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Alex Villegas worked as a physical therapy aide for more than a year after graduating from UC Davis, unsure how to make the most of his hard-earned 20mg levitra price biology degree. Then one day, his boss summoned him into the office. New medical student Alex Villegas at the 2021 induction ceremony thanks his 20mg levitra price parents for their supportThe manager was concerned that Villegas, who is highly empathetic to patients and savvy about musculoskeletal health, wasnâÂÂt living up to his career potential. He wanted to know what Villegas saw himself doing in five years. Villegas, unprepared for the question, blurted out that he was considering going back to school for physical therapy.
But the manager, Edgar 20mg levitra price Villanueva, had another vision. ÃÂÂI looked him straight in the eye and said, âÂÂyou should not be a physical therapist, you should be a doctor.âÂÂâ That conversation in 2017 propelled Villegas on a new journey â one that brought him to the UC Davis School of Medicine where he started classes this month as a first-year medical student.His path to Sacramento has been an unconventional one. Along the way, Villegas worked in construction, labored as a farmworker and was first in his extended Mexican-immigrant family to complete high school. Lately, he has dedicated 20mg levitra price quality time to being with his father who is on hospice care with cancer. The experience has reinforced his decision to study medicine.âÂÂMy path toward medical school has been everything but a straight line,â Villegas said.
ÃÂÂInitially, I couldnâÂÂt believe 20mg levitra price I had gotten accepted, and I got really emotional, because of all the sacrifices my parents had done to get me to this point.â Work ethic instilled at an early ageVillegas, 28, was born in Modesto, the oldest of three boys. The family moved to Turlock, where the kids spent weekends operating power tools with their dad, a farmworker and truck driver who supplemented his income by repairing homes. ÃÂÂWork was my dadâÂÂs way of showing me the value of what he calls âÂÂganas,â or the desire to succeed,â Villegas said. Whenever Villegas contemplated his future, college was never in the 20mg levitra price picture, not even in a thought bubble. His parents had cut short their own schooling by the seventh grade in the rural Mexican state of Michoacán, and nobody in the family, including aunts, uncles and cousins, had ever been to college.
Alex Villegas puts on the School of MedicineâÂÂs white coat with help from his parents at a special induction ceremonyVillegas was a bookworm. His family called him el estudioso, the studious 20mg levitra price one. At Turlock High School, teachers steered Villegas into AVID, short for Advancement Via Individual Determination, a school-based organization that provides college track resources for students from diverse and underrepresented demographic groups. Following his 20mg levitra price junior year, Villegas sought summer work at McDonalds and Taco Bell to earn money to pay for college applications, but got rejected. Instead, he took a job as a farmworker picking blueberries, onions and chili peppers.
His parents tried to talk him out of agricultural work. Deep inside, though, he had a personal reason for 20mg levitra price wanting to work in the fields. ÃÂÂPart of me wanted to see the work that my parents did, to know what theyâÂÂve gone through.â On his first day in a Stanislaus County field, Villegas earned just $26 in a seven-hour shift. The job paid by the quantity of crops he picked instead of a minimum wage. He empathized with coworkers who suffered from pesticide-related medical issues, 20mg levitra price no health insurance, lack of transportation and low wages.
ÃÂÂAt the time I didnâÂÂt know I wanted to be a doctor,â Villegas said, âÂÂbut I knew I had to do something for my community.â Deciding to study at UC DavisDuring his senior year, Villegas received a generous scholarship from California State University, Stanislaus. He chose UC Davis instead 20mg levitra price for its strong reputation in science education. In doing so, he became the first student from Turlock HighâÂÂs AVID program to attend a University of California school. ÃÂÂMy path towards medical school has been everything but a straight line. Initially, I couldnâÂÂt believe I had gotten accepted, and I got really emotional, because of all the sacrifices my parents had done to get me to this point.âÂÂâ Alex VillegasMoving 100 miles 20mg levitra price to Davis was bittersweet.
He would pursue a higher education, yet far from his father, his role model. Villegas apologized to his dad for no longer being able to help with construction jobs. He recalled how his 20mg levitra price father put his arm around him and said everything would be fine and encouraged his son to study hard, despite the distance between them. ÃÂÂSomething my dad told me that kind of stuck to me is that to keep growing as a person, you have to take risks and step out of your comfort zone,â Villegas said. ÃÂÂLike when my parents came 20mg levitra price from Mexico.â Villegas taught his parents how to text, then left for college.
At UC Davis, he took science courses and worked in research for Chicana/o Studies, surveying the needs of farmworkers. On weekends, he volunteered in the Knights Landing One Health Center, a student-run clinic that provides free health care in rural Yolo County. At one 20mg levitra price point he thought about a career in medicine, but the goal seemed unattainable. Villegas graduated in 2016, debt free, thanks to federal and state grants, and gravitated back home. He took the job as an entry-level physical therapy aide in the hospital where he was born, DoctorâÂÂs Medical Center in Modesto.
He loved every aspect of 20mg levitra price patient care and didnâÂÂt really aspire to a higher career goal. But that one heart-to-heart conversation with his manager motivated Villegas to realize the potential others saw in him. ÃÂÂUltimately, my boss wanted whatever I would be happy with, whether that was physical therapy or medical school, but he always encouraged me to 20mg levitra price reach for the stars and not give up on my initial aspirations.â Villegas gave serious thought to studying medicine. Eventually, he enrolled in the UC Davis yearlong Postbaccalaureate Program. The well-regarded program, heavy on science curriculum, also offers study tips and test-taking strategies for college graduates who want to apply to medical school.
Afterward, Villegas returned to 20mg levitra price Modesto and to his therapy aide job, while also studying for the Medical College Admissions Test. He took a leadership role in MiMentor, an organization that supports students from diverse backgrounds to become health professionals. Tragedy prompts him to rethink medical schoolThen tragedy struck the family. His father was diagnosed with liver cancer 20mg levitra price. Alex Villegas with his father Joe Villegas, his role model who is on hospice care with cancerVillegas drove his dad from one specialist to another while debating whether to apply to medical school or wait until later.
ÃÂÂI wanted to be around my family,â he 20mg levitra price said. He discussed his dilemma with his parents. His father, as usual, encouraged Villegas to continue pursuing his goals. And his supportive brothers promised to look after 20mg levitra price their dad. Villegas then decided to apply.
On Dec. 15, 2020, 20mg levitra price Villegas received an unforgettable phone call from an unknown 916 number that flashed on his cell phone screen. It was Charlene Green, the UC Davis School of Medicine admissions director. Villegas was one of nearly 10,000 20mg levitra price students who applied to the school. Suddenly, he was one of only 132 who would enroll.
ÃÂÂI just froze,â Villegas recalled of his conversation with Green. ÃÂÂI didnâÂÂt say anything for maybe 10 seconds, a good 15 seconds.â Villegas couldnâÂÂt believe he had been accepted, but 20mg levitra price soon realized it was affirmation for his hard work. ÃÂÂI am worthy of being a medical student.â Not only did he get into the UC Davis School of Medicine, and had offers from other schools, but Villegas also gained acceptance into a competitive academic track. REACH (Reimagining Education to Advance Central California Health) tailors medical education to UC Davis students who desire to practice in the Central Valley, one of the stateâÂÂs most medically underserved areas. ÃÂÂI look forward to Alex becoming a physician, returning to CaliforniaâÂÂs Central Valley and caring 20mg levitra price for a diverse and underserved patient population,â said Olivia Campa, an internal medicine physician and director of the post-bacc program, where she was a mentor to Villegas.
ÃÂÂI am so proud Alex is well on his way to becoming an excellent physician and truly represents the values of our institution.â This past spring, Villegas learned about the medical schoolâÂÂs upcoming induction ceremony, a meaningful event where first-year students receive their white coats and stethoscopes. Villegas envisioned his mom and dad in the 20mg levitra price audience. But his dadâÂÂs health was deteriorating. ÃÂÂIt got me thinking whether he would be able to attend my white coat ceremony,â Villegas said. ÃÂÂThatâÂÂs something I really wanted to share with him.â Villegas 20mg levitra price reached out to Green and explained his situation.
He asked if he could borrow a white coat to hold his own ceremony at home. A special induction ceremonyGreen enlisted other medical school staff and faculty members and mailed a box to Villegas containing a white coat. The school also sent personalized video greetings from key faculty members, in English and Spanish, which allowed Villegas to hold his own ceremony 20mg levitra price with his father in late May. ÃÂÂIt was a very emotional moment for me,â recalled Villegas, whose girlfriend videotaped the ceremony. It was perhaps even more special for his father 20mg levitra price.
ÃÂÂI feel so proud because heâÂÂs accomplished all this, despite the difficulties weâÂÂve been through,â his father, Joe Villegas, said. ÃÂÂHe gave it a lot of ganas, a lot of ganas,â the father repeated, for emphasis. ÃÂÂWe were so happy to put the white coat on him.â As it turned out, 20mg levitra price the schoolâÂÂs induction was an in-person event only for students. Families had to watch from home because of the levitra. On that morning, July 31, Villegas walked up to the stage, received his stethoscope from Associate Dean for Students Sharad Jain and headed to the microphone to address his family watching via Facebook.
With his hands clasped together, he said in Spanish, âÂÂIâÂÂd like to thank my parents for all their support, my brothers and my partner, and to all my mentors who have been supporting and guiding me during 20mg levitra price this entire journey.â He then looked at the audience and proclaimed, âÂÂGo Ags!. àand pumped his left fist into the air. Following the ceremony, Villegas drove to Turlock to celebrate with his father and the rest of the family..
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IntroductionIdentification of a jason levitre germline pathogenic TP53 (MIM. *191170) variant in a patient with cancer has drastic medical impacts.1 Indeed, in TP53 variant carriers, chemotherapy and radiotherapy have been shown to contribute to the development of subsequent primary cancers, the incidence of which is remarkably high (above 40%).1âÂÂ4 Therefore, in these patients, jason levitre surgical treatment should be prioritised and radiotherapy and chemotherapy avoided, if possible, or at least carefully discussed in terms of benefit:risk ratio between risk of recurrence and risk of inducing second primary tumours. Furthermore, TP53 variant carriers should have specific surveillance protocols, including annual whole-body MRI,5 6 whose efficiency for early tumour detection has recently been shown by numerous studies.5âÂÂ14Interpretation of germline TP53 variants, which are mainly missense variants, remains particularly complex.
Whereas germline jason levitre variants of TP53 were initially detected in Li-Fraumeni syndrome (LFS, MIM#151623),15âÂÂ17 our perception of cancers related to germline alterations of TP53 has drastically evolved over time.1 2 18 19 The presence of a disease-causing germline variant should be considered in patients fulfilling Chompret criteria, which were sequentially updated and extended.1 The question of germline TP53 variant interpretation is becoming a growing concern in the field because the TP53 gene is currently included in many cancer gene panels, and the number of TP53 tests performed in patients not fulfilling the criteria mentioned earlier has increased exponentially. 20 21Classification of TP53 variants, in agreement with the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, is based on several items, including frequency of the variant in the general population (gnomAD. Https://gnomad.broadinstitute.org/), segregation data, bioinformatics predictions jason levitre and functional assays developed in yeast or human cancer cell lines.22 One of the first assays commonly used for TP53 missense variant interpretation was developed in yeast and is based on the expression of TP53 cDNA in strains containing reporter plasmids with different p53 binding sites.23 In this assay, p53 variants are classified as functional, not functional or partially functional if the transcriptional activity is conserved for some but not all yeast reporter plasmids (http://p53.iarc.fr/).
More recently, two teams have developed in human cancer cell lines high throughput p53 functional assays.24 25 Kotler et al24 generated a synthetic library of TP53 variants located within the p53 DNA-binding domain and quantified the antiproliferative activity of these variants in the p53-null H1299 cancer cell line. In this jason levitre assay, TP53 variants are categorised as âÂÂwild-type TP53-like variantâ (functional) or âÂÂdisruptingâ (non-functional). In another assay, Giacomelli et al25 generated by saturation mutagenesis a TP53 library and tested the ability of the variants (1) to restore the survival of the p53-null A459 cell line exposed to high doses of DNA damaging agents, in order to detect loss of function (LOF) variants and (2) to induce in p53-wild-type A459 cells resistance to Nutlin-3, in order to detect variants with dominant negative effect (DNE).We previously developed, in Epstein-Barr levitra-immortalised lymphocytes, a p53 functional assay exploring the transcriptional activity of the protein underlying its tumour suppressor activity.26 This assay is based on the exposure of cells to DNA damaging agents followed by the measurement of the p53 transcriptional response.27 28 With this assay, we showed that pathogenic TP53 variant carriers exhibit a constitutive defect in the transcriptional response to DNA damage, establishing a biological endophenotype associated with germline pathogenic variants.27 jason levitre 28 Compared with the other assays, its main advantage is to evaluate the impact of heterozygous variants in the genetic context of the patients.
Its main disadvantage is that it requires EBV immortalisation, which is time-consuming and, therefore, not suited for a rapid classification and interpretation of TP53 variants in medical practice.Therefore, despite the different tools indicated previously and before the completion in the future of curated international databases, interpretation of germline TP53 variants remains challenging in clinical practice. This prompted us to develop jason levitre a p53 functional assay derived from the previous one but performed on fresh blood samples and suitable for rapid interpretation and medical management of patients. We show here that this assay can accurately detect pathogenic variants and can be used to reallocate unclassified variants by integrating the results to the classification strategy.22 Furthermore, this assay revealed that a TP53 polymorphism (rs78378222), present in 1.7% of the European population, compromises p53 functional activity with the same magnitude as a heterozygous null variant, when carried on both alleles.MethodsCell culture and treatmentEBV-immortalised cell lines were maintained in RPMI 1640 medium (GIBCO.
Life Technologies, Carlsbad, California, USA) with jason levitre 10% fetal calf serum (Invitrogen, Life Technologies) and 1% L-glutamine (Invitrogen) at 37ðC with 5% CO2. Cells were seeded in duplicate in 12-well plates (Corning, New York, USA) at a density of 106âÂÂcells/well. Cells were treated or not with 200âÂÂng/mL (0.3âÂÂõM final concentration) of doxorubicin jason levitre (Sigma Aldrich, St.
Louis, Missouri, USA) for 8âÂÂhours. Cells were washed with 1àPBS and harvested for RNA extraction.Peripheral blood mononuclear cell (PBMC) isolation and cultureBlood samples were collected in EDTA tubes and kept for 2 days at room temperature jason levitre before PBMC isolation on a lymphocyte separation medium (Eurobio, Evry, France). From 2.5 to 10.0âÂÂmL of blood per jason levitre patient was used for PBMC isolation.
Cell number and cell viability were assessed on a NanoEnTek Adam automatic cell counter with the AccuChip Kit (ScienceTEC, Villebon-sur-Yvette, France). One million cells were seeded per well in a 24-well plate and were let to grow for 48âÂÂhours in a lymphocyte activating medium jason levitre (Chromosome Medium P, AmpliTech, Compiègne, France). At least two wells were seeded per patient (treated and untreated) and duplicates or triplicates were performed whenever possible.
Cells were treated with 800âÂÂng/mL of doxorubicin for 8âÂÂhours, washed with 1àPBS, harvested and RNA extraction was performed using the NucleoSpin RNA XS kit (Macherey Nagel, Düren, Germany) according to the manufacturerâÂÂs instructions and quantified using a UV-VIS ND-1000 spectrophotometer (Biocompare, Nanodrop Technologies, USA).RNA-SeqFour control EBV cell lines wild-type for TP53 and four heterozygous TP53-mutant cell lines, corresponding to three canonical dominant negative missense variants (p.(Arg175His), p.(Arg248Trp) and p.(Arg273His)) and one complete deletion of the TP53 locus, were treated jason levitre or not with doxorubicin. RNA was extracted using the Nucleospin RNAII kit (Macherey Nagel). Libraries were prepared using the NEBNext Ua Directional RNA Library Kit for Illumina (NEB, jason levitre Ipswich, USA) and NGS sequencing of the libraries was performed on an Illumina NextSeq500 (Illumina, San Diego, USA) using 2*75âÂÂbp sequencing to generate 50M read pairs on average per sample.
Experiments were performed in jason levitre triplicates. Bioinformatic analysis was carried out using an in-house automated pipeline AURIGA that uses the STAR V.2.5.3a tool for alignment, FeatureCounts tool V.1.5.2 for read counting and DESeq2 V.1.18.1 for statistical analysis.Selection of biomarkers indicative of p53-transcriptional activityNew biomarkers were selected among the transcripts strongly up-egulated by doxorubicin in control cells but not in the cells harbouring heterozygous TP53 alterations. CEP170B (NM_015005), PODXL (MIM*602632, NM_001018111), RRAD (MIM*179503, NM_004165), GLS2 (MIM*606365, NM_013267), CABYR (MIM*612135, jason levitre NM_012189), TP53I3 (MIM*605171, NM_004881), EPS8L2 (MIM*614988, NM_022772), SULF2 (MIM*610013, NM_001161841), SESN1 (MIM*606103, NM_014454) and FHL2 (MIM*602633, NM_201555).
Three control transcripts with a steady expression across all conditions and genotypes and expressed at the same level as the selected targets were also selected. TBP (MIM*600075, NM_003194), RIC8B (MIM*609147, jason levitre NM_001330145) and MPP5 (MIM*606958, NM_022474.3). An internal control of treatment efficacy was included.
PLK1 (MIM*602098, NM_005030.5), whose transcript is downregulated by doxorubicin treatment both in wild-type and jason levitre mutant cells.Reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment (RT-QMPSF)Reverse transcription (RT) was performed on 100âÂÂng of total RNA using the Verso cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). RT-QMPSF was performed on 1.5âÂÂõL of RT using Diamond Taq DNA polymerase (Kaneka Eurogentec, Seraing, Belgium), 6% Dymethyl sulfoxide and 26 PCR cycles (94ðC. 30âÂÂs/58ðC.
1âÂÂmin/72ðC. 30âÂÂs). Primer sequences are listed in online supplemental table 1.
Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using GeneScan 3.7 software.Supplemental materialReverse transcriptionâÂÂmultiplex ligation probe amplification (RT-MLPA)RT-MLPA probes were pooled at a concentration of 1âÂÂfmol/õL each in 10âÂÂmM Tris/1âÂÂmM EDTA. Probe sequences are given in online supplemental table 1. RT (6.5âÂÂõL), probe mixture (1.5âÂÂõL) and SALSA-MLPA buffer (1.5âÂÂõL, MRC-Holland, Amsterdam, The Netherlands) were mixed before denaturation (95ðC, 2âÂÂmin) and hybridisation (60ðC, 1âÂÂhour).
Ligation was performed at 54ðC for 15âÂÂmin, adding 32âÂÂõL of ligation mixture, and heated 5âÂÂmin at 98ðC. Then, 2.5âÂÂõL of the ligation was added to 7.5âÂÂõL of a Q5Hot Start High-Fidelity 2X Master Mix (NEB) supplemented with universal fluorescent PCR primers. PCR was performed using 35 cycles (94ðC.
30âÂÂs/58ðC. 30âÂÂs/72ðC. 30âÂÂs).
Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer using GeneScan V.3.7 software.Calculation of p53 functionality score and p53 mRNA ratioThe RT-MLPA or RT-QMPSF profiles of doxorubicin-treated and untreated cells were superimposed after adjusting the control amplicons to the same height. In the treated condition, the peak height of each of the 10âÂÂp53 target genes was measured and divided by the sum of the heights of the three control genes. This value was then divided by the same ratio calculated in the untreated condition.
In the assay, the mean of the 10 values defines the p53 functionality score. The final p53 functionality score is the mean of the scores obtained in RT-MLPA and RT-QMPSF assays. The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals.
The efficacy of the genotoxic treatment was assessed by calculating a PLK1 (MIM*602098) ratio (treated/untreated) normalised with the three controls, which should be less than 0.5.ResultsDevelopment of a rapid p53 functional assay performed on bloodThe rationale of the assay is that p53 acts as a powerful transcriptional inductor when DNA damage occurs and that the common deleterious impact of pathogenic variants is the alteration of this transcriptional activity.26 To develop a functional assay directly performed on patientâÂÂs fresh blood, we first optimised the quantitative assay that we had previously developed in EBV-immortalised cell lines.27 28 To this aim, we performed a new comparative transcriptomic analysis using RNA-Seq, including non-polyadenylated RNAs. Four control EBV cell lines wild type for TP53 and four patients with LFS EBV cell lines were compared in the context of genotoxic stress induced by doxorubicin treatment. We selected 10 biomarkers corresponding to p53 targets involved in different biological pathways controlled by p53, such as cell adhesion and migration, cellular response to stress, apoptosis, cytoskeleton organisation, glycolysis or regulation of other metabolic pathways.
To normalise the results, we selected three transcripts with a steady expression across all conditions and genotypes. All these biomarkers were then included in two quantitative assays based on RT-MLPA and RT-QMPSF. To detect in the same assay the potential effect of variants on the TP53 transcript levels, we added different amplicons or probes corresponding to TP53 cDNA.
As a defect in treatment efficacy would result in a low functionality score leading to the misinterpretation of a wild-type genotype as a mutant one, we also integrated in the assays an internal control of treatment efficacy. After exposure to doxorubicin, cells were harvested and the RT-MLPA and RT-QMPSF assays were performed in parallel for each sample to increase the robustness of the assay. An arbitrary functionality score was calculated from the induction score of the 10âÂÂp53 targets.
The p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals. This new quantitative assay, based on both RT-QMPSF and RT-MLPA, was first validated on 31 lymphoblastoid cell lines derived from patients with LFS harbouring different germline heterozygous TP53 variants (online supplemental table 2).Supplemental materialWe then set up the conditions allowing the assay to be performed directly on the patientsâ peripheral blood. Blood was collected in conventional EDTA tubes and kept at room temperature for 2 days to mimic sample shipping delays.
PBMCs were isolated and cultured for 48âÂÂhours in a lymphocyte activating medium. Under these conditions, a strong p53 transcriptional response could be monitored in wild-type individuals (figure 1), indicating that testing p53 function directly on patientsâ blood cells was feasible.P53 functional assay on peripheral blood. (A) Schematic representation of the blood p53 functional assay workflow.
(B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells.
Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5). In the treated condition, the peak height of each of the 10âÂÂp53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score.
The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell. RT-MLPA, reverse transcriptionâÂÂmultiplex ligation probe amplification.
RT-QMPSF, reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment." data-icon-position data-hide-link-title="0">Figure 1 P53 functional assay on peripheral blood. (A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype.
The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells. Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5).
In the treated condition, the peak height of each of the 10âÂÂp53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals.
PBMC, peripheral blood mononuclear cell. RT-MLPA, reverse transcriptionâÂÂmultiplex ligation probe amplification. RT-QMPSF, reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment.p53 functional analysis of patientâÂÂs blood cells with different TP53 genotypesWe then applied the p53 functional assay on blood samples sent to our laboratory for TP53 molecular analysis (NGS screening of the 11 exons complemented by QMPSF).
Molecular and functional analyses were performed in parallel, in double blind conditions. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.
This sample reflects the real-life recruitment of our diagnostic laboratory as it includes unaffected individuals as well as individuals affected by cancer who may have undergone different chemotherapy treatments. Molecular analyses revealed that 51 individuals had no detectable germline TP53 variant. For these 51 individuals, the mean p53 functionality score measured was 12.7 (13.6 for the RT-QMPSF assay and 11.9 for the RT-MLPA assay) with a range of 7.5âÂÂ22.8 (online supplemental table 3 and figure 2).
The mean observed p53 mRNA levels were 93% with a range of 74%âÂÂ125% (online supplemental table 3). In eight tested individuals, molecular analysis revealed seven distinct TP53 variants which could be considered as likely pathogenic or pathogenic based on their ClinVar classification or their truncating nature (table 1). All the variants tested were confirmed to be germline heterozygous variants.
For these eight patients, the assay yielded a reduced score compared with the wild-type individuals (mean 4.8, range 3.1âÂÂ7.1. Table 1 and figure 2). In the patients with missense variants, p53 mRNA levels were above 75%.
In contrast, p53 mRNA was clearly reduced in patients harbouring frameshift or splice variants (mean 58%, table 1 and figure 2) probably reflecting the activity of the nonsense-mediated mRNA decay.Supplemental materialView this table:Table 1 Interpretation of germline TP53 variants integrating the blood p53 functional assayp53 functional scores and mRNA level ratios in individuals with wild-type TP53 or with germline TP53 variants. (A) p53 functionality scores obtained in 51 wild-type TP53 individuals, compared with the scores obtained for nine samples from eight individuals carrying a classified TP53 variant (online supplemental table 3) using a Mann-Whitney non-parametric test. (B) Comparison of the p53 mRNA ratios obtained in 51 wild-type TP53 individuals and in samples carrying a missense (five samples) or a truncating variant of TP53 (four samples), using a Kruskal-Wallis test with Dunns post-test (p=0.0031).
***PFigure 3 Impact of the heterozygous and homozygous TP53 c.*1175A>C variation on p53 pre-mRNA 3â² end processing. (A) Schematic representation of the TP53 3â² end region. The c.*1175A>C variant is predicted to yield at least two different transcripts.
The upper one corresponds to the normal transcript with pre-mRNA cleavage and polyadenylation, and the lower one to longer transcript that extends after the poly-A signal. ÃÂÂExon 11â primers amplify both transcripts, while âÂÂpostpoly-Aâ primers specifically amplify the longer transcripts. As postpoly-A primers could also amplify gDNA, primers âÂÂexon 7â and âÂÂexon 10âÂÂ, which are specific to gDNA, were added to the reaction in order to monitor DNA contamination.
(B) RT-QMPSF result obtained for the index caseâÂÂs father (individual 58, S1. Table 1 and online supplemental table 3) carrying the variant TP53 c.723del, p.(Cys242Alafs*5). The profile (in red) was superimposed on the profile of a control individual wild type for TP53 (in blue), using the control amplicons RIC8B and TBP.
(C) RT-QMPSF result obtained for the index caseâÂÂs mother (individual 76, S1. Table 1 and online supplemental table 3) carrying the c.*1175A>C variant at the homozygous state. (D) RT-QMPSF result for the index case (individual 77, online supplemental table 3) carrying the c.723del, p.(Cys242Alafs*5) variant and the c.*1175A>C in trans.
Red arrows indicate the appearance of longer p53 transcripts. The horizontal bars show the reduction of the normal p53 transcript level, as compared with the control. RT-QMPSF, reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment.DiscussionThe interpretation of germline TP53 variants in patients with cancer is critical and should be performed before starting treatment considering their medical impact.
The main objective of our assay was to provide a fast functional classification of rare uncharacterised variants in order to help clinicians with decision-making. Compared with the previous assay that we developed in EBV-immortalised lymphocytes,27 28 this blood assay does not require long-term cell culture and the results can be obtained within 1âÂÂweek, fulfilling the timing required for diagnostic practice. The only constraint is to perform it within 48âÂÂhours after blood sampling in order to obtain robust results.
Under these conditions, we were able to successfully analyse samples sent from other European countries.Our assay fulfils most of the recommendations recently published by the Clinical Genome Resource Sequence Variant Interpretation working group regarding the clinical validity of functional assays29. (1) compared with the previously described p53 functional assays that test in vitro either cloned cDNA in yeast or artificial mutant libraries in cancer cell lines,23âÂÂ25 this blood assay is performed in clinical samples in the patientsâ genetic context. (2) the assay evaluates the transcriptional activity of p53 and not a specific domain of the protein.
(3) it analyses simultaneously the impact of the variant on protein function and mRNA levels. (4) it was validated using 51 wild-type TP53 controls and 8 patients with seven distinct pathogenic or likely-pathogenic TP53 variants. And finally, (5) results show the robustness of the assay.
Indeed, as shown in table 1, for 12 tested variants, we were able to perform the assay on EBV-immortalised cell lines and the results were very similar. Moreover, for five individuals, two different blood samples were tested and yielded similar results (table 1), and two variants (c.844C>T, p.(Arg282Trp). C.847C>T, p.(Arg283Cys)) were tested on two different individualsâ blood with concordant results (4.8 vs 5.0 and 5.3 vs 6.4).We observed among the wild-type TP53 individuals a wide range of functionality scores (7.5âÂÂ22.8).
This probably suggests that there is a variability of the p53-mediated transcriptional response to DNA damage in the general population, although no obvious impact of age, clinical status or sex could be observed. The thresholds used in this study could be refined by testing additional deleterious variants. Despite this variability, all pathogenic/likely pathogenic variants generated low p53 functionality scores, and variants resulting in premature stop codons were also detected by a clear reduction of p53 mRNA levels.
In addition, our assay allows testing of non-missense variants such as in frame indels. It should be highlighted that none of the previously published functional assays can be considered as a gold-standard method to classify germline TP53 variants.23âÂÂ25 Therefore, no available p53 functional assay can be used to calibrate the blood assay. Indeed, as illustrated in table 1, discordant results were obtained for variants unambiguously classified in ClinVar as pathogenic or likely pathogenic.
In particular, the founder Brazilian p.(Arg337His), an example of a variant with low penetrance, highlights the limits of the available tools. Whereas segregation data performed on large Brazilian pedigrees have clearly shown that this variant is pathogenic,34 bioinformatic predictions and functional analyses35 are conflicting (table 1). Our blood functional assay clearly shows that this variant alters the transcriptional activity of p53, although to a lesser extent than DNE missense variations, highlighting the limits of functional assays based on overexpression of cDNA.
This result was confirmed in four additional patients carrying this variant using EBV cell lines (table 1).The blood functional assay performed on PBMC harbouring unclassified variants led us to consider 12 variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del)) as âÂÂfunctionally abnormalâÂÂ, some with high impact. The interpretation is particularly challenging for p.(Pro72His), p.(Arg110His), p.(Arg158Cys), p.(Arg283Cys) and p.(Asp352Tyr) variants, as they were considered in yeast assays as functional or partially functional, and the Giacomelli assay classified them as not LOF_not DNE or was not conclusive. The low functionality score observed for p.(Arg110His) was confirmed in an EBV cell line derived from the patient and confirmed in two EBV cell lines from other patients carrying this variant.
The result for the p.(Asp352Tyr) variant was confirmed on a second blood sample and with an EBV cell line derived from another patient also carrying this variant. The effect of p.(Arg283Cys) was also confirmed in EBV cell lines derived from the patient and from three additional patients with the same variant (table 1).The clinical utility of the p53 functional assay is highlighted by the p.(Pro191Arg) variant. This variant was initially detected in a child with medulloblastoma at 2 years of age and whose brother died from a fibrosarcoma.
Presymptomatic testing revealed that an unaffected brother (18 months), the mother and two maternal aunts were also carriers. We were then requested to evaluate this variant, and the functional assay performed in the maternal aunt (individual 65, online supplemental table 3) clearly showed that this variant does not alter the p53 transcriptional activity (table 1 and online supplemental table 3). Considering this result, segregation analysis was performed on the brotherâÂÂs fibrosarcoma sample, revealing the absence of the variant and consolidating the conclusion of a non-pathogenic variant.Our results show that this blood functional assay is also able to detect TP53 variations outside the coding regions, which are the only regions commonly analysed.
Thanks to this assay, we discovered that the unaffected mother of an index case was homozygous for the polymorphic c.*1175A>C variant, and we show that this variant decreases p53 mRNA by altering the polyadenylation signal and produces longer transcripts extending beyond the poly-A site, as previously reported.30 When present on both alleles, this variant impacts p53 functionality with the same magnitude as a germline pathogenic TP53 variant. This prompted us to recommend breast MRI every year for this unaffected adult relative. We had the opportunity to perform the assay on EBV-immortalised lymphocytes harbouring only this heterozygous variant, and we observed a normal score (data not shown), suggesting that the heterozygous c.*1175A>C variant alone is insufficient to alter p53 function.
The comparison of the p53 functional scores observed in the index case who developed a high-grade glioma at 5 years of age and harbours the null c.723del, p.(Cys242Alafs*5) variant and in trans the polymorphic c.*1175A>C variant, and in her father carrying only the TP53 null variant suggests that the c.*1175A>C variant may act as a genetic modifier in pathogenic TP53 variant carriers and could increase the risk of glioma in carriers, as previously shown in the general population.30âÂÂ33In summary, we suggest that our blood p53 functional assay should be a useful tool not only for the rapid interpretation of germline TP53 variants of unknown significance in clinical practice, in complement to the previously developed assays, but also for the indirect detection of cryptic alterations within regulatory regions impacting p53 function.Data availability statementAll data relevant to the study are included in the article or uploaded as supplementary information. Deidentified participant data are available from thierry.frebourg@chu-rouen.fr.Ethics statementsPatient consent for publicationNot required.AcknowledgmentsThe authors are grateful to their French and European colleagues for providing clinical information and sending blood samples for TP53 analysis. The authors are indebted to Philippe Ruminy (Inserm U1245, Comprehensive Cancer Centre Becquerel, Rouen) for advices on the reverse transcriptionâÂÂmultiplex ligation probe amplification experiments and to Nikki Sabourin-Gibbs (Rouen University Hospital) for her assistance in editing the manuscript..
IntroductionIdentification of 20mg levitra price a germline pathogenic TP53 (MIM. *191170) variant in a patient with cancer has drastic medical impacts.1 Indeed, in TP53 variant carriers, chemotherapy and radiotherapy have been shown to contribute to the development of subsequent primary cancers, the incidence of which is remarkably high (above 40%).1âÂÂ4 Therefore, in these patients, surgical treatment should be prioritised and radiotherapy and chemotherapy avoided, if possible, or at 20mg levitra price least carefully discussed in terms of benefit:risk ratio between risk of recurrence and risk of inducing second primary tumours. Furthermore, TP53 variant carriers should have specific surveillance protocols, including annual whole-body MRI,5 6 whose efficiency for early tumour detection has recently been shown by numerous studies.5âÂÂ14Interpretation of germline TP53 variants, which are mainly missense variants, remains particularly complex. Whereas germline variants of TP53 were initially detected in Li-Fraumeni syndrome (LFS, MIM#151623),15âÂÂ17 our perception of cancers related to germline alterations of TP53 has drastically evolved over time.1 2 18 19 The presence of a disease-causing germline variant should be considered 20mg levitra price in patients fulfilling Chompret criteria, which were sequentially updated and extended.1 The question of germline TP53 variant interpretation is becoming a growing concern in the field because the TP53 gene is currently included in many cancer gene panels, and the number of TP53 tests performed in patients not fulfilling the criteria mentioned earlier has increased exponentially. 20 21Classification of TP53 variants, in agreement with the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines, is based on several items, including frequency of the variant in the general population (gnomAD.
Https://gnomad.broadinstitute.org/), segregation data, bioinformatics predictions and functional assays developed in yeast or human cancer cell lines.22 One of the first assays commonly used for TP53 missense variant interpretation was developed in yeast and is based on the expression of TP53 cDNA in strains containing reporter plasmids with different p53 binding sites.23 In this assay, p53 variants are classified as functional, not functional or partially functional if the transcriptional activity is conserved for 20mg levitra price some but not all yeast reporter plasmids (http://p53.iarc.fr/). More recently, two teams have developed in human cancer cell lines high throughput p53 functional assays.24 25 Kotler et al24 generated a synthetic library of TP53 variants located within the p53 DNA-binding domain and quantified the antiproliferative activity of these variants in the p53-null H1299 cancer cell line. In this assay, TP53 variants are 20mg levitra price categorised as âÂÂwild-type TP53-like variantâ (functional) or âÂÂdisruptingâ (non-functional). In another assay, Giacomelli et al25 generated by saturation mutagenesis a TP53 library and tested the ability of the variants (1) to restore the survival of the p53-null A459 cell line exposed to high doses of DNA damaging agents, in order to detect loss of function (LOF) variants and (2) to induce in p53-wild-type A459 cells resistance to Nutlin-3, in order to detect variants with dominant negative effect (DNE).We previously developed, in Epstein-Barr levitra-immortalised lymphocytes, a p53 functional assay exploring the transcriptional activity of the 20mg levitra price protein underlying its tumour suppressor activity.26 This assay is based on the exposure of cells to DNA damaging agents followed by the measurement of the p53 transcriptional response.27 28 With this assay, we showed that pathogenic TP53 variant carriers exhibit a constitutive defect in the transcriptional response to DNA damage, establishing a biological endophenotype associated with germline pathogenic variants.27 28 Compared with the other assays, its main advantage is to evaluate the impact of heterozygous variants in the genetic context of the patients. Its main disadvantage is that it requires EBV immortalisation, which is time-consuming and, therefore, not suited for a rapid classification and interpretation of TP53 variants in medical practice.Therefore, despite the different tools indicated previously and before the completion in the future of curated international databases, interpretation of germline TP53 variants remains challenging in clinical practice.
This prompted us to develop a p53 functional assay derived from the previous one but performed on fresh blood samples and suitable for rapid 20mg levitra price interpretation and medical management of patients. We show here that this assay can accurately detect pathogenic variants and can be used to reallocate unclassified variants by integrating the results to the classification strategy.22 Furthermore, this assay revealed that a TP53 polymorphism (rs78378222), present in 1.7% of the European population, compromises p53 functional activity with the same magnitude as a heterozygous null variant, when carried on both alleles.MethodsCell culture and treatmentEBV-immortalised cell lines were maintained in RPMI 1640 medium (GIBCO. Life Technologies, 20mg levitra price Carlsbad, California, USA) with 10% fetal calf serum (Invitrogen, Life Technologies) and 1% L-glutamine (Invitrogen) at 37ðC with 5% CO2. Cells were seeded in duplicate in 12-well plates (Corning, New York, USA) at a density of 106âÂÂcells/well. Cells were treated or 20mg levitra price not with 200âÂÂng/mL (0.3âÂÂõM final concentration) of doxorubicin (Sigma Aldrich, St.
Louis, Missouri, USA) for 8âÂÂhours. Cells were washed with 1àPBS and harvested for RNA extraction.Peripheral blood mononuclear cell (PBMC) isolation and cultureBlood samples were collected in EDTA tubes and kept for 2 days at room temperature before PBMC isolation on a lymphocyte separation medium (Eurobio, Evry, France) 20mg levitra price. From 2.5 20mg levitra price to 10.0âÂÂmL of blood per patient was used for PBMC isolation. Cell number and cell viability were assessed on a NanoEnTek Adam automatic cell counter with the AccuChip Kit (ScienceTEC, Villebon-sur-Yvette, France). One million cells were seeded per well in a 24-well plate and were let to grow for 48âÂÂhours in a lymphocyte activating medium 20mg levitra price (Chromosome Medium P, AmpliTech, Compiègne, France).
At least two wells were seeded per patient (treated and untreated) and duplicates or triplicates were performed whenever possible. Cells were treated with 800âÂÂng/mL of doxorubicin for 8âÂÂhours, washed with 20mg levitra price 1àPBS, harvested and RNA extraction was performed using the NucleoSpin RNA XS kit (Macherey Nagel, Düren, Germany) according to the manufacturerâÂÂs instructions and quantified using a UV-VIS ND-1000 spectrophotometer (Biocompare, Nanodrop Technologies, USA).RNA-SeqFour control EBV cell lines wild-type for TP53 and four heterozygous TP53-mutant cell lines, corresponding to three canonical dominant negative missense variants (p.(Arg175His), p.(Arg248Trp) and p.(Arg273His)) and one complete deletion of the TP53 locus, were treated or not with doxorubicin. RNA was extracted using the Nucleospin RNAII kit (Macherey Nagel). Libraries were prepared using the NEBNext Ua Directional RNA Library Kit for Illumina (NEB, Ipswich, USA) and NGS sequencing of the libraries was performed on an Illumina NextSeq500 (Illumina, San Diego, USA) 20mg levitra price using 2*75âÂÂbp sequencing to generate 50M read pairs on average per sample. Experiments were performed 20mg levitra price in triplicates.
Bioinformatic analysis was carried out using an in-house automated pipeline AURIGA that uses the STAR V.2.5.3a tool for alignment, FeatureCounts tool V.1.5.2 for read counting and DESeq2 V.1.18.1 for statistical analysis.Selection of biomarkers indicative of p53-transcriptional activityNew biomarkers were selected among the transcripts strongly up-egulated by doxorubicin in control cells but not in the cells harbouring heterozygous TP53 alterations. CEP170B (NM_015005), PODXL (MIM*602632, NM_001018111), RRAD (MIM*179503, NM_004165), GLS2 (MIM*606365, NM_013267), CABYR (MIM*612135, NM_012189), TP53I3 (MIM*605171, NM_004881), EPS8L2 (MIM*614988, NM_022772), SULF2 20mg levitra price (MIM*610013, NM_001161841), SESN1 (MIM*606103, NM_014454) and FHL2 (MIM*602633, NM_201555). Three control transcripts with a steady expression across all conditions and genotypes and expressed at the same level as the selected targets were also selected. TBP (MIM*600075, NM_003194), RIC8B (MIM*609147, 20mg levitra price NM_001330145) and MPP5 (MIM*606958, NM_022474.3). An internal control of treatment efficacy was included.
PLK1 (MIM*602098, NM_005030.5), whose transcript is downregulated by doxorubicin treatment 20mg levitra price both in wild-type and mutant cells.Reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment (RT-QMPSF)Reverse transcription (RT) was performed on 100âÂÂng of total RNA using the Verso cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). RT-QMPSF was performed on 1.5âÂÂõL of RT using Diamond Taq DNA polymerase (Kaneka Eurogentec, Seraing, Belgium), 6% Dymethyl sulfoxide and 26 PCR cycles (94ðC. 30âÂÂs/58ðC. 1âÂÂmin/72ðC. 30âÂÂs).
Primer sequences are listed in online supplemental table 1. Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using GeneScan 3.7 software.Supplemental materialReverse transcriptionâÂÂmultiplex ligation probe amplification (RT-MLPA)RT-MLPA probes were pooled at a concentration of 1âÂÂfmol/õL each in 10âÂÂmM Tris/1âÂÂmM EDTA. Probe sequences are given in online supplemental table 1. RT (6.5âÂÂõL), probe mixture (1.5âÂÂõL) and SALSA-MLPA buffer (1.5âÂÂõL, MRC-Holland, Amsterdam, The Netherlands) were mixed before denaturation (95ðC, 2âÂÂmin) and hybridisation (60ðC, 1âÂÂhour). Ligation was performed at 54ðC for 15âÂÂmin, adding 32âÂÂõL of ligation mixture, and heated 5âÂÂmin at 98ðC.
Then, 2.5âÂÂõL of the ligation was added to 7.5âÂÂõL of a Q5Hot Start High-Fidelity 2X Master Mix (NEB) supplemented with universal fluorescent PCR primers. PCR was performed using 35 cycles (94ðC. 30âÂÂs/58ðC. 30âÂÂs/72ðC. 30âÂÂs).
Amplicons were analysed on an ABI Prism 3500 Genetic Analyzer using GeneScan V.3.7 software.Calculation of p53 functionality score and p53 mRNA ratioThe RT-MLPA or RT-QMPSF profiles of doxorubicin-treated and untreated cells were superimposed after adjusting the control amplicons to the same height. In the treated condition, the peak height of each of the 10âÂÂp53 target genes was measured and divided by the sum of the heights of the three control genes. This value was then divided by the same ratio calculated in the untreated condition. In the assay, the mean of the 10 values defines the p53 functionality score. The final p53 functionality score is the mean of the scores obtained in RT-MLPA and RT-QMPSF assays.
The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals. The efficacy of the genotoxic treatment was assessed by calculating a PLK1 (MIM*602098) ratio (treated/untreated) normalised with the three controls, which should be less than 0.5.ResultsDevelopment of a rapid p53 functional assay performed on bloodThe rationale of the assay is that p53 acts as a powerful transcriptional inductor when DNA damage occurs and that the common deleterious impact of pathogenic variants is the alteration of this transcriptional activity.26 To develop a functional assay directly performed on patientâÂÂs fresh blood, we first optimised the quantitative assay that we had previously developed in EBV-immortalised cell lines.27 28 To this aim, we performed a new comparative transcriptomic analysis using RNA-Seq, including non-polyadenylated RNAs. Four control EBV cell lines wild type for TP53 and four patients with LFS EBV cell lines were compared in the context of genotoxic stress induced by doxorubicin treatment. We selected 10 biomarkers corresponding to p53 targets involved in different biological pathways controlled by p53, such as cell adhesion and migration, cellular response to stress, apoptosis, cytoskeleton organisation, glycolysis or regulation of other metabolic pathways. To normalise the results, we selected three transcripts with a steady expression across all conditions and genotypes.
All these biomarkers were then included in two quantitative assays based on RT-MLPA and RT-QMPSF. To detect in the same assay the potential effect of variants on the TP53 transcript levels, we added different amplicons or probes corresponding to TP53 cDNA. As a defect in treatment efficacy would result in a low functionality score leading to the misinterpretation of a wild-type genotype as a mutant one, we also integrated in the assays an internal control of treatment efficacy. After exposure to doxorubicin, cells were harvested and the RT-MLPA and RT-QMPSF assays were performed in parallel for each sample to increase the robustness of the assay. An arbitrary functionality score was calculated from the induction score of the 10âÂÂp53 targets.
The p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals. This new quantitative assay, based on both RT-QMPSF and RT-MLPA, was first validated on 31 lymphoblastoid cell lines derived from patients with LFS harbouring different germline heterozygous TP53 variants (online supplemental table 2).Supplemental materialWe then set up the conditions allowing the assay to be performed directly on the patientsâ peripheral blood. Blood was collected in conventional EDTA tubes and kept at room temperature for 2 days to mimic sample shipping delays. PBMCs were isolated and cultured for 48âÂÂhours in a lymphocyte activating medium. Under these conditions, a strong p53 transcriptional response could be monitored in wild-type individuals (figure 1), indicating that testing p53 function directly on patientsâ blood cells was feasible.P53 functional assay on peripheral blood.
(A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells. Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5).
In the treated condition, the peak height of each of the 10âÂÂp53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell. RT-MLPA, reverse transcriptionâÂÂmultiplex ligation probe amplification.
RT-QMPSF, reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment." data-icon-position data-hide-link-title="0">Figure 1 P53 functional assay on peripheral blood. (A) Schematic representation of the blood p53 functional assay workflow. (B,C) Typical RT-QMPSF (B) and RT-MLPA (C) results obtained for individual 15 with a wild-type TP53 genotype. The fluorescent profiles of doxorubicin-treated cells (red line) and untreated cells (blue line) were superimposed using the three control amplicons (RIC8B, TBP and MPP5). The horizontal bars indicate for each p53 target gene the level of expression in untreated cells.
Treatment efficacy was evaluated by the transcriptional repression of the PLK1 marker (Plk1 treated/untreated ratio below 0.5). In the treated condition, the peak height of each of the 10âÂÂp53 target genes was measured and divided by the sum of the heights of the three control genes. This value was divided by the same ratio calculated in the untreated condition to yield an arbitrary p53 functionality score. The p53 mRNA levels were expressed as a ratio of the normal values obtained for three control individuals. PBMC, peripheral blood mononuclear cell.
RT-MLPA, reverse transcriptionâÂÂmultiplex ligation probe amplification. RT-QMPSF, reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment.p53 functional analysis of patientâÂÂs blood cells with different TP53 genotypesWe then applied the p53 functional assay on blood samples sent to our laboratory for TP53 molecular analysis (NGS screening of the 11 exons complemented by QMPSF). Molecular and functional analyses were performed in parallel, in double blind conditions. We analysed a total of 82 blood samples derived from 77 individuals (online supplemental table 3). These 77 individuals corresponded either to new index cases suspected to harbour a pathogenic TP53 variant or to relatives of index cases harbouring TP53 variants.
This sample reflects the real-life recruitment of our diagnostic laboratory as it includes unaffected individuals as well as individuals affected by cancer who may have undergone different chemotherapy treatments. Molecular analyses revealed that 51 individuals had no detectable germline TP53 variant. For these 51 individuals, the mean p53 functionality score measured was 12.7 (13.6 for the RT-QMPSF assay and 11.9 for the RT-MLPA assay) with a range of 7.5âÂÂ22.8 (online supplemental table 3 and figure 2). The mean observed p53 mRNA levels were 93% with a range of 74%âÂÂ125% (online supplemental table 3). In eight tested individuals, molecular analysis revealed seven distinct TP53 variants which could be considered as likely pathogenic or pathogenic based on their ClinVar classification or their truncating nature (table 1).
All the variants tested were confirmed to be germline heterozygous variants. For these eight patients, the assay yielded a reduced score compared with the wild-type individuals (mean 4.8, range 3.1âÂÂ7.1. Table 1 and figure 2). In the patients with missense variants, p53 mRNA levels were above 75%. In contrast, p53 mRNA was clearly reduced in patients harbouring frameshift or splice variants (mean 58%, table 1 and figure 2) probably reflecting the activity of the nonsense-mediated mRNA decay.Supplemental materialView this table:Table 1 Interpretation of germline TP53 variants integrating the blood p53 functional assayp53 functional scores and mRNA level ratios in individuals with wild-type TP53 or with germline TP53 variants.
(A) p53 functionality scores obtained in 51 wild-type TP53 individuals, compared with the scores obtained for nine samples from eight individuals carrying a classified TP53 variant (online supplemental table 3) using a Mann-Whitney non-parametric test. (B) Comparison of the p53 mRNA ratios obtained in 51 wild-type TP53 individuals and in samples carrying a missense (five samples) or a truncating variant of TP53 (four samples), using a Kruskal-Wallis test with Dunns post-test (p=0.0031). ***PFigure 3 Impact of the heterozygous and homozygous TP53 c.*1175A>C variation on p53 pre-mRNA 3â² end processing. (A) Schematic representation of the TP53 3â² end region. The c.*1175A>C variant is predicted to yield at least two different transcripts.
The upper one corresponds to the normal transcript with pre-mRNA cleavage and polyadenylation, and the lower one to longer transcript that extends after the poly-A signal. ÃÂÂExon 11â primers amplify both transcripts, while âÂÂpostpoly-Aâ primers specifically amplify the longer transcripts. As postpoly-A primers could also amplify gDNA, primers âÂÂexon 7â and âÂÂexon 10âÂÂ, which are specific to gDNA, were added to the reaction in order to monitor DNA contamination. (B) RT-QMPSF result obtained for the index caseâÂÂs father (individual 58, S1. Table 1 and online supplemental table 3) carrying the variant TP53 c.723del, p.(Cys242Alafs*5).
The profile (in red) was superimposed on the profile of a control individual wild type for TP53 (in blue), using the control amplicons RIC8B and TBP. (C) RT-QMPSF result obtained for the index caseâÂÂs mother (individual 76, S1. Table 1 and online supplemental table 3) carrying the c.*1175A>C variant at the homozygous state. (D) RT-QMPSF result for the index case (individual 77, online supplemental table 3) carrying the c.723del, p.(Cys242Alafs*5) variant and the c.*1175A>C in trans. Red arrows indicate the appearance of longer p53 transcripts.
The horizontal bars show the reduction of the normal p53 transcript level, as compared with the control. RT-QMPSF, reverse transcriptionâÂÂquantitative multiplex PCR of short fluorescent fragment.DiscussionThe interpretation of germline TP53 variants in patients with cancer is critical and should be performed before starting treatment considering their medical impact. The main objective of our assay was to provide a fast functional classification of rare uncharacterised variants in order to help clinicians with decision-making. Compared with the previous assay that we developed in EBV-immortalised lymphocytes,27 28 this blood assay does not require long-term cell culture and the results can be obtained within 1âÂÂweek, fulfilling the timing required for diagnostic practice. The only constraint is to perform it within 48âÂÂhours after blood sampling in order to obtain robust results.
Under these conditions, we were able to successfully analyse samples sent from other European countries.Our assay fulfils most of the recommendations recently published by the Clinical Genome Resource Sequence Variant Interpretation working group regarding the clinical validity of functional assays29. (1) compared with the previously described p53 functional assays that test in vitro either cloned cDNA in yeast or artificial mutant libraries in cancer cell lines,23âÂÂ25 this blood assay is performed in clinical samples in the patientsâ genetic context. (2) the assay evaluates the transcriptional activity of p53 and not a specific domain of the protein. (3) it analyses simultaneously the impact of the variant on protein function and mRNA levels. (4) it was validated using 51 wild-type TP53 controls and 8 patients with seven distinct pathogenic or likely-pathogenic TP53 variants.
And finally, (5) results show the robustness of the assay. Indeed, as shown in table 1, for 12 tested variants, we were able to perform the assay on EBV-immortalised cell lines and the results were very similar. Moreover, for five individuals, two different blood samples were tested and yielded similar results (table 1), and two variants (c.844C>T, p.(Arg282Trp). C.847C>T, p.(Arg283Cys)) were tested on two different individualsâ blood with concordant results (4.8 vs 5.0 and 5.3 vs 6.4).We observed among the wild-type TP53 individuals a wide range of functionality scores (7.5âÂÂ22.8). This probably suggests that there is a variability of the p53-mediated transcriptional response to DNA damage in the general population, although no obvious impact of age, clinical status or sex could be observed.
The thresholds used in this study could be refined by testing additional deleterious variants. Despite this variability, all pathogenic/likely pathogenic variants generated low p53 functionality scores, and variants resulting in premature stop codons were also detected by a clear reduction of p53 mRNA levels. In addition, our assay allows testing of non-missense variants such as in frame indels. It should be highlighted that none of the previously published functional assays can be considered as a gold-standard method to classify germline TP53 variants.23âÂÂ25 Therefore, no available p53 functional assay can be used to calibrate the blood assay. Indeed, as illustrated in table 1, discordant results were obtained for variants unambiguously classified in ClinVar as pathogenic or likely pathogenic.
In particular, the founder Brazilian p.(Arg337His), an example of a variant with low penetrance, highlights the limits of the available tools. Whereas segregation data performed on large Brazilian pedigrees have clearly shown that this variant is pathogenic,34 bioinformatic predictions and functional analyses35 are conflicting (table 1). Our blood functional assay clearly shows that this variant alters the transcriptional activity of p53, although to a lesser extent than DNE missense variations, highlighting the limits of functional assays based on overexpression of cDNA. This result was confirmed in four additional patients carrying this variant using EBV cell lines (table 1).The blood functional assay performed on PBMC harbouring unclassified variants led us to consider 12 variants (p.(Pro72His), p.(Gly105Asp), p.(Arg110His), p.(Phe134Leu), p.(Arg158Cys), p.(Pro278Arg), p.(Arg283Cys), p.(Leu348Ser), p.(Asp352Tyr), p.(Gly108_Phe109delinsVal), p.(Asn131del), p.(Leu265del)) as âÂÂfunctionally abnormalâÂÂ, some with high impact. The interpretation is particularly challenging for p.(Pro72His), p.(Arg110His), p.(Arg158Cys), p.(Arg283Cys) and p.(Asp352Tyr) variants, as they were considered in yeast assays as functional or partially functional, and the Giacomelli assay classified them as not LOF_not DNE or was not conclusive.
The low functionality score observed for p.(Arg110His) was confirmed in an EBV cell line derived from the patient and confirmed in two EBV cell lines from other patients carrying this variant. The result for the p.(Asp352Tyr) variant was confirmed on a second blood sample and with an EBV cell line derived from another patient also carrying this variant. The effect of p.(Arg283Cys) was also confirmed in EBV cell lines derived from the patient and from three additional patients with the same variant (table 1).The clinical utility of the p53 functional assay is highlighted by the p.(Pro191Arg) variant. This variant was initially detected in a child with medulloblastoma at 2 years of age and whose brother died from a fibrosarcoma. Presymptomatic testing revealed that an unaffected brother (18 months), the mother and two maternal aunts were also carriers.
We were then requested to evaluate this variant, and the functional assay performed in the maternal aunt (individual 65, online supplemental table 3) clearly showed that this variant does not alter the p53 transcriptional activity (table 1 and online supplemental table 3). Considering this result, segregation analysis was performed on the brotherâÂÂs fibrosarcoma sample, revealing the absence of the variant and consolidating the conclusion of a non-pathogenic variant.Our results show that this blood functional assay is also able to detect TP53 variations outside the coding regions, which are the only regions commonly analysed. Thanks to this assay, we discovered that the unaffected mother of an index case was homozygous for the polymorphic c.*1175A>C variant, and we show that this variant decreases p53 mRNA by altering the polyadenylation signal and produces longer transcripts extending beyond the poly-A site, as previously reported.30 When present on both alleles, this variant impacts p53 functionality with the same magnitude as a germline pathogenic TP53 variant. This prompted us to recommend breast MRI every year for this unaffected adult relative. We had the opportunity to perform the assay on EBV-immortalised lymphocytes harbouring only this heterozygous variant, and we observed a normal score (data not shown), suggesting that the heterozygous c.*1175A>C variant alone is insufficient to alter p53 function.
The comparison of the p53 functional scores observed in the index case who developed a high-grade glioma at 5 years of age and harbours the null c.723del, p.(Cys242Alafs*5) variant and in trans the polymorphic c.*1175A>C variant, and in her father carrying only the TP53 null variant suggests that the c.*1175A>C variant may act as a genetic modifier in pathogenic TP53 variant carriers and could increase the risk of glioma in carriers, as previously shown in the general population.30âÂÂ33In summary, we suggest that our blood p53 functional assay should be a useful tool not only for the rapid interpretation of germline TP53 variants of unknown significance in clinical practice, in complement to the previously developed assays, but also for the indirect detection of cryptic alterations within regulatory regions impacting p53 function.Data availability statementAll data relevant to the study are included in the article or uploaded as supplementary information. Deidentified participant data are available from thierry.frebourg@chu-rouen.fr.Ethics statementsPatient consent for publicationNot required.AcknowledgmentsThe authors are grateful to their French and European colleagues for providing clinical information and sending blood samples for TP53 analysis. The authors are indebted to Philippe Ruminy (Inserm U1245, Comprehensive Cancer Centre Becquerel, Rouen) for advices on the reverse transcriptionâÂÂmultiplex ligation probe amplification experiments and to Nikki Sabourin-Gibbs (Rouen University Hospital) for her assistance in editing the manuscript..
VARDENAFIL is used to treat erection problems in men. Vardenafil works faster than Sildenafil (Viagra®) and it is less likely to have visual disturbance side effect.
To the Editor generic levitra 40mg Cialis 5mg price in canada. Figure 1 generic levitra 40mg. Figure 1 generic levitra 40mg. erectile dysfunction Variants among Symptomatic generic levitra 40mg Health Workers.
Shown is the distribution of the B.1.1.7 (alpha), delta, and other erectile dysfunction variants according to vaccination status and month of diagnosis among health workers at University of California San Diego Health, March through July 2021. The number of workers indicates those who were symptomatic and had available variant data, and the number of positive tests indicates those that generic levitra 40mg included data on variants. In December 2020, the University of California San Diego Health (UCSDH) workforce experienced a dramatic increase in severe acute respiratory syndrome erectile dysfunction 2 generic levitra 40mg (erectile dysfunction) s. Vaccination with mRNA generic levitra 40mg treatments began in mid-December 2020.
By March, 76% of the workforce had been fully vaccinated, and by July, the percentage had risen to 87%. s had decreased dramatically by early February 2021.1 Between March and June, fewer than 30 health care workers tested positive each month generic levitra 40mg. However, coincident generic levitra 40mg with the end of CaliforniaâÂÂs mask mandate on June 15 and the rapid dominance of the B.1.617.2 (delta) variant that first emerged in mid-April and accounted for over 95% of UCSDH isolates by the end of July (Figure 1), s increased rapidly, including cases among fully vaccinated persons. Institutional review board approval was obtained for use of administrative data on vaccinations and case-investigation data to examine mRNA generic levitra 40mg SARS CoV-2 treatment effectiveness.
UCSDH has a low threshold for erectile dysfunction testing, which is triggered by the presence of at least one symptom during daily screening or by an identified exposure, regardless of vaccination status. From March generic levitra 40mg 1 to July 31, 2021, a total of 227 UCSDH health care workers tested positive for erectile dysfunction by reverse-transcriptaseâÂÂquantitative polymerase-chain-reaction (RT-qPCR) assay of nasal swabs. 130 of the 227 workers (57.3%) were fully vaccinated generic levitra 40mg. Symptoms were present in 109 of the 130 fully vaccinated workers (83.8%) generic levitra 40mg and in 80 of the 90 unvaccinated workers (88.9%).
(The remaining 7 workers were only partially vaccinated.) No deaths were reported in either group. One unvaccinated person was hospitalized generic levitra 40mg for erectile dysfunctionâÂÂrelated symptoms. Table 1 generic levitra 40mg. Table 1 generic levitra 40mg.
Symptomatic erectile dysfunction and mRNA treatment Effectiveness among UCSDH Health Workers, March through July 2021. treatment effectiveness was calculated for generic levitra 40mg each month from March through July. The case definition was a positive PCR test and one or more generic levitra 40mg symptoms among persons with no previous erectile dysfunction treatment (see the Supplementary Appendix). treatment effectiveness exceeded 90% from March generic levitra 40mg through June but fell to 65.5% (95% confidence interval [CI], 48.9 to 76.9) in July (Table 1).
July case rates were analyzed according to the month in which workers with erectile dysfunction treatment completed the vaccination series. In workers completing vaccination in January or February, the attack rate was 6.7 per 1000 persons (95% CI, 5.9 to 7.8), whereas the attack rate was 3.7 generic levitra 40mg per 1000 persons (95% CI, 2.5 to 5.7) among those who completed vaccination during the period from March through May. Among unvaccinated persons, the July attack rate was 16.4 per 1000 persons (95% CI, 11.8 to 22.9) generic levitra 40mg. The SARS CoV-2 mRNA treatments, BNT162b2 (PfizerâÂÂBioNTech) and mRNA-1273 (Moderna), have previously shown efficacy rates of 95% and 94.1%,2 generic levitra 40mg respectively, in their initial clinical trials, and for the BNT162b2 treatment, sustained, albeit slightly decreased effectiveness (84%) 4 months after the second dose.3 In England, where an extended dosing interval of up to 12 weeks was used, Lopez Bernal et al.
Reported a preserved treatment effectiveness of 88% against symptomatic disease associated with the delta variant.4 As observed by others in populations that received mRNA treatments according to standard Emergency Use Authorization intervals,5 our data suggest that treatment effectiveness against any symptomatic disease is considerably lower against the delta variant and may wane over time since vaccination. The dramatic change in treatment effectiveness from June to July is likely to be due to both the emergence of the delta variant and waning immunity over time, compounded by the end of masking requirements in generic levitra 40mg California and the resulting greater risk of exposure in the community. Our findings underline the importance of rapidly reinstating nonpharmaceutical interventions, such as indoor masking and intensive testing strategies, in addition to continued efforts to increase vaccinations, as strategies to prevent avoidable illness and deaths and to avoid mass disruptions generic levitra 40mg to society during the spread of this formidable variant. Furthermore, if our findings on waning immunity are generic levitra 40mg verified in other settings, booster doses may be indicated.
Jocelyn Keehner, M.D.Lucy E. Horton, M.D., M.P.H.UC San Diego Health, San Diego, generic levitra 40mg CANancy J. Binkin, M.D., generic levitra 40mg M.P.H.UC San Diego, La Jolla, CALouise C. Laurent, M.D., Ph.D.David Pride, M.D., generic levitra 40mg Ph.D.Christopher A.
Longhurst, M.D.Shira R. Abeles, M.D.Francesca J generic levitra 40mg. Torriani, M.D.UC San Diego generic levitra 40mg Health, San Diego, CA [email protected] Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org. This letter generic levitra 40mg was published on September 1, 2021, and updated on September 3, 2021, at NEJM.org.
Dr. Laurent serves as an author on behalf of the SEARCH Alliance generic levitra 40mg. Collaborators in the SEARCH Alliance are listed in the Supplementary generic levitra 40mg Appendix, available with the full text of this letter at NEJM.org. Drs generic levitra 40mg.
Keehner and Horton and Drs. Abeles and Torriani contributed equally to this letter generic levitra 40mg. 5 References1 generic levitra 40mg. Keehner J, Abeles generic levitra 40mg SR, Torriani FJ.
More on erectile dysfunction after vaccination in health care workers. Reply. N Engl J Med 2021;385(2):e8.2. Baden LR, El Sahly HM, Essink B, et al.
Efficacy and safety of the mRNA-1273 erectile dysfunction treatment. N Engl J Med 2021;384:403-416.3. Thomas SJ, Moreira ED Jr, Kitchin N, et al. Six month safety and efficacy of the BNT162b2 mRNA erectile dysfunction treatment.
July 28, 2021 (https://www.medrxiv.org/content/10.1101/2021.07.28.21261159v1). Preprint.Google Scholar4. Lopez Bernal J, Andrews N, Gower C, et al. Effectiveness of erectile dysfunction treatments against the B.1.617.2 (Delta) variant.
N Engl J Med 2021;385:585-594.5. Israel A, Merzon E, Schäffer AA, et al. Elapsed time since BNT162b2 treatment and risk of erectile dysfunction in a large cohort. August 5, 2021 (https://www.medrxiv.org/content/10.1101/2021.08.03.21261496v1).
Preprint.Google Scholar10.1056/NEJMc2112981-t1Table 1. Symptomatic erectile dysfunction and mRNA treatment Effectiveness among UCSDH Health Workers, March through July 2021.* MarchAprilMayJuneJulyUCSDH workforce â no. Of persons18,96418,99219,00019,03519,016Vaccination status â no. Of personsFully vaccinatedâ 14,47015,51016,15716,42616,492mRNA-1273 (Moderna)6,6087,0057,3407,4517,464BNT162b2 (PfizerâÂÂBioNTech)7,8628,5058,8178,9759,028Unvaccinated3,2302,5092,1872,0591,895Percentage of workers fully vaccinated76.381.785.086.386.7Symptomatic erectile dysfunction treatmentFully vaccinated workers343594Unvaccinated workers1117101031Percentage of cases in fully vaccinated workers21.419.023.133.375.2Attack rate per 1000 (95% CI)Fully vaccinated workers0.21 (0.21âÂÂ0.47)0.26 (0.26âÂÂ0.50)0.19 (0.21âÂÂ0.40)0.30 (0.31âÂÂ0.53)5.7 (5.4âÂÂ6.2)Unvaccinated workers3.4 (2.1âÂÂ5.9)6.8 (4.5âÂÂ10.6)4.6 (2.6âÂÂ8.2)4.9 (2.9âÂÂ8.7)16.4 (11.8âÂÂ22.9)treatment effectiveness â % (95% CI)93.9 (78.2âÂÂ97.9)96.2 (88.7âÂÂ98.3)95.9 (85.3âÂÂ98.9)94.3 (83.7âÂÂ98.0)65.5 (48.9âÂÂ76.9)Study Sample A total of 103,199 hospitalizations of patients with erectile dysfunction treatmentâÂÂlike illness who were 50 years of age or older were identified by the seven VISION partners.
Of these hospitalizations, 64,400 (62%) occurred after the dates of age-specific erectile dysfunction treatment eligibility and the time required for vaccination records to be updated (Table S3). The hospitalizations occurred during the period from January 1 through June 22, 2021. Among unvaccinated patients who were hospitalized, the median duration from treatment eligibility to the index date was 39 days (interquartile range, 16 to 70) (Table S4). erectile dysfunction testing with a molecular assay ordered by clinicians was conducted for 74% of the patients who were hospitalized (range across network partners, 55 to 99).
During the period from January 1 through June 22, a total of 121,709 visits to emergency departments or urgent care clinics for erectile dysfunction treatmentâÂÂlike illness were identified by three partners. 76,220 visits (63%) occurred after treatment age eligibility and updates to vaccination records (Table S5). Among the patients who visited an emergency department or urgent care clinic, the median duration from treatment eligibility to the index date was 39 days (interquartile range, 15 to 70). 30% (range, 25 to 41) of these patients were tested by means of molecular assay.
Across the partners, 1872 hospitalizations and 1350 emergency department or urgent care clinic visits were excluded because the index dates occurred 1 to 13 days after the patient received the first dose of erectile dysfunction treatment and immunity was considered indeterminant. Table 2. Table 2. Characteristics of the Patients According to erectile dysfunction Test Results and Vaccination Status.
Our analytic sample included 41,552 hospitalizations and 21,522 emergency department or urgent care clinic visits. 3% of the hospitalizations and 14% of the emergency department or urgent care clinic visits were repeat medical visits by the same patient (Table 2). Characteristics of the patients are listed in Table 2, and characteristics of the patients according to network partner are provided in Tables S6 through S11. The median age was 74 years (interquartile range, 66 to 82) among hospitalized patients and 70 years (interquartile range, 61 to 78) among those who visited an emergency department or urgent care clinic.
Black patients and Hispanic patients accounted for a larger percentage of medical visits in the hospitalization sample (9% and 11%, respectively) than in the emergency department or urgent care sample (4% and 5%). These findings reflect in part the differing demographic characteristics of the network partners that contributed data on emergency department or urgent care clinic visits. The percentage of patients with underlying medical conditions was higher among hospitalized patients than among those who visited an emergency department or urgent care clinic. erectile dysfunction treatmentâÂÂAssociated Medical Care We identified 4321 patients with erectile dysfunction treatment who had laboratory-confirmed erectile dysfunction among 41,552 patients who were hospitalized (10%.
Range across network partners, 5 to 21). The remaining 37,231 hospitalized patients (90%) had discharge codes for erectile dysfunction treatmentâÂÂlike illness but were erectile dysfunctionâÂÂnegative. Laboratory-confirmed erectile dysfunction was identified in 3251 of 21,522 patients who visited an emergency department or urgent care clinic (15%. Range across network partners, 9 to 19).
The remaining 18,271 patients who visited an emergency department or urgent care clinic (85%) were erectile dysfunctionâÂÂnegative (Table 2). The percentage of erectile dysfunctionâÂÂpositive patients also varied among network partners (Tables S12 and S13). The percentage of patients with laboratory-confirmed erectile dysfunction decreased with age among hospitalized patients and among those with emergency department or urgent care clinic visits. In both care settings, the percentage of infected patients was higher among unvaccinated patients and lower among White patients, non-Hispanic patients, and those with chronic nonrespiratory conditions.
The numbers of both erectile dysfunctionâÂÂpositive patients and erectile dysfunctionâÂÂnegative patients with medical visits on each day are provided in Figures S1 through S10. erectile dysfunction treatment Vaccination Status On the index date, unvaccinated patients composed approximately half the patients who were hospitalized (49%. Range across network partners, 26 to 73) or visited an emergency department or urgent care clinic (55%. Range, 45 to 65) (Table 2).
In both samples, the largest differences between vaccinated and unvaccinated patients were age, network partner, calendar time, and local erectile dysfunction circulation on the index date. These same differences were noted when the sample was limited to erectile dysfunctionâÂÂpositive patients only (Tables S14 and S15). As described in the Supplementary Appendix, the application of inverse propensity-to-be-vaccinated weighting reduced the differences between vaccinated and unvaccinated patients with respect to these factors and other patient characteristics to a standard mean difference of less than 0.2. Among vaccinated patients, 53.4% of those who were hospitalized and 53.7% of those who visited an emergency department or urgent care clinic had received the BNT162b2 treatment, 43.3% and 41.6%, respectively, had received the mRNA-1273 treatment, and 3.3% and 4.7%, respectively, had received the Ad26.COV2.S treatment.
The median days from full vaccination to the index date were similar with the three types of erectile dysfunction treatments and with both samples (hospitalization and emergency department or urgent care clinic) (range, 42 to 53). Among the patients who received the BNT162b2 treatment, the median duration from partial vaccination (one dose) to the index date of hospitalization was 21 days and the median duration from partial vaccination to the index date of an emergency department or urgent care visit was 20 days. Among patients who received the mRNA-1273 treatment, these durations were 26 days and 24 days, respectively. These findings reflected the different dosing schedules of these treatments.
MRNA-Based treatment and Hospitalization Figure 1. Figure 1. Estimated treatment Effectiveness against erectile dysfunction Leading to Hospitalization or an Emergency Department or Urgent Care Clinic Visit, According to the Type of treatment. Patients who were partially vaccinated with one dose of a messenger RNA (mRNA)âÂÂbased treatment received the first dose at least 14 days before the index date for the medical visit and had not received the second dose by the index date.
Patients who were partially vaccinated with two doses of an mRNA-based treatment received the second dose 1 to 13 days before the index date. Fully vaccinated patients received a single dose of the Ad26.COV2.S treatment or the second dose of an mRNA-based treatment at least 14 days before the index date. CI denotes confidence interval, and erectile dysfunction severe acute respiratory syndrome erectile dysfunction 2.Figure 2. Figure 2.
Estimated Effectiveness of Full Two-Dose mRNA Vaccination against erectile dysfunction Leading to Hospitalization, According to Age, Race or Ethnic Group, and Underlying Medical Conditions. Among adults who were 50 years of age or older, the effectiveness of full two-dose mRNA-based vaccination (âÂÂ¥14 days after the second dose) was 89% (95% confidence interval [CI], 87 to 91) against laboratory-confirmed erectile dysfunction leading to hospitalization. The treatment-effectiveness point estimates were similar (differences, â¤5 percentage points) with the BNT162b2 and mRNA-1273 treatments (Figure 1 and Figure 2). The effectiveness of full mRNA-based vaccination was 83% (95% CI, 77 to 87) among patients who were at least 85 years of age, 86% (95% CI, 75 to 92) among Black patients, 90% (95% CI, 85 to 93) among Hispanic patients, 90% (95% CI, 88 to 92) among patients with chronic respiratory conditions, and 88% (95% CI, 86 to 90) among patients with chronic nonrespiratory conditions (Figure 2).
When the hospital sample was limited to 7283 admissions to an ICU, the effectiveness of full mRNA-based vaccination against laboratory-confirmed erectile dysfunction leading to ICU admission was 90% (95% CI, 86 to 93) (Table S16). Patients who were partially vaccinated with one dose of mRNA-based treatment received the first dose at least 14 days before the index date and had not received the second dose by the index date. Patients who were partially vaccinated with two doses of mRNA-based treatment received the second dose 1 to 13 days before the index date. Among patients who received an mRNA-based treatment, the effectiveness of partial one-dose vaccination (âÂÂ¥14 days after the first dose, but without the second dose) was 54% (95% CI, 47 to 61) against erectile dysfunction leading to hospitalization, and the effectiveness of partial two-dose vaccination (1 to 13 days after the second dose) was 73% (95% CI, 66% to 79).
With both the BNT162b2 and mRNA-1273 treatments, the effectiveness of full vaccination with respect to erectile dysfunction treatmentâÂÂassociated hospitalization was higher than that of partial vaccination (first dose) (with 95% confidence intervals that did not overlap) (Figure 1). A similar pattern of higher treatment-effectiveness point estimates for full mRNA-based vaccination than for partial mRNA-based vaccination was noted in all stratified analyses (Table S17). The effectiveness after partial vaccination (first dose) was lower with BNT162b2 than with mRNA-1273 (Figure 1). The estimates of the effectiveness of full mRNA-based vaccination were similar when stratified according to the six network partners that contributed the most data on hospitalizations (range, 82 to 97%).
However, heterogeneity was observed among the partners in the estimates of effectiveness of partial vaccination (first dose). treatment effectiveness also remained consistent in the other sensitivity analyses (Section S5). Our simulation model suggested that if both misclassification of outcome and of exposure occur, treatment effectiveness could be underestimated by as much as 10 percentage points, given the rates of clinical testing, percent positivity, and vaccination coverage observed in our hospitalization sample. Figure 3.
Figure 3. Estimated Effectiveness of mRNA-Based Vaccination against erectile dysfunction Leading to Hospitalization or an Emergency Department or Urgent Care Visit, According to the Days since the Most Recent Dose Was Administered. The total number of hospitalizations shown is higher than the total number in the main analysis because this secondary analysis was conducted weeks after the main analysis and incorporated updated information from vaccination records and registries. Specifically, an additional 212 hospitalizations among unvaccinated patients and 831 hospitalizations among vaccinated patients with confirmed vaccination status were included.In secondary analyses, we stratified mRNA-based treatment exposure according to 14-day intervals after administration (Figure 3) and according to type of treatment (Table S18).
treatment effectiveness with respect to erectile dysfunction treatmentâÂÂassociated hospitalization was null 0 to 13 days after the first dose, and treatment-effectiveness point estimates increased through 55 days after the first dose. treatment-effectiveness point estimates for full mRNA-based vaccination remained consistently high (>80%) through at least 112 days after the second dose. MRNA-Based treatment and Emergency Department and Urgent Care Visits Figure 4. Figure 4.
Estimated Effectiveness of Full Two-Dose mRNA-Based Vaccination against erectile dysfunction Leading to an Emergency Department or Urgent Care Clinic Visit, According to Age, Race or Ethnic Group, and Underlying Medical Conditions. The effectiveness of full two-dose mRNA-based vaccination was 91% (95% CI, 89 to 93) against laboratory-confirmed erectile dysfunction leading to emergency department or urgent care clinic visits (Figure 4). The treatment-effectiveness point estimates were similar (3 percentage points) with the BNT162b2 and mRNA-1273 treatments (Figure 1). The effectiveness of full mRNA-based vaccination was 84% (95% CI, 73 to 91) among adults who were 85 years of age or older, 95% (95% CI, 84 to 98) among Black patients, 81% (95% CI, 70 to 88) among Hispanic patients, and 90% (95% CI, 86 to 93) and 90% (95% CI, 87 to 92) among patients with chronic respiratory conditions and those with chronic nonrespiratory conditions, respectively (Figure 4).
The effectiveness of partial (one-dose) mRNA-based vaccination (both types) against erectile dysfunction leading to emergency department or urgent care clinic visits was 68% (95% CI, 61 to 74), and the effectiveness of partial (two-dose) vaccination was 80% (95% CI, 73 to 85) (Table S19). With both the BNT162b2 and mRNA-1273 treatments, the effectiveness of full vaccination against erectile dysfunction leading to emergency department or urgent care clinic visits was higher than the effectiveness with partial vaccination (one dose) (Figure 1). In sensitivity analyses, treatment-effectiveness point estimates for full mRNA-based vaccination against erectile dysfunction leading to emergency department or urgent care clinic visits ranged from 89 to 97% across the three network partners. Estimates of treatment effectiveness also remained consistent in other sensitivity analyses (Section S5).
In secondary analyses, treatment effectiveness against erectile dysfunction leading to emergency department or urgent care clinic visits was null 0 to 13 days after the first dose, and then treatment-effectiveness point estimates increased through 55 days after the first dose. treatment-effectiveness point estimates for full mRNA-based vaccination remained consistently high (âÂÂ¥86%) through at least 112 days after the second dose (Figure 3). Estimates of effectiveness according to the type of erectile dysfunction treatment are provided in Table S20. Effectiveness of Ad26.COV2.S treatment Estimates of the effectiveness of Ad26.COV2.S treatment were limited to five network partners with Ad26.COV2.S treatment recipients (CUIMC, Intermountain Healthcare, KPNC, KPNW, and Regenstrief Institute).
These analyses included 11,468 hospitalizations and 8917 emergency department or urgent care clinic visits that occurred after the index date for the first patient who was fully vaccinated with Ad26.COV2.S for each network partner (Figure 1). The effectiveness of the full one-dose Ad26.COV2.S treatment was 68% (95% CI, 50 to 79) with respect to erectile dysfunction treatmentâÂÂassociated hospitalization. The effectiveness of full vaccination against erectile dysfunction leading to emergency department or urgent care clinic visits was 73% (95% CI, 59 to 82) (Figure 1).To the Editor. Pregnant persons are at risk for severe erectile dysfunction disease 2019 (erectile dysfunction treatment), and with severe acute respiratory syndrome erectile dysfunction 2 (erectile dysfunction) during pregnancy is associated with increased risks of preterm birth and other adverse maternal and neonatal outcomes.1 Although spontaneous abortion (pregnancy loss occurring at less than 20 weeks of gestation) is a common pregnancy outcome affecting 11 to 22% of recognized pregnancies (see Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org),2-4 data to inform estimates of the risk of spontaneous abortion after receipt of an mRNA erectile dysfunction treatment either before conception (30 days before the first day of the last menstrual period through 14 days after) or during pregnancy are limited.
We analyzed data from the Centers for Disease Control and Prevention (CDC) v-safe erectile dysfunction treatment pregnancy registry to determine the cumulative risk of spontaneous abortion from 6 to less than 20 weeks of gestation. Participants with a singleton pregnancy who had received at least one dose of an mRNA erectile dysfunction treatment either before conception or before 20 weeks of gestation and who did not have a pregnancy loss before 6 weeks of gestation were included in this analysis. Inclusion of pregnant participants at 6 weeks of gestation is consistent with literature estimating the risk of spontaneous abortion in the general population.2-4 Life table methods were used to calculate the cumulative risk of spontaneous abortion according to gestational week, with appropriate left truncation (i.e., with adjustment for gestational age at entry). Data were right-censored at the time of the most recent contact for participants with ongoing pregnancies who were not contacted at 20 weeks of gestation or later and at the time of the outcome for participants who reported pregnancy outcomes other than spontaneous abortion (induced abortions or ectopic or molar pregnancies) before 20 weeks of gestation.
The cumulative risk of spontaneous abortion was also age-standardized with the use of data on the risk of spontaneous abortion according to maternal age group.3 We conducted a sensitivity analysis to estimate the maximum possible risk of spontaneous abortion, using an extreme assumption that all participants whose most recent contact was during the first trimester (i.e., at less than 14 weeks of gestation) and whom we were unable to reach during the second trimester experienced a spontaneous abortion immediately after the most recent contact (see the Supplementary Appendix for details). Table 1. Table 1. Risk of Spontaneous Abortion among Participants in the v-safe erectile dysfunction treatment Pregnancy Registry, December 14, 2020, through July 19, 2021.
A total of 2456 participants who were enrolled in the CDC v-safe erectile dysfunction treatment pregnancy registry met the inclusion criteria for this study. 2022 participants reported ongoing pregnancies at 20 weeks of gestation, 165 participants reported a spontaneous abortion (154 participants before 14 weeks of gestation), 65 participants with most recent contact during the first trimester could not be reached for second trimester follow-up, 188 participants completed second trimester follow-up before 20 weeks of gestation, and 16 participants reported another pregnancy outcome before 20 weeks (induced abortion or ectopic or molar pregnancy) (Fig. S1). Most participants were 30 years of age or older (77.3%), were non-Hispanic White (78.3%), and worked as health care personnel (88.8%).
Slightly more than half the participants (52.7%) had received the BNT162b2 treatment (PfizerâÂÂBioNTech) (Table S2). The cumulative risk of spontaneous abortion from 6 to less than 20 weeks of gestation was 14.1% (95% confidence interval [CI], 12.1 to 16.1) in the primary analysis (Table 1) and 12.8% (95% CI, 10.8 to 14.8) in an analysis using direct maternal ageâÂÂstandardization to the reference population. The cumulative risk of spontaneous abortion increased with maternal age (Table S3). In the sensitivity analysis, under the extreme assumption that all 65 participants with most recent contact during the first trimester had a spontaneous abortion, the cumulative risk of spontaneous abortion from 6 to less than 20 weeks of gestation was 18.8% (95% CI, 16.6 to 20.9).
After age standardization, the cumulative risk was 18.5% (95% CI, 16.1 to 20.8). Figure 1. Figure 1. Cumulative Risk of Spontaneous Abortion in the v-safe erectile dysfunction treatment Pregnancy Registry and in Two Historical Cohorts.
Data from Mukherjee2 were presented as race-specific rates and are provided here for White women to maximize comparability with the v-safe pregnancy registry.As compared with data from two historical cohorts that represent the lower and upper ranges of spontaneous-abortion risk,2,4 the cumulative risks of spontaneous abortion from our primary and sensitivity analyses were within the expected risk range (Figure 1). Limitations of our study include the lack of a control group of unvaccinated pregnant persons, the homogeneity of the participants in terms of racial and ethnic groups and occupation, the voluntary enrollment of the population, and the use of data reported by the participants themselves, including some data collected retrospectively. Nonetheless, our findings suggest that the risk of spontaneous abortion after mRNA erectile dysfunction treatment vaccination either before conception or during pregnancy is consistent with the expected risk of spontaneous abortion. These findings add to the accumulating evidence about the safety of mRNA erectile dysfunction treatment vaccination in pregnancy.5 Lauren H.
Zauche, Ph.D., M.S.N.Bailey Wallace, M.P.H.Ashley N. Smoots, M.P.H.Christine K. Olson, M.D., M.P.H.Titilope Oduyebo, M.D., M.P.H.Shin Y. Kim, M.P.H.Emily E.
Petersen, M.D.Jun Ju, M.S.Jennifer Beauregard, Ph.D., M.P.H.Centers for Disease Control and Prevention (CDC), Atlanta, GAAllen J. Wilcox, M.D., Ph.D.National Institutes of Health, Durham, NCCharles E. Rose, Ph.D.Dana M. Meaney-Delman, M.D., M.P.H.Sascha R.
Ellington, Ph.D., M.S.P.H.CDC, Atlanta, GAfor the CDC v-safe erectile dysfunction treatment Pregnancy Registry Team Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org. The findings and conclusions in this letter are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC). Mention of a product or company name is for identification purposes only and does not constitute endorsement by the CDC or the Food and Drug Administration. The authors do not have any material conflicts of interest.This letter was published on September 8, 2021, at NEJM.org.5 References1.
Allotey J, Stallings E, Bonet M, et al. Clinical manifestations, risk factors, and maternal and perinatal outcomes of erectile dysfunction disease 2019 in pregnancy. Living systematic review and meta-analysis. BMJ 2020;370:m3320-m3320.2.
Mukherjee S, Velez Edwards DR, Baird DD, Savitz DA, Hartmann KE. Risk of miscarriage among black women and white women in a U.S. Prospective cohort study. Am J Epidemiol 2013;177:1271-1278.3.
Magnus MC, Wilcox AJ, Morken N-H, Weinberg CR, HÃÂ¥berg SE. Role of maternal age and pregnancy history in risk of miscarriage. Prospective register based study. BMJ 2019;364:l869-l869.4.
Goldhaber MK, Fireman BH. The fetal life table revisited. Spontaneous abortion rates in three Kaiser Permanente cohorts. Epidemiology 1991;2:33-39.5.
Shimabukuro TT, Kim SY, Myers TR, et al. Preliminary findings of mRNA erectile dysfunction treatment safety in pregnant persons. N Engl J Med 2021;384:2273-2282.10.1056/NEJMc2113891-t1Table 1. Risk of Spontaneous Abortion among Participants in the v-safe erectile dysfunction treatment Pregnancy Registry, December 14, 2020, through July 19, 2021.
Gestational AgeParticipants at RiskParticipants Who Reported Spontaneous AbortionWeek-Specific RiskCumulative Risknumber of personspercentpercent (95% CI)6 to <7 weeks904151.71.7 (0.8âÂÂ2.5)7 to <8 weeks982181.83.5 (2.3âÂÂ4.6)8 to <9 weeks1032373.66.9 (5.4âÂÂ8.5)9 to <10 weeks1087393.610.3 (8.4âÂÂ12.0)10 to <11 weeks1118191.711.8 (9.9âÂÂ13.7)11 to <12 weeks1184121.012.7 (10.7âÂÂ14.6)12 to <13 weeks127490.713.3 (11.3âÂÂ15.2)13 to <14 weeks139450.413.6 (11.6âÂÂ15.6)14 to <15 weeks15340013.6 (11.6âÂÂ15.6)15 to <16 weeks163220.113.7 (11.7âÂÂ15.7)16 to <17 weeks174220.113.8 (11.8âÂÂ15.8)17 to <18 weeks184820.113.9 (11.9âÂÂ15.9)18 to <19 weeks194130.214.0 (12.0âÂÂ16.0)19 to <20 weeks205220.114.1 (12.1âÂÂ16.1).
To the Cialis 5mg price in canada Editor 20mg levitra price. Figure 1 20mg levitra price. Figure 1 20mg levitra price. erectile dysfunction Variants among Symptomatic Health Workers 20mg levitra price.
Shown is the distribution of the B.1.1.7 (alpha), delta, and other erectile dysfunction variants according to vaccination status and month of diagnosis among health workers at University of California San Diego Health, March through July 2021. The number of workers 20mg levitra price indicates those who were symptomatic and had available variant data, and the number of positive tests indicates those that included data on variants. In December 2020, the University of California San Diego Health (UCSDH) workforce experienced a dramatic increase 20mg levitra price in severe acute respiratory syndrome erectile dysfunction 2 (erectile dysfunction) s. Vaccination with mRNA treatments began in mid-December 20mg levitra price 2020.
By March, 76% of the workforce had been fully vaccinated, and by July, the percentage had risen to 87%. s had decreased dramatically by early February 2021.1 Between March and June, fewer than 30 health care workers tested 20mg levitra price positive each month. However, coincident with 20mg levitra price the end of CaliforniaâÂÂs mask mandate on June 15 and the rapid dominance of the B.1.617.2 (delta) variant that first emerged in mid-April and accounted for over 95% of UCSDH isolates by the end of July (Figure 1), s increased rapidly, including cases among fully vaccinated persons. Institutional review 20mg levitra price board approval was obtained for use of administrative data on vaccinations and case-investigation data to examine mRNA SARS CoV-2 treatment effectiveness.
UCSDH has a low threshold for erectile dysfunction testing, which is triggered by the presence of at least one symptom during daily screening or by an identified exposure, regardless of vaccination status. From March 20mg levitra price 1 to July 31, 2021, a total of 227 UCSDH health care workers tested positive for erectile dysfunction by reverse-transcriptaseâÂÂquantitative polymerase-chain-reaction (RT-qPCR) assay of nasal swabs. 130 of the 227 workers (57.3%) were fully vaccinated 20mg levitra price. Symptoms were present in 109 of the 130 fully vaccinated workers (83.8%) and 20mg levitra price in 80 of the 90 unvaccinated workers (88.9%).
(The remaining 7 workers were only partially vaccinated.) No deaths were reported in either group. One unvaccinated person was hospitalized for 20mg levitra price erectile dysfunctionâÂÂrelated symptoms. Table 1 20mg levitra price. Table 1 20mg levitra price.
Symptomatic erectile dysfunction and mRNA treatment Effectiveness among UCSDH Health Workers, March through July 2021. treatment effectiveness was calculated for each month from March through July 20mg levitra price. The case definition was a positive PCR test 20mg levitra price and one or more symptoms among persons with no previous erectile dysfunction treatment (see the Supplementary Appendix). treatment effectiveness exceeded 90% from March through June but fell to 65.5% (95% confidence interval [CI], 20mg levitra price 48.9 to 76.9) in July (Table 1).
July case rates were analyzed according to the month in which workers with erectile dysfunction treatment completed the vaccination series. In workers completing vaccination in January or February, the attack rate was 6.7 per 1000 persons (95% CI, 5.9 to 7.8), whereas the attack rate was 3.7 per 1000 persons (95% CI, 2.5 to 5.7) among those who completed vaccination during the period from March through May 20mg levitra price. Among unvaccinated persons, the July attack 20mg levitra price rate was 16.4 per 1000 persons (95% CI, 11.8 to 22.9). The SARS CoV-2 mRNA treatments, BNT162b2 (PfizerâÂÂBioNTech) and mRNA-1273 (Moderna), have previously shown efficacy rates of 95% and 94.1%,2 respectively, in 20mg levitra price their initial clinical trials, and for the BNT162b2 treatment, sustained, albeit slightly decreased effectiveness (84%) 4 months after the second dose.3 In England, where an extended dosing interval of up to 12 weeks was used, Lopez Bernal et al.
Reported a preserved treatment effectiveness of 88% against symptomatic disease associated with the delta variant.4 As observed by others in populations that received mRNA treatments according to standard Emergency Use Authorization intervals,5 our data suggest that treatment effectiveness against any symptomatic disease is considerably lower against the delta variant and may wane over time since vaccination. The dramatic change in treatment effectiveness from June to July is likely to be due to both the emergence of the delta variant and waning immunity over time, compounded by 20mg levitra price the end of masking requirements in California and the resulting greater risk of exposure in the community. Our findings underline the importance of rapidly reinstating nonpharmaceutical interventions, such as indoor masking and intensive testing strategies, 20mg levitra price in addition to continued efforts to increase vaccinations, as strategies to prevent avoidable illness and deaths and to avoid mass disruptions to society during the spread of this formidable variant. Furthermore, if our findings on waning immunity are verified in other settings, booster doses 20mg levitra price may be indicated.
Jocelyn Keehner, M.D.Lucy E. Horton, M.D., M.P.H.UC San 20mg levitra price Diego Health, San Diego, CANancy J. Binkin, M.D., M.P.H.UC San 20mg levitra price Diego, La Jolla, CALouise C. Laurent, M.D., 20mg levitra price Ph.D.David Pride, M.D., Ph.D.Christopher A.
Longhurst, M.D.Shira R. Abeles, M.D.Francesca J 20mg levitra price. Torriani, M.D.UC San Diego Health, San Diego, CA [email protected] Disclosure forms provided by the authors are 20mg levitra price available with the full text of this letter at NEJM.org. This letter 20mg levitra price was published on September 1, 2021, and updated on September 3, 2021, at NEJM.org.
Dr. Laurent serves as an author on behalf of 20mg levitra price the SEARCH Alliance. Collaborators in the 20mg levitra price SEARCH Alliance are listed in the Supplementary Appendix, available with the full text of this letter at NEJM.org. Drs 20mg levitra price.
Keehner and Horton and Drs. Abeles and Torriani contributed equally 20mg levitra price to this letter. 5 References1 20mg levitra price. Keehner J, Abeles SR, Torriani 20mg levitra price FJ.
More on erectile dysfunction after vaccination in health care workers. Reply. N Engl J Med 2021;385(2):e8.2. Baden LR, El Sahly HM, Essink B, et al.
Efficacy and safety of the mRNA-1273 erectile dysfunction treatment. N Engl J Med 2021;384:403-416.3. Thomas SJ, Moreira ED Jr, Kitchin N, et al. Six month safety and efficacy of the BNT162b2 mRNA erectile dysfunction treatment.
July 28, 2021 (https://www.medrxiv.org/content/10.1101/2021.07.28.21261159v1). Preprint.Google Scholar4. Lopez Bernal J, Andrews N, Gower C, et al. Effectiveness of erectile dysfunction treatments against the B.1.617.2 (Delta) variant.
N Engl J Med 2021;385:585-594.5. Israel A, Merzon E, Schäffer AA, et al. Elapsed time since BNT162b2 treatment and risk of erectile dysfunction in a large cohort. August 5, 2021 (https://www.medrxiv.org/content/10.1101/2021.08.03.21261496v1).
Preprint.Google Scholar10.1056/NEJMc2112981-t1Table 1. Symptomatic erectile dysfunction and mRNA treatment Effectiveness among UCSDH Health Workers, March through July 2021.* MarchAprilMayJuneJulyUCSDH workforce â no. Of persons18,96418,99219,00019,03519,016Vaccination status â no. Of personsFully vaccinatedâ 14,47015,51016,15716,42616,492mRNA-1273 (Moderna)6,6087,0057,3407,4517,464BNT162b2 (PfizerâÂÂBioNTech)7,8628,5058,8178,9759,028Unvaccinated3,2302,5092,1872,0591,895Percentage of workers fully vaccinated76.381.785.086.386.7Symptomatic erectile dysfunction treatmentFully vaccinated workers343594Unvaccinated workers1117101031Percentage of cases in fully vaccinated workers21.419.023.133.375.2Attack rate per 1000 (95% CI)Fully vaccinated workers0.21 (0.21âÂÂ0.47)0.26 (0.26âÂÂ0.50)0.19 (0.21âÂÂ0.40)0.30 (0.31âÂÂ0.53)5.7 (5.4âÂÂ6.2)Unvaccinated workers3.4 (2.1âÂÂ5.9)6.8 (4.5âÂÂ10.6)4.6 (2.6âÂÂ8.2)4.9 (2.9âÂÂ8.7)16.4 (11.8âÂÂ22.9)treatment effectiveness â % (95% CI)93.9 (78.2âÂÂ97.9)96.2 (88.7âÂÂ98.3)95.9 (85.3âÂÂ98.9)94.3 (83.7âÂÂ98.0)65.5 (48.9âÂÂ76.9)Study Sample A total of 103,199 hospitalizations of patients with erectile dysfunction treatmentâÂÂlike illness who were 50 years of age or older were identified by the seven VISION partners.
Of these hospitalizations, 64,400 (62%) occurred after the dates of age-specific erectile dysfunction treatment eligibility and the time required for vaccination records to be updated (Table S3). The hospitalizations occurred during the period from January 1 through June 22, 2021. Among unvaccinated patients who were hospitalized, the median duration from treatment eligibility to the index date was 39 days (interquartile range, 16 to 70) (Table S4). erectile dysfunction testing with a molecular assay ordered by clinicians was conducted for 74% of the patients who were hospitalized (range across network partners, 55 to 99).
During the period from January 1 through June 22, a total of 121,709 visits to emergency departments or urgent care clinics for erectile dysfunction treatmentâÂÂlike illness were identified by three partners. 76,220 visits (63%) occurred after treatment age eligibility and updates to vaccination records (Table S5). Among the patients who visited an emergency department or urgent care clinic, the median duration from treatment eligibility to the index date was 39 days (interquartile range, 15 to 70). 30% (range, 25 to 41) of these patients were tested by means of molecular assay.
Across the partners, 1872 hospitalizations and 1350 emergency department or urgent care clinic visits were excluded because the index dates occurred 1 to 13 days after the patient received the first dose of erectile dysfunction treatment and immunity was considered indeterminant. Table 2. Table 2. Characteristics of the Patients According to erectile dysfunction Test Results and Vaccination Status.
Our analytic sample included 41,552 hospitalizations and 21,522 emergency department or urgent care clinic visits. 3% of the hospitalizations and 14% of the emergency department or urgent care clinic visits were repeat medical visits by the same patient (Table 2). Characteristics of the patients are listed in Table 2, and characteristics of the patients according to network partner are provided in Tables S6 through S11. The median age was 74 years (interquartile range, 66 to 82) among hospitalized patients and 70 years (interquartile range, 61 to 78) among those who visited an emergency department or urgent care clinic.
Black patients and Hispanic patients accounted for a larger percentage of medical visits in the hospitalization sample (9% and 11%, respectively) than in the emergency department or urgent care sample (4% and 5%). These findings reflect in part the differing demographic characteristics of the network partners that contributed data on emergency department or urgent care clinic visits. The percentage of patients with underlying medical conditions was higher among hospitalized patients than among those who visited an emergency department or urgent care clinic. erectile dysfunction treatmentâÂÂAssociated Medical Care We identified 4321 patients with erectile dysfunction treatment who had laboratory-confirmed erectile dysfunction among 41,552 patients who were hospitalized (10%.
Range across network partners, 5 to 21). The remaining 37,231 hospitalized patients (90%) had discharge codes for erectile dysfunction treatmentâÂÂlike illness but were erectile dysfunctionâÂÂnegative. Laboratory-confirmed erectile dysfunction was identified in 3251 of 21,522 patients who visited an emergency department or urgent care clinic (15%. Range across network partners, 9 to 19).
The remaining 18,271 patients who visited an emergency department or urgent care clinic (85%) were erectile dysfunctionâÂÂnegative (Table 2). The percentage of erectile dysfunctionâÂÂpositive patients also varied among network partners (Tables S12 and S13). The percentage of patients with laboratory-confirmed erectile dysfunction decreased with age among hospitalized patients and among those with emergency department or urgent care clinic visits. In both care settings, the percentage of infected patients was higher among unvaccinated patients and lower among White patients, non-Hispanic patients, and those with chronic nonrespiratory conditions.
The numbers of both erectile dysfunctionâÂÂpositive patients and erectile dysfunctionâÂÂnegative patients with medical visits on each day are provided in Figures S1 through S10. erectile dysfunction treatment Vaccination Status On the index date, unvaccinated patients composed approximately half the patients who were hospitalized (49%. Range across network partners, 26 to 73) or visited an emergency department or urgent care clinic (55%. Range, 45 to 65) (Table 2).
In both samples, the largest differences between vaccinated and unvaccinated patients were age, network partner, calendar time, and local erectile dysfunction circulation on the index date. These same differences were noted when the sample was limited to erectile dysfunctionâÂÂpositive patients only (Tables S14 and S15). As described in the Supplementary Appendix, the application of inverse propensity-to-be-vaccinated weighting reduced the differences between vaccinated and unvaccinated patients with respect to these factors and other patient characteristics to a standard mean difference of less than 0.2. Among vaccinated patients, 53.4% of those who were hospitalized and 53.7% of those who visited an emergency department or urgent care clinic had received the BNT162b2 treatment, 43.3% and 41.6%, respectively, had received the mRNA-1273 treatment, and 3.3% and 4.7%, respectively, had received the Ad26.COV2.S treatment.
The median days from full vaccination to the index date were similar with the three types of erectile dysfunction treatments and with both samples (hospitalization and emergency department or urgent care clinic) (range, 42 to 53). Among the patients who received the BNT162b2 treatment, the median duration from partial vaccination (one dose) to the index date of hospitalization was 21 days and the median duration from partial vaccination to the index date of an emergency department or urgent care visit was 20 days. Among patients who received the mRNA-1273 treatment, these durations were 26 days and 24 days, respectively. These findings reflected the different dosing schedules of these treatments.
MRNA-Based treatment and Hospitalization Figure 1. Figure 1. Estimated treatment Effectiveness against erectile dysfunction Leading to Hospitalization or an Emergency Department or Urgent Care Clinic Visit, According to the Type of treatment. Patients who were partially vaccinated with one dose of a messenger RNA (mRNA)âÂÂbased treatment received the first dose at least 14 days before the index date for the medical visit and had not received the second dose by the index date.
Patients who were partially vaccinated with two doses of an mRNA-based treatment received the second dose 1 to 13 days before the index date. Fully vaccinated patients received a single dose of the Ad26.COV2.S treatment or the second dose of an mRNA-based treatment at least 14 days before the index date. CI denotes confidence interval, and erectile dysfunction severe acute respiratory syndrome erectile dysfunction 2.Figure 2. Figure 2.
Estimated Effectiveness of Full Two-Dose mRNA Vaccination against erectile dysfunction Leading to Hospitalization, According to Age, Race or Ethnic Group, and Underlying Medical Conditions. Among adults who were 50 years of age or older, the effectiveness of full two-dose mRNA-based vaccination (âÂÂ¥14 days after the second dose) was 89% (95% confidence interval [CI], 87 to 91) against laboratory-confirmed erectile dysfunction leading to hospitalization. The treatment-effectiveness point estimates were similar (differences, â¤5 percentage points) with the BNT162b2 and mRNA-1273 treatments (Figure 1 and Figure 2). The effectiveness of full mRNA-based vaccination was 83% (95% CI, 77 to 87) among patients who were at least 85 years of age, 86% (95% CI, 75 to 92) among Black patients, 90% (95% CI, 85 to 93) among Hispanic patients, 90% (95% CI, 88 to 92) among patients with chronic respiratory conditions, and 88% (95% CI, 86 to 90) among patients with chronic nonrespiratory conditions (Figure 2).
When the hospital sample was limited to 7283 admissions to an ICU, the effectiveness of full mRNA-based vaccination against laboratory-confirmed erectile dysfunction leading to ICU admission was 90% (95% CI, 86 to 93) (Table S16). Patients who were partially vaccinated with one dose of mRNA-based treatment received the first dose at least 14 days before the index date and had not received the second dose by the index date. Patients who were partially vaccinated with two doses of mRNA-based treatment received the second dose 1 to 13 days before the index date. Among patients who received an mRNA-based treatment, the effectiveness of partial one-dose vaccination (âÂÂ¥14 days after the first dose, but without the second dose) was 54% (95% CI, 47 to 61) against erectile dysfunction leading to hospitalization, and the effectiveness of partial two-dose vaccination (1 to 13 days after the second dose) was 73% (95% CI, 66% to 79).
With both the BNT162b2 and mRNA-1273 treatments, the effectiveness of full vaccination with respect to erectile dysfunction treatmentâÂÂassociated hospitalization was higher than that of partial vaccination (first dose) (with 95% confidence intervals that did not overlap) (Figure 1). A similar pattern of higher treatment-effectiveness point estimates for full mRNA-based vaccination than for partial mRNA-based vaccination was noted in all stratified analyses (Table S17). The effectiveness after partial vaccination (first dose) was lower with BNT162b2 than with mRNA-1273 (Figure 1). The estimates of the effectiveness of full mRNA-based vaccination were similar when stratified according to the six network partners that contributed the most data on hospitalizations (range, 82 to 97%).
However, heterogeneity was observed among the partners in the estimates of effectiveness of partial vaccination (first dose). treatment effectiveness also remained consistent in the other sensitivity analyses (Section S5). Our simulation model suggested that if both misclassification of outcome and of exposure occur, treatment effectiveness could be underestimated by as much as 10 percentage points, given the rates of clinical testing, percent positivity, and vaccination coverage observed in our hospitalization sample. Figure 3.
Figure 3. Estimated Effectiveness of mRNA-Based Vaccination against erectile dysfunction Leading to Hospitalization or an Emergency Department or Urgent Care Visit, According to the Days since the Most Recent Dose Was Administered. The total number of hospitalizations shown is higher than the total number in the main analysis because this secondary analysis was conducted weeks after the main analysis and incorporated updated information from vaccination records and registries. Specifically, an additional 212 hospitalizations among unvaccinated patients and 831 hospitalizations among vaccinated patients with confirmed vaccination status were included.In secondary analyses, we stratified mRNA-based treatment exposure according to 14-day intervals after administration (Figure 3) and according to type of treatment (Table S18).
treatment effectiveness with respect to erectile dysfunction treatmentâÂÂassociated hospitalization was null 0 to 13 days after the first dose, and treatment-effectiveness point estimates increased through 55 days after the first dose. treatment-effectiveness point estimates for full mRNA-based vaccination remained consistently high (>80%) through at least 112 days after the second dose. MRNA-Based treatment and Emergency Department and Urgent Care Visits Figure 4. Figure 4.
Estimated Effectiveness of Full Two-Dose mRNA-Based Vaccination against erectile dysfunction Leading to an Emergency Department or Urgent Care Clinic Visit, According to Age, Race or Ethnic Group, and Underlying Medical Conditions. The effectiveness of full two-dose mRNA-based vaccination was 91% (95% CI, 89 to 93) against laboratory-confirmed erectile dysfunction leading to emergency department or urgent care clinic visits (Figure 4). The treatment-effectiveness point estimates were similar (3 percentage points) with the BNT162b2 and mRNA-1273 treatments (Figure 1). The effectiveness of full mRNA-based vaccination was 84% (95% CI, 73 to 91) among adults who were 85 years of age or older, 95% (95% CI, 84 to 98) among Black patients, 81% (95% CI, 70 to 88) among Hispanic patients, and 90% (95% CI, 86 to 93) and 90% (95% CI, 87 to 92) among patients with chronic respiratory conditions and those with chronic nonrespiratory conditions, respectively (Figure 4).
The effectiveness of partial (one-dose) mRNA-based vaccination (both types) against erectile dysfunction leading to emergency department or urgent care clinic visits was 68% (95% CI, 61 to 74), and the effectiveness of partial (two-dose) vaccination was 80% (95% CI, 73 to 85) (Table S19). With both the BNT162b2 and mRNA-1273 treatments, the effectiveness of full vaccination against erectile dysfunction leading to emergency department or urgent care clinic visits was higher than the effectiveness with partial vaccination (one dose) (Figure 1). In sensitivity analyses, treatment-effectiveness point estimates for full mRNA-based vaccination against erectile dysfunction leading to emergency department or urgent care clinic visits ranged from 89 to 97% across the three network partners. Estimates of treatment effectiveness also remained consistent in other sensitivity analyses (Section S5).
In secondary analyses, treatment effectiveness against erectile dysfunction leading to emergency department or urgent care clinic visits was null 0 to 13 days after the first dose, and then treatment-effectiveness point estimates increased through 55 days after the first dose. treatment-effectiveness point estimates for full mRNA-based vaccination remained consistently high (âÂÂ¥86%) through at least 112 days after the second dose (Figure 3). Estimates of effectiveness according to the type of erectile dysfunction treatment are provided in Table S20. Effectiveness of Ad26.COV2.S treatment Estimates of the effectiveness of Ad26.COV2.S treatment were limited to five network partners with Ad26.COV2.S treatment recipients (CUIMC, Intermountain Healthcare, KPNC, KPNW, and Regenstrief Institute).
These analyses included 11,468 hospitalizations and 8917 emergency department or urgent care clinic visits that occurred after the index date for the first patient who was fully vaccinated with Ad26.COV2.S for each network partner (Figure 1). The effectiveness of the full one-dose Ad26.COV2.S treatment was 68% (95% CI, 50 to 79) with respect to erectile dysfunction treatmentâÂÂassociated hospitalization. The effectiveness of full vaccination against erectile dysfunction leading to emergency department or urgent care clinic visits was 73% (95% CI, 59 to 82) (Figure 1).To the Editor. Pregnant persons are at risk for severe erectile dysfunction disease 2019 (erectile dysfunction treatment), and with severe acute respiratory syndrome erectile dysfunction 2 (erectile dysfunction) during pregnancy is associated with increased risks of preterm birth and other adverse maternal and neonatal outcomes.1 Although spontaneous abortion (pregnancy loss occurring at less than 20 weeks of gestation) is a common pregnancy outcome affecting 11 to 22% of recognized pregnancies (see Table S1 in the Supplementary Appendix, available with the full text of this letter at NEJM.org),2-4 data to inform estimates of the risk of spontaneous abortion after receipt of an mRNA erectile dysfunction treatment either before conception (30 days before the first day of the last menstrual period through 14 days after) or during pregnancy are limited.
We analyzed data from the Centers for Disease Control and Prevention (CDC) v-safe erectile dysfunction treatment pregnancy registry to determine the cumulative risk of spontaneous abortion from 6 to less than 20 weeks of gestation. Participants with a singleton pregnancy who had received at least one dose of an mRNA erectile dysfunction treatment either before conception or before 20 weeks of gestation and who did not have a pregnancy loss before 6 weeks of gestation were included in this analysis. Inclusion of pregnant participants at 6 weeks of gestation is consistent with literature estimating the risk of spontaneous abortion in the general population.2-4 Life table methods were used to calculate the cumulative risk of spontaneous abortion according to gestational week, with appropriate left truncation (i.e., with adjustment for gestational age at entry). Data were right-censored at the time of the most recent contact for participants with ongoing pregnancies who were not contacted at 20 weeks of gestation or later and at the time of the outcome for participants who reported pregnancy outcomes other than spontaneous abortion (induced abortions or ectopic or molar pregnancies) before 20 weeks of gestation.
The cumulative risk of spontaneous abortion was also age-standardized with the use of data on the risk of spontaneous abortion according to maternal age group.3 We conducted a sensitivity analysis to estimate the maximum possible risk of spontaneous abortion, using an extreme assumption that all participants whose most recent contact was during the first trimester (i.e., at less than 14 weeks of gestation) and whom we were unable to reach during the second trimester experienced a spontaneous abortion immediately after the most recent contact (see the Supplementary Appendix for details). Table 1. Table 1. Risk of Spontaneous Abortion among Participants in the v-safe erectile dysfunction treatment Pregnancy Registry, December 14, 2020, through July 19, 2021.
A total of 2456 participants who were enrolled in the CDC v-safe erectile dysfunction treatment pregnancy registry met the inclusion criteria for this study. 2022 participants reported ongoing pregnancies at 20 weeks of gestation, 165 participants reported a spontaneous abortion (154 participants before 14 weeks of gestation), 65 participants with most recent contact during the first trimester could not be reached for second trimester follow-up, 188 participants completed second trimester follow-up before 20 weeks of gestation, and 16 participants reported another pregnancy outcome before 20 weeks (induced abortion or ectopic or molar pregnancy) (Fig. S1). Most participants were 30 years of age or older (77.3%), were non-Hispanic White (78.3%), and worked as health care personnel (88.8%).
Slightly more than half the participants (52.7%) had received the BNT162b2 treatment (PfizerâÂÂBioNTech) (Table S2). The cumulative risk of spontaneous abortion from 6 to less than 20 weeks of gestation was 14.1% (95% confidence interval [CI], 12.1 to 16.1) in the primary analysis (Table 1) and 12.8% (95% CI, 10.8 to 14.8) in an analysis using direct maternal ageâÂÂstandardization to the reference population. The cumulative risk of spontaneous abortion increased with maternal age (Table S3). In the sensitivity analysis, under the extreme assumption that all 65 participants with most recent contact during the first trimester had a spontaneous abortion, the cumulative risk of spontaneous abortion from 6 to less than 20 weeks of gestation was 18.8% (95% CI, 16.6 to 20.9).
After age standardization, the cumulative risk was 18.5% (95% CI, 16.1 to 20.8). Figure 1. Figure 1. Cumulative Risk of Spontaneous Abortion in the v-safe erectile dysfunction treatment Pregnancy Registry and in Two Historical Cohorts.
Data from Mukherjee2 were presented as race-specific rates and are provided here for White women to maximize comparability with the v-safe pregnancy registry.As compared with data from two historical cohorts that represent the lower and upper ranges of spontaneous-abortion risk,2,4 the cumulative risks of spontaneous abortion from our primary and sensitivity analyses were within the expected risk range (Figure 1). Limitations of our study include the lack of a control group of unvaccinated pregnant persons, the homogeneity of the participants in terms of racial and ethnic groups and occupation, the voluntary enrollment of the population, and the use of data reported by the participants themselves, including some data collected retrospectively. Nonetheless, our findings suggest that the risk of spontaneous abortion after mRNA erectile dysfunction treatment vaccination either before conception or during pregnancy is consistent with the expected risk of spontaneous abortion. These findings add to the accumulating evidence about the safety of mRNA erectile dysfunction treatment vaccination in pregnancy.5 Lauren H.
Zauche, Ph.D., M.S.N.Bailey Wallace, M.P.H.Ashley N. Smoots, M.P.H.Christine K. Olson, M.D., M.P.H.Titilope Oduyebo, M.D., M.P.H.Shin Y. Kim, M.P.H.Emily E.
Petersen, M.D.Jun Ju, M.S.Jennifer Beauregard, Ph.D., M.P.H.Centers for Disease Control and Prevention (CDC), Atlanta, GAAllen J. Wilcox, M.D., Ph.D.National Institutes of Health, Durham, NCCharles E. Rose, Ph.D.Dana M. Meaney-Delman, M.D., M.P.H.Sascha R.
Ellington, Ph.D., M.S.P.H.CDC, Atlanta, GAfor the CDC v-safe erectile dysfunction treatment Pregnancy Registry Team Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org. The findings and conclusions in this letter are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC). Mention of a product or company name is for identification purposes only and does not constitute endorsement by the CDC or the Food and Drug Administration. The authors do not have any material conflicts of interest.This letter was published on September 8, 2021, at NEJM.org.5 References1.
Allotey J, Stallings E, Bonet M, et al. Clinical manifestations, risk factors, and maternal and perinatal outcomes of erectile dysfunction disease 2019 in pregnancy. Living systematic review and meta-analysis. BMJ 2020;370:m3320-m3320.2.
Mukherjee S, Velez Edwards DR, Baird DD, Savitz DA, Hartmann KE. Risk of miscarriage among black women and white women in a U.S. Prospective cohort study. Am J Epidemiol 2013;177:1271-1278.3.
Magnus MC, Wilcox AJ, Morken N-H, Weinberg CR, HÃÂ¥berg SE. Role of maternal age and pregnancy history in risk of miscarriage. Prospective register based study. BMJ 2019;364:l869-l869.4.
Goldhaber MK, Fireman BH. The fetal life table revisited. Spontaneous abortion rates in three Kaiser Permanente cohorts. Epidemiology 1991;2:33-39.5.
Shimabukuro TT, Kim SY, Myers TR, et al. Preliminary findings of mRNA erectile dysfunction treatment safety in pregnant persons. N Engl J Med 2021;384:2273-2282.10.1056/NEJMc2113891-t1Table 1. Risk of Spontaneous Abortion among Participants in the v-safe erectile dysfunction treatment Pregnancy Registry, December 14, 2020, through July 19, 2021.
Gestational AgeParticipants at RiskParticipants Who Reported Spontaneous AbortionWeek-Specific RiskCumulative Risknumber of personspercentpercent (95% CI)6 to <7 weeks904151.71.7 (0.8âÂÂ2.5)7 to <8 weeks982181.83.5 (2.3âÂÂ4.6)8 to <9 weeks1032373.66.9 (5.4âÂÂ8.5)9 to <10 weeks1087393.610.3 (8.4âÂÂ12.0)10 to <11 weeks1118191.711.8 (9.9âÂÂ13.7)11 to <12 weeks1184121.012.7 (10.7âÂÂ14.6)12 to <13 weeks127490.713.3 (11.3âÂÂ15.2)13 to <14 weeks139450.413.6 (11.6âÂÂ15.6)14 to <15 weeks15340013.6 (11.6âÂÂ15.6)15 to <16 weeks163220.113.7 (11.7âÂÂ15.7)16 to <17 weeks174220.113.8 (11.8âÂÂ15.8)17 to <18 weeks184820.113.9 (11.9âÂÂ15.9)18 to <19 weeks194130.214.0 (12.0âÂÂ16.0)19 to <20 weeks205220.114.1 (12.1âÂÂ16.1).
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202-693-1999 Advisory Committee bayer levitra 20 on Construction Safety and Health meetingwill be held virtually on Wednesday, Aug. 11Stakeholders must pre-register to join the event WASHINGTON, DC â The U.S. Department of LaborâÂÂs Occupational Safety and bayer levitra 20 Health Administration reminds stakeholders that the next meeting of the Advisory Committee on Construction Safety and Health is scheduled from 1 p.m.
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Attendance at this meeting 20mg levitra price will be virtual only. Register to attend the meeting. Once registration is approved, OSHA will send 20mg levitra price individuals an email with information on how to join to connect.
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