Background: We hypothesized that SRX246, a vasopressin V1a receptor antagonist, blocks the effect of intranasally administered vasopressin on brain processing of angry Ekman faces. An interaction of intranasal and oral drug was predicted in the amygdala.Methods: Twenty-nine healthy male subjects received a baseline fMRI scan while they viewed angry faces and then were randomized to receive oral SRX246 (120 mg PO twice a day) or placebo. After an average of 7 days of treatment, they were given an acute dose of intranasal vasopressin (40 IU) or placebo and underwent a second scan. The primary outcome was BOLD activity in the amygdala in response to angry faces. Secondary analyses were focused on ROIs in a brain regions previously linked to vasopressin signaling.Results: In subjects randomized to oral placebo-intranasal vasopressin, there was a significantly diminished amygdala BOLD response from the baseline to post-drug scan compared with oral placebo-intranasal placebo subjects. RM-ANOVA of the BOLD signal changes in the amygdala revealed a significant oral drug × intranasal drug × session interaction  = 4.353, p < 0.05). Follow-up tests showed that antagonism of AVPR1a with SRX246 blocked the effect of intranasal vasopressin on the neural response to angry faces. Secondary analyses revealed that SRX246 treatment was associated with significantly attenuated BOLD responses to angry faces in the right temporoparietal junction, precuneus, anterior cingulate, and putamen. Exploratory analyses revealed that the interactive and main effects of intranasal vasopressin and SRX246 were not seen for happy or neutral faces, but were detected for aversive faces (fear + anger) and at a trend level for fear faces.Conclusion: We found confirmatory evidence that SRX246 has effects on the amygdala that counter the effects of intranasal vasopressin. These effects were strongest for angry faces, but may generalize to other emotions with an aversive quality. Vasopressin (AVP) is a mediator of social and emotional behavior in many species  with a high degree of selectivity for the AVPR1a has undergone preclinical and early clinical testing. SRX246 crosses the blood brain barrier and binds to V1a receptors with a high degree of selectivity . AVP challenge was used to maximize the chance of finding an effect of AVPR1a blockade in a sample of healthy subjects, who presumably did not exhibit excessive central AVP signaling. We then looked for evidence that brain regions were activated when patients looked at angry faces vs. a fixation point, that vasopressin affected such activation, and that SRX246 blunted the responses seen in the presence or absence of AVP. Because the amygdala is known to express vasopressin AVPR1a receptors . The second session (Session 2) followed randomization, treatment for a minimum of 5 days with oral SRX246 or placebo, and 45 min after acute administration of intranasal vasopressin or matching placebo  or equivalent dosing of the matching pill placebo (n = 14). The mean of number of days between the first and second scanning session was 7.3 (SD = 1.3); the SRX246 and placebo treatments continued until the day of the second scanning session. The second session began with randomization to either IN AVP or IN placebo. Vasopressin was prepared by the research pharmacy of the University of Chicago General Clinical Research Center. Forty IU synthetic vasopressin  was dispensed using Good Clinical Practices into two intranasal atomizers . IN placebo was prepared from a commercial nasal saline solution to mask the mild scent of Pitressin solution. IN drug was administered in 4 puffs (0.2 mL) per nostril over 15 min by the research staff to subjects reclining on an exam table with their heads tilted back. Subjects rested on the examination table by themselves with the examination room door open until the beginning of the second fMRI scanning session, timed to begin 45 min after IN drug administration. This time point was chosen based on the time course of CSF levels of vasopressin after IN administration  vasopressin, both with placebo counterparts. After the first scanning session , subjects were randomized to receive 5 days of oral SRX246 . The stimuli were presented over 4 runs. To preserve statistical power, analyses focused on the neural responses to angry faces, based on a priori hypotheses regarding the relevance of angry faces to vasopressin function. Each emotion block lasted 20 s and consisted of 5 faces displayed for 4 s in the center of the screen, with no interstimulus interval. The behavioral task was to identify the valence of the emotion  by button press. An explicit emotion paradigm was employed based on a large amount of high resolution fMRI data demonstrating that this type of task evokes a readily detectable amygdala response . fMRI images were obtained with high-field functional MRI utilizing T2*-weighted echo planar imaging with BOLD (blood oxygenation level dependent) contrast . A modified high efficiency z-shim compensation was applied to the 4 slices covering the orbitofrontal cortex . Images were band pass filtered to remove very low frequency drift artifact and high frequency, non-physiologic noise. Images acquired during excessive movement  were excluded from the analysis. Motion in the three planes was recorded and images were motion corrected relative to the first image of the first run, normalized to a Montreal Neurological Institute template, resampled to 2 mmp < 0.05, cluster size > 10 contiguous voxels). Analyses were focused on angry face contrasts given the limited power of the study. An exploratory analysis of other facial emotion conditions is provided in Supplementary Material.T-statistical images were generated for the first and second fMRI sessions separately to confirm that the task led to the expected pattern of regional brain activation. To control for Type I error, Family Wise Error (FWE) was utilized for the entire brain region in voxelwide analyses (n = 14). One-Way ANOVA was conducted on whole-brain contrast images of angry faces vs. fixation point, with the factors of session and IN drug; the hypothesized IN drug effect was tested with the statistical interaction between the two factors by confirming significant clusters of activation within the anatomical amygdala. To balance concerns of Type I and Type II error, correction for multiple comparisons on significant clusters utilized familywise error correction (FWE) within the small volume of the anatomic amygdala as defined by Wake Forest University (WFU) Pickatlas . Parameter estimates (β weights) of average activation were extracted from the anatomical amygdala ROI (WFU Piackatlas) and exported to SPSS 18 (IBM) for statistical analysis. Two separate paired t-tests were then conducted in the seven subjects receiving intranasal vasopressin and the seven subjects receiving intranasal placebo. To account for baseline differences, follow up tests were repeated using ANCOVA to compare IN vasopressin vs. IN placebo on the extracted Session 2 amygdala BOLD signal, covarying for Session 1 in the 14 subjects receiving oral placebo.To establish the effect of the IN vasopressin probe in the principal ROI of the left and right amygdala, data were compared between the IN vasopressin and IN placebo group within the sub-sample of subjects randomized to oral placebo  ANOVA for the interaction of the factors of oral drug , intranasal drug (IN vasopressin vs. IN placebo), side (left vs. right), and session (Session 1 vs. Session 2) on the extracted parameter estimates of average amygdala BOLD response to angry faces. Repeated Measures (RM) ANOVA was conducted with the between-subjects factors of oral drug and intranasal drug, with the within-subjects factor session. The statistical significance threshold was set at p < 0.05, FWE corrected for the anatomical search volume. Individual differences in the tortuous gyral/sulcal morphology of the temporoparietal region make spatial definitions of it unreliable for group analyses . To relate the behavioral measures with brain response, change in reaction time and accuracy (Session 2 – Session 1) was correlated with change in amygdala BOLD (Session 2 – Session 1).Accuracy and reaction time were recorded and analyzed by RM-ANOVA, with the within subjects factor of session and between subjects factors of oral and intranasal drug. Significant interactions were followed up with The Adverse Events Questionnaire (AEQ) was used to measure a range of possible somatic and psychological side effects. Suicidal symptoms were assessed with the Columbia Suicide Severity Rating Scale  = −2.560, p < 0.05] and at a trend level in the average of both sides , but no effect was seen in the left amygdala. No significant pairwise difference in amygdala BOLD between the two sessions was seen in the subjects receiving IN placebo. ANCOVA of the amygdala BOLD response in subjects receiving oral placebo produced similar results, with IN vasopressin associated with significantly decreased BOLD response at Session 2, controlling for Session 1 BOLD response, in the right amygdala  and at a trend level for the combined left and right amygdala . No effect was found in the left amygdala. See Supplementary Material One-Way ANOVA in the subsample of 14 subjects who did not take SRX246 revealed a significant interaction of session × IN drug within both the left and right amygdala  = 4.353, p < 0.05]. Follow up testing of the interaction in the four drug subgroups with paired t-tests comparing Session 1 to Session 2 BOLD response in the left and right amygdala confirmed that a significant Session 1 vs. Session 2 difference was found only in the subgroup of subjects randomized to oral placebo and IN vasopressin (as described in the Effect of IN Vasopressin). Subjects randomized to oral SRX246 and IN vasopressin did not show a Session 1 to Session 2 difference , right amygdala , or left amygdala . There was no main effect of SRX or vasopressin on amygdala BOLD response. See Supplementary Material The primary hypothesis that SRX246 blocks effects of vasopressin on the amygdala was confirmed. RM ANOVA of extracted parameter estimates from the left and right amygdala in the entire sample revealed a significant 3 way interaction of session × oral drug × IN drug in contrasts of angry faces vs. fixation point .SRX246 compared with oral placebo was associated with significantly diminished BOLD signal intensity in the right temporoparietal junction .SRX246 was associated with significantly diminished BOLD activity in a cluster within the right precuneus  = 4.34, cluster size = 20 voxels; MNI coordinates = 6, 46, 22; Figure F = 14.473, p = 0.001].SRX246 was associated with reduced BOLD response in a cluster of the right anterior cingulate .SRX246 was associated with reduced BOLD response in two clusters within the right putamen , with reaction time to Angry faces decreasing from Session 1  to Session 2 . A trend level interaction was detected for session × oral drug . Follow up testing with paired t-tests comparing Session 1 to Session 2 revealed that subjects randomized to oral placebo showed a significant decrease in reaction time to angry faces from Session 1 to Session 2 ; subjects randomized to SRX246 also showed a decrease but the difference was not significant .For accuracy, RM-ANOVA revealed no main effects or interactions for session, IN drug, or oral drug. For reaction time, RM-ANOVA revealed a significant effect of time [r = −0.520, p = 0.005, n = 28), for the left amygdala  and the right amygdala . Thus, the decreased BOLD response from Session 1 to Session 2 seemed to predict improved performance. There was no relationship between change in amygdala BOLD response to angry faces and reaction time measures.Change in amygdala BOLD response to angry faces (Session 2 – Session 1) was negatively correlated with the change in number of hits . This was true for the average of left and right amygdala together (There were no serious or unexpected adverse events. SRX246 was not associated with change in AEQ subscores, BDI-II, vital sign parameters, laboratory parameters, CSSR-S score, or urine specific gravity.Chronic treatment with SRX246, a novel AVPR1a antagonist, blunts the effect of acute vasopressin administration on the functional response of the amygdala to angry faces in healthy males. An effect of intranasal vasopressin on subcortical processing of emotional facial expressions was confirmed: in healthy males, vasopressin enhanced accommodation, as reflected in a decrease from Session 1 to Session 2, of the amygdala to angry faces. This effect was effectively blocked by pretreatment with SRX246. Additional main effects of SRX246 were found on cortical processing of angry faces in the right side of the brain within the ROIs of the temporoparietal junction, precuneus, anterior cingulate, and putamen. These novel findings provide the first evidence for AVPR1a signaling in a neural circuit that mediates processing of social and emotional information (Saxe and Kanwisher, The findings add to a growing literature regarding the role of vasopressinergic AVPR1a signaling in human social and emotional behavior. Genetic variation in the promoter region of AVPR1a has been associated with risk for autism, in which social deficits are the core symptom (Kim et al., Preclinical studies have demonstrated that vasopressin plays an important role in social recognition. We observed that IN vasopressin was associated with accommodation of the BOLD response of the amygdala to angry faces and that SRX246 pre-treatment blocked this effect. These results are consistent with a role for vasopressin in social recognition in humans and provide the first evidence for the involvement of AVPR1a in this process. The direction of the effect of IN vasopressin, specifically decreased BOLD signal, differed with a previous animal model finding suggesting excitation as an expected outcome (Huber et al., Secondary analyses of the main effects of SRX246 showed that treatment with the AVPR1a antagonist reduced the response to angry faces in the right sided temporoparietal junction, precuneus, anterior cingulate, and putamen. The inhibitory effect of AVPR1a blockade on temporoparietal junction activation to angry faces is consistent with previous findings of vasopressinergic modulation of this brain region during processing of social and emotional information (Zink et al., AVPR1a modulation of the precuneus is consistent with previous findings that IN vasopressin increased precuneus activity during a simulated aggressive social interaction (Brunnlieb et al., That the AVPR1a antagonist treatment resulted in blunting of the anterior cingulate response to angry faces is consistent with previous research. IN vasopressin increases anterior cingulate activity during processing of social interactions (Brunnlieb et al., Given the design of the study and results from the exploratory analyses on the processing of emotions other than anger, the effects of vasopressin modulation and SRX246 may extend beyond only angry faces to the processing of aversive emotional expressions more generally. Such an interpretation would be consistent with the findings of Thompson and others , who fouin vitro studies that demonstrate exceptional selectivity and selectivity for the V1a receptor and in vivo results in preclinical models (e.g., Ferris et al., Our study presents the first translational investigation of a novel, first-in-class AVPR1a antagonist in a vasopressin challenge, emotional processing paradigm. Some limitations of the study are worth mentioning regarding the interpretation of the results. To optimize reliability, the sample was made as homogenous as possible in terms of age range, gender, and psychiatric profile. To preserve statistical power for the primary comparisons, the analysis focused on the amygdala and contrasts involving angry facial expressions. For feasibility reasons, the study was not powered to detect emotion specific effects of SRX246. Secondary analyses regarding main effects of SRX246 were similarly restricted to a subset of brain regions previously identified to be affected by vasopressin signaling. The limited power of the study makes it likely that significant effects of vasopressin and SRX246 on other brain regions were not detected. Finally, interpretation of the fMRI BOLD signal as responsive to pharmacological manipulations requires the inference that the drug has a direct effect on regional brain activity. While the results, when combined with extensive In conclusion, the results provide the initial demonstration in humans that blockade of AVPR1a with SRX246 significantly reduces the effect of intranasally administered vasopressin on the response of the amygdala to angry face stimuli as measured by the fMRI BOLD response. Additional effects of SRX246 were observed on responses in the temporoparietal junction, putamen, precuneus, and anterior cingulate. These findings extend a growing body of evidence establishing the importance of vasopressin signaling in the processing of social and emotional stimuli. Because exaggerated responses to negatively valenced emotional stimuli in circuits that include these structures are characteristic of several stress-related psychiatric disorders, the ability of SRX246, a novel AVPR1a antagonist, to attenuate the response to angry faces supports the potential of AVPR1a antagonism as a new approach to the treatment of these indications.Royce J. Lee conceptualized the experiment, designed the study, devised the intranasal approach, and wrote the manuscript. Emil F. Coccaro provided advisory support, helped to design the experiment, and contributed to the manuscript. Henk Cremers played a key role in processing of fMRI data, choice of analysis parameters, and assistance with critical revisions. Rosemary McCarron designed the fMRI experiment and played a key role in fMRI data analysis. Shi-Fang Lu contributed to the design and the study. Michael J. Brownstein helped to design the experiment and contributed to the manuscript. Neal G. Simon helped to design the experiment and contributed to the manuscript.Emil F. Coccaro is on the scientific advisory board for Azevan. Shi-Fang Lu, Michael J. Brownstein, and Neal G. Simon hold equity in Azevan Pharmaceuticals. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While a variety of registered therapies exist for Cutaneous T Cell Lymphoma, no such therapy is available for Cutaneous B Cell Therapy. In this context we performed a phase II, open label, multicenter, non-comparative study to evaluate the efficacy and safety of repeated intra-lesional administrations of TG1042 (adenovirus-interferon-γ) in patients with relapsing primary cutaneous B-cell lymphomas (CBCL).10 viral particles into up to six lesions simultaneously. Injections were performed on days 1, 8 and 15 of each of four consecutive 28 day cycles.Thirteen patients have been enrolled and received intralesional injections of TG1042 containing 5×10Eleven (85%) out of 13 enrolled patients showed an objective response after injections of TG1042. Seven patients (54%) exhibited complete and four (31%) displayed partial response. The median time to disease progression in the study population was 23.5 months (range 6.25 to 26+). Most commonly observed adverse events were minor to moderate flu-like symptoms, fatigue and injection site reactions.Our study showed that treatment with TG1042 was associated with a clinical benefit in the majority of the patients with relapsing CBCL, including tumor regression, a clinically meaningful duration of response and a good treatment tolerance.www.clinicaltrials.govNCT00394693 Cutaneous lymphomas (CL) represent a heterogeneous group of Non-Hodgkin's lymphomas and are the second most frequent extranodal lymphomas While a variety of registered therapies exist for CTCL, no such therapy is available for CBCL. For the management of relapsing CBCL, after first line radiotherapy or surgery, various off-label therapeutic options are being explored. Curative treatments include radiotherapy and surgery, but disease relapses justify to seek alternative therapeutics TG1042 is an adenovirus 5 expressing the cDNA of human IFN-γ gene. In the previously conducted phase I/II, open label, multicenter, dose-escalation study, 38 CTCL and CBCL patients received intra-tumoral injections of TG1042. Local tumor response could be observed in all 5 treated CBCL patients, with 3 complete responses and 2 partial responses. TG1042 was well tolerated with minor to moderate injection side reactions and flu-like symptoms as most frequent adverse events Considering clinical responses in CBCL patients and absence of approved medicinal therapy in this indication, we conducted a multicenter phase II study in patients with relapsing primary CBCL to evaluate the efficacy and safety of intra-lesional administrations of TG1042 in this population of patients. Our results show high response rates of CBCL to intra-lesional TG1042 treatment as well as good tolerability making this gene-therapy based immunotherapy a reasonable option for these cutaneous lymphomas.The protocol for this trial and supporting checklist are available as supporting information; see 10 viral particles (vp) per lesion into up to six lesions treated simultaneously on days 1, 8 and 15. Patients received no treatment during the fourth week of the cycle. The cycle length was 4 weeks (1 cycle), at the end of the cycle the tumors were evaluated for response (Day 29). In the absence of progression an additional cycle was to be delivered up to a maximum of four cycles. In the event of local response and consequent disappearance of the initially-treated lesion(s), the investigator was to decide if applicable whether or not to treat a second series of one or several additional lesion(s) that were not previously treated.Patients received from the participating investigators intralesional injections of TG1042 at the dose of 5×10The primary objective of the study was the response rate of the treated lesions in the group of study participants. The response to the treatment was defined in a given patient as the best objective tumor response, which was recorded from the first cycle evaluation until the last follow up visit on study. The response was to be confirmed by a repeated assessment performed no less than 4 weeks after the criteria for response were first met. Complete response (CR) was defined as the clinical disappearance of all treated lesions. Partial response (PR) was defined as the clinical disappearance of at least half of all treated lesions. Minor response (MR) was defined as a decrease of at least 50% in the sum of the products of all treated lesions with the clinical disappearance of less than half of all treated lesions. Progressive disease (PD) was defined as an increase of at least 25% in the sum of the products of all treated lesions. Stable disease (SD) was defined as any response that did not meet the criteria for CR, PR, MR or PD. Total response assessment used the same definitions applied to all the lesions but also integrated for the definition of PD the appearance of new cutaneous lesions, lymph nodes, hematological or visceral involvement.An evaluation of all lesions (treated and not treated) was performed at baseline and on Day 29 of each cycle, at the last treatment cycle visit and at each follow-up visit or up to tumor progression. The treatment efficacy in patients that had shown a CR as best objective tumor response was confirmed by pathological analysis of skin biopsies at the site of the treated lesions.An independent histo-pathology review was performed on the biopsies obtained from the 13 patients, in order to confirm the initial diagnosis of primary cutaneous B-cell lymphomas and the treatment efficacy of TG1042 for patients who achieved a CR. Secondary endpoints included the assessment of safety as well as quality of lifehttp://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf (accessed January 14th 2013)).The condition of the patient was monitored for adverse events (AEs) throughout the study by clinical safety assessments by the investigator. The intensity of AEs was graded according to the NCI-CTCAE version 3.0  questionnaire at each visit. The DLQI questionnaire allows a scoring from 0 to 30 corresponding respectively to no effect at all on patient's life and an extremely large effect on patient's life The purpose of the trial was to reject the experimental treatment from being further studied, if TG1042 was shown to be insufficiently active in the considered indication and to accept it for further studying, if it was shown to be active. The study was designed as a two-stage ‘optimum’ phase II trial with the following assumptions: the inactivity cut-off was chosen equal to 50%, the activity cut-off equal to 75%.0: r≤50% against HA: r≥75%, where r is the response rate. The type I error rate  was set to 5%. The type II error rate  was set to 10%. Under these assumptions, the first step of the optimal design consisted in treating 13 evaluable patients, if at most 7 responses were observed, the trial was to be stopped and the drug declared insufficiently active. If at least 8 responses were observed, the drug was to be declared active and additional patients were to be treated up to a total of 41 evaluable patients with the aim of obtaining a total of 26 responses to confirm TG1042 is active. After the first step of the study achieved its endpoint and showed a sufficient level of activity, the sponsor decided to not initiate the second step of the statistical design for company reasons.Hence the hypotheses of interest were HThe Intent-To-Treat population (ITT) (N = 13) included all patients enrolled in the study including the one non-evaluable patient. The Per-Protocol population (PP) (N = 12) included all patients of the ITT population but excluded patients with major protocol violations (0), the non-evaluable patients (1) and the wrongly included patients (0). The Safety Population included all patients enrolled in the study and who received at least one injection of TG1042 (N = 13). The efficacy analyses were done on ITT and PP populations. The ITT analysis was considered as the primary analysis of the study. All data analyses were performed using SAS® version 8.1 or higher. The best response rates were calculated with their exact 95% confidence interval using the Pearson-Clopper method. Duration of response was calculated for each patient with CR, PR or MR. The median time to progression was determined by Kaplan-Meier method. Its 95% confidence interval was calculated by the Brookmeyer-Crowley method.Thirteen patients were enrolled and treated by seven study centers between March 2007 and July 2008, last patient last visit was in January 2010 . PatientThe primary efficacy endpoint on study was the response rate of the treated lesions. Of the 13 patients treated with TG1042 intra-tumoral injections 12 were evaluable for response to treatment, all patients completed their follow-up as planned per-protocol. Eleven  evaluable patients in the intent-to-treat population showed complete or partial response. Seven (54%) patients had a complete response and 4 (31%) patients had a partial response. One additional patient (8%) was considered as having a stable disease after the treatment. The secondary efficacy points of the study included duration of response for treated lesions, global response rate of treated and non-treated lesions and time-to-progression  patients who received treatment with TG1042 were included in safety population. All patients experienced one or more Adverse Event (AEs) that was considered possibly or probably related to study drug . One patWith the exception of a single grade 3 event (increased lipase considered not related to TG1042 and which resolved without a treatment) all AEs were mild or moderate (grade 1 or 2) in severity. The most commonly reported AEs were fatigue (8/8 patients), headache (6/7 patients), pyrexia (6/6 patients), injection site irritation (5/5 patients), chills, influenza-like illness, injection site erythema and injection site pain (4/4 patients each), all of which were expected events associated with injections of TG1042. All reported adverse drug reactions resolved after treatment discontinuation. No clinically significant hematology or serum chemistry laboratory abnormalities were reported by the investigators.This clinical study shows that repeated injections of TG1042 (adenovirus-IFNγ) into CBCL lesions lead to a reliable partial or total regression of the treated disease. This observation represents evidence that gene delivery based immunotherapy is feasible and can lead to sustained clinical tumor responses, which are of clinical benefit. The clinical improvement was supported by the improvement of the lesions seen at the pathological review of pre- and post-treatment biopsies. As expected, most commonly observed adverse events were injection site reactions and transient flu-like syndromes of mild or moderate intensity, demonstrating a good tolerability of the product.The high response rate observed with TG1042 in this population of patients with CBCL raises the question of the mechanism of action of TG1042. It has been shown previously in patients treated with TG1042 for cutaneous lymphomas that not only the transgene IFNγ is expressed, but also a type I IFN response is induced due to the adenoviral vector itself. In addition, this type of combined type I (IFNα) and type II (IFNγ) IFN-response was predictive of the objective  clinical responses observed in the course of the treatment with TG1042 This study has confirmed initial observation of the previous clinical trial, in which patients with CBCL already displayed a high response rate in general and as compared to what was observed in patients with CTCL. The high responsiveness of CBCL lesions to this approach in the absence of any cytotoxic treatment implies a high sensitivity of this tumor type to type I and II IFN-response. This argues in favor of immunologic approaches for the treatment of CBCL especially, since the treatment options for CBCL are rather limited and there are currently no registered drugs for this indication. The most commonly used approaches are still radiation therapy or surgery . The immediate response rate is high, close to 100% but is associated with a high rate of early recurrences, 42% in the year following treatment. The most frequently adverse events observed with rituximab are fever, chills, nausea, headache, urticaria and asthenia, which are mainly of grade 1 or 2. However one of the main concerns with rituximab, because of the long lasting complete depletion of B lymphocytes, is the risk of serious viral infections and especially progressive multifocal leukoencepahalopathy Checklist S1TREND Statement Checklist for this manuscript.(PDF)Click here for additional data file.Protocol S1Clinical study protocol n°TG1042.06, final version, study phase II.(PDF)Click here for additional data file.


FLT1 gene, based on knowledge of the generating model. Using all 200 replicate data sets, we similarly analyzed Q1 and Q4 (which is known to be free of association with FLT1) to evaluate relative efficiency, type I error, and power. Simulation study results suggest that the QT-dependent selection designs generally yield greater than 50% relative efficiency compared to using the entire cohort, implying cost-effectiveness of 50% sample selection and worthwhile reduction of sequencing costs.Use of trait-dependent sampling designs in whole-genome association studies of sequence data can reduce total sequencing costs with modest losses of statistical efficiency. In a quantitative trait (QT) analysis of data from the Genetic Analysis Workshop 17 mini-exome for unrelated individuals in the Asian subpopulation, we investigate alternative designs that sequence only 50% of the entire cohort. In addition to a simple random sampling design, we consider extreme-phenotype designs that are of increasing interest in genetic association analysis of QTs, especially in studies concerned with the detection of rare genetic variants. We also evaluate a novel sampling design in which all individuals have a nonzero probability of being selected into the sample but in which individuals with extreme phenotypes have a proportionately larger probability. We take differential sampling of individuals with informative trait values into account by inverse probability weighting using standard survey methods which thus generalizes to the source population. In replicate 1 data, we applied the designs in association analysis of Q1 with both rare and common variants in the  We assume an existing cohort of unrelated individuals, well phenotyped for a quantitative trait (QT) that is approximately normally distributed. At present, the cost of whole-genome sequencing of an entire cohort to test for association remains prohibitive, and selection of a subset of informative individuals for sequencing is a reasonable strategy to reduce costs. In the absence of relevant genetic information, such as family history or single-nucleotide polymorphism (SNP) association identified through genome-wide association studies, one might hypothesize that individuals at the extremes of the QT distribution would be more informative for the detection of association with sequence variants. For example, individuals with high QT values are more likely to carry high-risk variants, whereas those with low values do not. In contrast, taking a simple random sample from the cohort will tend to select individuals from the central part of the trait distribution and can fail to sample individuals with low-frequency variants. Because ascertainment by the phenotype needs to be taken into account, simple selection from the tails of the trait distribution complicates inference. The resulting trait distribution is bimodal, violating the usual linear regression assumptions and can produce distorted effect estimates and type I errors greater or less than nominal. In addition, the resulting sample is not representative of the original cohort, making it more difficult to design validation studies in nonselected populations. Finally, if both extremely high and extremely low QT values are “pathological” then excluding “normal” individuals would reduce the ability to detect protective variants associated with favorable QT values. As an alternative, we propose a sampling design in which all individuals have a nonzero probability of being selected into the sample but in which individuals with extreme phenotypes have a proportionately larger probability of being selected. We account for differential sampling in the association analysis by inverse probability weighting (IPW) with standard survey methods for variance estimation and hypothesis testing. The purpose of our analyses of the Genetic Analysis Workshop 17 (GAW17) mini-exome data is to compare alternative sampling strategies with respect to relative efficiency. We also examine type I error and power of hypothesis testing.iY. For individual i, we use the distance from the median of the trait distribution (Ymed) in the original cohort:We consider the analysis of sequencing data for the entire cohort as the ideal against which to compare sampling designs in which only 50% of the cohort is sequenced. Four alternative sampling designs are summarized in Table In particular, we specify the IPW weight as:where the sampling probabilities under design 5 are:or:which we refer to as 5.1 and 5.2, respectively. These probabilities are scaled by the maximum distance from the median, with:Ymax and Ymin are the maximum and minimum of the iY, respectively. In design 5.2 extreme observations are selected more frequently.where For common variants, it is feasible to analyze one SNP at a time. For rare variants, however, methods that accumulate multiple SNP genotypes across a region are necessary to improve the power to detect association. We calculate a rare variant score, defined as the total count of rare alleles within a gene ,2. AssocFLT1 gene to compare methods under a true alternative hypothesis of genetic association and use analysis of Q4 for comparisons under the null hypothesis. To reduce heterogeneity and potential for population stratification bias, we chose the Asian subpopulations for study: Chinese + Japanese (n = 321).We analyze quantitative traits Q1 and Q4 in replicates 1 to 200 of the GAW17 simulated unrelated mini-exome data [FLT1 gene that were monomorphic within the Asian subpopulation. Of the 20 remaining SNPs, 18 were classified as rare ; but according to the generating model, only 6 of these had functional variants. We calculated a rare variant score  from the 18 rare SNPs. The total count of rare alleles per person ranged from 0 to 3 . One SNP with a common functional variant, C13S523, was analyzed separately (MAF = 8.72% with no rare homozygotes).We excluded SNPs in the QT-dependent sampling and QT analysis for Q1 and for Q4 were based on residuals from a linear regression on Age, Sex and Smoking status. We conducted an association analysis under each of the five sampling designs listed in Table FLT1 was detected at near genome-wide significance levels for both the common SNP and the rare variant score . For the rare variant score , defined as 100 times the ratio of the empirical variance of design 1 to that of the alternative design Table . This meAs expected, use of a 50% SRS design is not a cost-effective approach for reducing sequencing costs: The empirical relative efficiency of estimation was less than 50% in the GAW17 simulations we examined. However, designs 3–5, in which RE generally exceeded 50% when conditioning on the QT and could be substantially higher, appeared to offer a net benefit. The more extreme IPW sampling design 5.2, which is similar to designs 3 and 4, behaves similarly, at least for the regression analyses that condition on the phenotype. However, the simple IPW linear regression analysis that we applied here may be less than optimal in finite samples under the alternative hypothesis. Although RE results for design 5 are encouraging, for hypothesis testing the source of parameter underestimation under the alternative hypothesis needs to be addressed and SE estimation improved. Evaluations reported here are limited, and it is quite possible that sampling and/or weighting schemes could be improved.In our extreme-phenotype implementation of designs 3 and 4, we used the observed QT values, in contrast to more common case-control approaches. Because the linear regression assumption of normal residuals is grossly violated, the observed exaggeration of regression coefficients is not surprising. The lack of elevated type I error may be ascribed to the use of symmetric tail selection, but for rare variant analysis there is a clear loss of efficiency. In the GAW17 simulation, in which rare and common variant effects were generated under an additive model, differences between designs 3 and 4 were trivial. In other settings, design 4 may be more robust.Although it has not been commonly applied, Poisson regression of allele counts in the rare variant score appears to be useful as a general approach for single-gene analysis in many designs, provided that the tendency toward elevated type I error is resolved. For design 5, Poisson regression avoids the complications of weighting, because the model conditions on the QT value.There is established literature on optimal design for continuous outcomes in experimental settings and survey samples, and sampling has been well exploited in the context of time-to-event outcomes in epidemiological designs, such as the case-cohort study . In compThe authors declare that there are no competing interests.YEY and SBB conceived of the study. YEY carried out the analyses and wrote the first draft of the manuscript. Both authors revised and approved the final manuscript.


Manipulation of dystrophin pre-mRNA processing offers the potential to overcome mutations in the dystrophin gene that would otherwise lead to Duchenne muscular dystrophy. Dystrophin mutations will require the removal of one or more exons to restore the reading frame and in some cases, multiple exon skipping strategies exist to restore dystrophin expression. However, for some small intra-exonic mutations, a third strategy, not applicable to whole exon deletions, may be possible. The removal of only one frame-shifting exon flanking the mutation-carrying exon may restore the reading frame and allow synthesis of a functional dystrophin isoform, providing that no premature termination codons are encountered. For these mutations, the removal of only one exon offers a simpler, cheaper and more feasible alternative approach to the dual exon skipping that would otherwise be considered. We present strategies to by-pass intra-exonic dystrophin mutations that clearly demonstrate the importance of tailoring exon skipping strategies to specific patient mutations. Although there are two deletion hotspots in the Thirty-nine of the seventy-seven “skippable” dystrophin exons , if one Intra-exonic mutations within frame-shifting exons will normally require two or more exons to be excised, the lesion-carrying exon and a flanking exon(s) to maintain the reading frame. Some such cases present alternative options in that either of the flanking exons may be excised . HoweverAlthough promising results have been reported for the Phase 2 exon 51 clinical trials using the morpholino oligomer AVI-4658 22.1Splice switching AOs, designed to excise human dystrophin exons, have been described previously 2.2All patient biopsy samples were collected with informed consent and de-identified. The use of human tissue was approved by the University of Western Australia Human Ethics Committee . Myogenic cells were propagated as previously described 2.3DMD transcripts were predicted using ESEFinder3(c) http://www.rulai.cshl.edu/new_alt_exon_db2/HTML/score.html.Genomic DNA was isolated using Trizol reagent as per the manufacturer’s instructions . PCR amplicons were isolated for sequencing using the band stab protocol 2.42OMe AOs were transfected into differentiated myotubes in Opti-MEM (Invitrogen) as cationic lipoplexes with Lipofectamine 2000 reagent at 1:1 w:w ratio, according to the manufacturer’s instructions (Invitrogen). Concentrations stated are total AO concentrations, where a cocktail of AOs is used; the concentration is the total of all AOs at 1:1 ratio. AOs used are listed in 33.1Before AOs were transfected into cells from DMD patients, their efficacy was confirmed in normal human myogenic cells . Cocktai3.2DMD gene. One in exon 51,  a single G duplication and two in exon 20: a 2 bp deletion  and an 8 bp deletion , all of which induce a reading frame-shift and dystrophin protein truncation, resulting in a DMD phenotype. The mutations were confirmed by sequencing genomic DNA and RT-PCR amplicons  are illustrated in 3.3A DMD patient was identified with a point mutation  at the exon 22 acceptor splice site A. This bAnother patient diagnosed with DMD was found to carry a single base change , at the acceptor splice site of exon 21. This mutation changed the normal splice site score from 4.2 to −6.3, and created an alternative splice site with a score of 3.1. This induced retention of the last 2 AG nucleotides of intron 20 in the mature dystrophin mRNA F. The tw4DMD is a serious muscle wasting condition for which there is no treatment. The application of splice switching oligomers to remove or correct frame-shifting mutations that would otherwise lead to premature termination of translation has progressed from a concept to clinical trials. Proof-of-concept has been demonstrated in animal models and recently in DMD patients, with completion of Phase I intramuscular studies and Phase I/IIa systemic studies with two different oligonucleotide analogues in vitro? Which strategy will produce the most functional truncated dystrophin isoform, determined by BMD patient data and transient inducible mouse model DMD is regarded as an orphan disease, with an estimated 420,000 affected individuals worldwide in vitro, by skipping of exon 51 + 52. This is in contrast to the result in normal myogenic cells where exons 50 + 51 were more efficiently removed. ESE analysis did not show any alterations to ESEs as a result of the mutation, yet this is not conclusive evidence that the exon 51 disease-causing variant does not influence splicing. We previously demonstrated that a nonsense mutation in dystrophin exon 17 did not alter the efficiency of oligomers targeted to excise exon 17, but did influence exon 18 skipping This study reports DMD mutations that can be addressed by multiple strategies to restore the reading frame and facilitate translation of a BMD-like dystrophin isoform. This includes intra-exonic mutations in exons 51 and 20. In these cases, the mutated exon must be excised with either flanking exon. The mutation in exon 51, was marginally more efficiently addressed, at least http://www.dmd.nl), although detailed clinical data is not publicly available for these patients.In cells from the patients with small intra-exonic deletions in exon, exons 20&21 were consistently removed more efficiently than exons 19&20. This is in agreement with the results obtained in normal myogenic cells. Both these mutations did cause small changes in the predicted ESE sites, but this does not appear to have affected the efficiency of the AO cocktails. Again, the effect on the functionality of the resultant dystrophin must be considered. In an AO induced transient mouse model, exons 19&20 could be removed from 100% of transcripts in the mouse diaphragm, with no detectable detrimental effects on the muscle phenotype in vitro in patient cells. Some large in-frame DMD deletions are associated with a mild phenotype, however this is greatly dependent upon the location of the DMD mutation We also report on single base changes that alter splicing at particular exon boundaries and present unique opportunities for simpler, alternative exon skipping strategies. In these cases, additional factors should be considered, other than which is the most efficient strategy and which results in the most functional dystrophin isoform. We have demonstrated that for two splice site mutations an opportunity exists to restore the reading frame by excision of a single exon, rather than the two exons suggest by conventional exon skipping strategies. For the exon 22 acceptor splice site mutation, the results demonstrate that skipping both exons 21&22 is inefficient in the patient’s cells, while skipping exon 21 alone is a much simpler strategy and is more efficient In normal myoblasts, skipping exon 20&21 or exon 21&22 was comparable, with the 20&21 cocktail proving marginally more efficient. In MyoD converted fibroblasts from the patient carrying the exon 21 splice site mutation, exon 20&21 were effectively removed by an AO cocktail. Exon 20 was also effectively excised on its own. In this case the inserted bases at the beginning of exon 21 (AG) are identical to the last two bases of exon 20, so excision of exon 20 does not alter the remaining amino acid sequence. Unexpectedly, it appears that the mutation facilitates skipping of exon 21 when exon 20 only is targeted, possibly because the exon 21 acceptor splice site is compromised. However, as both these transcripts are in-frame and allow dystrophin translation, targeting exon 20 only remains a viable option for this patient, despite this unexpected effect on exon selection.We propose a set of guidelines to be followed when dealing with splice site mutations that may be amenable to splice manipulation. The “minimalist” exon skipping strategies will be more applicable to mutations that occur near exon boundaries, and to minimise the possibility of encountering in-frame stop codons, will be very dependent on the precise position and nature of the mutation. Therefore, careful analysis of the effect of the mutation on splicing of the exon is required and it must also be determined if the mutation affects the AO binding sites. Many exons are most efficiently removed by an AO targeted near the 3′ acceptor splice site and mutations in this region would therefore disrupt AO binding, and it may be necessary to use sub-optimal AOs that avoid the mutation, or design additional sequences specific to the mutation. Once these considerations have been taken into account, the other factors that impact upon the optimal strategy; efficient removal of the target exon, functionality of the resultant dystrophin, potential off-target effects, applicability to other DMD patients and cost-benefit analysis of the alternative strategies must be considered, before the optimal strategy is selected.5As splice switching oligomer therapies progress through early clinical studies, it will be necessary to demonstrate unequivocal benefits in response to the treatment and then expedite application to the widest possible cohort of DMD patients. We report DMD mutations that present multiple exon skipping strategies to correct the reading frame. In addition, we have shown that splice site mutations can present unique opportunities that reduce the number of exons that must be excised, producing a simpler, more efficient and less costly strategy that could potentially result in a more functional dystrophin. This highlights the need to carefully characterise all DMD mutations to determine if they are suitable for treatment by exon skipping, and detailed investigation may be required to determine which will be the optimal strategy for each patient. This strengthens the case for splice-switching oligomers as a personalised genetic treatment for DMD, and emphasises aspects that should be considered as this therapy becomes available to amenable patients.


Six volunteers wore palatal removal appliances with four polished dentin specimens protected from intra-oral mechanical forces for up to 14 days. Each volunteer applied a 50% sucrose solution (four times a day) on the specimens and performed a daily mouthwash with 0.05% NaF. Samples were removed after 2, 5, 9 and 14 daysin situ. Demineralization was analyzed by stereomicroscopy and SEM (secondary electrons and backscattered electrons modes) and calculus was analyzed by energy dispersive spectroscopy and fluorescence spectroscopy. Seventeen samples, at least one sample from each volunteer, presented dental calculus on both carious and non-carious ones, detected in all time intervals. Ca/P ratios of dental calculus ranged from 1.1 to 1.7. Some large calculus deposits on carious surfaces were confirmed by fluorescence. In conclusion, concomitant caries and calculus formation can be found in dentin caries formedin situ. This has important repercussions on the study of surface phenomena on the interface between hard dental tissues and dental plaque.The aim of this study was to test the possibility of the concomitant formation of calculus deposits and caries from In situ caries models are not expected to give rise to calculus formation. However, the formation of loosely mineralized deposits in dental plaque concomitantly with carious dissolution of enamel, in an environment with fluoridated mineralizing solution, has been reported1. In a morphological study with transmission electron microscopy, those deposits were shown not to be identical to calculus as they were never present within intact bacterial cells2. In addition, it was shown by scanning electron microscopy (SEM) that calculus  could develop on dentin specimens subjected to anin situ carious attack with a daily application of a 50% sucrose solution in individuals living in a water-fluoridated area for periods of four weeks3. While demineralization is likely in such models, no clear-cut evidence was presented. In this study, some samples with calculus had the cariogenic challenge disturbed by weekly mechanical plaque removal, raising the hypothesis that the concomitant development of caries and calculus may occur during shorter periods under the given conditions.in situ caries model (using sucrose and fluoride) for short time periods (2–14 days).The aim of this study is to report concomitant formation of calculus deposits and caries on dentin surfaces submitted to anHuman dentin specimens (3 × 3 mm) from non-erupted third molars, previously autoclavated, had their surfaces consecutively polished with slurries of decreasing grain size until the alumina was 1 mm. Then, they were mounted in customized hand-made resinous removable palatal appliances , each one containing four specimens distributed in two pairs each in a different side of the appliance, where they were protected from intra-oral mechanical forces by means of plastic perforated tape. A space of up to 2 mm was left between the specimen and the tape. In the mouth, specimens were located near the palatal surfaces of the first permanent molar and the first premolar.Six volunteers, aged 20–29 years, were selected on the basis of the following criteria: (i) to be otherwise healthy (no medication regimen), (ii) no calculus treatment in the last two years and no clinical calculus deposits at the time of the study, and (iii) to present normal stimulated salivary flow. All volunteers were dental students who lived in non water-fluoridated areas, and were given fluoridated dentifrices (1500 ppm of the same brand) daily. All volunteers provided signed informed consent. Ethical approval was obtained from the Universidade do Vale do Itajaí Human Research Ethics Committee.3 was applied onto the specimens only, four times a day to mimic meals, and a 0.05% NaF solution was provided to perform one daily mouthwash for about one minute with the appliance in the mouth (at a different time to the sucrose rinsing). Instructions were given to replace the appliance in the mouth just after the sucrose rinsing and to not brush on the specimens. For each volunteer, specimens were removed after 2, 5, 9 and 14 days of intra-oral period. Six other polished specimens prepared in the same way described above, not exposed to the oral cavity, were also selected for reproducibility during morphological analysis, to make a total of 30 specimens. After thein situ period, specimens were stored in 0.1% timol prior to further analysis.Intra-oral periods lasted fourteen days and the volunteers were instructed to use the appliances all day long, except during meals. Two drops of a 50% sucrose solution (the same applied previously)2-atmosphere of 10-2 Torr- and coated with a 10 nm-thick layer of gold. The secondary electron detector of a scanning electron microscope JEOL 5900HV (voltage of 15 KeV) was used. Each specimen was analyzed under tilting angles of 0° and 40°. EDS analyses were performed in representative areas by a VOYAGER system attached to the microscope using the following parameters: spatial resolution of 1 mm, electron beam area of 10 mm, working distance of 8–9 mm, voltage of 15 KeV and a live time of 60 s. Ca/P (mol/mol) ratios (mean of 5 measurements) were analyzed using a standardless method - intensity of the standard calculated on the basis of the spectral properties of the sample4-with corrections for atomic number, absorption and fluorescence (ZAF correction). SEM analysis of surface changes  was performed twice by blinded examiners with a time interval of seven days between the two analyses. A set of 3 images from each sample was chosen. The Kappa test was used to calculate agreement. Only Schroeder’s type A dental calculus mineralization centers 5 were considered.All samples were treated with NaOCl 5% for 15 minutes, to remove organic coatings, then critical point dried with COAfter SEM, all samples were treated with NaOCl 5% for 15 minutes to remove the gold coating and then hemi-sectioned centrally on the exposed surface. The surfaces created after cutting were gently polished with alumina 10 mm and then analyzed under the stereomicroscope . Image analysis for the automated identification of demineralized areas and lesion depth measurements were performed using Leica QWIN Plus software . A standard margin of color discrepancy detection in the software was used for all samples when the software was asked to differentiate sound and carious dentin. The only influence of the operator was to choose a 1 mm-wide central area for automated lesion depth measurement.excit) was 416 nm and the emission (λemis) was collected from 425 to 800 nm (mean of 15 readings). Fluorescence of the other two saturated solutions in HCl 27%, one with human dental calculus and another with human dentin, were also analyzed.In samples that presented thick (> 100 mm in height) and extensive calcified deposits, a scalpel was used to remove the surface by scraping after stereomicroscopy. The scraped material was dissolved in a solution of HCl 27% (~ 0.2 ml), dispensed in a quartz cuvette and submitted to fluorescence spectroscopy in a commercial photon-counting spectrofluorometer  equipped with a xenon arc lamp operating at 10 mA, using 1 mm slits (bandwidth of 8 nm). Excitation wavelength  for SEM analysis using a backscattered electron detector (voltage of 15 KeV) in order to confirm thein situ periods of all volunteers with regard to lesion depth were evaluated by paired t test (significance limit at 5%).The differences betweenin situ periods, respectively . BSE-SEM analysis confirmed the presence of demineralization (lower gray levels due to lower mean atomic number) detected by stereomicroscopy.The occurrence of demineralization and calcified deposits was depicted from SEM examination7Seventeen samples presented calculus deposits  has a structure that is not easily distinguished from β-tricalcium phosphate or magnesium-containing β-tricalcium phosphate, and this is the reason why they have been referred as synonyms9. However, it is known that they are distinguishable by careful powder diffractometry10, although both present a Ca/P ratio of 1.57. While β-tricalcium phosphate does not form in biological systems, whitlockite is found in many biological mineralizations9. Whitlockite precipitates at low, neutral or high pH, with magnesium stimulating whitlockite precipitation at the expense of hydroxyapatite7. The presence of hydroxyapatite most probably reflects pH fluctuations  during the periods of time employed. In this context, it is possible to chemically explain the concomitant calculus and caries formation in our model.The crystalline structure of young calculus is reported to be mainly of dicalcium phosphate in the early stages, after which octacalcium phosphate develops and, with further growth, whitlockite and hydroxyapatite are formed11.Crystallite morphology and Ca/P ratios resembling hydroxyapatite in 10-day old calculus deposits have been reported previouslyin situ period proceeded, calculus deposits  with increased height, macroscopic in some cases, were seen on demineralized areasAs thein situ times of 9 and 14 days) that presented “large” deposits of calculus and caries were submitted to fluorescence spectroscopy. All of them presented fluorescence of dental calculus 12. A lower fluorescence emission band below 580 nm has also been reported for human dental calculus13. Human dentin fluoresces with a λemis peak at 440 nm and does not present any emissions in the region of 580–700 nm14, which is in accordance with our fluorescence data for λexcit at 416 nm15) showed a mixture of fluorescence bands of human dental calculus and human dentinFluorescence of human dental calculus in acid solution has been shown to present the highest emission with λin situ caries model reported here occurred under conditions known to cause the formation and growth of dental plaque. Combining SEM, fluorescence spectroscopy and stereomicroscopyin situ experiment. Our model was able to promote time-dependent demineralisation (seen from lesion depth data), which means that volunteers followed instructions properly. To our knowledge, this is the first time that this phenomenon has been reported on the basis of the combination of such mutually validating techniques.The concomitant development  of caries lesions and calculus deposits using anin situ dentin caries to the identification of calculus on hard dental tissues exposed toin situ caries models.In conclusion, calculus formation in active cariogenic dental plaque has important repercussions on the study of surface phenomena on the interface between hard dental tissues and dental plaque. It is also important in the development and the evaluation of anti-tartar and caries-preventative agents. Our study shows that more attention should be paid by those studying It was noted that all dentin samples were exposed to sucrose solutions, fluoride and were protected by tape. Only the unexposed slabs were used as controls and none of these three conditions were controlled. A more complete picture would have been possible with their inclusion.The authors claimed “It is known that dicalcium phosphate and octacalcium phosphate can be precipitated in acidic environments whereas hydroxyapatite is dissolved.” They listed references 7 and 8 to support this statement. However, I was unable to access reference 7 and could not find supporting data in reference 8. While DCP is slightly less soluble than HAP around pH4 I would not consider that to mean that either DCP or OCP readily precipitate at that pH.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I thank the reviewer for his valuable comments and his opinion to accept the article is greatly acknowledged by us. Regarding the first paragraph of the reviewer’s comment, we understood that the critique was that some control was lacking. We think that the inclusion of samples that were exposed to the oral environment but did not develop caries would no add relevant information for the aim of the study. The in situ caries induction aimed at producing caries lesions with known history and with severity directly proportional to the in situ time. Mean lesion depth increased with in situ time, confirming the prediction. In an environment where caries develops, surface features of the samples were analyzed in order to verify whether calculus deposits could have been formed under the same conditions. Such conditions are those common to in situ caries models. Because the length of the article is short, we focused on the more relevant data . If we had exposed dentin samples that remained sound after the in situ period, the presence of absence of calculus deposits on those samples would have not contributed to prove that calculus deposits can be formed concomitantly with caries lesions in short time intervals in an in situ caries model. et al. 1992Johnsson describes evidence indicating that formation of both DCP and OCPin vivo, during normal mineralization, is quite different from the conditions operating under pathological mineralization. The authors cite reports of the precipitation of DCP and OCP in slightly acidic conditions . The critical pH of dentin is between 6.5 and 7 . There is a pH range below the critical pH of dentin that can be considered as slightly acidic, and so it is consistent with the possible precipitation of both DCP and OCP in our samples. Regarding formation of DCP and OCP in acidic conditionsin vitro, evidence can be found in the following papers;et al. 1984De Rooij andet al. 1991Lundager Madsen.In the case of precipitation of dicalcium phosphate (DCP) and octacalcium phosphate (OCP) in acidic environments, I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


To examine the potential contribution of herpes simplex virus 2 (HSV-2) infection to female/male and racial/ethnic disparities in HIV among non-injecting heroin and cocaine drug users. HSV-2 infection increases susceptibility to HIV infection by a factor of two to three.Subjects were recruited from entrants to the Beth Israel drug detoxification program in New York City 2005-11. All subjects reported current use of heroin and/or cocaine and no lifetime injection drug use. A structured questionnaire was administered and serum samples collected for HIV and HSV-2 testing. Population-attributable risk percentages (PAR%s) were calculated for associations between HSV-2 infection and increased susceptibility to HIV.1745 subjects were recruited from 2005-11. Overall HIV prevalence was 14%. Females had higher prevalence than males (22% vs. 12%) (p<0.001), African-Americans had the highest prevalence (15%), Hispanics an intermediate prevalence (12%), and Whites the lowest prevalence (3%) (p<.001). There were parallel variations in HSV-2 prevalence , HSV-2 prevalence was strongly associated with HIV prevalence (OR  =  3.12 95% CI 2.24 to 4.32). PAR%s for HSV-2 as a cause of HIV ranged from 21% for Whites to 50% for females. Adjusting for the effect of increased susceptibility to HIV due to HSV-2 infection greatly reduced all disparities .Female/male and racial/ethnic variations in HSV-2 infection provide a biological mechanism that may generate female/male and racial/ethnic disparities in HIV infection among non-injecting heroin and cocaine users in New York City. HSV-2 infection should be assessed as a potential contributing factor to disparities in sexually transmitted HIV throughout the US. There are substantial racial/ethnic disparities in HIV/AIDS in the United States, with African-Americans comprising 46% of newly diagnosed HIV infections despite comprising only 12% of the US population There are also important female/male disparities among the heterosexual contact cases in the US. In 2010, there were a total of 12,875 newly reported heterosexual contact cases, of whom 4416 (34%) were among males and 8459 (66%) were among females. This disparity differs from that seen among female commercial sex work networks, in which a small number of females transmit HIV to large numbers of males, and differs from that seen in the “mature” heterosexual generalized epidemic in which HIV seropositive females outnumber HIV seropositive males, but typically only by a modest percentage, e.g., Given the size of these disparities, it is likely that multiple causal factors are operating. The different causal factors: social structural factors such as male/female ratios, social network factors such as HIV prevalence among sexual networks, partnership factors such as the rate of sexual partner change, individual level factors such as the frequency of condom use, and biological factors such as the prevalence of sexually transmitted infections that may facilitate HIV transmission, may also be operating at different levels of analysis. Of note, individual level behaviors such as condom use vary little across racial/ethnic groups and typically do not explain racial/ethnic disparities In the US general population, HSV-2 seroprevalence among African-Americans is approximately three times as high (39.2%) as among White Americans (12.3%) and approximately twice as high among females (20.9%) compared to males (11.5%) The data reported here are derived from ongoing analyses of data collected from drug users entering the Beth Israel Medical Center drug detoxification program in New York City. The methods for this “Risk Factors” study have been previously described in detail Both injecting and non-injecting drug users entering the detoxification program are eligible to participate in the study. The present analyses include only persons who reported that they have never injected illicit drugs (never-injecting drug users or NIDUs). As we were interested in the relationship of HSV-2 infection to sexual transmission of HIV, including persons who might have become infected with HIV through drug injecting transmission would have introduced considerable measurement error into the analyses. Hospital records and the questionnaire results are checked for consistency on route of drug administration and subjects are examined for physical evidence of injecting. The data presented here are from subjects who participated in the study from 2005 to 2011.Research staff visited the general admission wards of the program in a preset order and examined all intake records of a specific ward to construct lists of patients admitted within the prior three days. All of the patients on the list for the specific ward were then asked to participate in the study. Among patients approached by our interviewers, willingness to participate has been more than 95%. After all the patients admitted to a specific ward in the three-day period had been asked to participate and interviews have been conducted among those who agreed to participate, the interviewers moved to the next ward in the preset order. Because there was no relationship between the assignment of patients to wards and the order that the staff rotated through the wards, these procedures should produce an unbiased sample of persons entering the detoxification program.A structured questionnaire covering socio-demographic characteristics, drug use, sexual risk behavior, and use of HIV prevention services was administered by a trained interviewer. Race/ethnicity was assessed by asking participants with which racial/ethnic group they identified. African-American, Hispanic and White were the most common groups given. A small number (<0.05%) of participants gave other responses, but they were not included in the analyses presented here. Most HIV risk behavior questions referred to the six months prior to the interview.After completing the interview, the participant was seen by an HIV counselor for pretest counseling and serum collection. HIV testing was conducted at the New York City Department of Health Laboratory using a commercial, enzyme-linked, immunosorbent assays (EIA) test with Western blot confirmation . HSV-2 testing was conducted for all subjects beginning in 2005 and was performed by BioReference Laboratories using the Focus HerpeSelect 1 and 2 ELISA. We used an index value of 1.1 or greater for classifying a subject as HSV-2-seropostive.never injected illicit drugs. As we were primarily interested in the relationship of HSV-2 infection to heterosexual transmission of HIV, subjects who reported male with male sexual behavior without also reporting male with female sexual behavior were excluded from the analyses. We did include subjects who reported both male with male and male with female sexual behavior, as these subjects may well be contributing to heterosexual transmission. As we were interested in racial/ethnic disparities in HIV infection, we limited the analyses to subjects who reported their race/ethnicity as White, African-American and Hispanic. The numbers of subjects who reported other specific ethnicities were too small for statistical testing. Subjects were permitted to participate in the study multiple times, though only once per year. For these analyses, if subjects had multiple interviews during 2005-2011, we used the first interview. The design of the study is thus a cross-sectional survey.Participants were included in this analysis if they reported that they had We calculated “population attributable risk percentages” (PAR%s)The Stata statistical programs  were used for statistical analyses.The study was approved by the Beth Israel Medical Center Institutional Review Board.We observed significant sex and racial/ethnic disparities in HSV-2 infection and in HIV infection among non-injecting heroin and cocaine users in New York City. Adjusting for the biological effect of HSV-2 increasing susceptibility to HIV infection greatly reduced the female/male disparity and the disparities between Whites versus African-Americans and Hispanics. The disparities in HSV-2 infection preceded the HIV/AIDS epidemic in the US, so that the biological mechanism of increased susceptibility can thus explain much of the disparities in HIV infection It is likely that there are multiple causes of the female/male and racial/ethnic disparities in HIV/AIDS. HSV-2 is not a complete explanation for these disparities, but the data presented here for heterosexual non-injecting drug users suggest that HSV-2 should be considered when examining female/male and race/ethnic disparities in sexual transmission of HIV in the US.Limitations. Several limitations of the present study should be considered. First, the data presented here are from subjects recruited at a single site. However, the data are consistent with HIV prevalence among non-injecting drug users recruited from community settings in New York in 2004 The data presented here are also consistent with the recent study showing unexpectedly high HIV incidence among women in six US cities Second, the research design used in this study was a cross sectional survey, so it was not possible to observe new HSV-2 and new HIV infections in individual subjects or identify the specific risk behaviors associated with seroconversion. Data on risk behavior at the time of HIV seroconversion would permit examining whether racial/ethnic differences in risk behaviors contribute to the racial/ethnic disparities in HIV prevalence which we were not able to do in this study. Seroconversions and associated risk behaviors could have been observed in a cohort study however an extremely large study would be needed. We estimate that incidence among these NIDUs was approximately 1-2/100 person-years overall, so that a very large number of subjects would have to be followed over a long time period to detect significant racial/ethnic differences in incident HIV infections.Third, the effect of HSV-2 on increased susceptibility to HIV infection is undoubtedly an important contributing factor to the high HIV prevalence  we observed in these NIDUs, but clearly is not a complete explanation for this relatively high prevalence. Other factors, including a biological effect of HSV-2 on the transmission of HIV and transmission from HIV seropositive drug injectors to non-injecting drug users are likely to be operating. Unsafe sexual behavior would also be a necessary causal factor for acquisition of HIV among our subjects.These limitations are important, but it is difficult to envision how they would have created the sex and racial/ethnic patterns in HSV-2 and HIV serostatus that we observed. Rather it is much more likely that these patterns are sufficiently strong that we observed them despite the limitations of the study.Adjusting for a causal pathway of HSV-2 increasing susceptibility to HIV essentially eliminates the female/male disparities and greatly reduces the racial/ethnic disparities in HIV infection among these New York non-injecting heroin and cocaine users. The higher rates of HSV-2 among racial/ethnic minorities have been observed since the late 1970s in the US Finally, there is a clear need to implement interventions to reduce HSV-2 related HIV transmission among non-injecting drug users and their sexual partners. There are therapies that suppress HSV-2 infection and reduce the frequency of outbreaks Locating and working with HSV-2 seropositive drug users at risk for HIV and locating and working with their possibly HIV seropositive partners, would require substantial resources, but given the increased susceptibility to HIV associated with HSV-2 infection, and its relationship to racial/ethnic disparities in HIV infection, the additional costs would be fully justified.


VEGFC. The causal variant had minor allele frequency of 0.0717% in the simulated unrelated individuals and explained about 0.1% of the phenotypic variance. However, it explained 7.9% of the phenotypic variance in the eight simulated pedigrees and 23.8% in the family that carried the minor allele. The carrier’s family was selected in all 200 replications. Thus our results show that family-specific linkage information is useful for selecting families for sequencing, thus ensuring that rare functional variants are segregating in the sequencing samples.Genome-wide association studies have been successful in identifying common variants for common complex traits in recent years. However, common variants have generally failed to explain substantial proportions of the trait heritabilities. Rare variants, structural variations, and gene-gene and gene-environment interactions, among others, have been suggested as potential sources of the so-called missing heritability. With the advent of exome-wide and whole-genome next-generation sequencing technologies, finding rare variants in functionally important sites  becomes feasible. We investigate the role of linkage information to select families enriched for rare variants using the simulated Genetic Analysis Workshop 17 data. In each replicate of simulated phenotypes Q1 and Q2 on 697 subjects in 8 extended pedigrees, we select one pedigree with the largest family-specific LOD score. Across all 200 replications, we compare the probability that rare causal alleles will be carried in the selected pedigree versus a randomly chosen pedigree. One example of successful enrichment was exhibited for gene  Genome-wide association studies (GWAS) have achieved great success in recent years, with about 4,000 single-nucleotide polymorphisms (SNPs) found to be associated with hundreds of common complex traits . HoweverIn this work, we investigate a strategic approach for studying rare variants in families. A real example of our approach has been demonstrated recently . Using tWe used the simulated family data set provided by GAW17, which consists of 697 subjects in 8 extended pedigrees. The 202 founders were chosen from 697 unrelated individuals participating in the 1000 Genomes Project, and their 495 descendants were simulated. See the GAW17 answers  for moretrends[trends program was developed to conduct variance component linkage analysis with interactions; we used a regular variance component model without interactions for this analysis. We analyzed each of the 200 replications; in each replication, we selected one pedigree that had the largest family-specific LOD score. To evaluate the efficacy of enriching rare causal variants using linkage information, we compared the relative frequency among the 200 replications of selecting the correct family  with the probability that a randomly selected family will carry the causal minor allele. To save computational burden, we conducted analyses on only 9 and 13 causal genes used for simulating phenotypes Q1 and Q2, respectively. The variants in gene FLT4 for Q1 and in genes INSIG1 and RARB for Q2 are monomorphic and thus do not contribute to any phenotypic variation; hence they were deemed noncausal in our evaluations. Analyses on these three genes were actually carried out under the null hypothesis.We first adjusted the phenotypes Q1 and Q2 for age, sex, and smoking effects and used the standardized residuals in the analysis. We conducted variance component linkage analysis on Q1 and Q2. Family-specific logarithm of odds (LOD) scores were computed using QTLtrends using thβ) provided in the answers, assuming an additive model; the effect of each gene was the sum of all causal variants in it. The percentage of explained phenotypic variance was computed from the average ratio of the variance of the gene effect to the total phenotypic variance from 200 replications.We also computed effect sizes of the causal genes in terms of phenotypic variances explained within each family and in the total sample. The effect of each variant for each subject was computed from the number of rare alleles and effect  carry rare alleles, some of the rare alleles were transmitted to descendants and some were not. For example, the founder in family 3 did not transmit a copy of the rare allele of the SNP C1S3181 in gene ELAVL4 to any descendants. On the other hand, 30 descendants in family 7 inherited the rare allele of the causal SNP C4S4935 in gene VEGFC from one common founder. In a sequencing study of rare variants, it is desirable to have families that not only carry the causal rare alleles but also have multiple copies transmitted to the descendants.In Table P-values comparing the relative frequency of families carrying rare alleles when selected using linkage information versus randomly selecting a family are shown in the last column of Table We evaluated the utility of linkage information for selecting families that are enriched for rare variants for a sequencing study. We computed family-specific LOD scores for each family and examined whether any rare allele existed in the family with the largest family-specific LOD score. Results from analyzing 200 replications are presented in Table VEGFA and VEGFC, both of which have a single causal SNP and carrier families selected in all 200 replications. Comparing the strategy across different genes and variants, we can see that this method works well when rare alleles not only exist in founders but also are transmitted to multiple descendants. The limited number of rare variant copies in a family where founders are carriers but not descendants contributes little to the phenotypic variance, even though the individual genetic effects may be large. For instance, only one founder from family 4 has rare alleles of the two causal SNPs (C12S181 and C12S211) in gene VWF. The frequency of picking this family by the family-specific LOD criterion is 0.105, which is close to the random chance of 0.125. For SNPs with only a few copies of the rare alleles transmitted to descendants , the results are similar.As can be seen from Table max) are also presented in Table max values represent the averaged largest family-specific LOD scores, which could come from different families across replications. The best linkage evidence is from genes VEGFA and VEGFC. Gene VEGFA has the causal SNP C6S2981, whose rare alleles are carried in families 1, 2, and 7; the average overall LOD score is 3.32, and the LODmax score is 2.40. For gene VEGFC, only family 7 has rare alleles of the causal SNP C4S4935, and its average LODmax score is 4.44, which is larger than the overall LOD score of 3.87.LOD scores and maximum family-specific LOD scores  of 0.0717% and explains 0.1% of the phenotypic variance in the 697 unrelated individuals. However, it explains 7.9% of the phenotypic variance in the eight pedigrees and 23.8% in family 7, which carries the minor allele. This agrees well with a recent study of adiponectin levels that identified a low-frequency variant with MAF of 1.1% in the general population that explained 17% of the phenotypic variance in families and 63% in carriers’ families [FLT4, INSIG1, and RARB.Phenotypic variance explained by each gene is presented in Table We have shown that family-specific LOD scores provide valuable information for selecting families that carry rare causal variants. This method performs well even when the effects of rare variants are not large and the aggregate linkage evidence for the entire sample (a replication in this case) is weak. Although linkage analysis has low power to detect unknown small signals, it seems to work well for selecting samples in order to study a locus known to harbor causal variants, for example, a locus confirmed in GWAS. In a nuclear family in which one of the parents has one copy of a rare allele , half of the offspring are expected to carry this rare allele. Hence rare variants could be common in carriers’ families, which are ideal for evaluating associations with rare variants. On the other hand, because of the rarity of variants, not all founders or families carry the rare alleles; hence choosing families randomly from the general population will not guarantee enrichment of rare variants. Therefore an effective sampling strategy is crucial for enriching rare variants using family data.VEGFA and VEGFC, some other genes also showed the efficacy of using family-specific linkage information. For instance, the frequency of selecting a family carrying any rare causal allele in KDR is 0.965 compared to the frequency for a randomly chosen family, which is 0.75. As we can see in Table KDR reflects the fact that those families that do not carry any rare causal alleles are less likely to have the largest family-specific LOD scores compared with the carriers’ families. This is consistent with our other results. On the other hand, this finding does not represent the typical common disease/rare variant scenario that is being investigated by current exome-wide sequencing studies. In fact, in real studies there is probably no need to enrich causal variants to find this gene because it can easily be detected by GWAS.Besides FLT4, INSIG1, and RARB are monomorphic in the simulated family data, providing an opportunity for us to examine the method under the null hypothesis. With the nominal distribution of a half-half mixture of a chi-square with 1 degree of freedom and a point mass of 1 at 0, the average LOD score is expected to be 0.11 under the null hypothesis. The LOD score for both INSIG1 and RARB averaged over 200 replications is 0.09, which is close to the expected value. FLT4 has a much higher average LOD score of 0.24; this number is even larger than the average LOD score for FLT1, which has 74 causal minor alleles in five pedigrees and explains 5.8% of the phenotypic variance in the eight pedigrees. According to the GAW17 answers, there were no other causal genes in the proximity of FLT4. We were not able to identify the source of the inflated linkage evidence for this gene.As mentioned previously, all causal SNPs in genes Linkage analysis is able to test the effects of both common and rare variants and hence is immune to the synthetic correlations  in GWAS.Families that carry causal rare variants for common complex traits provide an invaluable resource for exome-wide and whole-genome sequencing studies. We evaluated a sampling strategy using family-specific linkage information to select samples that carry the rare alleles. This method works well even when the aggregate linkage evidence is small. Therefore this method can be a powerful approach for follow-up studies of loci identified from previous GWAS or other studies.The authors declare that they have no competing interests.GS developed the concept, performed the statistical analysis, interpreted the data and drafted the manuscript. JS participated the statistical analysis, interpreted the data and revised the manuscript critically. DCR interpreted the data, revised the manuscript critically and gave final approval for publication. All authors read and approved the final manuscript.


Although these metabolic disturbances are responsible for most nephrolithiasis cases, renal stone formation may occur in the absence of any metabolic disturbance. Previous evidence from our region (southern Iran) suggests that low 24-hour urine volume, hypercalciuria, and hyperuricosuria are the most common metabolic abnormalities associated with nephrolithiasis.3 As renal stones are the only gross evidence of these disorders, their composition can be used for proposing the pathogenesis leading to stone formation and can provide crucial information for the management of the patients. It has been recommended that the analysis of stone composition should be considered as an integral part of evaluation in those with urinary calculi. This approach would be specifically helpful in determining the cause of stone formation and for planning the prevention strategies.4 To the best of our knowledge, there are limited data on renal stone composition in the Iranian population.  We performed a 2-year cross-sectional study on patients with renal stones who were candidats for surgical intervention as a sample of patients with “complex” nephrolithiasis. From March 2009 to March 2011, all patients who underwent surgery for renal calculi were enrolled and their stones were analyzed. The predominant composition of each stone was considered as the basis of classification. Of 423 stones, calcium-oxalate stones were the most common type (67%), followed by uric acid stones (27%). These findings were consistent with previous reports from the Middle East region.5,6 In contrast, the only previous study to have addressed renal stone composition in Iran showed that the most common components were whewellite (81.5%), weddellite (40.7%), apatite (69%), and ammonium acid urate.7 The mentioned study was done more than 3 decades ago in Tehran, northern Iran. Compared to our findings, it seems that some factors, including changes in the population’s life style and diet, global warming, and geographic factors, may contribute to the differences in the results.Nephrolithiasis is a common urinary problem with a worldwide estimated prevalence rate of 4–20% and a 5-year recurrence rate of 50%. It accounts for significant expense and morbidity. Recurrent stone disease is a major cause of end-stage renal disease, which may eventually lead to renal transplantation. The prevalence rate of stone disease, in the Middle East region and in Iran, is estimated between 1-20% and 5-7%, respectively.Although we did not evaluate patients who were managed with medical therapy or shock wave lithotripsy, we recommend stone analysis as a basic and cost-effective method for the evaluation of stone-forming patients. It may help to understand the mechanism of lithogenesis and may help care providers to take preventive measures for these patients.