the Affymetrix HG-U133 2.0 Plus microarray. All processing was done according to manufacturer's instructions. Globin reduction was performed followed by cRNA target amplification using the Affymetrix In-vitro Transcription (IVT) Kit. Standard pre- and post-hybridization quality control metrics were used to assess sample processing and hybridization quality. Microarrays have been deposited within GEO (Accession number GSE12711).Multiplexed primer and competitive template designs were created using the MassARRAY QGE Assay Design software v1.0  for random hexamer priming, such that at least one PCR primer spanned an exonic boundary per each transcript assayed. The 20 gene panel assayed for this study was designed as a single 20-plex reaction.Copy number determination for each transcript was conducted using real-time competitive PCR coupled with product resolution via Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry , as previously described . ProductNormalization of copy numbers between samples for the different assays was conducted using a multiplexed set of ten well-characterized human housekeeping  transcripts plus an 18s RNA assay and geNorm software. Normalization factors per sample were calculated using the geometric mean of the most stable combination of these normalization assays, determined by the measure of their pairwise variation as calculated by geNorm.Microarray normalization and preprocessing was performed as follows: Log-transformed Affymetrix microarray intensity values were processed using the 'threestep' function in the R/Bioconductor affyPLM package; backgroThe normalized data from the experiments were fit into linear models using the Bioconductor package limma. The priPair-wise comparisons of interest were performed using moderated t-statistics to test for significant differential expression. The Benjamini-Hochberg multiple comparison adjustment was usedSequenom QGE analysis was performed using the Mann-Whitney test to identify differentially expressed transcripts between samples with and without PHA stimulation using Tempus™ and PAX platforms respectively.ALA contributed to the study design, data analysis and drafting of the manuscript. SAK contributed to the study design, management of samples, and drafting the manuscript. ZG and RW performed the gene expression analysis. KR processed the samples for PHA stimulation and performed flow cytometry analysis. KB and VS contributed to the study design, interpreting the results in the context of immune profiling, and drafted and reviewed the manuscript.Transcripts up-regulated by PHA using Tempus ABI and PAXgene™ collection systems.Column headings are:Probe Affymetrix IDABI 0 = Failed statistical criteria1 = Passed statistical criteriaPAX 0 = Failed statistical criteria1 = Passed statistical criteriaABI-log2ratio log 2 ratios for Tempus ABI tubeABI-p value p-value for Tempus ABI tube, FDR correctedPAX-log2ratio log 2 ratios for PAXgene tube tubePAX-p value p-value for PAXgene tube, FDR correctedSymbol Gene symbolDescription Gene descriptionUniGene UniGene IDOMIM OMIM IDPathway Pathways associatedClick here for fileTranscripts down-regulated by PHA using Tempus ABI and PASGene collection systemsColumn headings are:Probe Affymetrix IDABI 0 = Failed statistical criteria1 = Passed statistical criteriaPAX 0 = Failed statistical criteria1 = Passed statistical criteriaABI-log2ratio log 2 ratios for Tempus ABI tubeABI-p value p-value for Tempus ABI tube, FDR correctedPAX-log2ratio log 2 ratios for PAXgene tube tubePAX-p value p-value for PAXgene tube, FDR correctedSymbol Gene symbolDescription Gene descriptionUniGene UniGene IDOMIM OMIM IDPathway Pathways associatedClick here for file


Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of microarrays. Recently, there have been significant improvements in whole blood RNA isolation techniques which, through stabilization of RNA at the time of sample collection, avoid bias and artifacts introduced during sample handling. Despite these improvements, current human whole blood RNA stabilization/isolation kits are limited by the requirement of a venous blood sample of at least 2.5 mL. While fingerstick blood collection has been used for many different assays, there has yet to be a kit developed to isolate high quality RNA for use in gene expression studies from such small human samples. The clinical and field testing advantages of obtaining reliable and reproducible gene expression data from a fingerstick are many; it is less invasive, time saving, more mobile, and eliminates the need of a trained phlebotomist. Furthermore, this method could also be employed in small animal studies, i.e. mice, where larger sample collections often require sacrificing the animal. In this study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via a fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit.2 values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r2 values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.94 to 0.96 for fingerstick collection 2.From two sets of fingerstick collections, about 70 uL whole blood collected via finger lancet and capillary tube, we recovered an average of 252.6 ng total RNA with an average RIN of 9.3. The post-amplification yields for 50 ng of total RNA averaged at 7.0 ug cDNA. The cDNA hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips had an average % Present call of 52.5%. Both fingerstick collections were highly correlated with rOur comparisons of RNA quality and gene expression data of the fingerstick method with traditionally processed sample workflows demonstrate excellent RNA quality from the capillary collection as well as very high correlations of gene expression data. Whole genome gene expression profiling has revolutionized research in the past decade especially with the advent of DNA microarrays -3. This Helicobacter pylori infection [However, one limitation of such RNA stabilization systems in humans is the requirement for the collection of a venous blood sample, which involves a venipuncture. Fingerstick capillary blood collection has been widely used and has been shown to be very reliable. A classic example of this methodology is blood glucose monitoring. Fingerstick blood has also been used to test for nfection ,6, cholenfection ,8, glyconfection  and syphnfection . Howevernfection . Anothernfection . In addiObtaining reliable and reproducible gene expression data from a fingerstick has obvious advantages in clinical as well as field testing applications. A fingerstick is arguably a less invasive, less time consuming and a more mobile method of blood sample collection, eliminating the need of a trained phlebotomist. The utility of such a method is obvious in studies designed to collect blood samples from physically active subjects (i.e. soldiers and athletes) or for field studies in remote and under-developed areas. Fingerstick blood collection would also be of immense value in several types of subjects where it is commonly difficult to collect venous blood via venipuncture: infants and young children, intravenous drug addicts, and very obese individuals. The value of fingerstick capillary collection as opposed to venipuncture can also be appreciated in study designs where there is a need for serial sample collections or for pharmacokinetic studies that involve gene expression assays. This method could also be employed in small animal studies or nonhuman primates. Therefore, a fingerstick method of blood collection will broaden the current range of genomic profiling possible. We also believe that it will be an integral part of diagnosis and serial monitoring of disease states in the future where one can envision a hand-held device that could monitor expression levels of validated panels of genomic biomarkers, much like the glucose monitoring systems of today.In the present study, we offer a rapid and simple method to extract sufficient amounts of high quality total RNA from approximately 70 μl of whole blood collected via fingerstick using a modified protocol of the commercially available Qiagen PAXgene RNA Blood Kit. RNA amplification, labeling, and fragmentation were performed using the Nugen Ovation kits . This approach hybridizes biotinylated cDNA onto the microarray and has been shown to perform with superior sensitivity especially with smaller amounts of input RNA -14. Our http://www.scripps.edu/researchservices/dna_array[http://www.nugeninc.com/tasks/sites/nugen/assets/File/user_guides/userguide_encore_biotin.pdf[In order to test the success and reproducibility of the fingerstick method of RNA isolation we took two fingerstick capillary samples from 5 donors on two separate days . From each collection time point we chose one purified total RNA sample from each donor and went forward with the RNA amplification, fragmentation and hybridization protocols. A venous sample was also collected in parallel from each of the donors by a trained phlebotomist and processed according to standard protocols to represent the established method dna_array. All samiotin.pdf.http://www1.qiagen.com/Products/RnaStabilizationPurification/DSP/PaxGeneBloodRnaKitIVD.aspx#Tabs=t2[Approximately 70 μL of blood collected via fingerstick was combined with 200 μL PAXgene RNA stabilizing reagent (a PAXgene Reagent:blood ratio of 2.86) and left to incubate at room temperature for at least two hours as per manufacturer suggestions  protocol for RNA isolation and purification, with the exception of one modification. After the first spin, we washed the pellet with 1 mL RNase free water instead of 4 mL due to its small volume. We initially tested a "scaled down" version of the entire PAXgene protocol, but, through further testing, we found that using the standard volumes of buffers and washes had no effect on the yields and were easier to employ (data not shown). Furthermore, we also found the DNase step in the protocol was crucial for the yield and fidelity of the amplified cDNA. Without the DNase step, contaminating DNA was subsequently amplified causing the GAPDH and Actin ratios used as quality control metrics on the GeneChips to be abnormally high (data not shown).260/280 ratios, an average of 3.9 ± 1.35, and lower OD260/230 ratios, an average of 0.09 ± .07, we found that this did not affect downstream applications and was probably caused by the high concentration of salts in the elution buffer relative to the low concentration of RNA http://www.flychip.org.uk/protocols/gene_expression/rna_qc.php[From 20 samples of 70 μL fingerstick blood, the average total RNA yields ranged from 138 to 430 ng (average of 255.7 ± 72.6 ng) which was well above the maximum of 50 ng required for the Nugen Ovation RNA Amplification System v2 . While we did experience higher than normal ODna_qc.php. To assena_qc.php.260/280ratio of 2.0 ± 0.03 and an average OD260/230 ratio of 1.0 ± 0.45. To assess the quality and integrity of the RNA, the samples were then run on the Agilent 2100 BioAnalyzer using an RNA NanoChip. The average RIN of the 5 total RNA samples obtained by venipuncture was 9.2 ± 0.09.The total RNA from the 5 normal venipuncture PAXgene blood collection tubes was extracted and purified according to the PAXgene Blood RNA kit (product# 762164) protocol. From 2.5 mL of blood the yield ranged from 4.1 to 7.9 μg of total RNA (average 5.96 ± 1.5 μg) with an average OD® RNA Amplification System V2 (Cat.# 3100) and Ovation® WB Reagent (Cat.# 1300). The total RNA yields and Agilent traces for these 15 samples are shown in Figure ® PCR Purification Kit . As shown in Figure 260/280ratio of the 10 samples was 1.9 ± 0.01.50 ng total RNA from each donor was taken from all three sample sets  and subsequently amplified using the Nugen Ovation260/280 ratio of 1.9 ± 0.01. Before fragmenting and labeling the cDNA with the Nugen FL-Ovation™ cDNA Biotin Module V2 (Cat.# 4200), the BioAnalyzer was used again to determine quality of the amplified whole cDNA , the cDNA yields ranged from 8.2 to 9.3 μg (average of 8.6 ± 0.43 μg) . Following hybridization, the chip was then washed, stained and scanned according to standard Affymetrix protocol http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf[4.4 μg of the labeled cDNA was then hybridized to Affymetrix GeneChipanual.pdf.The Affymetrix quality metrics for the fingerstick samples are given in Table 2 values ranging from 0.94 to 0.97. Similarly both fingerstick collections were highly correlated to the venous collection with r2 values ranging from 0.88 to 0.96 for fingerstick collection 1 and 0.93 to 0.96 for fingerstick collection 2.We calculated correlation coefficients for each donor comparing fingerstick collection 1 vs. fingerstick collection 2 and also each fingerstick collection vs. the venipuncture collection Table . Both fiWe analyzed the degree of disagreement in Affymetrix present/absent calls between the fingerstick collections and also between the fingerstick and venous collections for each donor. Disagreement is described as a change in the present, marginal or absent calls between any two comparisons. Within each comparison we binned the average signal intensities of each probe set in the ranges between 0-100, 101-250, 251-500, 501-1000 and >1001. We got similar results for all three comparisons that we performed. There was an inverse correlation between the signal intensities and the disagreement calls for the probesets in each bin Figure  &3c. In We further analyzed the disagreement as the number of calls that changed from present to absent and vice versa to test the hypothesis that a change from absent to present would indicate higher sensitivity for the method used. On average, there was a higher number of disagreement calls, namely, change in call of a probe set from present in the venipuncture method to absent in the fingerstick method. However, there were also a number of calls that changed from present in the fingerstick collection to absent in the venipuncture collection Table . For exaDespite the fact that technologies for RNA isolation have shown tremendous improvements over the past decade with RNA isolation kits for tissue, cells as well as whole blood, there is still no commercially available methodology for the isolation of good quality RNA from microliter volumes of human whole blood suitable for gene expression profiling on DNA microarrays. To test the hypothesis that we can isolate excellent quality and sufficient quantities of RNA from small volumes of whole blood (<100 μl) we investigated a modified protocol of whole blood RNA isolation in the present study.Our results show that we were able to successfully isolate RNA that gave comparable results with the standard venous blood method when assayed on an Affymetrix GeneChip. The advantages of the fingerstick RNA isolation method were the ease of small volume blood collection, minimal modification of the standard PAXgene protocol, no requirement of a trained phlebotomist, and amenability to off-site studies as well as feasibility in studies involving serial blood collections on a large number of subjects. The fingerstick RNA was of very high quality, comparable to the venous blood RNA when assayed by the Agilent Bioanalyzer with no signs of degradation despite the technical differences between a venipuncture and a fingerstick collection. It is important to note that even though the volume of blood used in the fingerstick collection was small, the DNase treatment of the RNA was found to be crucial. This was especially necessary for the accurate quantitation of the RNA since the Nugen protocol calls for nanogram quantities of RNA as the starting material. The accurate quantitation of RNA could be biased due to the presence of contaminating DNA.The yields of amplified cDNA were only slightly higher in the venipuncture method compared to both fingerstick collections. However, the Agilent Bioanalyzer traces for the amplified cDNA from the Nugen protocol showed no differences between the two collection methods. The quality control metrics for the GeneChip data showed that the venous collections had slightly higher average percent present calls (3.5% more) but this could reflect the inherent biological differences between venous and capillary blood. In a recent publication, Schalk et al., show that there are significantly higher WBC and RBC counts in capillary blood but lower numbers of platelets. Such diOn further analysis we showed that the differences are mainly due to the flux between calls at the lower signal intensities (<100), which accounts for almost all the variation between the methodologies. On average only about 12% of the total probesets on the GeneChip disagreed between any two comparisons that we made, which is well within the range of expected variation between samples in any DNA microarray study (data not shown). A study which looked at the individuality and variation in gene expression patterns in human blood found that there were several significant variations in gene expression among 77 samples of peripheral blood collected from normal healthy volunteers. The varIn the present study we demonstrate a simple, modified RNA isolation protocol from small volumes of whole blood (70 μL by fingerstick) that is highly comparable to the standard method of RNA isolation (2.5 mL by venipuncture). The RNA from the fingersticks were further assayed on Affymetrix GeneChips and the results were very similar to the venipuncture collections. We effectively show that this fingerstick RNA isolation methodology can be used and should open up a broad range of applications for whole blood DNA microarray analysis ranging from pediatric studies to animal studies where there is access to only smaller volumes of blood. This methodology is especially suitable for serial monitoring, field testing, pharmacokinetic assays and rapid diagnosis of disease states.All samples were drawn from normal, healthy volunteers as part of The Scripps Research Institute's Normal Blood Drawing Service approved by the Office of Research Subjects Protection of the Scripps Health Human Subjects Committee. Both fingerstick and venous blood samples were collected by a trained phlebotomist.http://www1.qiagen.com/Products/RnaStabilizationPurification/DSP/PaxGeneBloodRnaKitIVD.aspx#Tabs=t2[Sterile, DNase and RNase free, 1.5 mL Eppendorf tubes pre-filled with 200 μL PAXgene RNA stabilizing reagent  were prepared and set aside. The donor finger was cleaned with an alcohol wipe and stuck with a single-use, spring loaded, retracting needle lancet (Unistick2 Super Lancet) according to the manufacturer's directions. Approximately 70 μl of blood was immediately collected into a capillary tube (Fisherbrand Heparinized Micro-Hematocrit). The sample was then transferred into the Eppendorf tube containing the PAXgene solution and the capillary tube was aspirated with a pipette to increase sample recovery. The sample was then mixed well with a pipette, given a quick spin, and left to incubate at room temperature; allowing complete lysis of blood cells in sample  at room temperature for 15 minutes. After carefully aspirating and discarding the supernatant, the pellet was washed with 1 mL RNase free water. From this point forward, we followed the processing guidelines given in the PAXgene Blood RNA kit (product# 762164) protocol. Note: As the pellet was often in the form of a streak on the back of the tube, we used the pipette tip to scrape it from the tube and fully re-suspend the pellet.Due to the small amount of total RNA in the eluate, the samples were dried in a SpeedVac  to about 20 μL (from the standard 80 μL elution) before any quality assessment was done. The samples were then measured using the NanoDrop 1000  to determine the concentration and purity. The Agilent 2100 BioAnalyzer and accompanying software was used to determine the integrity and quality of the total RNA.® RNA Amplification System V2 (Cat.# 3100) in conjunction with the Ovation® WB Reagent (Cat.# 1300). They were subsequently purified using the Qiagen QIAquick® PCR Purification Kit . Other purification methods can be used, according to the Nugen user guide, but we only tested the kit listed above. Again, the NanoDrop 1000 and Agilent 2100 BioAnalyzer were used to assess sample quantity and quality. The samples were then labeled and fragmented using the Nugen FL-Ovation™ cDNA Biotin Module V2 (Cat.# 4200).50 ng aliquots were amplified using the Nugen Ovationhttp://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf[All sample hybridization and GeneChip processing were done according to the Nugen standard array protocol for cDNA hybridization (Nugen user guide) and the standard Affymetrix protocols anual.pdf.Conceived and designed: DS, SK, AW, TM, ER, and SH. Performed experiments: SK, AW, ER, and TM. Analyzed data: SK, ER, TM, and AW. Wrote paper: SK, TM, and ER. Read and approved paper: DS, SK, AW, TM, ER, and SH.


Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the srv mutant (MGAS5005Δsrv) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δsrv to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δsrv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement (MGAS5005ΔsrvΔspeB) and tested its ability to form biofilms in vitro.Group A speB in the srv mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.Allelic replacement of srv mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of speB/SpeB.The constitutive production of SpeB by the  Streptococcus (GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease [covS, part of the two component regulatory system CovRS (CsrRS)[ speB[Listeria monocytogenes regulator PrfA, results in a mutant strain exhibiting a significant reduction in biofilm formation [srv mutant suggesting Srv contributes to the control of SpeB production [srv mutant was due to the constitutive production of SpeB. In support of this hypothesis, we demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of the srv mutant to form biofilms [in vitro biofilms by western immunoblot analysis [srv was due only to the constitutive production of SpeB or to other changes inherent in the srv mutant strain. To address this question, we constructed a ΔsrvΔspeB double mutant through allelic replacement . This muRS)[ speB,19. ReprRS)[ speB,20,21. Wormation ,22. Unlioduction . SpeB isoduction -30. Prevoduction -34. Givebiofilms . Furtheranalysis . Still, speB ORF was amplified from MGAS5005 genomic DNA using speBsrv UP FWD (Table speBsrv UP REV (Table BsrGI-XhoI site of pFW14 [speB-UP. Sequence located downstream of the speB ORF was amplified from MGAS5005 genomic DNA using speBsrv DOWN FWD (Table speBsrv DOWN REV (Table XmaI-AgeI site of pFW14ΔspeB-UP. The resulting plasmid (pFW14ΔspeB) was transformed into NovaBlue competent cells (Novagen). Electrocompetent MGAS5005Δsrv cells (200 μL) were incubated with pFW14ΔspeB  for 10 minutes on ice. The competent cells and DNA were placed in a pre-chilled 0.2 cm cuvette and electroporated . Electroporated cells were incubated for 10 minutes on ice. Cells were allowed to outgrow at 37°C with 5% CO2 for 3.5 h in Todd Hewitt broth supplemented with 2% Yeast extract (THY) . Selection for MGAS5005ΔsrvΔspeB occurred on THY agar supplemented with chloramphenicol (5 μg/mL) (Sigma) and incubated at 37°C with 5% CO2 for 48 hours. The speB deletion was verified in chloramphenicol resistant transformants using PCR and restriction digestion. A PCR utilizing internal srv and internal speB primers (Table srv (II), MGAS5005ΔspeB (III) and MGAS5005ΔsrvΔspeB (IV)  Figure  to validspeB mRNA was not produced by MGAS5005ΔsrvΔspeB, total RNA was isolated from MGAS5005 (control) and MGAS5005ΔsrvΔspeB and subjected to TaqMan real-time reverse transcriptase PCR (RT-PCR) analysis [srv or speB (data not shown) in the MGAS5005ΔsrvΔspeB strain. Transcript of prsA, a gene located immediately downstream of speB, was ~ 3 fold higher in MGAS5005ΔsrvΔspeB than MGAS5005, indicating that transcription of downstream genes was not disrupted. It should be noted that MGAS5005Δsrv [speB have previously been shown to be free of detectable polar effects [To verify that analysis ,38. Resu5005Δsrv  and MGAS effects ,34,39. A effects ,23.srv, MGAS5005ΔspeB [srvΔspeB cultures were grown under static conditions (0.5 h - 48 h); biofilm production was measured through crystal violet (CV) staining as previously described [speB in the srv mutant background restored biofilm formation to near wild-type levels after 24 h . Over time, biofilm formation of MGAS5005ΔsrvΔspeB closely resembled what we have previously reported for MGAS5005 with maximal formation occurring between 24 h and 30 h with a subtle decline in CV staining thereafter .To examine biofilm formation, MGAS5005, MGAS5005Δ005ΔspeB ,34,39 anescribed   3 times over the course of static biofilm development . CV staining was performed on treated and untreated samples at 18 h post-seeding Figure . SpeB adv Figure .srv is due to the constitutive production of mature SpeB. Inactivation of speB in the MGAS5005Δsrv background restored biofilm formation to wild-type levels. Complementation of MGAS5005ΔsrvΔspeB through the addition of exogenous SpeB significantly reduced biofilm formation to MGAS5005Δsrv levels. These results support a model in which the Srv mediated control of SpeB production regulates GAS biofilm formation  that mutation of covS  resulted in a strain with decreased biofilm formation due to increased capsule production[covS mutant[srv in a covS+ M1T1 background (as well as in other serotypes) to understand the role of Srv in biofilm formation and GAS disease.Taken together, the data indicate that the biofilm deficient phenotype of MGAS5005Δe profile,15. Receroduction. They shvS mutant. Thus, oThe authors declare that they have no competing interests.in vitro experiments, and drafted manuscript. RCH designed and developed MGAS5005ΔsrvΔspeB mutant and critically analyzed manuscript. SDR participated in the design of the study and helped to draft the manuscript. All authors read and approved of the final manuscript.ALR participated in the design of the study, conducted


Orthodontic management of anterior open bites is a demanding task for orthodontists. Molar intrusion as a primary means of open bite correction entails the need for appropriate anchorage. Orthodontic mini implants can provide the required mechanical support. The suggested procedure aims to reduce the risk of complications such as root damage or soft tissue irritations while minimizing overall complexity.Three female patients aged 14, 18 and 19 years who decided against a surgical correction were treated with a device consisting of mini implants in the palatal slope, a palatal bar and intrusion cantilevers.In all three patients, an open bite reduction of more than a millimeter occurred within four months. An anterior overbite of 2 mm or more could be established within 6 to 9 months.The method presented in this article enables the practitioner to use mini implants in an easily accessible insertion site. A lab-side procedure is optional but not required. The management of anterior open bites is considered one of the most difficult tasks in orthodontics.In growing patients, functional or orthopedic approaches can be applicable-3. WithoOrthodontic solutions may involve an unfavorable extrusion of the incisors and may result in an increased display of gingiva. In manyVarious mini implant insertion sites are available for the purpose of molar intrusion. Interradicular insertion bears the risk of root damage. Even after correct implant insertion, root contact may still occur later due to the progress of the intrusion, which may impair implant stability. Insertion beyond the attached gingiva may also lead to an increased failure rate. The palThe aim of this work is to present an approach that does not require incision, interradicular implant placement or placement in the movable alveolar mucosa while keeping the complexity of the orthodontic mechanics at a minimum.Instead of a gingival collar the Jet Screw (JS) type mini implants  used in this work have a long neck which widens towards the implant head. This makes them applicable in areas covered by thick soft tissue. They are advertised for use with the TopJet Distalizer . The insertion site recommended by the manufacturer is located at half of the distance of the perpendicular line segment from the raphe to the palatal cusp tip of the first bicuspid Figure.The mini implants were inserted according to the following protocol:•Informed consent regarding potential risks, complications and behavior via standardized documents•surface anesthesia with 1% lidocaine spray applied using a cotton ball•infiltration using 4% articaine solution•mouth rinse with 0.2% chlorhexidine digluconate•assessment of gingival thickness using a probe•choice of JS neck length: 3 mm neck for gingival thicknesses not exceeding 3.5 mm, otherwise 5 mm•insertion using a surgical handpiece or a hand screw driverPosterior intrusion was achieved through distally extended cantilevers fabricated out of 16×22 stainless steel wire. The connection between implant neck and wire was established by bending the anterior end of the wire into the shape of a clip Figure.A palatal bar served to avoid palatal tipping of the molars during intrusion. Also, it helped in preventing the cantilevers from interfering with the gingiva. Notably, the palatal bar was adjusted to leave at least 3 mm of space to the roof of the palate in order to obviate excessive soft tissue contact as the intrusion progresses.Once attached to the mini implant, the cantilevers rest on the palatal bar. To establish the desired intrusive force level, they were then tied to the molar bands with steel ligatures. An initial force level of 60 cN per side was chosen for the intrusion of the first molars and verified using a spring balance. A force of 200 cN per side was applied when second bicuspids, first molars and second molars were being intruded simultaneously. During subsequent appointments the ligatures were gradually tightened to maintain the intrusive force.An 18 year old woman with an anterior open bite was treated according to above method Figure. The negInitially, only the upper first molars were intruded Figure. After fAfter removal of the intrusion cantilevers three months later, treatment ceased for two months in order to estimate the amount of relapse. Panoramic x-ray images served to assess the apical situation and to identify possible root resorptions Figure.The amount of dentoalveolar change was assessed clinically as well as by means of digitized plaster casts. An optical 3D-scanner and a spezialzed software  served to obtain the according data and to overlay initial and post-intrusion maxillary casts using the best fit method Figure. The antIn a 19 year old woman, the intrusion procedure was commenced when a fixed appliance was already in situ and the leveling and aligning stages of treatment were almost complete (FigureA 14 year old girl received a similar treatment FigureAt the time of the most extensive over-correction, the anterior open bite was remediated and an iatrogenic lateral open bite of 3.5 mm on the right side and 4 mm on the left had occurred. In the following four months, the lateral open bite closed incompletely. A lateral open bite of 1.5 mm on each side remained. The anterior overbite persisted. It amounted to 1 mm at the right lateral incisors and to 4 mm in the left central incisor area.The x-ray images Figure showed nIn FigureIn addition to the vertical correction, an improvement of the molar relationship was observed. On the right side, a class I molar relationship was established. On the left side, a quarter of a unit class II remained.The transversal distance between the upper first molars increased by 2.5 mm which may be attributed to a slight over-activation of the palatal bar.Within five months, the anterior open bite was closed and a lateral open bite of 1 mm was established. Occlusal contacts remained between the second molars and first bicuspids. On both sides, a super class I occlusion resulted.The cephalometric tracings Figure reveal aFive months of intrusion resulted in an anterior overbite and a vertical distance of 2 mm between upper and lower first molars.The aspired treatment objective was reached in all cases. The clinically visible open bite reduction can be attributed to the intrusion mechanics since no intermaxillary elastics were used. Still, an incisor extrusion was observed in case 2. It may result from the fact that the intrusive force was applied distal to the center of resistance of the maxillary dentition entailing a clockwise rotation of the entire dental arch.In terms of implant placement, the suggested procedure benefits from an insertion site which is accessible with a surgical hand piece as well as with a straight hand screw driver unless pathological limitations of mouth opening are present. Slight deviations regarding the implants’ position as well as their angulation can be compensated for by adapting the cantilevers accordingly.Maxillary molar intrusion may also be conducted by using miniplates instead of mini implants. HoweverMini implant insertion in the buccal alveolus may bear the risk of root damage. Additionally, in order to achieve a favorable force vector, a high insertion location in the area of the movable mucosa would be necessary, which is suboptimal in terms of implant survival. The palThe suggested procedure can be performed chair side. The clip connectors can be pre-fabricated. A lab-side fabrication of the palatal bar is optional.Although the reliability of the presented method requires further investigation, the results appear propitious. The first case is especially insightful since the observed effect can be fully attributed to the intrusion mechanics. No brackets, elastics or other appliances were being used. It can, however, be argued that treatment might have been more efficient if molar intrusion had been performed simultaneously with leveling and aligning.The intrusion cantilevers were fabricated out of .016”×.022” stainless steel wire. The decision for this material was made because TMA wires are more prone to breakage whereas NiTi wires cannot as easily be bent. A hybrid construction with a steel clip on one end and a NiTi lever is conceivable but was not deemed necessary. The sheer length of the levers provided for a sufficient level of elasticity. Finally, with regard to the possibility of root resorption, dissipating forces may even be favorable.While additional research is required, present results indicate that the proposed method is suitable for treatment of anterior open bites. It is advantageous in terms of surgical difficulty and mechanical complexity.Written informed consent was obtained from all patients for publication of this report and the accompanying images. In all cases, a medical indication for the respective treatment was present. The surgical procedure constitutes a routine treatment. The authors declare that no ethical approval was necessary.The authors declare that they have no competing interests.TZ suggested the original idea for the paper. TZ, SF and JK wrote the main part of the manuscript. JK and DW reviewed the paper for content, and reviewed and contributed to the writing of all iterations of the paper, including the final version of the manuscript. All authors read and approved the final manuscript.


In the crystal, molecules are linked by N—H⋯O and C—H⋯π interactions.In the title compound, C N-acetamide derivatives, see: Barluenga et al.  Åb = 17.668 (8) Åc = 10.103 (5) Åβ = 107.914 (7)°V = 1555.0 (13) Å3Z = 4Kα radiationMo −1μ = 0.08 mmT = 153 K0.61 × 0.07 × 0.02 mmRigaku AFC10/Saturn724+ diffractometerCrystalClear; Rigaku, 2008Tmin = 0.954, Tmax = 0.998Absorption correction: multi-scan (12600 measured reflections3028 independent reflectionsI > 2σ(I)2386 reflections with Rint = 0.050R[F2 > 2σ(F2)] = 0.070wR(F2) = 0.156S = 1.003028 reflections197 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.22 e Å−3Δρmin = −0.24 e Å−3ΔρCrystalClear  used to solve structure: SHELXS97  I, global. DOI: Click here for additional data file.10.1107/S1600536813007320/lx2279Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536813007320/lx2279Isup3.cmlSupplementary material file. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


Rituximab has emerged as an alternative in the induction of remission in ANCA-associated vasculitis. Recent studies revealed a non-inferiority of a single dose rituximab in addition to a six month course of steroids compared to standard therapy after a follow-up period of 18 months. The results of the RAVE-ITN trial are encouraging and a special cohort of patients might benefit from this protocol.Randomized controlled trials have revolutionised the treatment options in small vessel vasculitis. From large genome wide association studies we have learned that granulomatosis with polyangiitis (Wegener’s) and microscopic polyangiitis are distinct diseases . These fRituximab (RTX), a monoclonal chimeric antibody targeting CD20 bearing B-cells, has been approved by the FDA and the EMEA for induction of remission in ANCA-associated vasculitis. The trials leading to the approval have been published with the aim to show non-inferiority of RTX when compared to cyclophosphamide (CYC). Both studies, the RAVE-ITN trial , includeThe RAVE-ITN study population was further followed over a period of 18 months. The results revealed that a single course of RTX along with a 6 month regimen of steroids is as effective as CYC followed by azathioprine  for the induction and maintenance of remission in ANCA-associated vasculitis. Remission could be maintained in 39% of the patients receiving RTX and in 33% of patients with CYC followed by AZA . These rReduction of relapses is one of the main outcomes relevant in daily clinical practice. Strategies with the aim to reduce relapses have been designed, but to date the rate of prolonged remission in ANCA-associated vasculitis is still unsatisfactory. More recently, a single center study revealed an efficacy of repeated RTX applications to maintain remission in ANCA-associated vasculitis. Interestingly, 73% of patients who received a single course of RTX to induce remission had a relapse over a period of 2 years, whereas 88% of the patients with RTX infusion on a regular basis  had sustained remission. Unfortunately, patients receiving RTX maintenance in this study showed a trend to relapse after cessation . ClinicaTaken together, the RAVE-ITN trial was important to approve RTX as treatment strategy to achieve remission in ANCA-associated vasculitis. The single dose strategy together with no maintaining immunosuppression may be an option for a small collective, i.e. elderly patients with co-morbidities when prolonged immunosuppression may increase the overall risk for mortality. RTX is increasingly used in the therapy of autoimmune disorders  and futuAK is the single author of this manuscript.Dr. Kronbichler received unrestricted research grants from GlaxoSmithKline and Roche/Genentech.Ethical issues  have been completely observed by the authors.None declared.


Genetic testing is increasingly used for clinical diagnosis, although variant interpretation presents a major challenge because of high background rates of rare coding-region variation, which may contribute to inaccurate estimates of variant pathogenicity and disease penetrance.To use the Exome Aggregation Consortium (ExAC) data set to determine the background population frequencies of rare germline coding-region variants in genes associated with hereditary endocrine disease and to evaluate the clinical utility of these data.Cumulative frequencies of rare nonsynonymous single-nucleotide variants were established for 38 endocrine disease genes in 60,706 unrelated control individuals. The utility of gene-level and variant-level metrics of tolerability was assessed, and the pathogenicity and penetrance of germline variants previously associated with endocrine disease evaluated.In silico variant prediction tools demonstrated low clinical specificity. The frequency of several endocrine disease‒associated variants in the ExAC cohort far exceeded estimates of disease prevalence, indicating either misclassification or overestimation of disease penetrance. Finally, we illustrate how rare variant frequencies may be used to anticipate expected rates of background rare variation when performing disease-targeted genetic testing.The frequency of rare coding-region variants differed markedly between genes and was correlated with the degree of evolutionary conservation. Genes associated with dominant monogenic endocrine disorders typically harbored fewer rare missense and/or loss-of-function variants than expected. Quantifying the frequency and spectrum of rare variation using population-level sequence data facilitates improved estimates of variant pathogenicity and penetrance and should be incorporated into the clinical decision-making algorithm when undertaking genetic testing. The frequency of germline coding-region variation was evaluated in 38 endocrine disease genes in 60,706 individuals. The clinical utility of these data in improving genetic diagnosis is demonstrated. IncreasTherefore, to address these challenges, we used the ExAC cohort to quantify the spectrum and frequency of rare germline missense and loss-of-function (LOF) SNVs in 38 genes associated with hereditary endocrine disease and explored the utility of these data when applied to several clinical settings. Our results demonstrate the value of large control cohorts such as ExAC and illustrate how estimates of cumulative rare variant frequencies, together with additional gene- and variant-level factors, may be incorporated into the workflow for clinical genetic testing.http://exac.broadinstitute.org; accessed March 2016 to October 2017). Details of the contributing populations, sequencing methods, and variant filtering and calibration methods have been reported  constraint metrics were obtained directly from the ExAC browser . Detailed descriptions of these metrics are reported elsewhere, and a brief overview is provided in the Supplemental Appendix  were evaluated in the ExAC cohort. Disease-associated mutations were identified from publicly available sources (details are provided in delines) . For eacAIP SNVs with an AF <0.5% were established for the ExAC cohort and compared with those observed in 1866 individuals with apparently sporadic pituitary tumors reported in nine earlier studies. Odds ratios with 95% confidence intervals were established.Individual and cumulative frequencies of all missense Four disease-targeted gene panels were formulated to model estimated background frequencies of rare missense and LOF SNVs when undertaking multiple-gene sequencing. These represented PPGL, calcium-/parathyroid-related disorders, pituitary tumor disorders, and MEN syndromes. Cumulative rare variant frequencies were used to establish the likelihood that a given individual would harbor a rare variant in at least one of the panel genes.i.e., single-nucleotide substitutions resulting in missense or nonsense amino acid changes or directly affecting donor or acceptor splice sites) in each of the 38 genes revealed that the overwhelming majority were rare, with ~60% occurring as singletons , whereas ~92% of gene-specific SNVs had an AF <0.05%  (chromodomain helicase DNA-binding protein 7 (CHD7) and neurofibromin (NF1) demonstrated the highest frequencies of singleton variants , whereas the lowest frequency was observed for adaptor-related protein complex 2 sigma 1 subunit (AP2S1) . Of note, removal of the TCGA subgroup (n = 7601) from the analysis had a minimal impact on cumulative rare SNV frequencies of the genes evaluated , with the exception of von Hippel-Lindau (VHL), for which reduced sequence coverage of part of the gene reduced the reliability of the estimates .Thirty-eight genes were selected for study, representing a range of endocrine disorders reported to be associated with heterozygous germline missense and/or LOF SNVs . The ideviduals) . The cumviduals) . For exae.g., for singleton SNVs2, r= 0.84; P < 0.0001) , marked differences persisted after correcting for gene size . Furthermore, a significant correlation was observed between rare nonsynonymous SNV frequency and the degree of amino acid sequence identity between orthologs of the encoded protein, with the most highly conserved genes  demonstrating the lowest rates of rare variation .Although a positive correlation was observed between rare nonsynonymous SNV frequency and coding-region nucleotide length  . Of the 38 genes, 20 (53%) were categorized as missense and/or LOF intolerant  or X-linked‒dominant disorders , thereby indicating appropriate constraint against nonsynonymous heterozygous variation. In contrast, many disease-associated genes were categorized as missense and/or LOF tolerant, including several in which the role of heterozygous variation and endocrine disease is less established  or was previously associated with reduced disease penetrance . For other genes in the tolerant groups , their small size reduced the reliability and utility of the respective constraint metrics , the cumulative estimates of LOF SNV allele frequencies were established for each gene . Although many genes displayed an absence or very low number of LOF SNV alleles consistent with their known haploinsufficiency function , some genes harbored cumulative LOF SNV frequencies considerably higher than the associated disease phenotype , indicating a reduced penetrance of such variants. For other genes, , the apparent high LOF SNV frequency observed was consistent with the known disease prevalence . Similarly, small indels resulting in an LOF  were absent or very rare in the majority of genes , although higher frequencies were observed in several genes in which LOF indels would be anticipated to be disease causing, including NF1 , CASR , SDHB , and CDC73  . However, it is important to note that the reliability of indel variant calls is most likely reduced compared with SNVs, whereas larger indels will not be identified by capture-based sequencing methods.To further quantify the variability in LOF SNV frequency . To assess the potential utility of such tools, we analyzed SIFT, Polyphen2, and CADD scores of all rare missense SNVs in a subset of 12 genes. In total, an average of 21% of all rare SNVs in each gene were categorized as deleterious using criteria encompassing all three tools, whereas 53% were described as possibly deleterious . This analysis revealed that for several genes, the frequency of alleles reported as pathogenic far exceeded the reported prevalence of the associated disease , giving rise to four possible explanations: The prevalence of disease is higher than reported; prior reports of SNV pathogenicity are incorrect ; the gene- or allele-specific disease penetrance is lower than reported; or the cohort is unknowingly enriched for hereditary endocrine disorders. For example, approximately one in 1750 individuals was observed to harbor a pathogenic ret proto-oncogene (RET) allele , in which approximately one in 3500 individuals harbored a variant previously associated with FHH type 1 , although in this setting it is plausible that the condition is more prevalent than currently recognized because of its typically asymptomatic phenotype [MEN1 and VHL variants in the ExAC cohort indicates that several of these variants were likely misclassified. However, it is important to note that in the case of VHL, four of the six likely pathogenic variants occurred in individuals from the TCGA cohort, which included 344 patients with a history of sporadic renal clear cell carcinoma who may be at an increased risk of harboring such germline variants .An apparent similar overrepresentation of disease alleles was observed for the calcium sensing receptor . Of note, this collection of disease-associated SNVs did not demonstrate a significant excess of alleles originating from the TCGA cohort (P > 0.1) .Next, the ExAC cohort was evaluated for individuals harboring clinically-actionable variants in one of seven hereditary endocrine tumor predisposition genes currently included in the ACMG guidelines . Only prAIP are reported in familial isolated pituitary adenoma kindreds but are associated with reduced penetrance, making it difficult to differentiate between hereditary and sporadic forms [AIP variants have also been reported in individuals with apparent sporadic pituitary tumors, although ascribing pathogenicity may be challenging because several AIP variants are observed in both disease and control populations [AIP in this setting, we compared the frequencies of rare germline missense AIP variants in 1866 individuals with sporadic pituitary adenomas  with those observed in the ExAC cohort. Prior analysis demonstrated the predicted frequency of likely cases in the ExAC control population to be negligible . Of note, only a small excess of rare missense AIP variants was observed in the tumor cohort compared with the ExAC cohort , whereas no overall excess was identified when compared with the European ExAC subpopulation, selected to represent the most relevant cohort for comparison  were observed in the tumor group, indicating that such variants are most likely benign or associated with very low penetrance . A small but notable excess of novel singleton SNVs was observed in the tumor group, predominantly in patients with acromegaly, suggesting genuine enrichment of pathogenic variants in this subgroup . Thus, modeling of four disease-targeted gene panels  revealed high cumulative estimates of identifying rare variants for each panel , with ~3% of control individuals harboring a novel singleton variant . Such estimates were then modified to allow direct comparison with literature reports. For example, a recent study evaluating 14 genes in patients with sporadic PPGL reported a rare germline mutation frequency of 7%, which did not exceed the background frequency predicted from our analysis of the ExAC cohort [Finally, the gene-level estimates of rare SNV frequency were used to model expected rates of background variation likely to be observed when performing simultaneous sequence-analysis of multiple genes . Therefore, these genes are likely to be under strong evolutionary selection pressure to conserve key cellular processes, thereby resulting in a relative intolerance to variation.In this study, we used the ExAC cohort to quantify the spectrum and frequency of rare nonsynonymous germline variants occurring in a broad range of genes associated with hereditary endocrine diseases and illustrate the potential utility of this information for improved variant interpretation, both in ascribing potential pathogenicity and in reevaluating estimates of disease penetrance. Of note, we observed marked differences in the frequency or rare nonsynonymous SNVs between genes after correcting for coding-region length, and this was correlated with the degree of evolutionary conservation. The observation that the lowest rare SNV frequencies were observed in genes with the highest degrees of evolutionary conservation is not unexpected, as several of the most highly conserved genes regulate essential cellular functions , current analyses often use a combination of variant-level features, such as the absence of a variant in a control population, as well as predictions from computational tools [i.e., enabling categorization only as a variant of uncertain significance), they are frequently cited as supporting evidence of pathogenicity and may ultimately result in a patient being managed as if the variant is disease causing. Our analyses indicate that variant rarity  together with computational prediction tools frequently have a low specificity for ascribing clinically relevant effects and that relying on such features likely overestimates pathogenicity. For example, we observed that across the 38 genes, the majority of individual variants were observed only once in the ExAC cohort and that a similar majority of rare SNVs were classified as potentially deleterious by at least one of the computational tools evaluated. However, our studies also demonstrated how the use of additional gene-specific factors, including cumulative rare variant frequencies together with metrics of missense and LOF constraint, may provide important additional context when incorporated into the variant interpretation workflow . Indeed, it is plausible that in the future such gene-specific constraint metrics and/or estimates of rare variant burden may be incorporated into bioinformatic or clinical computational algorithms to improve estimates of variant pathogenicity and/or disease penetrance.Although consensus guidelines have been established by the ACMG for the clinical interpretation of germline sequence variants, in many instances an unambiguous assignment of pathogenicity is not possible . In the al tools . Althougworkflow . For exaFurthermore, we demonstrated how quantifying the burden of rare variation in a given set of genes may be used to derive an expected frequency of background rare variants that should be anticipated when using disease-targeted gene panels. Of note, these studies illustrate how a failure to consider the high frequency of background rare variation arising from the use of such gene panels is likely to contribute to diagnostic uncertainty and may have confounded earlier genetic studies.e.g.,RET, VHL, and MEN1), indicating their likely prior misclassification and/or overestimates of disease penetrance. Indeed, the need to define allele-specific estimates of disease penetrance is an important concept, as recently illustrated for prion disease in which the disease-penetrance of individual PRNP variants ranged from <0.1% to ~100% [e.g.,RET), and quantifying these is essential to enable appropriate patient care  in a large pituitary tumor cohort compared with the ExAC population cannot exclude an etiological role in disease, although it suggests that any disease relationship is associated with extremely low penetrance and that the overwhelming majority of such variant carriers will not manifest clinical features. Indeed, establishing accurate estimates of disease penetrance should be a priority, and it is evident that for several hereditary endocrine genes, penetrance may have been overstated  because of unintentional ascertainment bias and/or the inclusion of index cases in such estimates [However, differentiating benign from low-penetrance alleles remains challenging, even with large disease and control cohorts . For exastimates .e.g., the presence of founder mutations in local populations). For example, although ExAC offers large numbers of individuals from specific ethnic groups , other populations are underrepresented, and for these groups, it may not provide a suitable comparator group.However, for accurate estimates of penetrance to be established, it is essential that the control population used be closely matched to the study population, thereby avoiding confounding from population-specific differences in variant frequencies  [e.g., in SDHB and SDHD). This not only highlights the potential clinical burden that increased genetic testing may bring but also emphasizes the need for accurate estimates of variant pathogenicity and penetrance because the potential for patient harm arising through tumor surveillance programs is not insignificant [Periodic clinical, biochemical, and/or radiological screening is generally recommended for carriers of pathogenic alleles associated with hereditary endocrine tumor syndromes (nd SDHB) , 27, 29.nificant , 30. Fure.g., PPGL, medullary thyroid cancer), it was reassuring that the TCGA samples did not contribute an excess of rare SNVs or likely pathogenic alleles in the genes investigated, with the possible exception of VHL, in which the inclusion of renal carcinoma cases may have introduced a risk of bias.Our analysis has several potential limitations. First, although not knowingly enriched for hereditary endocrine disorders, the ExAC cohort will contain individuals with these diseases as well as controls with polygenic disorders with accompanying disease-associated alleles. However, the inclusion of a broad range of genes and associated conditions reduced the likelihood of widespread enrichment for relevant disease phenotypes. For example, one potential concern was that the inclusion of germline samples from the TCGA cohort might result in an overrepresentation of alleles in genes associated with hereditary endocrine tumors; although this cohort does not include endocrine tumors relevant to the genes under study  could not be evaluated. In addition, high-quality SNV calls were assumed to be accurate, and although the performance of the ExAC variant calling pipelines has been extensively validated , confirmatory sequencing of individual variants was not undertaken. Furthermore, caution is required in interpreting SNVs in regions with reduced sequence coverage  or those presenting difficulties in the sequencing pipelines. For example, the reliability of sequence data for genes with multiple pseudogenes  may be reduced, although in these instances, visual inspection of individual sequence reads covering regions adjacent to the SNVs enabled increased confidence in the variant call .A further limitation of the current study is that individual rare variants were considered in isolation in our analysis, and potential interactions between variants or other modifying influences .Another limitation of our study is that we excluded indels from the main analysis because of their reduced reliability of detection ; althougIn summary, these studies demonstrate how quantifying rare germline variations in a large control cohort such as ExAC may be exploited to improve variant interpretation and clinical decision-making. Furthermore, this information may be incorporated into different stages of the clinical genetic testing workflow  and may


This case report exhibits a patient with generalized aggressive periodontitis who has been under maintenance for the past 12 years after being surgically treated in a single sitting and restored with dental implants. A 41-year-old systemically healthy male patient presented complaining of lower anterior teeth mobility and pain in the upper right quadrant. After clinical and radiographic examination, the upper right molars and lower anterior incisors were deemed unrestorable. Covered by doxycycline, the patient received a nonsurgical periodontal treatment. Three weeks later, teeth extraction, immediate implant placement, immediate nonloading provisional prosthesis, and a guided tissue regeneration were performed at indicated areas in a single sitting. The clinical decisions were based on patient compliance, the status of the existing periodontal tissues, and the prognosis of the remaining teeth. During the 12-year follow-up period, no residual pockets were observed and there was no exacerbation of the inflammatory condition. Marginal bone stability is present on all implants. For aggressive periodontal disease, a high risk of relapse as well as limited success and survival of dental implants should be considered. This case shows proper containment of the disease based on appropriate treatment planning and a strict maintenance program. In 1999, the term “aggressive periodontitis” (AgP) was introduced by the American Academy of Periodontology (AAP) to define a group of destructive periodontal diseases with a rapid progression. This definition was used to include previous terminologies of early-onset periodontitis, juvenile periodontitis, and rapidly progressive periodontitis, using “aggressive” nomenclature [Lang et al.  classifiAggregatibacter actinomycetemcomitan (AA) and, in some populations, Porphyromonas gingivalis (Pg), phagocyte abnormalities, and a hyper-responsive macrophage phenotype, including elevated levels of prostaglandin E2 and interleukin-1beta.Microbiological criteria were not mentioned as primary features separating chronic from aggressive periodontitis. However, for AgP, the secondary features that are generally, but not universally, present included elevated proportions of The following additional specific features were proposed for defining the localized and generalized forms. Localized aggressive periodontitis is usually circumpubertal onset, localized to first molar and incisor presentation with interproximal attachment loss on at least two permanent teeth (one of which is a first molar) and involving no more than two teeth other than first molars and incisors. Generalized aggressive periodontitis usually affects adult persons between 20 and 30 years of age, but patients may be older. There is generalized interproximal attachment loss affecting at least three permanent teeth other than first molars and incisors. The disease has a pronounced episodic nature of the destruction of attachment and alveolar bone with a history of relapse.Although success in implant dentistry depends on marginal bone stability and health, patient systemic factors and susceptibility to periodontal diseases play a role in achieving long-term stability.Many studies have shown the negative effect of previously treated and untreated periodontal disease on marginal bone stability around implants, including higher frequency of mucositis and peri-implantitis and lower success and survival rates of implants placed in patients with history of chronic or aggressive periodontitis –12.Swierkot et al.  reportedThis case report exhibits a 41-year-old systemically healthy male patient with GAgP who has been maintained for the past 12 years after being treated periodontally in a single sitting and restored with dental implants. He has been compliant for 10 years with supportive and maintenance therapy since the conclusion of his treatment in 2007.An engineer living abroad sought a second opinion at our office in 2005. The patient's oral surgeon had previously recommended extraction and immediate implantation of all compromised teeth including any tooth with a vertical defect. He sought an alternative treatment option after being shocked that at only 41 years of age, the only treatment will be losing 16 of his teeth. The patient's chief complaint was that he is not able to bite on his front teeth and that his esthetics are compromised due too increased crown length.Diagnostics included radiographic examination Figures  and 2 anA conventional supra gingival scaling and subgingival root planing was performed under the coverage of antibiotics having a significant action against AA (doxycycline (Doxylag)® 100 mg 2 tabs first day and 1 tab daily for 21 days) , 15 and Upon the patients' return, teeth 18, 17, and 16 all had class III furcation involvement with grade 3 mobility; they were extracted under full thickness flap allowing visibility of a 3-wall defect of 10 mm at the mesial of tooth number 13 which was then treated with guided tissue regeneration technique using bovine xenograft bone substitute Bioss® and resorbable collagen membrane Resolute®.At the same visit, extraction of tooth number 26 was performed as it presented with a 9 and 10 mm bone loss at the mesial and distal sites, respectively, with a class III furcation involvement.At the lower right quadrant, tooth number 48 was extracted, and scaling and root planing was performed at the distal of tooth number 47 which presented with a wide shallow bony defect.At the lower left quadrant, extraction of tooth number 38 was done, and the full thickness flap showed a narrow and deep bone defect until the apex of tooth number 46 distally maintaining the mesial bone peak at tooth number 47 which presented as well with a wide moderate bone defect distally. Scaling and root planing was done, due to the contained geometry of the defect on the 46; the bone substitute Bioss was used as a filling materiel, without the need for a membrane.Teeth 32, 31, 42, and 41 were extracted and immediately replaced with 3 narrow neck SLA Straumann dental implants (3.3 × 12 mm at site 41 and 31 and 3.3 × 10 mm at site 32). The choice of cantilevering was based on the presence of a wide intrabony defect surrounding tooth number 42; immediate nonloading temporization was provided on implants number 41 and 32 to maintain the esthetic appearance.No provisional prosthesis was delivered for molar areas, and an association of amoxicillin 500 mg and metronidazole 250 mg was prescribed 3 times a day for 8 days .∗ implants were used in the maxilla replacing the first and second molars:  implants were inserted on the right side, and  implants on the left side (Figures Almost 5 months later, the clinical exam showed a perfect soft tissue integrity, and the radiographic evaluation revealed total bone formation at the site of extracted molars and bone stability around Straumann mandibular implants . Four os Figures .The patient could not return until 16 months after the implant surgery, and for the realization of final fixed prosthesis, all prosthesis were delivered within 10 days and the case was documented radiographically and clinically and follow-up maintenance program was scheduled after this visit.The patient is placed on a strict maintenance schedule; prophylaxis is performed every 3 to 6 months and periapical follow-up radiographs are done every year to follow-up on the surgical sites. During the 12 year follow-up period, no residual pockets were observed, and there was no exacerbation of the inflammatory condition. Marginal bone stability is present on all implants. Since the case was done, there was no need for the adjunct use of antibacterial mouth rinses or systemic antibiotic use Figures  and 8.The high risk of relapse as well as limited success and survival rate of dental implants is considered as a severe complication related to aggressive periodontal disease, and this case showed a perfect bone stability, after guided tissue regeneration and around implants, over 10 years after treatment without any sign of inflammation, and the stability of such results is maybe related to the strict supportive therapy program or the choice of doing full mouth surgery in one day which may assure the complete eradication of bacteria and prevent the contamination of treated areas when surgeries are usually done at variable intervals. To the best of our knowledge, this is the first case report treating surgically a case of generalized aggressive periodontitis with GTR and immediate implantation in one single day. The successful treatment is maybe related to the choice of the treatment; however, additional clinical studies with more patients are necessary in order to support this choice.