Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells.

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the ß-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

A Digital PCR-Based Method for Efficient and Highly Specific Screening of Genome Edited Cells.

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs "mismatch nucleases" T7E1 or "Surveyor" that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an "all-in-one" CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.

Large multiallelic copy number variations in humans.

Thousands of genomic segments appear to be present in widely varying copy numbers in different human genomes. We developed ways to use increasingly abundant whole-genome sequence data to identify the copy numbers, alleles and haplotypes present at most large multiallelic CNVs (mCNVs). We analyzed 849 genomes sequenced by the 1000 Genomes Project to identify most large (>5-kb) mCNVs, including 3,878 duplications, of which 1,356 appear to have 3 or more segregating alleles. We find that mCNVs give rise to most human variation in gene dosage-seven times the combined contribution of deletions and biallelic duplications-and that this variation in gene dosage generates abundant variation in gene expression. We describe 'runaway duplication haplotypes' in which genes, including HPR and ORM1, have mutated to high copy number on specific haplotypes. We also describe partially successful initial strategies for analyzing mCNVs via imputation and provide an initial data resource to support such analyses.

Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

The somatic reproductive tissues of C. elegans promote longevity through steroid hormone signaling.

In Caenorhabditis elegans and Drosophila melanogaster, removing the germline precursor cells increases lifespan. In worms, and possibly also in flies, this lifespan extension requires the presence of somatic reproductive tissues. How the somatic gonad signals other tissues to increase lifespan is not known. The lifespan increase triggered by loss of the germ cells is known to require sterol hormone signaling, as reducing the activity of the nuclear hormone receptor DAF-12, or genes required for synthesis of the DAF-12 ligand dafachronic acid, prevents germline loss from extending lifespan. In addition to sterol signaling, the FOXO transcription factor DAF-16 is required to extend lifespan in animals that lack germ cells. DAF-12/NHR is known to assist with the nuclear accumulation of DAF-16/FOXO in these animals, yet we find that loss of DAF-12/NHR has little or no effect on the expression of at least some DAF-16/FOXO target genes. In this study, we show that the DAF-12-sterol signaling pathway has a second function to activate a distinct set of genes and extend lifespan in response to the somatic reproductive tissues. When germline-deficient animals lacking somatic reproductive tissues are given dafachronic acid, their expression of DAF-12/NHR-dependent target genes is restored and their lifespan is increased. Together, our findings indicate that in C. elegans lacking germ cells, the somatic reproductive tissues promote longevity via steroid hormone signaling to DAF-12.

Characterization of two melanin-concentrating hormone genes in zebrafish reveals evolutionary and physiological links with the mammalian MCH system.

Melanin-concentrating hormone (MCH) regulates feeding and complex behaviors in mammals and pigmentation in fish. The relationship between fish and mammalian MCH systems is not well understood. Here, we identify and characterize two MCH genes in zebrafish, Pmch1 and Pmch2. Whereas Pmch1 and its corresponding MCH1 peptide resemble MCH found in other fish, the zebrafish Pmch2 gene and MCH2 peptide share genomic structure, synteny, and high peptide sequence homology with mammalian MCH. Zebrafish Pmch genes are expressed in closely associated but non-overlapping neurons within the hypothalamus, and MCH2 neurons send numerous projections to multiple MCH receptor-rich targets with presumed roles in sensory perception, learning and memory, arousal, and homeostatic regulation. Preliminary functional analysis showed that whereas changes in zebrafish Pmch1 expression correlate with pigmentation changes, the number of MCH2-expressing neurons increases in response to chronic food deprivation. These findings demonstrate that zebrafish MCH2 is the putative structural and functional ortholog of mammalian MCH and help elucidate the nature of MCH evolution among vertebrates.

Distinct activities of the germline and somatic reproductive tissues in the regulation of Caenorhabditis elegans' longevity.

The two parts of the Caenorhabditis elegans reproductive system, the germ cells and the somatic reproductive tissues, each influence the life span of the animal. Removing the germ cells increases longevity, and this life span extension requires the somatic gonad. Here we show that the somatic gonad and the germ cells make distinct contributions to life span determination. The life span increase produced by loss of the germ cells requires the DAF-16/FOXO transcription factor. In response to germ-cell removal, DAF-16 accumulates in nuclei. We find that the somatic gonad is not required for DAF-16 nuclear accumulation or for the increased stress resistance that is produced by germ-cell removal. The somatic gonad is required, however, for expression of specific DAF-16 target genes. DAF-16 is known to be activated by reduced insulin/IGF-1 signaling in C. elegans. In certain insulin/IGF-1-pathway mutants, the somatic gonad is not required for germ-cell removal to extend life span. Our genetic experiments suggest that these mutations reduce insulin/IGF-1 signaling below a critical threshold level. At these low levels of insulin/IGF-1 signaling, factors normally provided by the somatic gonad are no longer needed for germ-cell removal to increase the expression of DAF-16 target genes.

Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling.

In C. elegans, removing the germ cells extends life span by triggering the nuclear localization and activation of the DAF-16/FOXO transcription factor in the intestine. In this study, we identify and analyze genes required for germline removal to extend life span. We find that the reproductive system communicates with the intestine through lipophilic-hormone signaling and that a gene called kri-1 is likely to act in the intestine to promote DAF-16 nuclear localization in response to this signal. This lipophilic-signaling pathway and kri-1 are not required for DAF-16's nuclear localization and life-span extension in animals with decreased insulin/IGF-1 signaling. Thus, this pathway specifically enables the integration of cues from the reproductive system with central DAF-16-activation pathways to influence the aging of the animal.

The role of androgens in hormone replacement therapy.

In recent years, increased attention to women's sexual health has propelled basic scientific research and clinical trials investigating treatment paradigms for improving sexual well-being. As the prevalence of female sexual dysfunction has become manifest, knowledge of the intricate pathophysiological role of androgens in maintaining sexual function has fostered a clearer understanding of the effect of age on androgen status, the role of androgens in the postmenopausal ovary, and aetiological mechanisms of androgen insufficiency in premenopausal and postmenopausal women. Understanding the long-term safety and efficacy of physiological androgen replacement and the development of sensitive testosterone assays for specific use in women will better characterise women who are most likely to respond to androgen therapy and, thereby, optimise their quality of life.

Sexual dysfunction in the older woman: an overview of the current understanding and management.

Sexuality is one of the most important quality of life issues for both men and women. Sexual dysfunction is a highly prevalent, age-related and progressive problem. The various physiological and psychological changes that occur with aging can have a significant impact on sexual function. The complexity of female sexual dysfunction remains distinct from that of a man. Thus, we cannot approach female patients or their sexual function problems in a similar fashion to that of male patients. A woman's motivation and ability to find and respond to sexual stimuli is largely influenced by her emotional intimacy with her partner. Frequently, the emotional and relationship well-being a woman experiences contributes more to her sexual enjoyment than does her physiological response. However, it is imperative to assess for possible physiological barriers a woman may have which impede a healthy and satisfying sexual life. Therefore, a comprehensive approach, addressing both the physiological and psychological factors is instrumental to the evaluation of female patients with sexual complaints. After years of ardent research and recent therapeutic advances in male sexual dysfunction, researchers have begun addressing the intricacy of female sexual complaints. Studies involving both pre- and postmenopausal women have reported that most women do experience some type of sexual dysfunction during their lifetime. The sexual complaints women experience in their younger years may follow them into older adulthood, but often times change considerably because of various age-related changes. In an effort to assist researchers and clinicians in designing studies and implementing appropriate evaluation and treatment options for women with sexual complaints, a classification system for female sexual dysfunction has been designed. The four categories of female dysfunction include: hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and sexual pain disorders. Evaluation of women with sexual complaints should include a detailed psychological, social and medical history and thorough physical examination including a hormonal profile. Current treatment options are dependent on the diagnosis and include physical therapy, psychological counselling, hormonal supplements, medication changes and sexual devices. There has also been a burgeoning interest in investigational medications for female sexual dysfunction, from centrally acting (e.g. serotonin agonists) to peripheral, localised treatment (e.g. vasodilating creams). The area of female sexuality and sexual dysfunction has been undergoing important critical changes within the last 10 years. Researchers and clinicians are continuing to recognise the need to try and understand both the psychological and physiological aspects of the female sexual experience and how they influence one another.

Safety and efficacy of sildenafil citrate for the treatment of female sexual arousal disorder: a double-blind, placebo controlled study.

PURPOSE: We evaluated the efficacy and safety of sildenafil citrate in spontaneously or surgically postmenopausal women with female sexual arousal disorder (FSAD). MATERIALS AND METHODS: Sildenafil (a 50 mg dose adjustable to 100 or 25 mg) was evaluated in a 12-week, double-blind, placebo controlled study in 202 postmenopausal women with FSAD who had protocol specified estradiol and free testosterone concentrations, and/or were receiving estrogen and/or androgen replacement therapy. Patients were excluded if emotional, relationship or historical abuse issues contributed significantly to sexual dysfunction. Primary end points were questions 2 (increased genital sensation during intercourse or stimulation) and 4 (increased satisfaction with intercourse and/or foreplay) from the Female Intervention Efficacy Index (FIEI). Secondary end points were the remaining questions from this index, the Sexual Function Questionnaire and sexual activity event log questions. RESULTS: Significant improvements in FIEI questions 2 (p = 0.017) and 4 (p = 0.015) were noted with sildenafil compared with placebo. For women with FSAD without concomitant hypoactive sexual desire disorder (HSDD) sildenafil was associated with significantly greater improvement in 5 of 6 FIEI items compared with placebo (p <0.02). No significant improvements were shown for women with concomitant HSDD. Most adverse events were mild to moderate with headache, flushing, rhinitis, nausea and visual symptoms reported most frequently. CONCLUSIONS: Sildenafil was effective and well tolerated in postmenopausal women with FSAD without concomitant HSDD or contributory emotional, relationship or historical abuse issues. All patients had protocol specified estradiol and free testosterone concentrations or were receiving estrogen and/or androgen replacement therapy.

Tissue-specific activities of C. elegans DAF-16 in the regulation of lifespan.

In C. elegans, the transcription factor DAF-16 promotes longevity in response to reduced insulin/IGF-1 signaling or germline ablation. In this study, we have asked how different tissues interact to specify the lifespan of the animal. We find that several tissues act as signaling centers. In particular, DAF-16 activity in the intestine, which is also the animal's adipose tissue, completely restores the longevity of daf-16(-) germline-deficient animals, and increases the lifespans of daf-16(-) insulin/IGF-1-pathway mutants substantially. Our findings indicate that DAF-16 may control two types of downstream signals: DAF-16 activity in signaling cells upregulates DAF-16 in specific responding tissues, possibly via regulation of insulin-like peptides, and also evokes DAF-16-independent responses. We suggest that this network of tissue interactions and feedback regulation allows the tissues to equilibrate and fine-tune their expression of downstream genes, which, in turn, coordinates their rates of aging within the animal.

Correlation of androgen receptors, aromatase, and 5-alpha reductase in the human vagina with menopausal status.

OBJECTIVE: To determine whether aromatase and 5alpha-reductase mRNAs are expressed in human vagina and to evaluate the presence of androgen receptors in human vaginal tissue based on age and menopausal status. DESIGN: Prospective study. SETTING: Specimens obtained from clinical renal urology practice. PATIENT(S): Premenopausal and postmenopausal women undergoing surgery for prolapse or incontinence. MAIN OUTCOME MEASURE(S): Expression of aromatase and 5alpha-reductase type 1 and 2 mRNAs was evidenced by reverse transcriptase-polymerase chain reaction (RT-PCR), and the density of androgen receptors was measured by semiquantitative immunohistochemistry. RESULT(S): The mRNAs for aromatase and 5alpha-reductase isotypes 1 and 2 were detected in vaginal specimens. Androgen receptors were present in vaginal mucosa, submucosa, stroma, smooth muscle, and vascular endothelium. Expression was significantly greater in vaginal submucosa. A negative correlation existed between age and androgen receptor density. CONCLUSION(S): Expression of genes encoding for enzymes involved in testosterone metabolism in the human vagina, as well as androgen receptor location, density, and changes with menopausal status, suggests that androgens may play a role in regulating vaginal smooth muscle and vaginal blood flow.

Impact of pelvic floor disorders and prolapse on female sexual function and response.

Pelvic floor disorders and FSD are prevalent and challenging problems. These disorders include prolapse of the uterus, cervix, vagina, bladder, and rectum and incontinence. These diseases likely affect women's sexual well-being through physical and emotional effects. Women with pelvic floor disorders often have co-existing urologic and sexual complaints. Patients who present with these urologic problems should be questioned about their sexual function. Surgical treatment in these patients may be curative of their sexual disorders (e.g., by repairing incontinence) but may also have undesired effects on sensation, blood flow, and the anatomy. These effects can affect sexual arousal and orgasm or cause dyspareunia. It is hoped that a better understanding of the anatomy of this area will guide us in a more targeted approach to management of these conditions.

Sexual side effects of SSRI medications: potential treatment strategies for SSRI-induced female sexual dysfunction.

Depression often coexists with sexual dysfunction, and the medical treatment of depression can further worsen sexual symptoms or cause de novo sexual dysfunction in a person who did not experience it prior to treatment. There are many drugs that can adversely affect sexual response. Among antidepressants, this effect is commonly observed with selective serotonin-reuptake inhibitors (SSRI). Various strategies for the treatment of SSRI-related sexual dysfunction have been studied, including: awaiting spontaneous remission of sexual dysfunction; reducing the dose of medication; taking a "drug holiday"; adding another drug to help reverse sexual symptoms; changing antidepressants; or initially starting with a different antidepressant that is known to have fewer or no sexual side effects. Overall, it is important to address sexual health when caring for a patient--to improve drug compliance and the patient's well being.

Safety and efficacy of topical nitroglycerin for treatment of vulvar pain in women with vulvodynia: a pilot study.

OBJECTIVE: To evaluate the safety and efficacy of topical nitroglycerin cream for the treatment of vulvar pain in women with vulvodynia. METHODS: A total of 34 women diagnosed with vulvodynia were included in this study. Patients were treated with 0.2% nitroglycerin cream in the clinic. The cream was applied directly to the skin at the genital/vulvar area where the pain was located. Patients who did not experience any adverse side effects were instructed to use the cream at home at least three times per week, 5-10 minutes prior to sexual relations. Patients completed a pretreatment pain scale at baseline and a posttreatment pain scale questionnaire 4-6 weeks later. RESULTS: Twenty-one patients completed both the pre- and posttreatment pain scale questionnaires, and 13 patients completed only the posttreatment pain questionnaire. Thirty-one patients (91.5%) stated that "overall" their pain had improved. Analysis of the pre- and posttreatment questionnaires revealed a significant decrease in pain intensity on a scale of 0 (no pain) to 5 (excruciating pain; 3.95-2.57; P < .000). There was also a significant decrease in the frequency of overall painful episodes on a scale of 0 (never) to 4 (always; 3.25-2.15, P < .006). All 21 patients reported "improvement" of pain during sexual activity (3.65-2.15; P < .005). CONCLUSION: Topical nitroglycerin is safe and effective in providing temporary relief of introital dyspareunia and vulvar pain in women with vulvodynia. Women who completed this study experienced significant improvement in their overall pain and pain with sexual activity after nitroglycerin use. A larger placebo-controlled study is necessary to establish the optimum dosage level and to minimize the side effects.

Physiology and pathophysiology of female sexual function and dysfunction.

Female sexual dysfunction is age-related, progressive, and highly prevalent, affecting 30%-50% of American women. While there are emotional and relational elements to female sexual function and response, female sexual dysfunction can occur secondary to medical problems and have an organic basis. This paper addresses the anatomy and physiology of normal female sexual function as well as the pathophysiology of female sexual dysfunction. Although the female sexual response is inherently difficult to evaluate in the clinical setting, a variety of instruments have been developed to assess subjective measures of sexual arousal and function. Objective measurements, used in conjunction with the subjective assessment, help diagnose potential physiologic/organic abnormalities. Therapeutic options for the treatment of female sexual dysfunction, including hormonal, and pharmacological, are also addressed.