Lef-1 in dermal papilla cells (DPC) restores the intact DPC transcriptional signature and significantly enhances the efficiency of HF differentiation in HSCs. Furthermore, vascularization of hair-bearing HSCs prior to engraftment allows for efficient human hair growth in immunodeficient mice. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia, as well as chronic wounds, which represent major unmet medical needs.Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening for skin diseases; however, the incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs remains a major challenge. Here, we demonstrate a biomimetic approach for generation of human HFs within HSCs by recapitulating the physiological 3D organization of cells in the HF microenvironment using 3D-printed molds. Overexpression of  Human skin constructs hold potential for regenerative medicine, but the incorporation of hair follicles into such constructs is a challenge. Here, the authors use 3D printed molds to pattern hair follicle cell types in a physiological organization, and achieve human hair growth on the back of a mouse. The ability to generate bioengineered human skin constructs (HSCs) has provided a promising skin replacement therapy for these patients2 and allowed for human-relevant drug screening to target skin disorders3.Skin is a complex organ that contains more than 50 different cell types that comprise the core epidermal, dermal, and hypodermal tissues, as well as various other components, including vasculature, sensory neurons, the skin immune system, and appendages, such as hair follicles (HFs). Each year, more than six million patients are hospitalized in the U.S. for significant skin loss or disfigurement due to thermal and pressure injuries, chronic diabetic ulcers or genetic blistering skin diseases4. Our group recently improved the viability of skin grafts by establishing a method to micropattern induced pluripotent stem cell (iPSC)-derived vasculature in HSCs5. However, the incorporation of HFs into engineered HSCs remains a major challenge and limits their potential for regenerative medicine and preclinical drug testing.Currently available HSCs still have significant limitations including poor long-term viability and lack of appendages, such as HFs, which play roles in thermoregulation, barrier function, and wound healing6. However, transforming this concept into a feasible therapeutic strategy requires large numbers of human DPCs, which raises a number of challenges due to the paradoxical rapid loss of hair inductivity of DPCs when expanded in 2D in vitro culture8. Various approaches have been used to restore the inductive characteristics of DPCs, such as co-culture with keratinocytes9, use of small molecules8, and hypoxia culture10. We previously showed that 3D-spheroid culture of DPCs could partially restore their intact transcriptional signature7. This method allowed us and other groups11 to induce human hair formation using cultured DPC spheroids in mice, albeit inefficiently due to high levels of variability in the hair inductive properties of DPCs.Dermal papilla cells (DPCs) are highly specialized mesenchymal cells that are indispensable for HF morphogenesis and cycling. Previous studies have shown proof-of-concept for inducing human hair growth in mice through intracutaneous transplantation of intact DPCs and epithelial cells7. Similarly, we recently reported that Jak inhibitors also directly restore hair inductivity in treated DPCs in culture12. Following these substantial steps towards restoring intact DPC identity, we postulated that generation of de novo HFs in HSCs requires both significant reprogramming of the DPC transcriptional signature, as well as accurate recapitulation of critical microenvironmental cues, such as epithelial−mesenchymal and cell− extracellular matrix interactions.Using a systems biology approach, we also identified several master regulator (MR) genes of inductive DPC identity, which could potentially be used to achieve complete restoration of hair inductive transcriptional signature of DPCs13), which was not possible with previous microfabrication techniques, such as soft lithography. Our approach permits controllable self-aggregating spheroid formation of DPCs in a physiologically relevant extracellular matrix and initiation of epidermal−mesenchymal interactions, which results in HF formation in HSCs in vitro. Further, vascularization of hair-follicle-bearing HSCs increases graft survival and enables efficient human hair growth in mice. Our method represents a novel bioengineering strategy for feasible generation of hair-bearing HSCs entirely ex vivo from cultured human cells.In this study, we present an innovative biomimetic approach for effective generation of human HFs within HSCs by recapitulating the physiological 3D conformation of cells in the HF microenvironment. We exploit the unique capability of 3D-printing technology to create structures with high aspect ratios  over the dermal constructs and allowed the cells to settle down and fill up the microwells, engulfing the DPC aggregates Fig.  and formExtending the culture period from 1 week to 3 weeks in vitro led to the elongation of the HFs down into the dermis and a better organization of the inner and outer root sheath layers Fig. . Interes7. Therefore, to further enhance hair inductivity, we leveraged two MR genes, Lef-1 and Fli-1, which we previously identified as the key regulators of the intact DPC gene signature7. Interestingly, our previous systems biology approach showed that both spheroid culture of DPCs and Fli-1 overexpression restored very similar expression profiles7. In contrast, Lef-1 targeted genes that were not restored by spheroid culture, suggesting that Lef-1 overexpression and spheroid culture are complementary for restoration of the DPC hair inductive transcriptional signature.In our HSCs, we found that the ratio of the HFUs demonstrating HF differentiation to the total number of HFUs (success rate) was as low as 19%, perhaps due in part to incomplete restoration of DPC hair inductive gene signature (~22%) achieved solely through 3D aggregate formationLef-1 in cultured DPCs (passage 3) , yielding a normalized enrichment score (NES) of 3.45 and a p value (two-tailed t test) of 2.8×10-4  in the expression of the specific hair lineage genes, including the outer and inner root sheath , and hair companion and medulla markers (K75), compared to the DPCs transfected with empty vector  also increased the success rate up to ~50%, albeit lower than that was achieved with Lef-1 overexpression, highlighting the Wnt-signaling-independent role of cellular reprogramming  and collagen 6 (COL6)   in the dermis of our HSCs together with the dermal fibroblasts Fig. . CulturiB4) Fig. , and alsB4) Fig. .2 onto immunodeficient nude mice, we observed substantial hair growth in the grafts, whereas the HSCs prepared with FB aggregates did not induce hair formation , which was not possible with previous microfabrication techniques, such as soft lithography. Utilizing this capability, we generated HF-like microwells in 3D-reconstructed dermis where genetically/extrinsically reprogrammed cells could be arranged easily into a physiologically relevant conformation, forming HFUs. This biomimetic approach led to KCs differentiation into specific hair lineages and allowed us to generate human HFs within HSCs in an entirely ex vivo context.Tissue engineering of human HFs has been a long-standing challenge and its progress has lagged behind other lab-grown tissues, such as vasculature18. The same group was the first to achieve hair growth in reconstructed follicles in culture19. Similarly, two recent studies established the growth of mouse hair in vivo through encapsulation of mouse iPSCs20 or mouse adult DPCs and epidermal cells21 in hydrogels followed by transplantation of these in vitro-conditioned structures into mice. Another recent study also demonstrated the spontaneous formation of HFs in embryoid bodies of mouse iPSCs22.Since the animal body offers a physiological environment for the development and engraftment of animal/human tissues, the first demonstration of fully regenerated organs typically involves transplantation of tissues onto animals. For instance, creating de novo adult HFs from cultured cells dates back to 1984, where cultured rodent HF DPCs induced hair growth in recipient skin23, but also in the hair inductive properties of their dermal cells. One striking difference is that cultured rodent DPCs can self-aggregate when transplanted onto rodent skin24; however, this aggregative behavior is not observed in human DPCs25. In a study from Toyoshima et al., human HFs were induced in mice by using a hair germ-like 3D culture method by mixing bulge region-derived epithelial cells and scalp HF-derived intact DPCs6. This study represented a valuable proof-of-concept that human DPCs can generate HFs in vivo. However, to translate and scale such an approach into a clinical reality, these cells must be expanded in vitro for several passages, which causes a rapid loss of hair inductive gene signature in DPCs8.Despite these successes, significant interspecies differences exist between human and mouse HFs not only in their hair cycles, stem cell characteristics, and hormonal dependencies11. To enhance the efficiency of hair induction properties, in this study, we combined genetic and microenvironmental reprogramming strategies by overexpressing the MR gene Lef-1 in combination with spontaneous DPC spheroid formation in the HSCs, which resulted in 70% success rate of HF formation ex vivo, compared to only 19% with the empty vector-transfected DPCs.We recently addressed this issue by 3D-spheroid culture of cells and thereby restored 22% of the hair inductive DPC gene signature. Subsequently, other groups also reported the use of this method to induce HFs in mice, albeit inefficientlyVascularizing and grafting these HSCs led to efficient induction of human HF formation in mice. Although our MR strategy significantly enhanced the hair lineage differentiation in vitro, the efficiency of hair growth with a fully developed hair shaft was much higher in the grafted HSCs compared to in vitro cultured HSCs, reflecting the limitations of the in vitro/ex vivo approach in terms of the culture conditions. This will be addressed in the future by leveraging appropriate growth factors, small molecules or proliferation enhancers.22, especially in terms of the ability to control the pattern and density of the follicles, and to use expanded primary cells (>passage 3) yielding sufficient number of cells for any type of application. For example, for third degree burn patients that require full-thickness skin grafts, spatial control over HFs may facilitate recreating the variations in hair density at the site of transplantation (e.g. 292 HF per cm2 in forehead vs.18 HF per cm2 in forearms13). Interestingly, Plikus et al. recently showed that BMP signaling from HFs can convert myofibroblasts into adipocytes and potentially reduce scar formation after wounding26. The presence of HFs in our HSCs may enable this function by promoting adipocyte differentiation in the wound bed, although this requires further investigation. In addition, the formation of a scar or nonfunctional skin is typically observed in skin grafts due to poor viability and integration of HSCs28. Our ability to vascularize hair-bearing HSCs will significantly promote the viability of both the skin and hair tissues, constituting a groundbreaking innovation for regenerative skin therapies.Tissue-engineered human HFs have many compelling applications including: (i) skin replacement therapy for full-thickness skin loss; (ii) hair restoration surgery; (iii) substituting/complementing animal models of hair disease for drug development; and (iv) testing of cosmetic products. Our method has several advantages over previous approaches, which rely on spontaneous formation of HFs29. Hair restoration surgery to treat male and female pattern baldness usually requires 100 cm2 of donor tissue site to deliver nearly 2000 grafts (~4400 hairs) per patient. Using our strategy, we typically generate 15 million DPCs (passage 3) from a strip of donor tissue as small as 0.5 cm2, yielding enough cells to produce more than 5000 HFs in our HSCs, making it a feasible and efficient strategy to integrate with hair restoration therapy. The improvements of these robotic systems to harvest hair follicular units (groups of 1–4 hairs), as opposed to single hairs, yield significantly more hair growth per harvest30. Using 3D-printing approaches, our goal is to engineer HFs as follicular units and/or in desired patterns that can be integrated with surgical robots and facilitate effective hair transplantation surgery. Moreover, from a technical perspective, harvesting HFs with an obtuse angle, compared to 90 degrees, is more challenging and typically requires larger incisions31. In our 3D-printing approach, we used a starting HF angle of 90 degrees, which is then self-reorganized within the constructs above 120 degrees after 3 weeks of culture or skin grafting. However, for hair restoration applications, we envision that HFUs in our hair constructs can be harvested as soon as a week after their preparation, providing a more practical starting material to harvest HFs with a straight incision perpendicular to the epidermis.Robotic hair restoration surgery has revolutionized modern hair transplantation by allowing for performing repetitive maneuvers within high precision and speedOrgan culture of HFs is still the current gold standard for drug testing on human HFs. This method has several limitations including its low-throughput, dependence on fresh, living HFs from human donors, difficult to standardize due to wide inter-individual variation in rates of hair growth (due to different human donors), inability to capture the signaling from the dermis , and the short life-span of the cultures. Our ability to incorporate engineered HFs into HSCs represents the first major step to circumvent some of these limitations. We are currently able to make nine skin constructs containing 255 HFs in a six-well-plate format starting from only one HF donor tissue, which is a substantial improvement on the throughput aspect of drug testing (1:1 in the organotypic assay). In addition, the contribution of the dermal compartment on the HFs can potentially be examined especially with the addition of other dermal components in the future such as immune cells.32. Moreover, in our analyses we did not examine the capability of these engineered HFs to undergo the hair cycle, which relies on the presence and maintenance of a reservoir of stem cells in our HSCs. Recently, De Luca’s group demonstrated that holoclonal epidermal stem cells33, a subpopulation of cultured KCs with a high self-renewal capacity, are capable of regenerating the entire human epidermis34. The ability to maintain the holoclonal epidermal stem cells in a feeder-free environment over several passages, as well as within HSCs over long periods, could provide greater regenerative capacity to engineered HFs for translational purposes. Alternatively, incorporation of HF stem cells36 or iPSC-derived folliculogenic epidermal cells37 separately into HSCs could potentially establish the hair cycle. However, adding a stem cell niche, such as the bulge region, containing HF stem cells into our constructs will require better resolution in the spatial control of cells, which is a current technical limitation of our method utilizing 3D-printed molds. In the future, 3D-bioprinting technology operating at a single cell resolution may permit the inclusion of other cell types, such as stem cells and melanocytes, to generate cycling and pigmented HFs.One of the immediate extensions of our approach will be the incorporation of melanocytes to generate pigmented HFs. We previously derived melanocytes from iPSCs, and showed their capability to transfer their melanin to neighboring keratinocytes21. In agreement with this finding, our approach exploits the epidermal−mesenchymal interactions during hair development while synthetically guiding the physiological conformation and reconstituting the gene signature of cultured cells to induce human hair growth in vitro and in mice.A recent study revealed that hair organoid formation of mouse cells in vitro follows a different self-organization mechanism than what occurs during hair morphogenesis, despite resulting in successful hair growth in vivoOur novel biomimetic developmental tissue engineering strategy represents a crucial step forward in generating a truly functional human skin and marks a dramatic conceptual advance in regenerative medicine approaches to disorders of the skin and HF, as well as improving the outcome of severe skin injuries leading to disfiguring scars. Adaptation of this new technology by hair researchers, hair restoration surgeons and the pharmaceutical and cosmetic industries will have overwhelming implications in the maintenance and regeneration of this complex human tissue.38, and cultured in DMEM with 10% FBS up to P.3. Culture medium was changed every other day for all cell types. The cells were kept in a humidified incubator at 37 °C and 5% CO2.Neonatal dermal keratinocytes and fibroblasts were isolated from human foreskin and cultured in CnT-07 (CELLnTEC) up to passage 3 (P.3) and in Dulbecco Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) up to P.5, respectively. GFP-tagged HUVECs (Angio-proteomie) were cultured in tissue culture flasks coated with a quick coating solution (Angio-proteomie) and maintained in endothelial cell growth medium . DPCs were isolated from discarded scalp tissues from hair restoration surgery (kindly provided by Dr. Robert Bernstein) using microdissection2) were 3D-printed using Objet24 3D-Printer (Stratasys) which uses a UV-curing material VeroWhite (Stratasys). 3D skin constructs were generated in six-well-plate transwell inserts similar to the method described previously39. The dermal compartment was prepared by adding 4 mL of type I collagen matrix containing 1.25 × 105 fibroblasts per ml into the transwell inserts and polymerized around the 3D-printed HF molds placed on top of the gel at 37 °C for 30 mins. After complete polymerization, the molds were removed and 100 μl of DPC cell suspension at a density to give 3000 DPCs per microwell was added on top of the gel (e.g. 7 million cells per ml for 255 HF per cm2). The constructs were cultured overnight in DMEM with 10% FBS for aggregate formation, after which 1 million KCs were added on top of the gel. The constructs were maintained in low calcium epidermilization medium39 submerged for 1–3 weeks.All 3D-printed molds were designed and drawn using the computer-aided design (CAD) software, Solidworks. Each HF-like extension on the molds was 500 μm in diameter and 4 mm in length. The molds with varying hair densities  for 3 days for capillary formation. The DPC and KCs cell seeding were performed as described above. After a day of culture in a 1:1 mixture of EGM-2 and the epidermilization medium, the constructs were either imaged or used for grafting experiments.l-lysine-coated slides, dried overnight at 55 °C, dewaxed in xylene and rehydrated through a graduated ethanol series  and distilled water (dH2O). Samples were rinsed briefly with phosphate-buffered saline (PBS) and blocked using 2% fish skin gelatin (Sigma) in PBS containing 0.025% Triton-X-100 for 90 min at RT. Samples were incubated with primary antibodies -fixed, paraffin wax-embedded tissues were cut 20 µm) onto poly-0 µm ontoDewaxed 20 µm sections were stained with Mayer’s Haematoxylin (Sigma) at RT for 3 min. Blue staining was performed by rinsing in tap water, while differentiation was achieved by rinsing in 1% acid ethanol. Counterstaining was performed by rinsing with eosin (Sigma) for 30 s and dehydration was performed by sequential washing with 95% ethanol, 100% ethanol and Histo-Clear . Slides were covered with cover-slips with DPX (Agar Scientific) and examined by light microscopy using a Zeiss Axioplan 2 microscope.2 of skin was removed from the dorsal antero-posterior midline surface of 8–10-week-old, male immunodeficient nude mice -Foxn1nu, Charles River, Wilmington, MA) using the pinch cutting technique. A silicone chamber with a diameter and height of 1 cm was inserted underneath the skin on the back of the mouse. Only one chamber was placed on each mouse. The chamber was hat shaped, with a hole in the top of the hat. The HSCs were placed into the chambers and maintained for 5 days by adding epidermalization medium everyday into the chamber. Subsequently the chamber was removed and the HSCs were secured with four sutures (7–0 Nylon) in a simple interrupted pattern around the edge of the graft. A Bandaid was then wrapped around the mice (an OpSite Flexifix Transparent Film) to hold the graft in place. The mice were euthanized after 4–6 weeks for analyses. To visualize the vascularization, the mice were injected intravenously through tail vein with mouse-specific rhodamine-conjugated GS-IB4  solution 20 min prior to euthanasia40. The engraftment experiments were performed using five biological replicates (separate batches of HSCs) on two mice per each biological replicate (n = 10) per condition.All experimental animal protocols were approved by the Institutional Animal Care and Use Committee at Columbia University Medical Center. 0.8 cmThe plasmids pBABE-puro LEF1  and pCMV3-FLI1  were purchased. DPCs at passage 3 were seeded in six-well plates at 100,000 cells per well and cultured overnight prior to transfection. Lipofectamine P3000 transfection reagent was used to transfect the cells. In each well, the cells were maintained in 250 µl of P3000 with 2.5 µg of DNA overnight and the medium was changed to normal culture medium for further use or analyses.p value (two-tailed t test) of less than 0.05 as well as a twofold change were chosen for further analysis. Gene expression data of the samples clustered in heatmaps and gene distance matrix were performed using unsupervised hierarchical gene clustering analyses.Total RNA was isolated from cells maintained at indicated conditions by using the RNeasy Mini Kit (Qiagen) in accordance with the manufacturer’s instructions. Only the total RNA that received an RNA integrity number (RIN) of 9 or higher was submitted for RNA-sequencing to the Columbia University sequencing facility. Samples were processed at 30 million reads per sample. For the sequencing analysis, reads were aligned to the human reference genome (hg19). Gene counts were calculated using HTSeq and were used as an input for differential gene expression analysis with DESeq (version 1.20.0). Comparing the 2D cultured DPCs with the intact DPCs, genes with a Lef-1-targeted genes previously detected by ARACNE algorithm7 to the gene expression data found experimentally by RNA-sequencing in Lef-1-transfected DPCs. A null distribution for calculating the NES and its associated p value was found by label shuffling to randomize the gene rankings. These randomized sets were then used to calculate null ESs over 10,000 iterations to generate a null distribution. The observed leading-edge ES was normalized to this null distribution, and a two-tailed p value was generated for this NES.We previously identified MRs of DPC gene signature by interrogating the skin transcriptome for panel enrichment and associations of differential expression using both Fisher’s Exact Test and GSEA, to identify transcription factors with an enriched set of targets within a particular territory. Here, the GSEA was used to compare the t test was used to calculate whether differences between samples, across three technical replicates, were significant, or p < 0.05. Primer sequences are available in Supplementary Table To collect RNA from HSCs, the whole tissue was first homogenized by a sonicator in Trizol prior to RNA extraction. cDNA was generated from RNA samples following Invitrogen protocols and using SuperScript III with a mixture of random hexamers and oligo dT primers. qPCR was performed using Sybr Green PCR mix on an Applied Biosystems 7300 Real-Time PCR System. Fold changes were calculated using the delta-delta CT algorithm. Error bars were calculated based on SD across three technical replicates within three biological replicates. An unpaired two-tailed 41. The captured tissue was immediately lysed and prepared for further PCR analyses.Laser capture microdissection was performed at Columbia University Cancer Center Core Facility using a laser capture microscope . OCT-embedded samples were sectioned on membrane slides at a thickness of 5 μm, fixed in 75% ethanol, and dehydrated in xylene prior to microdissectiont test with 95% confidence interval with the software GraphPad Prism. Since we used four different cell types in our engineered tissues and anticipated variabilities between different donors, we chose an adequate sample size by using at least three biological replicates and three technical replicates throughput the study. In particular, the experiments done for establishing HF differentiation in hair constructs in vitro were performed in triplicates using cells from the foreskin of three different donors . qPCR analyses and quantification of the success rate of hair differentiation in vitro was done in triplicates and with three biological replicates for each condition. For the calculations of in vivo success ratio, the engraftment experiments were performed using five biological replicates (separate batches of HSCs) on two mice per each biological replicate (n = 10) per condition, yielding a total of 20 mice. The hair regeneration success ratio was based on the vascularized grafts that were viable at the time of harvesting. The researcher who performed the quantification of success rates shown in Fig. p < 0.05 was considered significantly different, where the *p < 0.05 and **p < 0.005. Data were shown as means ± s.e.m.The statistical analysis was performed for all the experiments using two-tailed paired Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1


Cusp beams are one type of complex structured beams with unique multiple self-accelerating channels and needle-like field structures owning great potentials to advance applications such as particle micromanipulation and super-resolution imaging. The traditional method to generate optical catastrophe is based on cumbrous reflective diffraction optical elements, which makes optical system complicated and hinders the nanophotonics integration. Here we design geometric phase based ultrathin plasmonic metasurfaces made of nanoslit antennas to produce three-dimensional (3D) optical cusp beams with variable numbers of self-accelerating channels in a broadband wavelength range. The entire beam propagation profiles of the cusp beams generated from the metasurfaces are mapped theoretically and experimentally. The special self-accelerating behavior and caustics concentration property of the cups beams are also demonstrated. Our results provide great potentials for promoting metasurface-enabled compact photonic devices used in wide applications of light-matter interactions. Catastrophe theory bridges geometry interpretation to the physical world and its manifestation in optics appears in high-intensity caustics with defined geometries of different orders5. According to the increasing dimensionality of the control parameter space, seven elementary catastrophes have been hierarchically classified including four cuspoid catastrophes  and three umbilic catastrophes 2. Subsequent to the Airy beam with typical fold caustics which is the first realized optical catastrophe7, the regular polygon beam (RPB) emerges as a paraxial beam with multiple cusp points equally spaced in a circle and propagating along a bending trajectory. The triple RPB corresponds exactly to the elliptic umbilic catastrophe11. In contrast to the fold caustics, this kind of cusp caustics owns not only the simultaneous self-healing and self-bending propagation properties but also has the remarkable caustics concentration property which leads to the generation of needle-like field structure14. Such novel beam properties of cusp beams will enable many spectacular applications such as optical particle micromanipulation and transportation16, super-resolution imaging18 and curved plasma channels19. Optical catastrophe is traditionally generated by using reflective diffraction optical elements like SLM 16 or DMD 12. However, these bulky devices usually have micrometer pixel size and the limited working-wavelength range, which inevitably hinder the further exploration of optical catastrophe integrated in miniaturized photonic devices and systems.Catastrophe theory mathematically describes the formation and dramatic change of bifurcations as geometrically stable structures in nonlinear potential functions under the perturbations of external control parameters21. Different from bulky optical components with the phase shift depending on the optical path, metasurfaces can introduce the Pancharatnam-Berry geometric phase accompanied with polarization conversion, which can overcome narrow bandwidth limitation and make the optical device compact27. Nowadays metasurfaces have been widely designed to shape the phase front of light field in many applications such as structured beam generation35, flat lens imaging39, compact wave plates for polarization conversion42 and holographic image construction47.Recently, the manipulation of complex light field is getting rapid growth with the development of ultrathin geometric phase based metasurfacesIn this work, we investigate and analyze the generated 3D cusp beams with geometric phase based ultrathin plasmonic metasurfaces constructed from nanoslit antennas. The Pancharatnam-berry (PB) geometric phase profile encoded in the metasurface is taken from the Fourier spectrum of cusp beam, and the local geometric phase is realized by varying the orientation angle of nanoslit antenna in each unit cell. Here three kinds of cusp beams with different numbers of self-accelerating channels have been studied. We have analyzed the whole beam propagation profile of cusp beams accompanied with their novel self-accelerating property and caustics concentration property at three different wavelengths of 633 nm, 808 nm and 980 nm, covering a broadband wavelength range. Furthermore, it is shown that the generated 3D cusp beams from the metasurface gets robust self-healing property over obstacles during propagation. Our demonstrated results will provide promising possibilities for building metasurface-enabled ultrathin photonic devices used in many applications of light-matter interactions such as complex light field conversion, optical particle manipulation, and super-resolution imaging.θ. When the incident circularly polarized beam normally transmits through the anisotropic nanoslit antenna, the original spin component has no phase shift, while the converted spin component will have the induced geometric phase shift of 2θ based on the general principle of Pancharatnam-Berry optical elements (PBOEs)33. The overall phase distribution from the metasurface is obtained by arranging the nanoslit antennas into the designed spatially inhomogeneous array. Figure E| under circular polarization basis , showing the polarization anisotropy of the nanoslit antenna. A SEM image of the fabricated homogeneous array of nanoslit antennas is shown in Fig. The designed plasmonic metasurface based on the PB geometric phase with compact size of 50 μm by 50 μm contains 150 by 150 subwavelength nanoslit antennas with specified orientation angles. As shown in Fig. 11. Here the targeted geometric phase profiles for generating the cusp beams with three different numbers of self-accelerating channels are determined by the beam Fourier spectra. The integral representation for the electric field of cusp beams in the Cartesian coordinates can be written as11Recently the class of RPBs has been extended with both odd and even numbers of cusp points, and such accelerating cusp beams with four or five cusp points are derived from Thom’s elliptic umbilics. These cusp beams naturally focus in free space to yield caustics with a non-trivial morphology that does not correspond to those described by standard catastrophe opticsψA is a constant and 11. Then the orientation angles 10C and w represent a constant scaling number and the initial phase, respectively. Specially, the polynomial order m determines the number of self-accelerating channels with the intensity maximum of the cusp beams.The phase function in the integral can be written as m = 3, 4, 5 are designed. Figure m = 3, C = 3.3 and In order to illustrate the special characteristics of cusp beams having various number of self-accelerating channels, three metasurfaces with m = 3, 4, 5 are first measured at the wavelength of 633 nm. In order to keep the similar beam sizes for the generated optical cusp beams with different m, the initial phase is m = 3, 4, 5 is set to be C = 3.3, 1.2, 0.52 respectively. Figure E|2 skeleton of cusp beams with m =  3, 4, 5 based on Eq.  system . To achieve the targeted metasurface, the orientation angle for the nanoslit antenna in each unit cell is determined by the designed phase profile for the cusp beam.To achieve the transmission spectra measured from the metasurface shown in Fig.


The AMP-activated protein kinase (AMPK) is a heterotrimeric complex with central roles in cellular energy sensing and the regulation of metabolism and exercise adaptations. AMPK regulatory β subunits contain a conserved carbohydrate-binding module (CBM) that binds glycogen, the major tissue storage form of glucose. Research over the past two decades has revealed that the regulation of AMPK is impacted by glycogen availability, and glycogen storage dynamics are concurrently regulated by AMPK activity. This growing body of research has uncovered new evidence of physical and functional interactive roles for AMPK and glycogen ranging from cellular energy sensing to the regulation of whole-body metabolism and exercise-induced adaptations. In this review, we discuss recent advancements in the understanding of molecular, cellular, and physiological processes impacted by AMPK-glycogen interactions. In addition, we appraise how novel research technologies and experimental models will continue to expand the repertoire of biological processes known to be regulated by AMPK and glycogen. These multidisciplinary research advances will aid the discovery of novel pathways and regulatory mechanisms that are central to the AMPK signaling network, beneficial effects of exercise and maintenance of metabolic homeostasis in health and disease. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of a catalytic α subunit and regulatory β and γ subunits, which becomes activated in response to a decrease in cellular energy status. Activation of AMPK results in metabolic adaptations such as increases in glucose uptake and glycolytic flux and fatty acid (FA) oxidation. AMPK activation simultaneously inhibits anabolic processes including protein and FA synthesis. AMPK can also translocate to the nucleus where it regulates transcription factors to increase energy production, meet cellular energy demands and inhibit cell growth and proliferation. Conversely, when energy levels are replete, AMPK activity returns to basal levels, allowing anabolic processes to resume. Given its central roles in cellular metabolic and growth signaling pathways, AMPK remains an appealing target for treating a range of pathologies associated with obesity and aging, including metabolic diseases such as obesity and type 2 diabetes (T2D).In response to changes in energy supply and demand, glycogen, predominately stored in the liver and skeletal muscle, serves as an important source of energy to maintain metabolic homeostasis. Glycogen is synthesized by the linking of glucose monomers during periods of nutrient excess. In response to energy stress and decreased arterial glucose concentration, rising glucagon levels induce increased hepatic glucose output by promoting the breakdown of glycogen and the conversion of non-glucose substrates into glucose. The newly formed glucose is released into the bloodstream to help restore blood glucose levels. Skeletal muscle glycogen serves as an accessible source of glucose to form adenosine triphosphate (ATP) and to reduce equivalents via glycolytic and oxidative phosphorylation pathways during muscle contraction.A significant body of evidence demonstrates that AMPK binds glycogen. This physical interaction is mediated by the carbohydrate-binding module (CBM) located within the AMPK β subunit and is thought to allow AMPK to function as a sensor of stored cellular energy. While glycogen is stored in multiple tissues throughout the body, this review will primarily focus on the physical interactions underlying the AMPK β subunit binding to glycogen and its potential functional links to glycogen storage dynamics in the liver and skeletal muscles, as these tissues are central to metabolic and exercise-regulated biological processes. In addition, as the majority of research on this topic has been undertaken in human and mouse model systems, studies in these species will be highlighted. Following a brief background on the regulation of AMPK and glycogen, this review will critically assess the recent advances and focus primarily on studies within the past two decades that have added to our understanding of the physical basis of AMPK-glycogen binding and its potential functional interactions in exercise and metabolism. Key remaining biological questions related to the interactive roles of AMPK and glycogen will be posed along with a discussion of research advancements that are feasible in the next decade with new technologies and experimental models to determine how AMPK-glycogen binding may be therapeutically targeted in health and disease.2+ concentrations via the upstream kinase calcium/calmodulin-dependent protein kinase kinase β (CAMKK2) in the absence of changes in adenylate energy charge [Structural biology-based studies over the past decade have provided new insights into the molecular mechanisms by which AMPK activation is regulated by nucleotides, including changes in AMP:ATP and ADP:ATP ratios  that occur in response to cellular energy stress . The biny charge ,12,13.Once activated, AMPK serves as a metabolic ‘switch’ to promote catabolic pathways and inhibit anabolic processes. For example, AMPK increases glucose uptake into skeletal muscle by phosphorylating and inhibiting Tre-2, BUB2, CDC16, 1 domain family, members 1 (TBC1D1) and 4 (TBC1D4), promoting glucose transporter 4 (GLUT4) vesicle translocation to the sarcolemmal membrane ,16,17,18The AMPK β subunit exists in two isoforms (β1 and β2) and serves as a scaffolding subunit that binds to the AMPK catalytic α and regulatory γ subunits, playing an important role in the physical stability of the heterotrimer  9,26,27,2726,27.2+, epinephrine and cAMP concentrations [A number of proteins are associated with glycogen particles and function as regulators of glycogen synthesis, breakdown, particle size, and degree of branching. Glycogenin initiates glycogen formation and functions as the central protein of the glycogen particle . Glycogetrations ,41. Whentrations . The glytrations . Furthertrations ,43.2+ release and reuptake. Therefore, it has been hypothesized that these different pools of glycogen play a significant role in muscle contraction and fatigue beyond their role as an energy substrate [Recently, there has been an increasing interest regarding the significance of glycogen’s subcellular localization in skeletal muscles ,46,47,48ubstrate ,47,48.Human skeletal muscles contain large stores of glycogen, which can exceed 100 mmol glucosyl units/kg wet weight (~500 mmol/kg dry weight) in the vastus lateralis muscle ,50 and aIn 2003, it was first demonstrated that recombinant AMPK β1 CBM bound glycogen using a cell-free assay system . StructuIn addition to its role in binding glycogen, the CBM of the β subunit also physically interacts with the kinase domain of the catalytic α subunit, forming a pocket, referred to as the allosteric drug and metabolite (ADaM) site. Small molecule AMPK activators such as A-769662, a β1 subunit specific activator, bind to this site and directly activate AMPK ,59. HoweRecent research has provided further structural insights into the affinity of the AMPK β subunits for carbohydrates. Isolated β2 CBM has a stronger affinity for carbohydrates than the β1 CBM, binding strongly to both branched and unbranched carbohydrates, with a preference for single α-1,6 branched carbohydrates . One posSeveral independent lines of evidence suggest that these physical AMPK and glycogen interactions also serve mechanistic functional roles in cellular energy sensing. A number of AMPK substrates are known to be directly involved in glycogen storage and breakdown, highlighting AMPK’s role as an important regulator of glycogen metabolism. In vitro, AMPK regulates glycogen synthesis directly via the phosphorylation and inactivation of GS at site 2 . In suppAn increase in GP activity has also been observed to be associated with AMPK activation induced by AICAR treatment of isolated rat soleus muscles ,71. HoweIt is important to consider additional factors that may impact physical and functional AMPK-glycogen interactions. In a proteomic screen utilizing purified glycogen from rat liver, AMPK was not included in the proteins detected to be associated with glycogen , and thiIn the fifteen years following the discovery of glycogen binding to the CBM on the β subunit, several studies utilizing AMPK isoform knockout (KO) mouse models have provided whole-body physiological evidence of AMPK’s interactive functional roles with glycogen. Collectively, studies using AMPK α and β subunit KO mouse models have found that the ablation of AMPK alters liver and skeletal muscle glycogen content, supporting the role of AMPK in the regulation of tissue glycogen dynamics in vivo. Specifically, whole-body β2 KO mice have reduced basal glycogen levels in both liver and skeletal muscles associated with reduced muscle AMPK activity and attenuated maximal and submaximal running capacity compared to wild-type (WT) mice . β2 KO mIn addition to mouse models targeting the AMPK β subunit(s), recent studies utilizing tissue-specific α1/α2 KO mice have provided support for the functional interactive roles of AMPK and glycogen. Liver-specific AMPK α1/α2 KO mice have an impaired ability to maintain euglycemia during exercise as a result of decreased hepatic glucose output due to decreased glycogenolysis . SpecifiA series of physiological studies have demonstrated that low glycogen availability can amplify the AMPK signaling responses and adaptations to exercise. This was originally described in rat skeletal muscles in which AICAR treatment resulted in increased AMPK α2 activity and a markedly reduced glycogen synthase activity in a glycogen-depleted state compared to a glycogen-loaded state . This obMetabolic diseases such as insulin resistance and T2D are associated with impairments in AMPK activity, signaling, and glycogen storage dynamics. Obese patients with T2D have reduced skeletal muscle AMPK, ACC, and TBC1D4 phosphorylation following an acute bout of exercise . In suppIn addition, glycogen storage diseases provide pathophysiological models that can help provide additional insights into the influence of glycogen dynamics on AMPK. McArdle’s disease is characterized by the accumulation of skeletal muscle glycogen due to a deficiency of GP. Individuals with McArdle’s disease display higher muscle glycogen both at rest and following exercise compared to healthy controls, and an increased AMPK α2 activity and reduced GS activity in response to exercise . PatientWhile much remains to be discovered with regard to the molecular and cellular roles and physiological relevance of AMPK-glycogen binding, recent multidisciplinary technical research advances can be used to help address remaining knowledge gaps. For example, global mass spectrometry-based phosphoproteomics have recently revealed a repertoire of new AMPK substrates, providing additional evidence regarding the complexity and interconnection of the AMPK signaling network. A recent phosphoproteomic analysis mapping the human skeletal muscle exercise signaling network before and immediately following a single bout of intense aerobic exercise, in combination with phosphoproteomic analysis of AICAR-stimulated signaling in rat L6 myotubes, identified several novel AMPK substrates . Other rNovel AMPK fluorescence resonance energy transfer (FRET)-based sensors have recently revealed heterogeneous activity and tissue-specific roles for AMPK. These AMPK FRET sensors have permitted the spatiotemporal and dynamic assessment of AMPK activity in single cells , 3D cellDespite the large body of research using in vitro models and physiological evidence indicating the potential functional roles for AMPK-glycogen binding, to date, there are no models that have been developed to disrupt and/or examine this physical binding directly in vivo. AMPK subunit KO models, while providing important insights into the functions of AMPK, are limited by the potential compensatory upregulation of other subunit isoforms or the disrupted stability of the AMPK heterotrimer complex . In addFinally, previous studies have primarily utilized centrifugation-based assays to detect and quantify physical AMPK-glycogen association. Novel biotechnological platforms and proximity assays will aid this investigation of AMPK-glycogen binding and AMPK’s proximity to glycogen with improved sensitivity and specificity across molecular, cellular, and physiological models. Furthermore, newly developed kinase activity reporters  and otheConsistent with the therapeutic relevance of the CBM, several lines of evidence demonstrate that the CBM may play a direct functional role in AMPK conformation and activation. The CBM contains the critical S108 autophosphorylation site required for drug-induced AMPK activation in the absence of AMP . DespiteAMPK is a central regulator of cellular metabolism and, therefore, possesses significant therapeutic potential for the prevention and treatment of a range of metabolic diseases. A growing body of evidence demonstrates that AMPK physically binds glycogen and this interaction can alter the conformation of AMPK, and subsequently, its activity and downstream signaling. AMPK activity subsequently regulates glycogen metabolism. Recent research has described experimental and physiological settings that impact functional AMPK and glycogen interactions, including AMPK β isoform affinity, glycogen availability, and particle size. Despite our understanding of AMPK’s relationship with glycogen, much remains to be elucidated. Further research using new technologies and experimental models can reveal additional mechanisms underlying AMPK and glycogen’s interactive roles in cellular energy sensing, exercise, and metabolism. Together, these findings will help provide insights into the physiological and therapeutic relevance of targeting AMPK and glycogen binding in health and disease.


We used track density imaging, which provides unprecedented anatomic details based on probabilistic tractography streamlines, to demonstrate apparent changes in the integrity of the dentato–rubro–olivary pathway (Guillain–Mollaret triangle) that were consistent with the diagnosis of hypertrophic olivary degeneration from the antecedent MS lesion involving the right superior cerebellar peduncle. Further medical therapy was avoided, and follow-up MRI 1 year later showed interval involution of the left olivary lesion. This case demonstrates the potential clinical utility of using track density imaging to detect lesion-induced alterations in brainstem connectivity and characterize neurodegeneration in patients.A 32-year-old female with relapsing–remitting multiple sclerosis (MS) presented with severe new onset ataxia and diplopia. MRI showed a new inflammatory MS lesion that involved the right dorsal pons and extended into the adjacent superior cerebellar peduncle. The patient improved with aggressive immunotherapy; however, repeat MRI 3 months later revealed a new non-enhancing lesion in the left inferior medullary olive. The differential diagnosis for this new lesion included an MS lesion  T2/fluid-attenuated inversion-recovery (FLAIR) hyperintense lesion in the right superior cerebellar peduncle (SCP)  presented with severe ataxia and diplopia requiring prolonged hospitalization. Examination at the time demonstrated rotary nystagmus when she was looking up and to the right, incomplete abduction of the right eye, decreased sensation on the right side of the face , left hemibody deficits and severe truncal ataxia . MRI of the head showed a new le (SCP) . Aggresse lesion . At thatT2/FLAIR hyperintensity—a new inflammatory MS lesion was considered most likely given the patient’s history. However, a new lesion would be atypical following aggressive treatment with medical therapy. Glial neoplasm seemed unlikely within the short 3-month interval and brainstem infarction would be unusual in this young female with no vascular risk factors and no corresponding changes in mean diffusivity. Hypertrophic olivary degeneration (HOD) was also a primary consideration, given the specific anatomic locations of the medullary lesion and the previously documented afferent demyelinating lesion involving the right SCP. The new medullary lesion may be attributed to degeneration of the dentato–rubro–olivary tract of the Guillain–Mollaret triangle secondary to pathologically altered dentato–rubral fibres, but direct demonstration of tract involvement was not possible with conventional MRI protocols. To help address this diagnostic dilemma, we performed a novel track density imaging (TDI).1The new expansile lesion in the left inferior medullary olive showed 2 to produce TDI images of the brainstem structures with a clinically feasible scan time at 3 T. The sequence parameters included 3 mm isotropic resolution, matrix = 80 × 80, field of view = 240 × 240 mm2, multiband accelerate factor = 2, 256 diffusion directions with b-value = 2500 s mm−2, eight images with b = 0, repitition time/echo time 3300/98 ms, 42 slices, parallel imaging integrated parallel acquisition technique = 2 with generalized autocalibrating partial parallel acquisition reconstruction . The DW images were corrected for eddy current distortions using Functional Magnetic Resonance Imaging of the Brain's (FMRIB) Toolbox .3The patient underwent a conventional contrast-enhanced MRI of the head using a 20-channel head and neck coil on a 3 T MRI . In addition, after informed consent was obtained, we used a novel combination of high angular resolution diffusion-weighted (DW) sequence obtained with simultaneous multislice acquisition diffusion MRIhttps://github.com/MRtrix3/mrtrix3), which combines the constrained spherical deconvolution (CSD) techniques4 with whole-brain probabilistic streamline reconstructions. The relevant fibre-tracking parameters were 100 million tracks randomly distributed throughout the whole brain, 1.5 mm step size, maximum curvature per step = 45°, maximum length = 20 mm and minimum length = 15 mm. The conventional colour scheme was used for track orientation in the patient’s brain: blue , green (anterior–posterior) and red (left–right). A short-track TDI method was utilized, where the maximum/minimum track length ratio approached 1, to visualize smaller fibre pathways for more accurate region of interest (ROI) placement in quantification.5The super-resolution TDI and direction-encoded colour (DEC) TDI maps were generated with 0.5 mm isotropic resolution by using MRtrix3 .TDI and DEC-TDI maps demonstrated a visually obvious unilateral loss of track density in the right dentate nucleus and corresponding dentato–rubral tract attributed to a combination of oedema, demyelination and/or axonal loss from the inflammatory MS lesion involving the right SCP . The ROI−3, while the normal contralateral side had 4410 tracks mm−3 (a 25% reduction in probabilistic streamlines). The TDI findings in the corresponding dentato–rubro–olivary pathway, known as the Guillain–Mollaret triangle, supported a final imaging and clinical diagnosis of HOD for the new lesion.The left rubro–olivary region and functionally connected inferior medullary olive, which was pathologically enlarged, also demonstrated unilateral track density loss. Qualitatively, the left inferior olive showed a loss of pixel intensity and blue streamlines on the TDI and DEC-TDI maps . The lefInitial treatment was directed at aborting MS relapse and included i.v. solumedrol courses, plasmapheresis and mitoxantrone chemotherapy. The initial lesion in the SCP resulted in delayed HOD imaging changes that were clinically silent and did not require further therapy upon imaging confirmation of the suspected diagnosis.The patient’s condition slowly improved and she was able to resume full-time employment 3 months after hospital discharge from the initial MS flare. At subsequent follow-up, she remained asymptomatic, except for episodic dizziness. She has been receiving monthly natalizumab infusions for the past 12 months and her MRI of the brain at 1-year follow-up showed the expected evolution of the olivary lesion.6 The hypertrophy correlates with an increased number of astrocytes and vacuoles in the affected neurons.7 The T2/FLAIR image hyperintensity within the olive correlates with gliosis. The clinical symptom of classical palatal myoclonus is due to the loss of inhibitory control. The typical imaging findings of HOD usually are present within 6 months following the initial insult. The apparent olivary hypertrophy resolves over 4 years, corresponding with a reduction in the normal number of neurons.8HOD is a unique form of neurodegeneration where loss of afferent input within the dentato–rubro–olivary pathway results in hypertrophy of the inferior medullary olive.via the dentato–rubral tract within the SCP. To complete the arc, efferent fibres from the olive cross the midline and ascend via the inferior cerebellar peduncle into the contralateral cerebellar cortex before projecting on the dentate. This triangle controls fine motor movements and was first described by Guillan and Mollaret9 in 1931. Only injury to either one of the two afferent limbs  results in HOD.The inferior olivary nucleus is situated in the ventrolateral medulla and receives fibres within the central tegmental tract from the ipsilateral red nucleus of the midbrain. The red nucleus receives input from the contralateral dentate nucleus of the cerebellum in vivo MRI post-processing technique based on high angular resolution diffusion acquisitions that generate super-resolution images derived by whole-brain probabilistic streamline tractography.1 Images of 500 μm isotropic resolution can be created where the pixel intensity reflects the number of probabilistic streamlines traversing the voxel and the colour reflects diffusion streamline orientations per conventional diffusiontensor imaging (DTI). These contrasts have been validated using histology in animal models.5TDI is a novel 2 to obtain TDI maps of brainstem structures in actual patients using clinically feasible scan times at 3 T. In addition, a sufficient number of DW imaging directions, higher b-values and signal-to-noise ratio were achieved to reduce uncertainty in estimating the white matter fibre tracts with whole-brain probabilistic tractography based on the CSD method. It is essential to use an MRI model that accounts for the presence of multiple fibre directions within a voxel, such as the CSD method, to resolve areas of “crossing fibres”.4We have combined a track density approach with the recently developed simultaneous multislice acquisition diffusion MRIThe super-resolution (0.5 mm isotropic resolution) of TDI provides high-quality fibre pathways with exquisite anatomical contrast. Smaller fibre pathways not directly visualized with conventional DTI, such as those found in the brainstem, are confidently identified. TDI offers the ability to alter the signal-to-noise ratio depending on the chosen sub-voxel resolution and the number of random seed points placed. Even though whole-brain TDI maps are generated from the same DW imaging raw data as routine DTI maps, whole voxel averaging leads to partial volume effects and poor resolution in DTI maps.4 There is debate over which post-processed track density contrast map  should be used to achieve white matter quantification for larger clinical studies. The short-track map was chosen here owing to its ability to illustrate right vs left differences of small structures in a single patient. A manual ROI was drawn around the elongated nature of the dentato–rubral tract and inferior olivary nuclei on axial slices and then compared to the contralateral ROI in the same axial slice. This manual approach helped in confirming tract loss in the expected regions, but it may be better to use automated ROI analysis in future clinical cases or research.TDI can also be used to quantify diseased local structure connectivity so long as diffusion acquisition and post-processing parameters are consistent across patients.10 Use of DTI also has been described for demyelination-induced HOD.11 To our knowledge, this is the first reported case of TDI being used to support a clinical diagnosis of HOD secondary to a demyelinating lesion. Owing to the super-resolution potential of the TDI methodology, more precise tractography and/or diffusion tract-based contrast of small brainstem structures can be obtained compared with conventional MRI protocols. This case clearly illustrates the potential TDI has for clinical characterization of brainstem pathology and future research investigations of neurodegeneration in the brainstem for other common brainstem pathologies, such as quantitative MRI correlates to parkinsonism.DTI has been used to diagnose the condition of HOD with increased radial and axial diffusivity and decreased fractional anisotropy (FA) in the affected inferior olivary nucleus.Understanding the underlying mechanisms and confident recognition of HOD on MRI are essential to prevent unnecessary medical interventions.TDI is a novel post-processing super-resolution technique that can generate 500 μm isotropic images based on whole-brain diffusion tractography using clinically feasible simultaneous multislice 3 T MRI diffusion acquisitions.TDI can depict and quantify important complex brainstem anatomy in healthy and diseased individuals, which is not apparent on conventional MRI.Written informed consent was obtained from the patient for publication of this case report, including accompanying images.


We aimed to examine whether back pain across adulthood was associated with spine shape at age 60–64 years. Data were from 1405 participants in the MRC National Survey of Health and Development, a nationally representative British birth cohort. Back pain was ascertained during nurse interviews at ages 36, 43, 53 and 60–64 years. Cumulative exposure to back pain was then derived by counting the number of ages at which back pain was reported. Statistical shape modelling was used to characterise thoracolumbar spine shape using lateral dual-energy x-ray absorptiometry images which were ascertained at age 60–64 years. Linear regression models were used to test associations of spine shape modes (SM) with: (1) cumulative exposure to back pain; (2) back pain reports during different periods of adulthood. After adjusting for sex, higher cumulative exposure to back pain across adulthood was associated with wedge-shaped L4-5 disc (lower SM4 scores) and smaller disc spaces (higher SM8 scores) in both sexes. In addition, reporting of back pain at ages 53 and/or 60–64 years was associated with smaller L4-5 disc space (lower SM6 scores) in men but not women. These findings suggest that back pain across adulthood may be associated with specific variations in spine shapes in early old age. It affects people of all ages3 and its consequences for both the individual and society are considerable7. Back pain is a complex multi-factorial disorder; biomechanical, biological, psychological and social factors play a role in its onset, persistence and severity8. Furthermore, there have been suggestions that variations in the anatomical shape of the spine may predispose some individuals to increased risk of back pain and spinal injury11.Back pain is the most prevalent musculoskeletal condition worldwide and a leading cause of years lived with disability13. Recent studies have shown that each individual has an intrinsic spine shape that is partly maintained during postural changes11. This influences the preferred manual lifting technique used11 raising the possibility that spinal shape may play a role in the susceptibility to and recovery from low back pain and may predispose individuals with certain variations in shape to increased risk of spinal injury.Within a population, spine shape varies with some individuals having curvier (lordotic) spines than others16. Similarly, pre-existing degenerative back disorders including spinal osteoarthritis, lumbar disc degeneration, osteoporosis and vertebral fractures are often accompanied by structural changes in the spinal shape as a consequence of disease processes and have been linked to back pain and reduced spine flexibility and mobility18. Other studies suggest that persistent back pain or trauma may be associated with decreased mobility and flexibility of muscles and joints19.Spine morphology is also likely to reflect age-related degenerative changes which may result in subsequent adverse health outcomes including back pain21 or sagittal spinal curves23. However, these studies face important limitations that lead to uncertainty about the relationship between back pain and intrinsic spine morphology in the general population23. For example, many studies are cross-sectional, and/or have assessed pain at a single prior age thereby failing to acknowledge that back pain is often a recurrent, intermittent problem with prior history predictive of future episodes and have not accounted for possible confounders24. They have also mainly used geometric features such as segmental angles to characterise spinal curvature which may not capture systematic variations in spine morphology22.To date most epidemiological studies of the relationship between back pain and spine shape have examined radiological abnormalities25 we also aimed to examine whether back pain reports during different periods of adulthood were associated with spine shape.Using data from a large, nationally representative sample, we aimed to address these gaps in the literature by examining whether cumulative exposure to back pain across adulthood is associated with spine shape at age 60–64 years. As different profiles of back pain across adulthood have been identifiedWomen had higher mean scores for spine mode (SM) 1, 3 and 8 than men but lower scores for SM6. Figure Table In both sexes, the prevalence of back pain increased until age 53 years and no sex differences were observed in the cumulative exposure or timing of back pain. In terms of covariates, men were taller, more likely to have a higher occupational class and less likely to report symptoms of anxiety and depression than women Table . Women wTable No associations were found between cumulative reports of back pain and modes which described total amount of curvature within L5 to T10 (SM1); differences in the distribution of curvature along the length of the spine (SM2); vertebral rotation at T10, L3 and L5 with resulting minor variations of lordosis and kyphosis and varying L4/L5 disc space (SM5); or minor variation in relative a-p diameter at T10-T12 and at L4-L5 (SM7).When the associations of timing of back pain with spine shape were examined Table , there wIn a large nationally representative population-based sample of adults aged 60–64 years, higher cumulative exposure to back pain across adulthood was associated with wedge-shaped L4-5 disc (lower SM4 scores) and smaller disc spaces (higher SM8 scores) in both sexes. In addition, back pain reports at one or two ages in adulthood were associated with smaller L4-5 disc space (lower SM6 scores) in men only. When we investigated back pain reports during different periods of adulthood, we found that back pain reports in later adulthood (at ages 53 and/or 60–64 years) were associated with lower SM6 scores (men only) whereas back pain reports in mid-life (at ages 36 and/or 43 years) and across adulthood (mid and later adulthood) were associated with higher SM8 scores. We found no associations between back pain and overall curviness (SM1) or uneven (or snaking) spinal curvatures (SM2).et al. found a higher frequency of degenerated discs at L4-L5 among 20–40 year old asymptomatic individuals but only in those with subtype II sagittal alignment (flatter lumbar spine)16, as described by Roussouly classifications13. Although the association between lumbar disc disease (LDD) and low back pain is widely reported to be weak (reviewed in24), in a separate study using a different SSM we found that asymptomatic subjects with total scores for LDD of greater than 5, using modified Pfirrmann grading, were also more likely to have smaller L4-S1 disc spaces26.To the best of our knowledge this is the first study to examine the relationship between back pain at multiple time points across adulthood and spinal shape in early old-age, characterised using statistical shape modelling (SSM). In this study, those who reported back pain at a greater number of ages across adulthood tended to have marginally smaller disc spaces, relative to vertebral heights, at the second to fourth lumbar levels (higher SM8 scores). This association was particularly evident among those who reported early and persistent back pain. In their cross-sectional study, Menezes-Reis We also found sex differences between back pain and smaller L4-5 disc space (lower SM6 scores). In men, associations were found among those who reported back pain in later adulthood but not among those who reported pain in mid-adulthood only or who had persistent back pain. No associations were found in women.27, onset of overweight at age 36 was also associated with larger relative anterior-posterior (a-p) vertebral diameters in both sexes but this association was fully attenuated after adjusting for current BMI, suggesting that the observed associations may be explained by tracking of BMI across adulthood.Our finding of a linear association between cumulative reports of back pain across adulthood and larger relative a-p vertebral diameters (lower SM3 scores) was fully attenuated after adjustment for covariates. This was mainly explained by the adjustment for current BMI. In previous NSHD analysesSimilarly, the weak linear relationship between cumulative reports of back pain across adulthood and wedge-shaped L4-5 disc (lower SM4 scores) was fully attenuated after adjustment for covariates. However, unlike for SM3, adjustment for leisure time physical activity, higher levels of which were independently associated with lower SM4 scores, had greater effect than adjustment for BMI suggesting a potentially different pathway of association for these two modes.23. One study has reported that individuals with Roussouly subtype II sagittal alignment (flat lordosis) had a higher frequency of degenerated discs at L4-L5 than those with subtype IV (long and curved lumbar spine), but found no significant differences in disc degeneration at other disc levels or with other shapes16. However, this sampled asymptomatic individuals and the relationship with low back pain is unclear. In a study of Modic changes identified using MRI among symptomatic patients with low back pain, 53% showed signs of loss of lumbar lordosis28; however, this study was limited by the lack of asymptomatic controls and by the use of visual examination of MRI scans to determine lordosis. In contrast, an MRI study which compared lumbar spine morphology (characterised using SSM) in 31 asymptomatic adults aged 45 to 70 years, those with Modic changes were significantly more lordotic, and had a more even curvature, than those without Modic changes26.We found no association between back pain and overall curviness (SM1) or uneven (or snaking) spinal curvatures (SM2). This is consistent with most existing literature reviews; these have reported no evidence of association between sagittal spinal curves (assessed using different measurement methods and position and at a varied number of vertebrae) and back pain29.A key strength of our study is the large population-based sample of adults with back pain ascertained at four time-points across adulthood. We also use SSM to quantify spinal morphology. This is not reliant on angles, which are difficult to measure precisely, and it provides a numerical assessment of subtle variations that might otherwise be overlooked. Limitations relate to the imaging methods employed, the definition of low back pain and bias that may have been introduced due to sample restrictions. DXA spinal images were captured at a single time point; therefore, we are unable to distinguish differences in spine shape due to developmental factors from those due to ageing-related degenerative changes. We are also unable to establish temporality in the relationship with back pain and so acknowledge that the observed associations between back pain and spine shape may reflect associations acting in both directions. Although two-dimensional DXA images are commonly used in large studies, it is conceivable that potential errors during positioning such as the tilt of the pelvis may have occurred; therefore images with extreme rotation were excluded from the SSM models. Similarly, images were acquired in a supine position at all CRFs except one where a fixed C-arm in the scanner was used. However, accounting for CRF had negligible effect on our findings. In addition, our binary measures of back pain, which were selected to facilitate longitudinal analyses, do not capture information on pain severity or its impact which may be a limitation given that greater pain intensity or pain-related disability may influence spinal morphology e.g. by affecting movement and posture. Finally, our analyses were restricted to the sample who attended a CRF as this is where DXA scans were undertaken. It is possible that this restriction may have introduced bias as participants who attended a CRF were less likely to be obese and more likely to be in better health than those who were visited at homeIn conclusion, this study found that higher cumulative exposure to back pain across adulthood was associated with wedge-shaped L4–5 disc (lower SM4 scores) and smaller disc spaces (higher SM8 scores) at age 60–64 in both sexes. In addition, reports of back pain in later adulthood (at ages 53 and/or 60–64 years) were associated with smaller L4–5 disc space (lower SM6 scores) in men but not women. These findings provide evidence which may help inform best strategy on the prevention of back pain.The MRC National Survey of Health and Development (NSHD) is a socially stratified population sample of 5362 single, legitimate births that occurred in England, Wales and Scotland in one week of March 1946 and participants have been prospectively followed regularly ever since. Between 2006 and 2010 (at age 60–64 years), eligible study members known to be alive and living in England, Wales and Scotland were invited for an assessment at one of six clinical research facilities (CRF) or to be visited at home by a research nurse. Of the 2856 invited, 2229 were assessed of whom 1690 attended a CRF.30. The study was carried out in accordance with relevant guidelines and regulations.All waves of data collection have complied with ethical standards. Ethical approval for the data collection at age 60–64 was obtained from the Central Manchester Research Ethics Committee (07/H1008/245) and the Scottish A Research Ethics Committee (08/MRE00/12) and written informed consent was obtained from each participant30. Images were obtained with the individuals lying in a supine position in all CRFs except one, where they were scanned in lateral decubitus due to a fixed C-arm in the scanner. All scans were centrally analysed and scrutinized by author JEA’s laboratory.During the visits to the CRF, dual-energy X-ray absorptiometry (DXA) images of the lumbar spine were acquired using the QDR 4500 Discovery  according to standard protocols31.For quality assurance, Hologic Spine Phantoms provided by the scanner manufacturer were scanned every day prior to participant scanning and in accordance with manufacturer’s protocols, and the results were sent to the coordinating centre once a month for scrutinyStandard Bone mineral density measures for the spine were derived using the scanner embedded software provided by the manufacturer.et al. and Faber et al.33. Prior to the placement two investigators  manually marked a random sample of 50 images from the cohort and intra-rater repeatability was measured as 1.4 pixels and inter-rater repeatability as 2.2 pixels34. Of the 1601 images, 72 were excluded due to poor quality, scanning artefacts, incomplete images, evidence of axial rotation or metalwork, leaving 1529 images for statistical shape modelling (SSM)27.Lumbar spine DXA scans were available for 1601 study participants. The Aberdeen team were responsible for image segmentation, point placement was performed by AVP and ambiguous images and statistical outliers were reviewed by the team in line with the approach outlined in Baird 34. Briefly, an 89-point template was constructed using custom SSM software  which described spine shape from the tenth thoracic (T10) to fifth lumbar (L5) vertebrae, marking the vertebral body outlines.The SSM modelling strategy and reasons for image exclusion have been described in detail elsewhere34 and from this eight modes of variance were selected for analysis; together these modes explained 84.9% of overall shape variance with the largest mode SM1 accounting for 53.0% and the smallest, SM8, 1.2%  to normalise the scale, thus removing size differences. Finally principal components analysis identified orthogonal modes of variation in spine shape; each mode describing a percentage of the total variation in shape (in descending order from mode 1). Each mode of variation has a mean of zero and a score in units of standard deviation. The variance was plotted against each mode in a scree plot.2% Fig. .24 by including participants with back pain data at three or more time points across adulthood in the analyses.Data on back pain were obtained at nurse interviews during home visits at ages 36, 43, 53 and 60–64 years. At these ages participants were asked whether they had sciatica, lumbago, or recurring/severe backache all or most of the time (ever at ages 36 and 43 years and in the previous 12 months at ages 53 and 60–64 years). We then considered the long-term course of back painA variable indicating cumulative exposure to back pain was derived by counting the number of ages at which back pain was reported. The derived score  to 4  was categorised into 4 groups .To distinguish between individuals who experienced back pain in different phases of adulthood, so that the importance of timing of exposure could be assessed, a variable was derived with 4 categories as follows: (i) no back pain (i.e. those who did not report back pain at any visit or at 3 visits if data at one visit were missing); (ii) back pain in mid-adulthood only (i.e. those who reported back pain at ages 36 and/or 43 years but not at later ages); (iii) back pain in later adulthood only (i.e. those who reported back pain at ages 53 and/or 60–64 years but not at earlier ages); (iv) back pain across adult life (i.e. those who reported back pain in both mid (36 and/or 43 years) and later adulthood (53 and/or 60–64 years)).2. Lumbar spine (L1-L4) areal bone mineral density (aBMD) was also obtained at this age27.Weight (kg) and height (m) were measured at age 60–64 years by a trained nurse and body mass index (BMI) was calculated as weight (kg)/(height (m))Own occupation at age 53 years was categorised according to the Registrar General’s social classification  into three groups: high (I or II); middle (IIINM or IIIM); low (IV or V).35.Symptoms of anxiety and depression were assessed at age 60–64 years using the 28-item General Health Questionnaire (GHQ-28). Each item was coded using the General Scoring Method then summed with a threshold for caseness of 5 or more selectedSelf-reported level of participation in sports, vigorous leisure activities or exercise was assessed at age 60–64 years and grouped as inactive, moderately active (1–4 times/month) or regularly active (≥5 times/month).Information on parity was obtained from study members prospective reports across adulthood updated to age 53 years and grouped as 0; 1; 2; ≥3 children.Using linear regression models, we first examined the associations between cumulative exposure to back pain across adulthood  and each spine shape mode (SM1–8) at age 60–64 (aim 1). We then investigated whether timing of back pain  was associated with spine shape (aim 2). Models were first adjusted for sex  and then for current BMI, spine BMD, own occupational class, physical activity, GHQ-28 and parity.All models included participants with complete data on back pain data (at least 3 visits), spine mode scores and covariates (N = 1405).All analyses were conducted using STATA v14.1. P-values (two-tailed) are reported.


Members of this gene family are extracellular matrix glycoproteins that play a role in neural cell adhesion and neurite outgrowth. Suggestive evidence of associations with ADHD was observed for an additional 111 SNPs (⩽9.91E−05). Although intriguing, the association between DNA variation in the TNR gene and ADHD should be viewed as preliminary given the small sample size of this discovery dataset.It is well-established that there is a strong genetic contribution to the aetiology of attention deficit hyperactivity disorder (ADHD). Here, we employed a hypothesis-free genome-wide association study (GWAS) design in a sample of 480 clinical childhood ADHD cases and 1208 controls to search for novel genetic risk loci for ADHD. DNA was genotyped using Illumina’s Human Infinium PsychArray-24v1.2., and the data were subsequently imputed to the 1000 Genomes reference panel. Rigorous quality control and pruning of genotypes at both individual subject and single nucleotide polymorphism (SNP) levels was performed. Polygenic risk score (PGRS) analysis revealed that ADHD case–control status was explained by genetic risk for ADHD, but no other major psychiatric disorders. Logistic regression analysis was performed genome-wide to test the association between SNPs and ADHD case–control status. We observed a genome-wide significant association ( The global prevalence of ADHD has been estimated at 5.2–7.2%4. Although the aetiology of ADHD is not well defined, genetic and environmental factors have been implicated in the disorder. Despite minor disparities across individual samples and study designs, the overall heritability of ADHD is estimated at 70–90%6.Attention deficit hyperactivity disorder (ADHD) is the most commonly diagnosed neurodevelopmental disorder of childhood. It is typically characterised by a persistent pattern of inattention, impulsivity and/or hyperactivity. Longitudinal studies indicate that approximately 66–77% of individuals who experience childhood ADHD continue to experience at least subthreshold symptoms of ADHD that significantly impact adulthood functioning7. Putative disruption of other monoamines such as noradrenaline and serotonin in ADHD has also been proposed. Accordingly, genetic markers mapped to these monoamine pathways have historically been pursued as candidate genes for ADHD. Although a number of replicated findings exist within this candidate gene literature9, a key limitation remains the a priori selection of genes based upon incomplete knowledge of the biology of ADHD, which may ultimately impede the identification of novel risk markers.The efficiency of indirect dopamine agonists in reducing the symptoms of ADHD led to the development of the ‘dopamine hypothesis of ADHD’, which postulated that dysregulated dopamine signalling is central to the pathophysiology of ADHD16, two were family-based analyses18, and three examined the association between quantitative ADHD symptom measures and genetic markers21. A GWAS meta-analysis was also performed in 2010 22. Overall, these previous ADHD–GWAS had limited success in identifying associations. However, preliminary evidence of associations  were identified for genes that function in biological processes relevant to ADHD aetiology. For example, pathway analysis highlights a potential role for potassium channel genes and activation Ras Homologue Family Member A (RhoA) signalling genes, lending further support to hypothesised dysregulation of neurotransmitter release in ADHD23. Additional pathway analysis utilising data arising from five ADHD–GWAS using the Ingenuity and BiNGO tools, showed significant enrichment of genes mapped to a network involved in neurite outgrowth whose targets are modulated by drugs used to treat ADHD24. Most recently, a large meta-analysis of GWAS data arising from 20,183 ADHD cases and 35,191 controls yielded the first 12 independently significant ADHD–GWAS loci25.Genome-wide association studies (GWAS) allow the identification of novel risk variants without prior knowledge of the biology of a trait or disorder. Further, this approach is aligned with the polygenic nature of complex disorders whereby the small role of individual single nucleotide polymorphisms (SNPs) can be considered in aggregate to better understand how genetic susceptibility may arise. To date, 13 ADHD–GWAS have been published. Of these, seven were case–control studiesHere, we conducted a GWAS on a rigorously diagnosed clinical ADHD cohort collected across Australia, England and Ireland in an attempt to clarify further the genetic architecture of ADHD and to potentially identify novel genetic risk factor(s). An additional important purpose of the current study was to contribute to the expansion of the international GWAS community. In this context, GWAS data derived from this study can be combined with those of the international ADHD–GWAS community  for subsequent GWAS analyses.N = 567) children with ADHD of European ancestry were recruited from Australia, the United Kingdom and Ireland. None of the ADHD probands were included in previously published GWAS. All cases met the DSM-IV diagnostic criteria for ADHD at the time of sample collection. ADHD status was determined through parental semi-structured interview and completion of the Conners’ Parent Rating Scale for ADHD, which has demonstrated internal reliability and criterion validity for use in assessing ADHD26. Children with an IQ less than 70 as determined using the Wechsler Intelligence Scale for Children version 4 27 were excluded. One thousand two hundred and ninety-six control (N = 1296) participants were also recruited. All patients and controls were European by descent based on self-report ethnicity of all four grandparents28. All control participants were recruited in Australia and had no self-reported personal history of psychiatric or neurological disorders including ADHD. Written informed consent was provided by the individual in the case of adults, or the primary caregiver or guardian in the case of children/adolescents.Five hundred and sixty seven  and supplemented with an additional ~50,000 SNPs implicated in psychiatric and neurodevelopmental disorders. To avoid spurious GWAS findings, we adopted a stringent quality control (QC) protocol using PLINK 1.9 software at both the individual subject and SNP levelThe following subject-level QC was employed: (1) We initially removed individuals with low-genotyping score by excluding participants with ⩾0.03 of missing data; (2) we performed identity by descent analysis to detect and remove possible sample contaminations, duplications as well as unknown familial relationships (such as relatedness); (3) we applied principal components analysis to identify any potential sources of population stratification and removed outlier subjects; (4) we tested for unusual heterozygosity, which refers to the presence of more or less heterozygous SNPs across the genome than would be expected by the population mean, and removed individuals displaying outlying mean heterozygosity (greater than ±3 SDs from the sample mean); and (5) disparities between recorded and observed sex status were determined through X-chromosome homozygosity were removed.M = 10.27 years, AgeSD = 3.03). Of the ADHD cases 87% were male and 13% were female. The high frequency of male participants in the ADHD cohort is reflective of the sex ratio in clinical populations31. For controls, 1208 participants aged between 7 and 60 years  were carried forward to the final statistical association analysis. Of these participants, 49% were male and 51% were female.Following the above rigorous QC, 480 ADHD probands  remained in the final set for analysis. All children were aged between 5 and 18 years  missing genotype rates between cases and controls were also removed. Finally, SNPs with a minor allele frequency (MAF) < 0.01 were removed leaving 290,265 in the final set taken forward to imputation.Prior to imputation, additional filtering steps were conducted by removing 3516 SNPs with genotyping call rate <95. In addition, genotyped SNPs departing from Hardy–Weinberg (H–W) equilibrium were also excluded. This resulted in the removal of 76 SNPs. A further 1855 SNPs with significantly different 34. PGRS for each subject and disorder were estimated as a sum of risk alleles weighted by their effect size as defined by data arising from the latest publically available GWAS38. For each disorder PGRS were calculated at a 1000 p thresholds (pT) ranging from 0.0005 to 0.5. To find the most predictive pT logistic regression was applied at each threshold using ADHD status as a regressor and Nagelkerke’s R2 and the corresponding p values were estimated. Analysis of PGRS here served to demonstrate that the current ADHD cohort replicated the published and publically available genetic risk profile for ADHD.Polygenic risk scores (PGRS) for the five major psychiatric disorders including ADHD, autism spectrum disorder (ASD), schizophrenia (SCZ), bipolar disorder (BD) and major depressive disorder (MDD) were calculated using PRSice software package2, age × gender, and the top five eigenvectors accounting for population stratification.Association analysis was performed in 480 ADHD cases and 1208 controls using logistic regression analysis implemented in PLINK 1.9. The following covariates were included: age, agepT values ranging from 0.0005 to 0.5. Age, gender, age2, age × gender, along with the top five eigenvectors accounting for population stratification were used as covariates. As expected, only ADHD PGRS was significantly predictive of ADHD case–control status . PGRS for the other four psychiatric disorders did not reach the recommended statistical significance threshold of p = 0.001at any p-threshold , MDD , ASD , SCZ .Here, we utilised PGRS analysis to determine whether our ADHD cohort replicated the published ADHD genetic risk profile. Logistic regression was used to examine the relationship between PGRS for each of the 5 major psychiatric disorders and ADHD case–control status, for 1000 p values (λ = 1.08) relative to expectation under the null distribution  and the distribution is skewed at the extreme tail of low p values, as expected. As can be seen from the Manhattan plot , was observed (p = 3.15E−08). The imputation quality of rs6686722 is very high (r2 = 0.98). Interestingly, and as presented in the regional association plot , which is mapped into a gene desert region . The second SNP is rs61975260 (p = 5.972E−07), which is mapped to the spermatogenesis associated 7 gene (SPATA7). This suggestive association signal is located within a subregion of Chr14 (88788507–89355721) that comprises several genes including SPATA7, ZC3H14, PTPN21, EML5 and TTC8. Some of these genes, such as SPATA7 have been implicated in psychiatric conditions, including schizophrenia.Three other genomic loci also showed strong trends towards association (p = 9.647E−07) which is mapped 58.4 kbp upstream of the Interferon Alpha and Beta Receptor Subunit 2 gene (IFNAR2). This region of the genome is enriched for genes that function in the immune system . Further, five SNPs from the current study were nominally significant in the PGC-iPSYCH meta-analysis (Supplementary Table The third of these SNPs is rs77224013 (p = 7.6E−15). These data demonstrate that the current dataset is enriched for genetic risk for ADHD and thus makes a worthwhile contribution to the international GWAS effort.Here, we report the results of a GWAS of 480 probands with childhood ADHD and 1208 controls. PGRS analysis revealed that our sample replicated the published genetic risk profile for ADHD. In fact, PGRS of ADHD, but not other major psychiatric disorders , explained 3.25% of variance in ADHD case–control status in our cohort (p = 3.15E−08). This SNP is located 22.8 kbp upstream of the Tenascin R (TNR) gene. The TNR gene is a member of the Tenascin family of the neural extracellular matrix glycoproteins and is highly expressed in the central nervous system39. TNR is known to function in biological processes such as neural cell adhesion, neurite outgrowth and modulation of sodium channel function40 that have been implicated in the aetiology of psychiatric disorders. In addition, TNR interacts with Fibronectin 1 (FN1), the latter being involved in cell adhesion and migration processes including embryogenesis. This interaction may modulate the adhesive properties of TNR during synapse maintenance, a process that is suggested as a risk mechanism for complex psychiatric disorders41. Moreover, genetic association studies have implicated the genomic region where TNR is located in several brain disorders including schizophrenia, Alzheimer’s disease, narcolepsy and neurological sleep disorder42. Further, an additional SNP (rs875326) mapped 2.5 kbp at the 3′untranslated region of the TNR was reported to marginally associate with drug response in schizophrenia43. Although indirect, our TNR ADHD–GWAS finding, combined with findings reported for other psychiatric disorders, provides tentative support for a role of TNR in the aetiology of psychiatric conditions.Genome-wide association analysis further revealed a significant association with rs6686722 that survived the stringent GWAS correction for multiple comparisons (DLC1). Recent studies have revealed common genetic susceptibilities to ADHD and smoking behaviour44. Adults with ADHD have higher rates of substance abuse, including higher rates of tobacco smoking44. In this context, a recent GWAS analysis of nicotine dependence reported genome-wide significant association with rs289519 (mapped to DLC1)45. Analysis of rare CNVs across two independent studies of ASD47, identified rare exonic loss within DLC1 as risk variants for the disorder. Specifically, Prasad et al.46 identified one ASD individual who possessed a rare CNV deletion of 22 kbp across a non-specified exonic region of DLC1. Furthermore, Woodbury-Smith et al.47 identified a 25 kbp deletion encompassing exon nine on DLC1 in two unrelated ASD-affected individuals. The above lines of evidence suggest that DLC1 is a candidate gene worthy of further investigation as a potential susceptibility locus for ADHD and its comorbid disorders (either ASD or substance abuse).The second top SNP in our ADHD–GWAS analysis is mapped 300.2 kbp upstream of the Rho GTPase Activating Protein  of the spermatogenesis associated 7 (SPATA7) gene which is expressed in the retina and cerebellum. This gene functions in the localisation of retinitis pigmentosa GTPase regulator interacting protein 1 to the photoreceptor connecting cilium (CC), as well as protein trafficking across the CC. Processing speed is a cognitive function that is compromised in psychiatric conditions including ADHD49. In this context, GWAS analysis49 of information processing speed measured using digit symbol, simple reaction time (RT), and 2 and 4-choice RT showed suggestive evidence of association between DNA variation in SPATA7 and 2-choice RT (p = 2.71E−06).An additional subthreshold association with ADHD was observed for rs61975260 (IFNAR2) and interleukin 10 receptor subunit beta (IL10RB), respectively. Other immune modulator genes such as IL10RB-As1 and IFNAR1 also map to this region. Immune imbalance has been suggested as a predisposing factor for ADHD in genetically susceptible individuals50. Significantly increased transmission of IL-1Ra 4-repeat allele and decreased transmission of 2-repeat allele of a variable number tandem repeat polymorphism to ADHD-affected children was reported by Segman et al.51 Further, immune dysregulation is supported by the finding that ADHD individuals have four times higher concentrations of Interleukins (IL-1 and IL-6) than typically developing children52. This led Verlaet et al.50 to hypothesise that “overproduction of these cytokines could lead to chronic inflammation in brain tissues”. This is consistent with brain anomalies in children with ADHD. For example, Nopoulous et al.53 reported increased frequency of gray-matter heterotropia (ectopic nodules of neurons) and posterior fossa abnormalities in ADHD patients compared to controls. Further, reduced cortical volume associated with reduced surface area and gyrification were also reported in ADHD compared to controls54. However, the correlation between overproduction of cytokines and brain anomalies in ADHD requires further evidence to establish a causal link.Finally, suggestive evidence of association between ADHD and rs77224013 (9.65E−07) was also observed. Rs77224013 is located 58.4 and 94.8 kbp upstream of the immune cytokine receptors interferon alpha and beta receptor subunit 2 , mapped to intron 3 of semaphorin 6D (SEMA6D) gene which sits within 70 kbp genomic region of significant LD. Members of the semaphorin gene family have been implicated as inhibitors or chemo-repellents in axon pathfinding and fasciculation and branching. More recently, Klein et al.55, examined if the genetic risk markers  mediate alteration in brain structure. They observed that rs281323 (in perfect LD with rs281324) is significantly associated with increased risk for ADHD and putamen volume. Further, the SEMA6D rs281323 is strongly associated with the expression level of SEMA6D55. Together, these finding clearly implicate SEMA6D as a susceptibility locus for ADHD.Our sample has provided replication evidence for some of the results arising from the recent and largest ADHD meta-analysis by the PGC-IPSYCH consortiaTNR (rs6686722) is not significant within the much larger PGC-IPSYCH meta-analysis of ADHD (p = 0.07). Notwithstanding these limitations, our ADHD cohort is clearly enriched for genetic risk for ADHD, as evinced by our strong PGRS results for ADHD (but not the other major psychiatric disorders). As such, our study therefore makes an important contribution to the international genetics effort for ADHD.Finally, it is important to emphasise that our study has a number of major limitations. First, the discovery sample is small and has limited statistical power to detect a reliable genome-wide association signal. Further, our leading SNP association for Supplimentry Table 1