Eccrine sweat glands play an essential role in regulating body temperature. Sweat is produced in the coiled secretory portion of the gland, which is surrounded by obliquely aligned myoepithelial cells; the sweat is then peristaltically transported to the skin surface. Myoepithelial cells are contractile and have been implicated in sweat transport, but how myoepithelial cells contract and transport sweat remains unexplored. Here, we perform ex vivo live imaging of an isolated human eccrine gland and demonstrate that cholinergic stimulation induces dynamic contractile motion of the coiled secretory duct that is driven by gap junction-mediated contraction of myoepithelial cells. The contraction of the secretory duct occurs segmentally, and it is most prominent in the region surrounded by nerve fibers, followed by distension-contraction sequences of the excretory duct. Overall, our ex vivo live imaging approach provides evidence of the contractile function of myoepithelial cells in peristaltic sweat secretion from human eccrine glands. Ex vivo imaging of eccrine sweat glands suggests that cholinergic stimuli trigger segmental contraction of myoepithelial cells through gap junction-mediated intercellular communication and facilitate peristaltic sweat secretion. Sweat from eccrine glands removes excess body heat from the skin surface through evaporative cooling. Eccrine gland dysfunction can cause either excessive or inadequate sweat secretion, commonly known as hyperhidrosis and hypohidrosis, respectively. Hyperhidrosis leads to physical discomfort, social stigmatization and even mental stress, whereas hypohidrosis can result in heatstroke, a form of life-threatening hyperthermia2. These abnormalities in sweat production are caused by failures in either the hypothalamic thermoregulatory center or local sweat glands4. Because the hypothalamus is a multimodal regulatory center, understanding the local mechanisms of sweat secretion in human skin is fundamental to developing site-specific treatments for sweating dysfunction.Humans have two types of sweat glands: eccrine and apocrine. Eccrine glands are essential thermoregulatory organs that secrete a watery fluid, i.e., sweat, while apocrine glands are evolutionary remnants of odor-producing organs that secrete thick, milky mucus5 , whereas nuclei in the secretory duct were rather sparse because of the elongated myoepithelial cells . In addition, the nuclei of the myoepithelial cells were typically elliptical , while those of the luminal cuboidal cells were rounded . These results indicate that myoepithelial cells are distinguishable from other cell types based on their cellular and nuclear morphologies on 3D live-tissue imaging.The secretory duct of the sweat gland is difficult to observe in vivo and ex vivo because the secretory duct, which forms the coiled portion of the sweat gland, is localized in the deep dermis. To visualize the shape and location of myoepithelial cells of the secretory duct, we harvested eccrine glands from human skin and live-stained the whole eccrine coiled structure. Because myoepithelial cells contain abundant filamentous actin, we used Acti-stain 488 (Acti-stain), a fluorochrome-conjugated phalloidin that directly binds to filamentous actin. The staining patterns of Acti-stain for excretory and secretory ducts were distinguishable Fig. . Acti-stnal Fig. . Furthernal Fig. ; arrows,5, we hypothesized that the myoepithelium in the secretory duct would synchronously contract by autonomic nerve stimulation to extrude sweat from the secretory duct. Thus, we attempted to record the motions of the isolated eccrine glands after labeling their nuclei and actin cytoskeletons with Hoechst 33342 and Acti-stain, respectively. Eccrine glands are innervated by atypical sympathetic terminals, which release acetylcholine8. We tracked the motions of both the nuclei and the tissue structure of the coiled portion of the eccrine gland upon stimulation with pilocarpine, a cholinergic agonist, to mimic autonomic nervous stimuli for sweating9. To quantitatively describe the motions from time-lapse images, we adopted the color aberration measurement of two merged images pseudocolored green or magenta at different time points  for both myoepithelial actin structures and nuclei , a cell membrane marker10, which should not interfere with physiological actin dynamics. CMDR staining visualized the αSMA-positive myoepithelial cells of the secretory duct as thick striped streaks . Previously, we reported that PGP9.5-positive nerve fibers were predominantly wrapped around the secretory portions of sweat glands5. Consistent with this observation, double immunostaining for PGP9.5 and αSMA confirmed that the PGP9.5-positive nerve fibers exclusively surrounded the secretory ducts transversely, while αSMA-positive myoepithelial cells were longitudinally aligned like thick stripes on the secretory duct  and a thick striped pattern with a faint contour of spindle-shaped myoepithelial cells . In sharp contrast, the excretory duct was visualized in a latticework pattern . Upon pilocarpine stimulation, only the concertina-like part of the secretory duct exhibited dynamic contractile motion . To further scrutinize the mode of nuclear displacement at the single-cell level, we also compared the direction of nuclear displacement in both myoepithelial and luminal cells. The elliptical myoepithelial nuclei diagonally slid along the perimeter of the duct . On pilocarpine administration, the narrow segment of the lumen started to shrink immediately  at t = 2’00”, Fig. open triangle (Δ) at t = 9’20”, Fig. open diamond (◊), Fig. closed triangles (▲), Fig. To further address the role of the contractile motions of the eccrine gland in sweat secretion, we analyzed the deformation of the CMDR-stained cellular latticework, focusing on the changes in the volume of the luminal space within the duct of the coiled structure. Cell membrane staining by CMDR revealed temporal changes in the latticework of the cellular structure and the lumen volume as signal voids inside the secretory coil. We extracted the areas of the signal voids from the 3D time-lapse data to trace the successive changes in the lumen volume in both the secretory and excretory ducts, thereby capturing the motion of sweat transport driven by the contraction of the secretory duct Fig. . First, 14, some smooth muscle cells are electrically interconnected via gap junctions to operate in a coordinated fashion as a functional cluster15. Accordingly, excitation can rapidly spread among unitary smooth muscle cells, resulting in segmental contraction. To determine whether the myoepithelial cells of the secretory duct are also interconnected by gap junctions, we examined the effects of carbenoxolone (CBX), a broad-spectrum inhibitor of gap junctions16, on the contractile motion of the secretory duct upon pilocarpine stimulation. In the presence of CBX, both luminal and myoepithelial nuclei scarcely moved from their initial positions after pilocarpine treatment , although no such punctate signals were detected on myoepithelial cells for other connexins, including Cx26, Cx30, Cx31, and Cx37 , in the myoepithelial cells of the secretory duct by quantitative reverse transcription polymerase chain reaction (qRT‒PCR). Because the myoepithelial cells in the eccrine sweat glands have been characterized as a CD29ant Fig. . Immunofx37 Fig. . Punctatx37 Fig. , raising18, although 2-APB has been known to exhibit complex pharmacology, inhibiting not only Cx43 but also inositol trisphosphate receptors (IP3Rs) and transient receptor potential (TRP) channels and having a dose-dependent bimodal effect on store-operated calcium entry19. Both inhibitors attenuated the pilocarpine-induced contractile motion of the secretory duct and the nuclear displacement of the luminal cells  and Gap27, both of which inhibit Cx43lls Fig. , 17, supFinally, we examined the contribution of gap junctions to sweating in vivo. We applied CBX to the human palm and measured the perspiration rate by the minor test, which visualizes the expulsion of sweat drops by the metachromasia of the iodine-starch reaction Fig. . Sweatin22, no study reported to date has captured the live motion of eccrine glands. In the present study, we established a method for ex vivo 3D live imaging to observe the overall contractile motions of isolated human eccrine glands. The dynamic motion of the coiled structure upon cholinergic stimulation was successfully captured, recapitulating the physiological contractile response to autonomic stimuli23. The dynamic motion was verifiably the direct consequence of cholinergic stimulation because atropine, a cholinergic antagonist, blocked the motion of the coiled structure. We found that some parts of the secretory duct were visualized on CMDR staining in a concertina-like pattern, which likely reflects sympathetic nerve fibers surrounding the secretory duct. The secretory coils of eccrine glands have been shown to be innervated by sympathetic nerves with varicose nerve terminals24. The concertina region in the secretory duct displayed more pronounced contractile motion upon cholinergic stimulation than the region with little or no concertina-like signal, consistent with previous ex vivo studies demonstrating glandular contraction22 and sweat secretion25 induced by cholinergic stimuli. Our results also revealed that contractile motion of the secretory duct was blocked by gap junction inhibitors, including CBX, 2-APB, and Gap27. The inhibitory effect of CBX was also confirmed by the in vivo perspiration test on human palms. These results raise the possibility that gap junction-mediated communication between myoepithelial cells controls the effective propagation of contraction in a single long tubular structure. Our ex vivo 3D live imaging is therefore a useful model for deciphering the mechanisms that operate during sweat secretion by eccrine glands and the role of myoepithelial cell contraction in transporting sweat to the skin surface.Although the motions of eccrine glands have been investigated by several methodologies29. Myoepithelial cells are enriched in actin filaments and arranged longitudinally in parallel around the secretory duct of the eccrine gland. We demonstrated that cytochalasin D, an inhibitor of actin polymerization, blocked the pilocarpine-induced contractile motion of the secretory duct of the isolated coiled structure. Acti-stain, a fluorochrome-conjugated phalloidin, also reduced the contractile response to cholinergic stimulation, supporting the role of myoepithelial cells in the contractile motion of the isolated coiled structure. Previously, Sato et al. examined the contractile properties of an isolated secretory duct of the primate palm sweat gland using a sensitive mechanical strain gauge transducer and demonstrated that acetylcholine, but not adrenergic agonists or the calcium ionophore A23187, induced glandular contraction of the secretory duct21. Consistent with this observation, our 3D live imaging detected shortening of the secretory duct in the region showing a concertina-like pattern upon CMDR staining. Such shortening of the secretory duct was not significantly detected in the region that did not show a clear concertina-like pattern. Furthermore, the high-resolution analysis focusing on the displacement of the nuclei of luminal and myoepithelial cells in cross-sections indicated that the curvature radii of the secretory duct decreased after pilocarpine treatment in the following manner: myoepithelial nuclei slid diagonally along the perimeter of the secretory duct, followed by sideways sliding and subsequent downward displacement of luminal nuclei. These observations indicate that contraction of myoepithelial cells causes not only shortening but also narrowing of the secretory duct.Several findings indicate that myoepithelial cells surrounding the exocrine secretory ducts and/or alveoli contract upon neuronal or hormonal stimulation31. Myoepithelial cells in mammary glands contract in response to the pulsatile release of oxytocin from the pituitary gland to eject milk. Mice deficient in αSMA (ACTA2), the most abundant actin isoform in the mammary duct, exhibited no gross anatomical defects but presented lactation failure26. The mammary gland myoepithelial cells from ACTA2-null dams contracted considerably less than those from wild-type dams26. Similar lactation defects with reduced contractility of myoepithelial cells have been reported in mice deficient in Orai1, a store-operated Ca2+ channel subunit required for milk ejection32. It should be noted that isolated sweat glands from Orai1-deficient mice exhibited a comparable calcium release from ER stores upon acetylcholine stimulation with that from wild-type mice33. In our study, we observed pilocarpine-induced contractile motion of isolated sweat glands in Ca2+-free PBS. Because intracellular calcium was not depleted by EGTA in our experimental conditions, such initial calcium release from ER stores by pilocarpine stimulation may be sufficient for the induction of the contractile motion of the isolated sweat glands. Notably, mammary myoepithelial cells have been shown to communicate with each other through gap junctions34. Given that gap junction inhibitors attenuate the contractile motions of the isolated eccrine secretory coil, it is conceivable that the coordinated contraction of myoepithelial cells via gap junctions operates in both mammary and sweat glands to expel secretory products.The contraction of myoepithelial cells is considered instrumental in the secretory functions of other exocrine organs, including mammary, salivary, and lacrimal glandsThe mechanistic basis of how sweat is transferred from the secretory duct to the excretory duct remains to be elucidated. Our 3D image reconstruction analyses focusing on the cholinergic stimulation-induced changes in luminal volume demonstrated that the lumen of the secretory duct was segmented by partition and underwent biphasic contraction with a rebound relaxation period upon cholinergic stimulation, followed by the progressive dilation-regression cycle of the excretory duct. These results raise the possibility that the secretory duct of eccrine glands is divided into segments and contracts segmentally upon cholinergic stimulation to transport sweat to the excretory duct.Consistent with the scheme, the nerve fibers wrap around the secretory coil in a discrete manner to yield CMDR staining patterns with and without (or less) concertina-like signals, thereby eliciting the segmental contraction of the highly entangled secretory coiled structure. Such segmental contraction may effectively summate the compression force of the segmented portion to transport the secreted sweat toward the excretory duct.36, which has been shown to occur in response to the firing rhythm of skin sympathetic nerve activity37. Such pulsatile sweat expulsion can be elicited either by rhythmic contraction of myoepithelial cells or by sweat production by secretory cells in a pulsatile rhythm. Segmental contraction of the secretory duct, together with intermittent cholinergic stimulation, is instrumental to ensure high-frequency pulsatile sweat expulsion from the eccrine gland.Previous in vivo studies demonstrated high-frequency pulsatile sweat expulsion in humans11. Both muscarinic and nicotinic acetylcholine receptors were detected in eccrine sweat glands by in situ hybridization and immunohistochemistry12. Our results show that nicotinic stimulation does not trigger the contractile motion of the secretory duct, making it likely that nicotinic stimulation governs sweat production from secretory cells but not the contraction of myoepithelial cells. Consistent with this conclusion, Sato and Sato (1981) reported that isolated eccrine sweat glands contracted in response to muscarinic agonists  but not in response to nicotine25. They concluded that sweating in response to nicotine was elicited through the axon reflex, as proposed by Coon and Rothman11.Perspiration can be induced not only by agonists of muscarinic acetylcholine receptors but also by an agonist of nicotinic acetylcholine receptors, i.e., nicotinehigh/CD49fhigh subpopulation indicate that the myoepithelial cells express Cx26, Cx30, Cx31, Cx37, Cx43, and Cx45, of which Cx43 is most abundant. Immunofluorescence staining of Cx43 in the secretory coil of the eccrine gland shows that Cx43 is expressed on the myoepithelial cells in small puncta that are frequently detected between the myoepithelial cells, making it likely that the gap junctions connecting the myoepithelial cells are predominantly composed of Cx43. In support of this possibility, Gap27, a peptide gap junction inhibitor derived from Cx43, blocked the pilocarpine-induced contractile motion of the secretory coil of the isolated eccrine gland. Consistent with this view, there is accumulating evidence that Cx43 is the major connexin type expressed in myoepithelial cells of various exocrine glands40. In mammary glands, while Cx26, Cx30 and Cx32 are expressed in luminal cells, Cx43 is primarily expressed in myoepithelial cells41 and is involved in myoepithelial contraction for milk ejection42. Thus, ectopic expression of Cx26 suppresses the expression of Cx43 in the myoepithelial cells of mammary glands, leading to impaired GJIC and defects in milk ejection34. A similar defect in milk ejection has been reported in a mouse model harboring a missense mutation in the Cx43 gene, which resulted in reduced Cx43 expression in myoepithelial cells40. Cx43 has also been observed at gap junctions connecting myoepithelial cells in sublingual glands and implicated in the contraction of myoepithelial cells39. Given that Cx43 is also predominantly expressed in the smooth muscle cells of the aorta43 and is involved in vasoconstriction by vasopressin44, it is conceivable that GJIC through Cx43 underlies the effective propagation of contractile signals within a segmented cluster of myoepithelial cells in the secretory duct of eccrine glands.Our results show that the segmental contraction of myoepithelial cells requires gap junctional intercellular communication (GJIC), as CBX, 2-APB, and Gap27 inhibited the contractile motions of the isolated secretory coil upon cholinergic stimulation. qRT‒PCR analyses of the connexins expressed in the myoepithelial cell–enriched CD2945. However, the sole antiperspirants available for the last century have been conventional metal colloid–type antiperspirants that mechanically plug sweat pores46. These mechano-plug antiperspirants are convenient, but they are clinically and esthetically disadvantageous because they irritate the skin and stain clothes48. Additionally, the sweat held in the ducts can pathologically leak into the dermis, causing acute exudative inflammation51. In contrast, selective gap junction inhibitors in the form of antedrugs could be safe antiperspirants with minimal adverse effects on the skin. CBX is such a candidate ingredient for next-generation antiperspirants. Notably, chemotherapeutic drugs acting on the hypothalamic‒pituitary‒adrenal (HPA) axis are known to have an impact on perspiration. For example, many antidepressants that act on the HPA axis have a side effect of causing excessive sweating52. Antiepileptic drugs sometimes cause sweating disorders by acting on the hypothalamic–pituitary–somatotropic axis, resulting in diminished sweating and a risk of heatstroke54. Conversely, it is known that anticholinergic drugs cause diminished sweating and have been used to treat hyperhidrosis, although these drugs cause side effects such as dry mouth, dry eyes and constipation55. CBX can be applied as a topical therapeutic agent that acts on the sweat glands directly by blocking GJIC, thereby eliminating the side effects of anticholinergic drugs.The requirement of GJIC in sweat secretion in vivo was also demonstrated by the perspiration test on a human palm, as topical administration of CBX suppressed sweat secretion by 30%. From a clinical viewpoint, selective inhibitors of Cx43 may be promising as antiperspirants to halt sweating without the drawbacks of conventional treatments. In everyday life, abnormally excessive sweating causes various social problems, including mental stress caused by self-hatred and unnoticed harassment by others in addition to daily inconvenienceIn conclusion, 3D live-tissue imaging of the whole eccrine coiled structure revealed the gap junction-mediated contraction of myoepithelial cells in the secretory duct and progressive dilation-regression cycle of the excretory duct for sweat expulsion. The contraction sequence is thus more complicated than simply compressing the secretory duct, requiring further investigation of the segmental contraction that occurs in the secretory duct.Fresh human postoperative skin specimens were obtained with written informed consent from all subjects at Osaka University Medical School , Kinugasa Clinic , Seishin Plastic and Aesthetic Surgery Clinic Co., Ltd  and Bizcom Japan . Experiments using human skin samples were approved by the Ethics Committee of Osaka University . Dissected skin specimens were stored at 4 °C in saline for up to 72 hours before use.56. The labeled eccrine glands were gently picked up manually from the skin by pinching the surrounding tissues with forceps to minimize damage to the ducts under a stereomicroscope  and placed in PBS(-) until use in subsequent experiments.Human eccrine glands were isolated from fresh skin specimens by microsurgery as follows. Eccrine glands in the dermis were labeled using 10 mM neutral red  in divalent cation-free PBS (PBS(-)) for 5–10 minutes prior to microsurgery18, except that the mounted eccrine glands were immersed in Ca2+/Mg2+-free PBS in the experiments shown in Fig. Freshly isolated eccrine glands were incubated for 30 minutes at 25 °C in vital stain solution supplemented with either Acti-stain 488 , CMDR  or Hoechst 33342 and then washed with PBS (-) for 10 minutes. The stained eccrine glands were mounted on glass-bottom dishes  coated with collagen gel , which stably held the isolated eccrine glands. To prevent the samples from drying, the mounted eccrine glands were immersed in Krebs–Ringer bicarbonate solution (KRS) supplemented with 11 mM glucose at 25 °CFor the time-lapse 3D visualization of eccrine gland dynamics, multicolor fluorescence images of labeled nuclei (excitation: 405 nm), cell membranes (excitation: 650 nm) and actin filaments (excitation: 488 nm) were recorded under a confocal microscope  with a 20× objective lens  at intervals of 30 or 40 seconds for 30 minutes, depending on the thickness of the samples. At each recording time, twelve to thirty Z slices at 2-µm steps were obtained and immediately stacked to reconstruct the 3D images of the ducts. The Z-stacked images were freely rotated, and any plane of interest was obtained as a single image using ImageJ . To induce eccrine gland contraction, 10 mM pilocarpine  was added 3 or 10 minutes after the initiation of image recording, thereby confirming the absence of any significant spontaneous contraction with the gland tested. To examine the nicotinic effect on sweat gland contraction, 6 mM nicotine  was added at 10 minutes to start the observation. To inhibit pilocarpine-induced contraction, the eccrine glands were treated with 10 µM atropine , 1 mM CBX , 50 µM 2-APB , or 50 µM Gap27  30 minutes before stimulation with pilocarpine. Eccrine gland dynamics were calculated by the object-tracking function of ImageJ, focusing on nuclei. Short-interval time-lapse images of eccrine gland dynamics were captured by confocal microscopy with a spinning disk system  and a 20× or 40× objective lens .To confirm whether myoepithelial contraction induces gland dynamics, actin dynamics were inhibited by cytochalasin D , which was dissolved in dimethyl sulfoxide  and used at 10 µM, where the DMSO concentration in the chamber was 0.1% after dilution with KRS. The mounted eccrine gland was pretreated with cytochalasin D 30 minutes before stimulation with pilocarpine. As the control, 0.1% DMSO was applied for pretreatment at the same time point.Original confocal images before Z-stacking were processed to detect the cellular latticework signals as follows. First, the CMDR cell membrane signals were selected and rendered into a continuous surface by Imaris software’s Surface tools . Within the rendered surface, the areas with continuous signal intensities corresponding to the cell-rich walls of the duct were digitally masked by the Imaris Masking tool. Next, the masked areas were cut out from the images. Then, the remaining cell-free luminal areas were selected and integrated along the depth to calculate the lumen volume of the inner duct in each 3D view along the entire length of the time-lapse movie. The nuclear signals provided information regarding the position of individual cells in motion. The 3D luminal views were imposed on the original 3D data, and any planes of interest were obtained by the Oblique-slicer tool in Imaris. The displacement of the nuclei was analyzed by the distance-calculation function in Imaris.Isolated eccrine glands with or without CMDR staining were fixed in 4% paraformaldehyde (PFA) in PBS  at 25 °C for 4 hours, washed three times with PBS supplemented with 0.5% Triton X-100 (PBS-T), and blocked with 5% bovine serum albumin  in PBS-T at 4 °C for 12 hours. Subsequently, the eccrine glands were incubated with anti-αSMA antibody  and anti-PGP9.5 antibody  in the same blocking solution at 4 °C for 12 hours. After washing with PBS-T three times, the eccrine glands were incubated with Alexa594-conjugated anti-rabbit antibody , Alexa 488-conjugated anti-mouse antibody  and Hoechst 33342  at 25 °C for 12 hours.Skin tissues were fixed in 4% PFA in PBS at 4 °C overnight, embedded in optimal cutting temperature compound  and frozen in liquid nitrogen–chilled 2-methylbutane. The samples were sectioned at 10 μm using a cryostat , fixed in 4% PFA in PBS and blocked with 5% BSA in PBS-T at 25 °C for 20 minutes. For Cx31 staining, frozen skin tissue blocks were used; after sectioning, they were fixed with ice-cold acetone for 15 minutes. Subsequently, the sections were incubated with primary antibodies in the same blocking solution at 4 °C for 12 hours. The antibodies used were anti-Cx26 , anti-Cx30 , anti-Cx31 , anti-Cx37 , anti-Cx43 , anti-Cx45  and anti-αSMA . After being washed with PBS-T three times, the sections were incubated at room temperature for 2 hours with the following secondary antibodies and Hoechst 33342 (1:1000): Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 594-conjugated donkey anti-rabbit or anti-goat IgG . After a final wash, the slides were mounted with Prolong Gold .57. All ethical regulations relevant to human research participants were followed. Signed informed consent was obtained from all participants.The activity of human eccrine glands was measured on the palm of six healthy volunteers  under passive heating by a foot bath with approval from the Ethics Committees of Mandom Corporation . Prior to the heat stress test, active sweat pores were stained by the starch-iodine method (Minor test). Absorbent cotton soaked in 16 mM CBX in 20% ethanol or in 20% ethanol was placed on the palm and held in position for 30 minutes at 25 °C. Then, the absorbent cotton was removed, and the surface of the palm was dried. The palms were stained with 3% w/v iodine  in ethanol and 10% starch solution in castor oil . To induce sweating, the volunteers immersed their feet in hot water (42 °C) for 30 minutesThe statistical analyses were performed using either Microsoft Excel  or KaleidaGraph 4.5 . All data are presented as the mean ± standard deviation (SD).https://www.wolframalpha.com/widgets/view.jsp?id=b0d28dd78e48b231c995d69b91666803).The coordinates of three continuous nuclei of luminal cells were chosen and plotted on an arbitrary Cartesian plane. These coordinates were applied to obtain the radius of curvature using the online calculation platform Wolfram Alfa  with 600 U/ml collagenase type II  on a tube rotator. After centrifugation at 300× g for 5 minutes, a single-cell suspension was obtained by repeated pipetting for 3 minutes in prewarmed 0.5% trypsin/1 mM EDTA in PBS, followed by repeated pipetting for 1 minute in prewarmed 5 mg/ml dispase /0.1 mg/ml DNase I (Sigma). The resulting suspension was filtered through a 40-µm mesh . Immunolabeling of cells was performed at 4 °C for 30 minutes in PBS containing 5% BSA with allophycocyanin-conjugated anti-CD29  and Brilliant Violet 421-conjugated anti-CD49f . After being washed twice with 5% BSA in PBS, the cells were resuspended in PBS with 7-AAD  for dead cell staining.high/CD49fhigh population was sorted for further qRT‒PCR analysis.Flow cytometric analysis and cell sorting were performed with a FACSAria2 (BD Biosciences), and the CD29Total RNA was isolated using TRI Reagent LS . cDNA was synthesized using the QuantiTect Reverse Transcription Kit . Real-time quantitative PCR was performed using THUNDERBIRD SYBR qPCR Mix  and a ViiA7 real-time PCR system . The expression levels of target genes were normalized to that of 18 S ribosomal RNA using a standard curve method. The primer sets for qRT‒PCR are listed in Table Further information on research design is available in the Peer review fileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary movie 1Supplementary movie 2Supplementary movie 3Supplementary movie 4Supplementary movie 5Supplementary movie 6Supplementary movie 7Supplementary movie 8Supplementary movie 9Supplementary movie 10Supplementary movie 11Supplementary movie 12Supplementary movie 13Supplementary movie 14Supplementary movie 15Supplementary movie 16Supplementary movie 17Reporting Summary


Cell Genomics, Maury et al.Detection of organismal mosaic states for variant alleles faces technical and analytical challenges, as does the association of such variant alleles with susceptibility to neurologic disease. In this issue of  Cell Genomics, Maury et al. reanalyze genotyping arrays of a schizophrenia cohort providing evidence for the contribution of somatic structural variant mutagenesis and rare variant alleles.Detection of organismal mosaic states for variant alleles faces technical and analytical challenges, as does the association of such variant alleles with susceptibility to neurologic disease. In this issue of  Mosaic mutations are now well established to underlie rare Mendelian disease.,Germline copy number variants (gCNV), including heterozygous deletion CNVs at 22q11.21, 15q13.3, and 1q21.1 and duplication CNV at 16p11.2, were among the first specific type of genome mutations robustly associated with susceptibility to schizophrenia. gCNVs were found to underlie cognitive phenotypes both in population studiesGenome-wide detection of somatic mutation events are dependent on: (1) the cell(s) or tissue source from which the genomic DNA was isolated; (2) the perspective and resolution of the assay applied to investigate that personal genome and alleles at a specific locus, e.g., SNP or CGH arrays, exome sequencing (ES), or genome sequencing (GS); and (3) the computational tools  applied to the analyses of the genome-wide data. For both ES and GS massively parallel DNA genome sequencing approaches, the read depth of coverage and allele fraction observed are important experimental parameters to measure inference of the organismal allelic state at a given locus.,de novo mutation for both sCNVs and sSNVs. Analyses of somatic mutations in humans have revealed asymmetric cellular dynamics in early human embryos and the cell population dynamics of peripheral blood, informing on clonal dynamics of early human embryogenesis,Parental genome unification of the paternal- and the maternal-derived constituents composing the diploid genome of the postzygotic somatic cell  is highly error prone in mammalian embryos.NRXN1 and ABCB11 to be recurrently involved in sCNVs in their dataset and a burden of such sCNVs in susceptibility to schizophrenia. Interestingly, three different-sized sCNV alleles detected by 40× GS resulted in deletions of NRXN1 exons 1–5. Breakpoint analyses from the available GS data implicated multiple SV mutagenesis mechanisms.A key aspect of the approach by Maury et al. was to filter sCNVs that involve gene loci mutated in clonal blood disorders and focus on recurrent sCNVs involving genes previously established to contribute to cognitive traits. This enabled enhancement of experimental signal-to-noise ratio and better visualization of early developmental sCNVs. They observed Alu or propensity to form unusual non-B-DNA conformers, making the locus more susceptible to genomic instability and DNA SV mutagenesis. This vulnerability to loss of genome integrity can potentially result from either a nick to the double helix backbone or a double-strand break (DSB), disrupting genome integrity. At a nicked locus, DNA replication through the gene locus can result in a collapsed fork and generation off a one ended, double-stranded DNA (oeDNA) molecule. The DNA DSB and oeDNA are likely to be repaired through different recombination or replication-based pathways.Systematic breakpoint junction studies of exon deletion and duplication variant alleles for all genes causing rare disease traits will likely be a rich data source both for unraveling the molecular basis of disease and for further understanding the mechanism of SV mutagenesis. Gene loci that are recurrently mutatedNRXN1 sCNV were explored by generating Hi-C data on neurons differentiated from induced pluripotent stem cells (iPSCs) with the heterozygous deletion versus control non-engineered iPSC-derived neurons. These studies suggest allele-specific compromise of topologically associating domain (TAD) structural integrity in schizophrenia. Their working model proposes de novo looping in 5′ NRXN1 deletion alleles that connects exon 6 to a putative regulatory element, promoting the formation of spurious pathogenic transcripts. Interestingly, such SV alleles could generate a potential gain-of-function mutation, as a hypermorphic or neomorphic allele, from copy number loss.The potential functional effects of the During the last decade, a better understanding of the molecular mechanisms of SV mutagenesis and the continuing establishment of allelic series of novel genes have contributed to some of the successes of re-analyses of extant genomic data but have also pointed out the limitations of such lower-resolution genome data to resolve variant alleles. One must first detect the SV allele and then identify the breakpoints to determine any predicted consequence to gene structure and phase to the paternal versus maternal allele. This pertains to both coding and non-coding variant alleles that may potentially contribute to perturbations in organismal biology. Some of these limitations in detecting and resolving variant alleles can be overcome by appropriate GS approaches. Family trio studies using long-read genome sequencing may enhance detection of both coding and non-coding variant alleles, enable phased genomes, identity of mutational signatures, and better resolution of SV, potentially uncovering more of both gCNV and sCNV variation contributing to disease.


Colorectal cancer with a global incidence of 10% has multiple pathways implicated in its carcinogenesis. WNT signaling is the principal underlying pathway via APC gene, while defective mismatch repair genes and epigenetic changes also are known to contribute.Here, we present an unusual case of rectal adenocarcinoma in a woman, with germline MSH6 and PMS1 mutations, and simultaneous somatic APC and TP53 mutations treated with surgery and adjuvant capecitabine.The case is unique suggesting a possible interaction between the two pathways and contributing to carcinogenesis in this patient. This also suggests need for a thorough germline and somatic mutation evaluation in select colorectal cancer patients to direct a tailored therapy. Colorectal carcinoma (CRC) has a global incidence of 10% ranking third among all cancers overall and accounts for 9.4% of cancer deaths worldwide. It ranks third in males and is second only to breast cancer in females . The significance of these variants however has not yet been established. But both being DNA mismatch repair genes, their protein expression may be defective, and hence, the patient might have an increased predisposition to cancer (Lynch syndrome). Family members including siblings and children were counselled for increased risk of malignancy and advised hereditary cancer risk gene panel.Owing to the presence of somatic APC and TP53, and germline MSH6 and PMS1 mutations in the patient, six cycles of single-agent capecitabine were given as adjuvant therapy. The patient was evaluated 2 months after completion of chemotherapy with PET scan and CEA which were normal. Patient was kept on follow-up at 3-month interval with CEA. Patient has been disease free for 36 months until date since the end of primary treatment.Carcinogenesis involves a series of events through acquisition of multiple genetic alterations which makes the milieu susceptible for further mutations and the precancerous cells genomically instable accelerating the process . MajoritIn our case, we identified somatic mutations in APC  and PMS1 (c.321A > C), whose significance has not yet been established. MMR system functions to identify and correct errors during DNA replication-single base pair mismatch and insertion-deletion loops. HNPCC is a genetically acquired autosomal dominant disease resulting from germline defect in MMR genes which increases susceptibility to CRC as well as extra-colonic malignancies. It has incomplete penetrance and a variety of expressions depending upon the gene mutated. Mutations in MSH2 and MLH1 account for 80%, with MSH6 gene mutated in 7–20% cases, PMS2 in < 5% cases, and other MMR genes are involved rarely [C > T andMSH6 mutations are associated generally with endometrial cancer and increase the likelihood of colorectal cancer by eightfold . PathogePMS1, another MMR protein, forms complex with MLH1 (hMutL beta), hMutS complex, and other proteins to facilitate excision of incorrect nucleotides and their repair . Its mutMicrosatellite instability (MSI) testing is essentially important in phenotyping colorectal cancers and is indicated in all colorectal tumors. Instability results either due to defect in MMR gene or epigenetic silencing of promoter region mostly MLH1. Tumors with MSI have better prognosis compared to microsatellite stable (MSS) tumors. Although they do not respond to 5-fluorouracil treatment, they are amenable to immunotherapy. It can be assessed by two ways: PCR and IHC. In the former, nowadays, instability at five quasi-monomorphic microsatellite sites can be detected simultaneously: BAT-26, NR-21, BAT-25, MONO-27, and NR-24 . In the The current case had somatic APC and TP53 pathogenic mutations while also possessing germline MSH6 and PMS1 mutations suggesting progression of cancer along somatic pathway with underlying defective mismatch repair pathway as in LS.The late onset and slow progression could probably be due to low penetrance of dMMR genes which affects the phenotype.The case is unique in its evolution predicting coexistence of three different pathways of carcinogenesis and their possible interplay which has not been reported.An extended panel should be used for evaluating MSS tumors.In select cases, both germline and somatic mutations should be looked for as they will help in understanding of the disease process better.MSI testing using IHC or PCR may not be accurate, and NGS may be a better alternative


Usefulness of an ultrathin endoscope with a newly developed knife for complex esophageal endoscopic submucosal dissection A 62-year-old woman underwent subtotal esophagectomy for esophageal cancer, local resection of pharyngeal cancer with postoperative radiotherapy, and bilateral cervical lymph node dissection owing to pharyngeal cancer metastasis. A follow-up endoscopy revealed a 10-mm brownish area in the cervical esophagus, consisting of type B1 vessels according to the magnifying endoscopic classification of the Japan Esophageal Society,www.giejournal.org). We started marking with a magnifying endoscope ; however, we could not mark the depressed area  was performed with the patient under general anesthesia because the lesion was located in the cervical esophagus (sed area . Thus, wsed area  and 3. Nsed area  to obtaised area  with incsed area  without sed area  without sed area .Figure 1,,The usefulness of ESD with an ultrathin endoscope with the tip of a snare for esophageal strictures has been reported.


CRLF2 locus that is often involved in chromosomal translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Latin Americans (LAs). To support this LA-specific identification of AID mutation signatures, we characterized DNA from immature B cells isolated from the bone marrow of ALL patients. Our ability to detect and quantify these mutation signatures will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.Activation-induced cytidine deaminase (AID) is a B cell-specific base editor required during class switch recombination and somatic hypermutation for B cell maturation and antibody diversification. However, it has also been implicated as a factor in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain types of blood cancers is critical in assessing disease severity and treatment options. Here, we have developed a digital PCR (dPCR) assay that allows us to track the mutational landscape resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this new assay showed that increased AID levels in immature B cells increases genome instability at loci linked to translocation formation. This included the  The consequences of aberrant AID expression and off-targeting are evident in the etiology of several B cell cancers.7 Further, that AID is B cell specific, and B cell cancers account for approximately 80–90% of hematopoietic malignancies,8 is a further indicator of the dangers of abnormal AID activity. Sequestering the RAG-induced DSBs of V(D)J recombination to pre-B cells and the AID-induced DSBs of CSR to mature B cells helps maintain genome stability. However, AID can be transiently expressed in pre-B cells concurrent with RAG expression when pre-B cells are stimulated by cytokines.9 Indeed, examination of the various chromosomal translocations that occur in many lymphoid malignancies show that one DSB was RAG-induced at the IGH locus adjacent to a D or J cassette while the other DSB locus shows evidence of AID activity,10 indicating the most likely mechanism for oncogenesis in a subset of cancers is AID expression in pre-B cells.11AID evolved to be an integral part of the adaptive immune system,BCL2 and CRLF2. 13 Unlike random DSB sites that can span tens or hundreds of kilobases, these AID-induced DSBs are focal with many occurring within 20–600 bp regions that we refer to as AID break clusters (ABCs).Understanding both why some RAG-induced DSBs in pre-B cells fail to be efficiently repaired by nonhomologous end-joining (NHEJ) and what leads to AID activity in pre-B cells will allow us to better predict populations susceptible to certain blood cancers and discern the etiology of these malignancies. Two loci that show mutations consistent with AID activity in pre-B cells are 14 For example, Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-Like ALL) has a higher cancer incidence in Latin Americans (LAs).17 Ph-like ALL shares several characteristics with Philadelphia-positive (Ph+) ALL regarding the gene expression profile, yet lacks the eponymous t BCR-ABL1 translocation.18 LAs specifically have a higher likelihood of developing Ph-like ALL and respond less favorably to current standard treatments.19 Genome rearrangements and mutations leading to Ph-like ALL are highly diverse, yet LAs are more likely to have Ph-like ALL characterized by a translocation between IGH and the ABC upstream of the CRLF2 locus.19 This is in contrast to Europeans and non-Latin Americans that more often have an interstitial deletion between CRLF2 and P2RY8.20 While some risk alleles have been identified that link LAs to higher Ph-like ALL risk,9 no molecular mechanism has been established to understand why CRLF2-IGH formation is more prevalent in this population.A critically understudied area in genome instability associated with diseases are cancer health disparities where one racial or ethnic group shows higher incidence and poorer outcomes of certain cancer types.CRLF2 ABC linked to LAs with Ph-like ALL and the non-LA linked BCL2 ABC linked to follicular lymphoma. In many cases, these indels are the “informational scars” left behind by NHEJ DSB repair due to processing factors that remove or add nucleotides prior to ligation.21 To deeply characterize these genetic changes, we developed a digital PCR (dPCR) assay to detect and quantify changes at the CpG sites in ABCs targeted by AID. We show that increased AID expression correlates with an increase in indels at these ABCs. Strikingly, these indels are also detected in ALL patient samples but not samples from healthy donors, indicating that dPCR can provide a novel diagnostic for early detection of patients with aberrant AID expression that would increase cancer risk.Here, using both human cell lines and patient samples we identify an increase in insertion and deletion events (indels) at two ABCs, the 2.Nalm6 and Reh cell lines were purchased from the American Type Culture Collection  and grown in RPMI-1640 supplemented with 10% FBS, 1% Penicillin/Streptomycin, 1% Glutamax (Gibco), and 50 μM b-mercaptoethanol. Cell lines with the transduced AID doxycycline (dox)-inducible cassette were grown under G418 selection (1 mg/mL of G418). Cells with constitutive Cas9 expression were grown in complete media further supplemented with 0.75 μg/mL of Puromycin. All cell lines stated above were grown at 37°C and 5% COSupplemental Table 1). PCR reactions were performed using Platinum Taq DNA Polymerase (Invitrogen). Amplified DNA fragments were purified using HighPrep PCR clean-up magnetic beads  at 1.8x concentration. SNP/INDEL detection analysis was performed by Genewiz’s Amplicon E-Z Next Generation Sequencing .DNA regions were amplified from Nalm6, Reh, and patient gDNA using primers with partial Illumina adapter sequences (BbsI sites of pSPgRNA22. Gateway cloning was used to generate pINDUCER-AID using the Invitrogen Clonase II kit to integrate AID into pINDUCER2023. Nucleofections were performed using the Amaxa Biosystems Nucleofector II with program T-001.Single guide RNAs (sgRNAs) targeting ABCs were designed using Benchling. Synthesized DNA oligos (Integrated DNA Technologies (IDT), Coralville, IA) were ligated into the 24 For constitutive Cas9, the lentiCRISPRv225 vector was used. Nalm6 and Reh cell lines with the dox-inducible AID cassette  were generated by transduction and selection in complete media supplemented with 1 mg/mL of G418. A similar process was done using virions that incorporate the Cas9 expression cassette and create Nalm6-Cas9 with selection done using 0.75 μg/mL of puromycin.Virions with pINDUCER-AID were generated using a 3rd generation lentiviral system in HEK293T cells.Total RNA was extracted from cells using TRIzol (Thermo Fisher Scientific) as described by the manufacturer’s instructions. cDNA was generated using the Maxima H Minus cDNA Synthesis Master Mix (Thermo Fisher Scientific) using 1 μg of total RNA according to the manufacturer’s instructions. The cDNA was then used as template in quantitative real-time PCR using a SYBR Green Master Mix (Applied Biosystems). Gene expression was normalized to 18S ribosomal RNA. Primer sequences used for gene expression analysis are listed in Supplemental Table 1.26 From aligned BAM files, PCR duplicates were removed using MarkDuplicates in gatk.27 Next, counts matrices were assembled from BAM files in using the summarizeOverlaps function from the GenomicAlignments package.28 Counts matrices were then filtered for genes with a rowSum of 10 counts across all samples then differential expression was performed using DESeq2.29Poly-A enriched RNA was isolated from patient samples stored at −80°C in TRIzol for library preparation and 150 bp paired-end sequencing was performed by Novogene . FASTQ files for RNA-seq have been deposited in Sequence Read Archive . FASTQ files were inspected for base quality scores using FASTQC and aligned to the human genome  using STAR aligner.Protein lysates containing an equal amount of protein were loaded and resolved on an SDS–PAGE gel and transferred to a nitrocellulose membrane. For imaging, membranes were treated with Immobilon Western Chemiluminescent Substrate (Millipore). Chemiluminescent images were obtained using the BioRad ChemiDoc System. Antibodies used for the Western blot were: AID (L7E7) , FLAG M2 , and p84 .30 Briefly, 9 μL reactions contained 1x Absolute Q DNA dPCR Master Mix (Thermo Fisher Scientific), 900 nM of each forward and reverse amplicon primer, 250 nM of FAM, TAMRA, Cy5 and/or SUN/HEX TaqMan probes, and 10–20 ng of gDNA prepared by phenol:chloroform extraction and ethanol precipitation. Primer and probe sequences used for dPCR drop-off assays are listed in Supplementary Table 1. Quantstudio software uses Poisson distribution that allows for quantification of each amplified product in copies/μL based on the fluorescent signal in each microchamber.dPCR samples were prepared as described previously.31 with modifications as described31 to quantify both single DSB rejoining as well as translocation to other DSBs (JoinT-seq). Briefly, 10 μg of isolated gDNA was used to prepare amplicons for Illumina Nova-seq 150 bp paired end sequencing. Sequence reads were aligned to the hg38 genome build and normalized to 2,914,360 and 793,219 each for IGHM-6 and IGHM-1 bait analyses, respectively. Hotspot determination used MACS2 with an FDR-adjusted P-value cutoff of 10− 9 as described.32HTGTS was performed using the linear amplification-mediated (LAM) platformPatient samples were obtained through two UCI IRB approved protocols. Samples were obtained from bone marrow or blood . De-identified healthy donor blood samples were provided by the UCI Institute for Clinical and Translational Science. Mononuclear cells were obtained by ficol gradient centrifugation followed by a brief red blood cell lysis step in ammonium chloride potassium (ACK) buffer. Cells were used fresh or thawed from frozen cells preserved in 90% Fetal Bovine Serum (FBS) + 10% DMSO.3 the major focus here is on the ABC upstream of the CRLF2 gene.13CRLF2 is located on a pseudoautosomal region (PAR) shared by both the X and Y chromosomes.33 We also include data on the ABC in the 3’ UTR of the BCL2 gene that is also called the BCL2 Major Breakpoint Region (MBR)   to demonCRLF2 can occur anywhere in the 27 kb region upstream of the gene, yet two specific DSB clusters are of note.13 Approximately 4.5 kb upstream of CRLF2 is a cluster that shows the CAC/CACA motif associated with being a cryptic recombination signal sequence (RSS) recognized by the RAG complex. Off-target RAG cutting at this site leads to an intra-chromosomal deletion that puts the CRLF2 gene under control of the P2RY8 promoter, leading to its overexpression.34 The other site is 16 kb upstream of CRLF2 and contains no cryptic RSS sites, but does have DSBs at or near a CpG, which is a hallmark of ABCs since AID has an affinity to deaminate cytosines within RCG sequences (WGCW>>>WRC > > RCG).37 For rearrangements involving ABCs, it has been shown that a translocation is 30% more likely to occur directly at a CpG dinucleotide and 70% more likely to occur within 8 bp of a CpG dinucleotide.10According to the mapping of chromosomal translocation junctions in human patients, a DSB that results in pathogenic rearrangements involving 35 While AID expression in the human pre-B cell lines Nalm6 and Reh is below the limit of detection,38 the ssDNA generated at these sites would still be vulnerable to damage. Thus, we were curious if ABCs show an inherent propensity for damage. This was tested using amplicon sequencing (AmpSeq). The detailed sequence for the CRLF2 region with the ABC is shown in Supplemental Fig. 1A. Strikingly, as with the reported patient data,13 there is a peak of deletions near the CpGs in the CRLF2 ABC in both Nalm6 and Reh cell lines (AID requires single-stranded DNA (ssDNA) as a substrate for deamination.ll lines . These dBCL2 ABC , we also see indels, yet observe a notably different pattern for each cell line . For CRLF2, three TaqMan probes bind directly at one of the three CpG sites . These are drop-off probes as DSB formation and repair would alter the sequence, preventing probe binding . The SUN reference probe binds where no DSBs have been mapped and acts as a detector for the amplicon and a baseline for the total fluorescent signal produced. Genomic DNA (gDNA) from cells or patient blood can then be collected and subjected to dPCR analysis to determine genome stability at specific loci  and were transfected into Nalm6-Cas9 cells . dPCR was performed on gDNA from untransfected Nalm6-Cas9 cells or cells transfected with sgCRLF2–1. 2D plots of the dPCR results are shown in i.e., an amplicon where the SUN probe binds but the TAMRA probed does not) (We tested the sensitivity of the dPCR assay using a CRISPR/Cas9 system to induce DSBs. Two single-guide RNAs (sgRNAs), sgCRLF2–1 and sgCRLF2–3, were designed to target Cas9 to sites bound by the TAMRA drop-off probe (oes not) . Comparioes not) . Interes41 An sgRNA that recognizes a region of the IGH gene that is unrearranged in Nalm6 cells  was used to create the bait DSB to map breakpoints genome-wide. Compared to no bait sgRNA controls, which showed very low numbers of random breakpoint junctions , sgIGH-6 transfection resulted in over a 1000-fold increase in junctions .unctions , but no unctions . Similarns  inducible AID expression cassette via lentiviral transduction to generate Nalm6-AID cells. While no protein was detectable with 0 ng/mL of dox, increases in both AID expression  and protRLF2 ABC . With noBCL2 ABC  and PLEKHA5 , by dPCR. Neither site shows any increased drop-off as AID levels increase in the cells showing that the RCG and WGCW sites at these control regions do not accumulate mutations  (Table 1) that contained ample cells to provide gDNA for AmpSeq. While the number of samples is small due to the relative rarity of these cases, it is adequate for the exploratory nature of this study as we continue to build our biorepository. Strikingly, for the CRLF2 ABC, there are remarkable similarities in terms of indels with each LA patient presenting a major peak centered over two CpGs that are proximal to each other (23 bp apart), yet not at a more distal CpG 150 bp away  and two LA patients with PhBCL2 ABC showed a more diverse pattern of indel formation in patients  also reveals that dPCR has a greater sensitivity to detecting indels. Patient 17–062, for example, shows elevated drop-off at the FAM and TAMRA sites, as shown in the AmpSeq data, yet the dPCR also detects indels at the more distal CpG covered by the Cy5 probe  or Phy5 probe . In conty5 probe . The dPC15 − 010  also sho+ ALL patient 17–061 is the most distinct and provides a direct example of how AmpSeq can miss crucial sequence information. When attempting to calculate the quantity of drop-off product, which relies on having nearly all amplicons bound by the SUN reference probe, we noted a significant decrease in the total SUN signal that was verified by calculating the total copies/μL based on the amplicon binding of all 4 probes . Indeed, when we look at the calculation of copies/μL for each probe, we see that the SUN signal is exactly half that of the FAM, TAMRA, and Cy5 probes in each case . This strongly suggests copy number variation (CNV), where this particular PAR sequence is only intact on either the X or Y chromosome in this patient. Relying on AmpSeq data alone would have led us to miss this instability, likely either because an amplicon was not able to be generated due to the sequence anomaly or the variant calling software was unable to align it with the reference and removed it as a low-quality read.The LA PhBCL2 ABC AmpSeq data to what was obtained from dPCR using the drop-off assay designed for this region. For the LA Ph-like patients, 17–062 and 19–021, most indels accumulate immediately after two CpG sites covered by a FAM probe  to provide a baseline of expression in a background with very low CRLF2-linked Ph-like ALL risk.19 All three LA Ph− patients, including the two that were Ph-like, showed high relative expression of CRLF2 , with analysis of differentially expressed genes (DEG) showing a number of differences in the expression pattern .Next, to compare global gene expression changes between Ph pattern . Notably− and Ph+ datasets to determine pathway enrichment of DEGs using the Gene Ontology Biological Process, Reactome, and Drug Signatures Database (DSigSB) databases. The analysis reveals very little overlap between enrichment in the Ph−  is a much more aggressive disease than Ph+ ALL.We further highlight the differences between these two cohorts by performing Gene Set Enrichment Analysis (GSEA) on the Ph the Ph−  and Ph+ e Ph− (+  patients44 We intersected significant (P < 0.01) DEGs between the two studies that allowed us to determine overlap between the 15 Ph-like ALL patients from Roberts, et al. with our Ph-like ALL cases as well as the ALL cases with no CRLF2 rearrangements. Importantly, we are only including the LA cases from this study in our analysis. As strongly suggested by CRLF2 rearrangements and the ones from Roberts, et al. Primarily, this demonstrates the complex gene expression patterns in these ALL patients that changes significantly depending on age and ethnicity, the uniqueness of different patient cohorts, and the challenge ahead for defining set characteristics among populations with significant genetic admixture that define Ph-like ALL risk.While rates of Ph-like ALL among LAs is increasing, it is still a relatively rare disease, making it difficult to collect a large cohort. To further explore the complex heterogeneity of this disease, we compared our RNA-seq data to a set previously published by Roberts, et al.13 Two such rearrangements are shown in CRLF2-IGH translocation presents in Ph-like ALL, a disease of immature B cells, and the rearrangement involves a DSB occurring between D and J cassettes of the IGH locus that can only consistently occur through the activity of the RAG complex. The BCL2-IGH translocation occurs in over 85% of follicular lymphoma and 20% of diffuse large B-cell lymphoma,45 which are both diseases of mature B cells. Yet, the translocation junction again shows that the IGH DSB was initiated by the RAG complex, thus indicating that the translocation is of pre-B cell origin. B cells harboring this translocation continue to mature and acquire additional mutations until it fully transforms into a mature B cell lymphoma.46 This evokes a model where high levels of RAG activity in pre-B cells combined with aberrant AID activity at ABCs generates the two DSBs required for a cancer initiating chromosomal translocation , flow cytometry for increased CRLF2 receptor at the cell surface, and FISH analysis of cytogenetically cryptic rearrangements. Despite evidence that AID is involved in the DSB leading to the CRLF2-IGH translocation, increased AID activity is not currently examined in any of these diagnostic assays. Likely, this is because increased AID levels occur transiently following exposure to external stimuli, and do not remain high in cells throughout the course of disease. As we demonstrate here, a peak of AID activity in immature B cells can increase mutations and indels at ABCs that will remain in the cells as information scars.Diagnosis of Ph-like ALL has remained difficult due to the lack of agreement on what type of gene expression profile or rearrangement constitutes Ph-like ALL, the heterogeneity of mutations and rearrangements in patients, and the lack of concordance regarding rearrangements in the United States versus Europe due to differences in genetic ancestry.JAK2 and IKZF1, particularly in cases where a CRLF2 rearrangement is also present.47 These additional mutations can indicate a more aggressive cancer that may be responsive to therapies other than the standard of care.48Overall, this analysis fills a critical gap between developing genetic assays in cells to monitor DSB formation and repair via expression of a selectable or detectable marker and NGS of primary or patient material to measure genome instability at a specific locus. In addition to using drop-off assays to examine mutations created at ABC sites as an indicator of instability caused by aberrant AID levels in pre-B cells, allele specific probes can also be designed for mutations associated with cancer subtypes. Ph-like ALL, for example, often has mutations in As the cancer field continues to move towards personalized medicine approaches to treatment, it is going to be important to not only take a holistic approach to studying cancer genomes that incorporates multiple races and ethnicities, but also to develop rapid and economical approaches to assessing cancer risk that are widely available to people across socioeconomical stratifications. We are currently lagging in both of these goals. Extensive analysis of a larger cohort of LA patients will allow us to develop diagnostics that can indicate risk for diseases such as Ph-like ALL and determine how factors such as genetic ancestry may be driving cancer health disparities and aid in the discovery of new therapies.


Peak incidence for many psychiatric disorders occurs in reproductive years. We don’t have FDA-approved drugs or clinical guidelines. The main cause of pharmacological suspension in pregnancy is the lack of knowledge of their effects on the product. Gathering information on this regard becomes crucial so we can avoid relapses or exacerbationDescribe neurodevelopmental effects in children ≥ 6 months exposed to antipsychotics prenatally..PROSPERO-CRD42020170314. Using MeSH terms in 5 databases, without language and time restriction, obtaining n=3932. After review and exclusion n=24 were obtained for qualitative review. (Table 1). In the cohort studies, exposure may cause short-term delay at 6 months but no significant difference at 12 months (Table 2).Of 20 case reports 4 had more ambivalence results Image:Most are case reports, hence the importance of collecting this information, not ignoring it due to lack of methodological rigor. The intent is not to conclude that prenatal exposure to antipsychotics doesn’t have long-term neurodevelopmental effects, rather documenting the available evidence contributing to an informed clinical decision.None Declared


When FGFR2 was eliminated in embryonic pluripotent precursors or in early postnatal astroglia, mice were hyperactive, and had small changes in working memory, sociability, and anxiety-like behavior. In contrast, FGFR2 loss in astrocytes starting at 8 weeks of age resulted only in reduced anxiety-like behavior. Therefore, early postnatal loss of FGFR2 in astroglia is critical for broad behavioral dysregulation. Neurobiological assessments demonstrated that astrocyte-neuron membrane contact was reduced and glial glutamine synthetase expression increased only by early postnatal FGFR2 loss. We conclude that altered astroglial cell function dependent on FGFR2 in the early postnatal period may result in impaired synaptic development and behavioral regulation, modeling childhood behavioral deficits like attention deficit hyperactivity disorder (ADHD).Fibroblast growth factor receptor 2 (FGFR2) is almost exclusively expressed in glial cells in postnatal mouse brain, but its impact in glia for brain behavioral functioning is poorly understood. We compared behavioral effects from FGFR2 loss in both neurons and astroglial cells and from FGFR2 loss in astroglial cells by using either the pluripotent progenitor-driven  Fibroblast growth factor (FGF) signaling has been characterized extensively for its role in the brain, particularly in neurons and their precursors –4. We anFGF receptors, and in particular FGFR2, are almost exclusively expressed in non-neurons in the postnatal forebrain –29. AlthAstrocyte dysfunction in the forebrain results in mixed behavioral outcomes but suggest a role for astroglia in regulation of activity—specifically disrupted astroglial SynCAMI or adenosine kinase increases locomotor activity , 37. AstHere, we specifically targeted FGF signaling in the brain at different times as important mechanisms underlying multiple behaviors by inducing the knockout of FGFR2 at different stages of development and in different cell types. We examined three different sets of mice that had a cell type-targeted knock-out of FGFR2: 1) knockout during embryogenesis, in radial glial cells and therefore both their neuronal and glial progeny 2) knockout during the rodent early postnatal period, largely in proliferating astroglial precursors and glial progeny and 3) knockout during adulthood, in largely post-mitotic astroglia during mature brain functioning. We hypothesized that early postnatal loss of FGFR2 in astrocytes would affect astrocyte biological function and impact the regulation of behaviors that are disrupted at early postnatal time points in psychiatric disorders.All experimental procedures involving animals were performed in accordance with the Yale University and University of Iowa Animal Resources Center and Institutional Animal Care and Use Committee (IACUC) policies. Sufficient animals or samples were generated for each assessments using a power analysis based on previous studies and α = 0.05 and β = 0.2hGFAP-cre fgfr2 knockout mice (referred to here as cKO) on a mixed background have been previously described [fgfr2 null allele harbors loxP recombination sites flanking regions encoding the Ig III binding and transmembrane domains of the fgfr2 gene (fgfr2f) [fgfr2f alleles were crossed with mice expressing the Cre recombinase transgene under the control of the human GFAP promoter (hGFAP) [hGFAP-cre transgene targets Cre recombination to radial glia progenitors of the dorsal telencephalon starting at E13.5 [Cre negative mice, littermates when possible, were used as control animals.Conditional escribed , 8. The fgfr2f) . Mice ho (hGFAP) . The hGFf . Mice fgfr2f alleles were crossed with GFAP-creERT2 (GCE) mice [T2) [GFAP promoter. Specifying gene knock outs to astroglia is challenging due to the overlapping nature of most genetic drivers in both postnatal neural stem cells and astroglia. Therefore, genetic approaches may have impacts on a small number of neural stem cells (which may impact neurons in the dentate gyrus and olfactory bulb) but will effect astroglia much more substantially; our approach must be considered with this caveat. Previous investigations of the GCE line demonstrate that it largely affects astrocytes; [fgfr2 loss in astrocytes. We used two different tamoxifen-induction protocols to target the knockout of fgfr2 at different time points. In the first neonatal protocol (referred to here as nKO), mother mice received intraperitoneal (IP) injections of 1 mg of tamoxifen dissolved in sunflower seed oil once daily for five consecutive days, beginning on postnatal day 1, 2, or 3 while Cre- and Cre+ experimental animals were nursing. In the second adult induction protocol (referred to here as iKO), Cre- and Cre+ adult mice received injections of 0.5 mg of tamoxifen dissolved in sunflower seed oil twice daily for five consecutive days at 2–4 months of age as previously [To assess the contribution of FGFR2 solely in the postnatal brain, mice homozygous for the CE) mice  also on ice [T2)  under throcytes;  in combieviously . BehavioAll behavior assessments were performed during the light cycle in a dedicated testing room with only one behavior assessment performed per day in the order described below, allowing mice to habituate to the testing room for 60 min prior to testing. Unless otherwise noted, mice remained in their home cage with cage-mates immediately before and after assessments. Each of the three types of knockout mice were tested alongside their control littermates. Only male mice were tested due to limited resources; childhood psychiatric disorders have a higher prevalence in males which allows for these data only in males, while limited, to be translationally informative . Behavio2 in area, mice were tested for locomotor activity for at least 50 min. Test mice were placed in the corner of the arena and their movements recorded using an overhead camera . The main measure of distance traveled was evaluated in 5 min epochs and repeated measures ANOVA was used to evaluate group differences over time.Open Field: In a square or rectangular plastic arena, at least 1500 cmt-test.Social Approach: In a three-chamber social approach apparatus , mice weSpontaneous Alternation: In a plastic Y maze with three 14 inch x 3 inch arms, mice were tested for working memory for 5 min. Mouse movement throughout the maze was monitored from live video recorded from above, noting arm entry order with experimenter blinded to group. For each set of three entries, the spontaneous alternation of those entries through all three arms was counted, and alternation percent calculated. Differences were evaluated with Student’s t-test.Radial Arm Water Maze: Due to lack of resources, this task was not assessed in FGFR2 cKO mice. A 6-foot maze was filled with room temperature water and fitted with 6 dividers to create 6 radial arms., Mice were tested for spatial memory with distant visual spatial cues across two days with experimenter blinded to group . BrieflyElevated Plus Maze: In a Stoelting  Elevated Plus Maze, mice were tested for anxiety-like behavior for 5 min. Mouse movement throughout the maze was recorded using an overhead camera. The amount of time spent in each arm and the center of the maze was assessed (Anymaze). Differences were evaluated with repeated measures ANOVA across zones and with a t-test of the ratio of time spent in the open to closed zones.fgfr2 gene and other genes related to glial regulation of brain function, dorsal forebrain, hippocampus, or medial frontal cortex was dissected out from brains of animals which were neonatally exposed to tamoxifen through maternal injection. RNA was isolated using standard Trizol methods and concentration assessed . cDNA  was used to evaluate relative gene expression to β-actin using TaqMan primers  and GeneAmp PCR Mastermix in a StepOne™ Instrument (Applied Biosystems).To evaluate the penetrance of Cre-mediated deletion of the At least 10 days after completion of behavioral testing, animals were anesthetized and perfused , then 4% paraformaldehyde), and brain tissue was post-fixed, cryoprotected with a sucrose solution in 1X PBS and embedded in OCT compound, and cryostat  sectioned at 50 µm thickness. Standard immunostaining methods were then used on free floating brain sections: blocking with 10% goat serum in 1XPBS, TritonX-100 and Tween20, incubation with primary antibodies GFAP , VGLUT1 , PSD95 , gephyrin , VGAT , glutamine synthetase  washing the primary antibodies 3x in 1XPBS, incubation with Alexa dye-conjugated secondary antibodies, anti guinea-pig or anti mouse Alexa 594, anti-rabbit or anti-goat Alexa 488 , and coverslipping using mounting medium with DAPI .Glutamine synthetase+ cells in coronal tissue sections were measured using fluorescent microscopy with a Zeiss Axiolmager M2 microscope. Stereological estimates of hippocampus and medial frontal cortex (mFC) cell densities were calculated using optical fractionator approach and unbiased counting rules with 3-dimensional 150 × 100 × 10 μm counting frames, on a 450 × 450 μm grid for mFC and 600 × 600 μm grid for hippocampal CA, using a 40× objective lens with experimenter blinded to group . Stereological counting to determine cell density, displayed as means and standard errors of the mean, was performed in 3–8 serial coronal sections (every 10th section) of the adult mFC and the hippocampus as previously described , 50.Astrocytes, GFAP + cells with well-delineated cell bodies and branches, in the mFC and hippocampus were randomly selected for morphology reconstruction. Ten z-stacks per cell were acquired at 100x and traced in each experimental group (Control and FGFR2 nKO) by using Neurolucida 11.03  The coordinate files obtained by the 3D reconstruction were analysed in Neuroexplorer .VGLUT1, PSD95, and colocalized puncta as well as VGAT, Gephyrin, and colocalized puncta were assessed by imaging the adult mFC of the FGFR2 nKO with a Zeiss Axioimager M2 microscope equipped with ApoTome2. Four mice per group were examined. Eight z-stacks spanning the entire cortical layers were imaged at 40x with experimenter blinded to group. The ImageJ software  Puncta Analyzer plugin for the estimation of these puncta was used as previously described .We performed assessments of glial and synaptic structure as previously . ElectroData normality and variance was assessed with GraphPad Prism8 to select appropriate statistical tests. Graphs were made and two-tailed Student’s t-tests were performed with Microsoft Excel. ANOVA for repeated measures outcomes were performed with GraphPad Prism8. Outliers were excluded if they were >2 standard deviations from the mean.fgfr2f alleles with the hGFAP-cre transgene, where Cre is expressed in radial glia beginning at embryonic day 13.5, therefore affecting all their neuronal and glial progeny in regions where the hGFAP-cre transgene is expressed, primarily the forebrain and cerebellum. An 80–91% loss of fgfr2 gene expression was previously found and a substantial reduction of FGFR2 protein level [fgfr2 gene expression, and was accomplished by recombination of the same fgfr2f alleles with the hGFAP-CreERT2 transgene, where Cre was expressed in postnatal GFAP+ glial cells after tamoxifen injection in adulthood [Mice with conditional loss of FGFR2 starting in embryonic radial glia (cKO) have been previously described , 8. In bfgfr2f alleles and the hGFAP-CreERT2 transgene received tamoxifen via the milk in the neonatal period by injecting the Cre negative dam with 1 mg tamoxifen once daily for 5 days. The reduction of fgfr2 assessed by qRT-PCR in juvenile or adult cortex or hippocampus varied between 29% and 43% , the same mice carrying p = 0.0001) [p = 0.04)), a phenotype suggesting reduced anxiety-like behavior.In FGFR2 cKO mice, multiple behavioral abnormalities were identified in addition to learning and memory deficits demonstrated previously . Locomot 0.0001) . Comparep = 0.0009). This higher sociability was true in approach behavior in close proximity to the novel mouse or object , as well as in larger chambers . This effect was not attributable to altered social recognition, as both cKO and control mice had similar interaction with a familiar versus a stranger mouse target .FGFR2 cKO mice also showed alterations in social approach. When tested on their social preference, they demonstrated a small but significantly higher preference than controls for interacting socially with a novel stranger versus spending time with a novel object , as shown by a small but significant 10% lower spontaneous alternation in a Y maze.Working memory was impaired in FGFR2 cKO mice . Mice demonstrated a small effect on anxiety-like behavior, with less time spent in the closed arms of the maze (p = 0.02) and a lower ratio of time across the elevated plus maze zones , but no difference in the ratio of entries across zones . Performance on the elevated plus maze, a different environment than an open field, also confirmed the higher activity level of these mice regardless of context .Behavior on the elevated plus maze was also significantly different from control littermates overall . We also examined activity level of FGFR2 nKO mice compared to an additional control group—a small sample of vehicle/oil injected Cre+ controls. Activity of FGFR2 nKO mice also trended increased by this comparison  = 4.574, p = 0.056, Supplementary Fig. The behavior of FGFR2 nKO mice, lacking FGFR2 signaling only in GFAPove Fig.  as well p = 0.04; sociability index: large chamber 31% increase: nKO: 0.76 vs controls: 0.58, p = 0.02; close proximity 21% increase: nKO: 0.75 vs controls: 0.62, p = 0.11) without a deficit in social recognition   Fig. .p = 0.07). Working memory was further measured by performance on the radial arm water maze .Working memory on the Y maze was trend deficient in FGFR2 nKO mice to the same small extent (12%) as shown for FGFR2 cKO mice . FGFR2 nKO mice showed a trend higher ratio of time in the open to closed zones , but no difference in the ratio of entries in the zones . We also examined these differences in the vehicle/oil injected animals and found that tamoxifen injection which induced the FGFR2 nKO was needed to see this effect .Measures of anxiety-like behavior on the elevated plus maze was also shown to be altered in FGFR2 nKO mice in a comparable fashion to the FGFR2 cKO mice, with a small reduction in anxiety-like behavior: less time in the closed arms of the maze and more time in the center  these FGFR2 iKO mice demonstrated trend less time spent in the closed arms (p = 0.05), more time in the open arms (p = 0.08), and higher ratio of time in the open to closed zones , indicative of less anxiety-like behavior, but no differences in overall activity level . There was no effect of adult oil injection itself on elevated plus maze performance .Differences of FGFR2 iKO mice from wild type littermates were found only for anxiety-like behavior. On the elevated plus maze . The volume of the hippocampus was also unchanged . In addition, the most broadly affected model used here, FGFR2 cKO mice, showed no deficit in GFAP + cell density either . This suggested that astrocyte numbers themselves were intact regardless of early loss of FGFR2.Given the multiple behavioral abnormalities induced when FGFR2 was lacking only beginning in neonatal life in primarily astrocytes, we performed pilot investigations of their neurobiology focused mainly on hippocampus as a major region implicated in regulation of the behaviors assessed here. We first assessed GFAP + astrocyte density in the hippocampus of FGFR2 nKO mice, finding no differences  analyses of the hippocampus in FGFR2 nKO mice. The analysis of the astrocytic coverage of the cell membrane of neurons in the principal cell layer of the CA3 region of the hippocampus showed reduced coverage by almost half Fig. . Of noteThe loss of FGFR2 signaling did influence the expression of glutamine synthetase (GS), a metabolic component of glial cells which is regulated by glutamate transport and synaptic activity. In FGFR2 nKO, the density of glutamine synthetase positive cells, as measured by stereological counts in adult brains, trended increased in hippocampus and was also increased to a greater extent in cerebral cortex of FGFR2 nKO mice Table . RegardlWith the greater change in astrocyte glutamine synthetase expression in the cortex of the FGFR2 nKO mice, we examined cortical density of synaptic proteins in adult FGFR2 nKO mice, as assessed by immunohistochemical puncta analysis. FGFR2 nKO mice were found to have increased puncta density after immunostaining for both the GABA neurotransmitter release protein, vGAT, and the glutamate neurotransmitter release protein, vGLUT1 Fig. . In contn = 8,6). GABA transporter vGat (by RT-qPCR) was increased and synaptic glutamate transporter vGlut1 trended increased in the juvenile hippocampus . These findings suggest that early postnatal glial FGFR2 loss in the dorsal forebrain induces a decrease in astrocyte-neuron contacts and potentially an increase in neuronal synaptic contacts, as shown by EM and level of presynaptic marker expression, particularly in hippocampus.Lastly, we examined gene expression of these same synaptic proteins in the hippocampus of another cohort of FGFR2 nKO mice and controls  , 61 in rfgfr. The 25–30% increase in the social preference index was similar in both these mouse lines and involved increased time spent with a novel stranger mouse relative to time spent with an empty social interaction cup. This may reflect a deficit in typical down-regulation of social approach over time or an increased drive for social reward. While models for the study of neuropsychiatric disorders commonly demonstrate decreased sociability [More distinct differences between these three FGFR2 KO lines included the presence of the deficits in hyperactivity, sociability, and working memory in only FGR2 cKO and FGFR2 nKO mice. Analysis of results from vehicle/oil injected mice and comparing mice tamoxifen-injected at different ages provided reassurance that differences were due to the knock out of iability , increasiability –71. Otheiability . DirectiFurther behavior similarities arose when FGFR2 loss began early in development. While working memory in FGFR2 cKO mice was only tested with the Y maze, this Y maze deficit was very similar to that in the FGFR2 nKO mice  which also showed a radial arm water maze working memory deficit. These deficits were distinct from the short and long term memory deficits previously identified with FGFR2 iKO and cKO mice respectively , but finGFAP-creERT2 also plays a role in behavioral impacts in the tamoxifen-induced lines. However, regardless of possible inclusion of neuronal FGFR2 signaling in these mechanisms, there were definitive impacts on astrocytes. Our neurobiological data suggest that the deficiency of FGFR2 has an impact on astrocyte differentiation and maturation, processes that occur in the early postnatal period in mouse brain, and which may result in abnormal astrocyte ultrastructure. Astrocyte maturation is regulated by multiple FGF ligands, including changes in protein levels that occur as astrocytes mature: for example, upregulation of the glutamate transporter, GLT-1, and downregulation of GFAP [The importance of glial cells during early developmental time periods has been suggested by other lines of research. Glial proliferation occurs predominantly in the last few embryonic days and first few postnatal weeks of mouse development which also may be a critical time period for establishing these cells’ own later functioning. Although FGF signaling regulates glial proliferation and fate , 76, ast of GFAP . Our datEarly postnatal astrocytes are in a distinct phase of differentiation;  as astroGiven that punctal density of vesicular proteins was increased in neonatally-induced FGFR2 knockout mice when assessed by immunocytochemistry, a disruption of astrocyte regulation of neuronal signaling is likely. Increased glial metabolism, as shown through increased glutamine synthetase positive cells, may reflect a response to a higher level of neuronal signaling from increased vesicular proteins. A similar reduction in FGFR2 signaling in glial cells just a few days later in juvenile brain  decreased vesicular proteins  suggestiAstrocyte functioning has also been implicated in psychiatric disorders –85, withThe range of behavioral alterations manifested by the glial-targeted FGFR2 mice resembles that which individuals with ADHD-combined type display; the diagnostic criteria include increased locomotor activity, poor attention—relevant to working memory deficits— and impulsivity which leads to dysregulated increased social interaction. Some other models for the study of ADHD consistently show all of these impacts, although social approach is not a task typically assessed . BecauseSupplemental Material


All BCL11A patients achieved the primary endpoint for NCT 03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects.We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT 03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects  that used post-transcriptional gene silencing of BCL11A expression to reverse the fetal to adult hemoglobin switch in sickle cell disease. They develop new single-cell flow cytometry and microfluidic techniques to predict the efficacy of HbF induction and show that red blood cells from these patients exhibit greater resistance to deoxygenation-induced polymerization than red blood cells from hydroxyurea-responsive patients. There are ~100,000 affected individuals in the United States, and despite improvements in SCD treatments including penicillin prophylaxis and hydroxyurea as standards of care, SCD is associated with significantly reduced life span due mainly to chronic and cumulative ischemia-induced end organ damage.Sickle cell disease (SCD) is an inherited hematologic disorder and an important genetic cause of childhood mortality in many countries, particularly in sub-Saharan AfricaSCD is caused by a single amino-acid substitution in the β-globin gene, replacing a glutamic acid with valine, resulting in the expression of sickle hemoglobin 6. Unfortunately, donors are available for only a small fraction of patients, with one US study estimating only 18% of patients had a suitable donor7. HSCT with unrelated matched or mismatched donors is associated with significantly higher complications including acute and chronic GVHD and mortality8.The only curative treatment for SCD currently is allogeneic hematopoietic stem cell transplantation (HSCT), ideally from an unaffected, HLA-identical sibling. For patients younger than 13 years, some studies indicate that this treatment has at least a 95% chance of cure, a 3-year survival >92%, and a 5-year probability of graft-vs-host disease (GVHD)-free survival of ~86–92%10. The presence of HbF  increases HbF through an undefined mechanism and mitigates clinical symptoms to a varying degree for individual patients15 and is the current standard of care for patients with severe SCD genotypes. Some patients continue to have significant vaso-occlusive events even with optimal HU dosage20. Gene therapy by gene addition to or gene editing of autologous hematopoietic stem cells (HSCs) has emerged as a promising curative treatment strategy25.The polymerization of HbS in a deoxygenated RBC is highly sensitive to the concentration of HbS and also depends on the fraction of fetal hemoglobin (HbF), the oxygen tension, and factors including 2,3-DPG concentration, temperature, and pH. The rate of HbS polymerization is extremely sensitive to small changes in HbS concentration22 leading to a multi-center phase 2 trial (NCT 05353647). Reversing the physiological fetal to adult globin switch may offer the advantage of simultaneously and coordinately increasing the expression of anti-sickling HbF and significantly reducing the expression of HbS in RBCs. This treatment approach may be distinguished from approaches that increase HbF expression pharmacologically or add sickling-resistant Hb transgenes without directly reducing HbS expression as well. The resulting combinatorial and coordinated reduction in HbS and elevation in HbF may further inhibit polymerization and increase the resistance of these RBCs to sickling at even lower physiologic oxygen tensions, such as in the renal medulla and bone marrow where oxygen tension may reach as low as 1% in healthy individuals27.Post-transcriptional silencing of BCL11A using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression has been shown to be safe and to induce high levels of total HbF expression that is broadly distributed in RBCs in a pilot clinical studyshmiR and HU subjects. Compared to HU patients, BCLshmiR patients had a higher percentage of RBCs rendered resistant to sickling at low physiologic oxygen tension.Here we used state-of-the-art assays to measure HbF and HbS at the single-RBC level to determine whether RBCs derived from HSCs in which BCL11A was molecularly targeted showed a measurable decrease in HbS with increased HbF as compared to RBCs in patients with high HbF responses to HU who achieved comparable levels of total blood HbF%. To determine the biological relevance of this switch, we also compared the level of hemoglobin polymer in RBCs from BCL+ HSPCs collection, transduction, and infusion are provided in ref. 22, and consent for exploratory research reported here was embedded within the trial written informed consent . The clinical trial (NCT 03282656) was performed under an approved Food and Drug Administration investigational new drug application (IND 17660) and was regularly reviewed by an independent data and safety monitoring board designated by the National Heart, Lung, and Blood Institute. Data reported were current as of April 2022. Participants did not receive compensation for their participation in this clinical trial.The pilot clinical trial aims to assess the safety and feasibility of gene therapy with the BCH-BB694 BCL11A shmiR-encoding lentiviral vector in patients with severe SCD. Details regarding the study protocol, eligibility criteria, and the CD340-thalassemia  were determined by a quantitative polymerase-chain-reaction (qPCR) performed on erythroid precursors, B-lymphoid cells, and myeloid cells from peripheral blood and bone marrow at 6 months for each of the 9 patients  who did not receive RBC transfusions after infusion and engraftment of the BCH-BB694 BCL11A shmiR drug product . Transfused BCLshmiR refers to one patient who maintained periodic RBC transfusions due to Moyamoya present prior to gene therapy (patient 03) and one patient who received at least one intercurrent transfusion and had a lower overall HbF induction associated with a low in vivo VCN from the infused product (patient 10).This exploratory research report includes data from the first 9 patients treated in the pilot trial, averaging 32 months follow-up [range 17–49 months], including 8 patients with genotype HbSS and one with HbS/βts Table . VCN wasn = 10, hereafter named HU High Responders), and the patients with HbF levels equal to or lower than 20% . HU High Responders were selected solely on the basis of HbF% and not based on clinical outcomes, age, or alpha-thalassemia status, and thus the cohorts are not explicitly matched for any characteristics other than HbF%. This study was designed specifically to compare the molecular and cellular effects of treatments and not to compare clinical outcomes. Clinical outcome data is therefore not reported, and no claims are made about the superiority of these single-cell assays for predicting clinical outcomes compared to current standards. The threshold of 20% HbF was defined by the lowest HbF level observed for the 7 BCLshmiR patients , namely patient 02 had 20.8% of HbF at the 3.5-year post-gene therapy study visit. The basic hematologic characteristics of the three groups are compared in Supplementary Fig. 29. See Table The hydroxyurea cohort included patients with sickle cell disease followed in one hematology clinic who were being treated with HU. Collection of samples from these patients was approved by the Boston Children’s Hospital Institutional Review Board (BCH protocol #P00027349) and written informed consent was obtained from all patients who did not receive compensation for their participation in this study. Samples from 18 HU-responsive patients  with more than 10 months of treatment  were obtained. We divided the HU cohort into two groups: patients with HbF higher than 20%  or capillary electrophoresis (CE), the most popular methods for identifying and quantifying fractions of Hb variantsshmiR subjects experienced an increase in overall fetal hemoglobin content to a median of 27.9%  fraction. Figure shmiR subjects had fewer RBCs with high HbS% than subjects in the HU-treated cohort (p < 0.004). To define this cellular-scale difference more precisely, we next compared the Untransfused BCLshmiR cohort to HU patients who had achieved blood HbF% > 20 (HU High Responders). Compared to this HU High Responders cohort with similar HbF content , the Untransfused BCLshmiR patients had fewer RBCs with more than 70% HbS  as well as in the peripheral microcirculation and in regions of the brain (~2.7%)27. We counted flowing RBCs with no detectable Hb polymer content , and 8% for the median HU Low Responders subject. A similar relationship was found at 2.7% , and all relevant information and study documentation are provided in the section “Participants and treatment”.32. The protocol involves calibrating the fluorescence intensity of an anti-HbF antibody  and quantifying its correlation and the mean single-cell HbF content using RBCs from individuals with homogeneous HbF distributions. The anti-HbF antibody was used as per the manufacturer’s instructions, using 50 μL of the antibody diluted with 50 μL of fixed and permeabilized cell suspension and incubated for 15 min, shielded from light at room temperature. The protocol constructs a standard curve for each batch of anti-HbF antibodies and a normalization of the fluorescence intensity by beads. Each standard curve was required to have a significant correlation with R² ≥ 0.95. Intracellular co-staining using anti-HbF and anti-HbS  was performed to assess the relative levels of HbS and HbF in single RBCs. For co-staining, anti-HbF was used diluted as above and anti-HbS was used at a final concentration of 20 μg/mL, incubated for 15 min, and shielded from light at room temperature. Control experiments were performed to ensure that the anti-HbS antibody and any HbS polymer did not interfere with the HbF measurement  system37. RBCs were fixed, permeabilized, and stained with anti-HbF antibody  at a final concentration of 12.5 μg/mL. Post incubation and washes, the samples were acquired using the BD FACSLyric RUO instrument and BD FACS FACSuite RUO v.1.5 (Beckton Dickinson). The F-cell reports were generated using the BD FACSuite software. The positive cutoff point was set at 0.1% above the negative population of unstained control RBCs. Fractions of whole blood HbF and HbS with respect to the total Hb were measured in each blood sample by high-performance liquid chromatography (HPLC). The gating strategy applied to exclude doublets and to determine the F cell fraction is described in Supplementary Fig. We assessed F-cell levels using flow cytometry (BDFACS Lyric) with a previously published protocolp-values shown in the box plots were computed by applying the Wilcoxon rank sum one-sided test, which is equivalent to the Mann–Whitney U one-sided test, with the subroutine ranksum from MATLAB42. All measurements used in this study were taken from different blood samples. Flow cytometry data was analyzed using GraphPad PRISM (version 6.07) and Python (version 3.7.9).Box plots compare the results between patients treated with gene therapy and HU. Boxes show median , 25th percentile (bottom edge), and 75th percentile (top edge). Top whiskers extend to the highest data point that is below or equal to the 75th percentile plus 1.5 times the IQR, while the bottom whiskers extend to the lowest data point that is above or equal to the 25th percentile minus 1.5 times the IQR. Black diamonds represent outliers. The shmiR as a function of time over 24 months after drug product infusion. The fitting was performed by using the MATLAB subroutine fitlmematrix42.Mixed effects linear regression modeling was used to estimate the evolution of the proportion of RBCs containing a minimum of 10 pg of HbF per cell in BCLFurther information on research design is available in the Supplementary InformationPeer Review fileReporting SummarySource Data