Zea mays L.) hybrids with enhanced nitrogen (N) use efficiency. Recent work has described the positive effects of an increased and extended expression of the zmm28 transcription factor (Event DP202216) on maize yield productivity. In this study, we expand on the previous findings studying maize N uptake and utilization in DP202216 transgenic hybrids compared to wild-type (WT) controls. Isotope 15N labeling demonstrates that DP202216 hybrids have an improved N uptake during late-vegetative stages (inducing N storage in lower leaves of the canopy) and, thus, N uptake efficiency (N uptake to applied N ratio) relative to WT. Through both greater N harvest index and reproductive N remobilization, transgenic plants were able to achieve better N utilization efficiency (yield to N uptake ratio). Our findings suggest the DP202216 trait could open new avenues for improving N uptake and utilization efficiencies in maize.Biotechnology has emerged as a valuable tool in the development of maize ( 15N labeling demonstrates that zmm28 transgenic maize have an improved N uptake during late-vegetative stages inducing N storage in lower leaves of the canopy and, thus, N uptake efficiency relative to wild-type maize.Isotope  The optimization of the nutrient resources to support crop development emerges as a prerequisite for sustaining high productivity while minimizing the environmental footprint of chemical fertilizers3. For maize, the largest staple crop in the world4, nitrogen (N) has long been recognized as a critical nutrient determining productivity and is expected to do so with the increasing global food demand6. Therefore, improved N use efficiency  is an important and desirable trait for modern hybrids to increase both the economic and environmental sustainability of maize-based cropping systems9.Today, agriculture faces the unprecedented challenge of providing enough safe and nutritious food to nourish an ever-increasing human population in a world threatened by climate change risks10. Plant NUE can be divided into two physiological mechanisms: (1) the uptake efficiency of N, and (2) the utilization efficiency of N11. During uptake, maize roots can acquire N from the soil as nitrate, ammonium, urea, or other organic N forms. The rate of N uptake is related to root architecture and uptake capacity of the plant12 and is determined by the N demand of the growing sinks13. The most widely adopted indicator of N uptake efficiency in N-fertilized crops is total N absorbed as a proportion of N applied (NUpE). Once incorporated into the plant, N is an essential source for growth as it is used for producing nucleotides, proteins, and numerous cellular components as well as supporting the fixation of carbon dioxide (CO2) via photosynthesis. The use of this N accumulated to produce grain yield is commonly evaluated through N utilization efficiency . Research over decades has resulted in substantial progress in understanding the mechanisms underlying both N uptake and N utilization in crops, including the close link between N uptake, assimilation, and remobilization processes14. However, the complexity and speed of these reactions at field scale make it difficult to validate promising traits for breeding programs15, and NUE improvement promoted by this knowledge is far from being fully exploited6. Future progress depends on the ability to elucidate the G × E interactions of candidate genes on N metabolic processes so that they can subsequently be deployed via breeding and/or biotechnology efforts.Breeding for NUE requires an understanding of physiological traits which can be prioritized as targets for genetic selection, but the genetic-environment (G × E) interactions on these traits bring substantial complexity to this process18. However, the validation of such early experimental successes in representative field environments will further aid plant biotechnology product development. Recently, the increased and extended expression of zmm28, a MADS-box transcription factor, in transgenic ZmGos2-zmm28 maize improved grain yield across a wide variety of field conditions20. Zmm28 is a native MADS-box maize transcription factor, with functions and physiological enhancements described previously in Wu et al.19 and Schussler et al.20. Since there is no previous publication of a single overexpressed transcription factor increasing NUE in field-grown maize, the extent to which these benefits can be transferred to a variety of environments would represent a major breakthrough for developing more productive and environmentally sustainable crops. In this work, we have investigated the impact of a transgenic maize event (hereafter referred to as DP202216) on N uptake and N utilization in maize crops and their agronomic adaptation to two levels of N supply in the field. These evaluations aim to highlight the connection between N dynamics and productivity in this novel transgenic event and to propose a mechanistic understanding of NUE improvements in maize.The introduction and regulation of N absorption, transport, metabolism, and signaling-related genes by genetic transformation have successfully improved the efficiency of N utilization in plants15N labeling to determine if DP202216 plays a direct role in N absorption and at which stage the corresponding effects were expressed as affected by N availability. Compared with the wild-type (WT) controls, the most prominent phenotypic trait of DP202216 plants was a higher rate of 15N uptake during crop growth stages up to flowering . This less pronounced effect of DP202216 as the crop advanced in age correlates with the lack of differences (against the WT controls) in 15N uptake rates during the grain filling period  was characterized at three canopy levels during the same V11 to R1 period. Differences in SLN between transgenic and WT controls were not consistent in the upper and middle sections of the canopy during stages prior to flowering. In contrast, the SLN in the lower leaves of the canopy  was often greater in DP202216 hybrids Fig. . This im15N allocation among fractions). It appears DP202216 hybrids achieve the critical period (time around flowering) with an improved source activity  until physiological maturity (R6)) revealed that transgenic plants had in general a higher N allocation to the ears relative to their WT controls Fig. . This waity Fig. , which pPost-flowering N uptake was similar between WT controls and DP202216 plants across most of the experimental conditions evaluated  was partially enhanced by DP202216 during pre-flowering stages . In addition, the larger response in NUtE to N rates in 2020 trials might be attributed to a greater N immobilization and lower soil N mineralization rates derived from wheat residues, although no substantial differences in the soil inorganic N pool were found at planting  than in the WT (V6), via control of a moderately constitutive GOS2 promoter19. We hypothesize that this earlier and increased expression of zmm28 triggers an early and enhanced uptake of N in the transgenic plants relative to WT, in part due to a differential source N demand. Evidence from this study reveals the DP202216 physiological response of altering the early vegetative N acquisition more efficiently converts N assimilates into grain yield  is proof that such an assumption tended to be satisfied in our experiments. Nevertheless, as a whole, both experimental sites concur that the altered and extended expression of zmm28 appears to confer major improvements in N uptake and storage during late-vegetative stages. More importantly, the physiological characterization of DP202216 represents a sustainable approach for recovering N from fertilizers in maize crops, especially for N applied during early vegetative stages.Members of the MADS-box gene family are known to be involved in vegetative growth and development in plants32), which suggests an ‘extra N’ pool presumably available for post-anthesis utilization. Likewise, Reyes Ponce33 showed that modern hybrids accumulate more N per unit leaf area, especially in leaves below the ear node, compared to older genotypes of the US Corn Belt. While it seems that this strategy has been a major physiological basis of genetic improvement over decades, our study demonstrates that the DP202216 trait may present novel genetic variation for improvement for this N storage and retrieval strategy in maize. This improved N status before flowering not only increases the amount of N available for translocation but also supports the formation of ear size and kernel set34. It is therefore expected that, especially under suboptimal N conditions, DP202216 can enhance the establishment of reproductive sink size, compared to WT controls. This is consistent with results from previous studies demonstrating the improved productivity of ZmGos2-zmm28 transgenic hybrids over multiple limited-N environments20.Increases in vegetative N assimilation resulted in a higher specific N content in the lower leaves of the canopy  in transgenic plants. Altogether, these results suggest that altered zmm28 expression plays a major role in N assimilation and translocation processes  or post-flowering N . Moreover, the distribution of 15N during the post-flowering phase and the N harvest index at maturity showed a strong preferential allocation of N to the ear in DP202216 hybrids, relative to their WT controls. This strategy allowed DP202216 hybrids to achieve a similar N concentration in grains at maturity, compared to their WT controls. This DP022216 maintenance of grain N should provide support for more effective use of plant N to produce grain in breeding programs targeting NUtE39.The ability of plants to absorb and store any excess of mineral N during vegetative growth and then translocate it to the grains has been previously identified as a good marker of productivity in maizeses Fig. . While t9. This trait has been poorly exploited to date but, at the same time, retains a high potential for future improvement. DP202216 transgenic plants have greater yields under sub-optimal N conditions and, perhaps even more importantly, may lower fertilizer requirements to optimize productivity at high N. These findings suggest that DP202216 hybrids could provide a path toward more sustainable maize production worldwide. Based on our analysis, DP202216 (1) augments pre-flowering N assimilation (better N uptake efficiency), and (2) improves post-flowering N use (better N utilization efficiency)  for maize, targeting as one of the future research priorities further crop breeding improvements on root traits for more effective use of N. In line with this concept, modulating the expression of zmm28 opens a new potential avenue for maize breeding programs around the world to increase the effective use of N under varying environmental conditions.Nitrogen use efficiency has been a challenging, and often overlooked, target breeding trait in many crops because of its complexity and large interaction with soil and environmental factorscy) Fig. . This, hThe field experiments were conducted during the 2019 and 2020 growing seasons at the Corteva Agriscience research station in York NE, USA . Soils consist of a silty clay loam  and analyses were conducted at pre-planting to characterize initial conditions  and non-irrigated (2020) conditions and the previous crops were soybean (2019) and wheat (2020). In 2019, irrigation was applied with a linear-move sprinkler system based on crop requirements to eliminate any significant plant water deficits. Seeds were planted on 14 May (2019) and 1 May (2020) at a target density of 82,000 (2019) and 70,000 (2020) plants ha19. Two N rates 0 and 225 kg ha−1 (N0 and N225) were applied two days after planting using 28% urea-ammonium nitrate (UAN) liquid N fertilizer as the source. Treatments were arranged in a split-plot design with three repetitions, with N supply as whole-plot and hybrids as subplot. Each experimental unit was an eight-row plot with a size of 5 m long and 6 m wide.Hybrids PH11V8W2Z and P1421 were selected to represent a sample of two elite Corteva Agriscience maize hybrids having similar relative maturity (114 days). Transformation and backcrossing procedures used to produce the DP202216 transgenic event and wild type (WT) hybrids are described in Wu et al.15N labeling was used to determine N uptake and allocation following the methodology employed in de Oliveira Silva et al.43 and Fernandez et al.44. In 2019, labeling was performed at three key developmental stages:45 10 days before flowering (V17), flowering (R1), and mid-grain-filling . In 2020, an additional earlier vegetative measurement was performed at the 11th expanded leaf (V11) stage in addition to samplings at R1, R3, and physiological maturity (R6). Briefly, at each stage, labeled fertilizer Ca(NO3)2 (10.15% 15N) was applied to the soil at the base of a five-plant microplot in each experimental unit at 0.7 g plant−1 using plastic syringes. Five days after labeling, the three center plants of each microplot were cut at the ground level and separated into leaves (green leaf blades), stem , and ears . The period between labeling and sampling was extended to 15 days for the sampling at R6, to account for the lower rates of N absorption at this stage and to ensure that the harvest was performed at physiological maturity in all entries. For all data reported, sampling stages for the labeling procedure refer to stages at which the biomass collection was performed. Samples were dried at 65 ˚C until constant weight and prepared for isotopic analyses.Short-term isotope Specific leaf N  in the canopy profile was determined at each sampling time  by sampling twelve leaf disks of 15.89 mm diameter in two (only at V11) or three canopy levels. At V11, the upper section included the three uppermost-expanded leaves while the lower section represented the remainder of plant green leaves. For samplings when the ear was visible, leaves were classified as from the middle section of the canopy (leaf blades from the ear node 0 and ±1 node positions), lower section (leaves from the −2 node and below), and upper (leaves from the +2 node and above). Samples were dried at 65 ˚C until constant weight, weighed on a 0.01 g precision scale, and prepared for N analyses.At harvest maturity, grain dry matter was collected for N use efficiency parameters on the four center rows that remained unaltered during the season. Grains were then dried at 65 ˚C until constant weight and prepared for laboratory N analyses.−2).All grain and leaf disk materials were ground through a 0.25-mm sieve, and N concentration was determined by combustion on an automated elemental analyzer  in two technical replicates. N concentration was multiplied by the dry weight of the fraction to obtain N content in grains and leaf disks. SLN was obtained as the ratio between leaf disk N content and its known area  to calculate tissue N content .For isotopic analysis, tissue samples were ground through a 0.10 mm sieve. Ground samples were weighed (3 mg) and packed in tin capsules with an A&D microbalance (BM-22) with an error index of 0.001 mg. Capsules were introduced into an automated elemental analyzer (PyroCube – Elementar Americas) coupled to an isotope ratio mass spectrometer . Nitrogen concentration of tissue was obtained along with the isotopic composition of δn = 2 independent years) and Block (n = 3 independent replicates) to recognize the experimental structure of the data and imbalances in the sampled growth stages across years. Inferences were based on four chains of the MCMC algorithm, with 4000 iterations and a warmup period of 2000 draws for the MCMC calibration on each. Sampling convergence of the chains was assessed using trace plots and the Gelman-Rubin diagnostics46. Medians of the posterior distributions were reported due to their robustness properties. The Bayesian modeling approach allowed determining the expected value of the predicted dynamics with a probabilistic component by means of their posterior distribution of samples47. An advantage of this approach is that it does not require a fixed measure of significance  but instead can provide an exact quantification of the likelihood for differences between treatments47. Therefore, the probabilities for differences between DP202216 and WT plants were estimated based on the posterior distribution of pairwise differences [derived from the samples produced by the Markov chain Monte Carlo (MCMC) algorithm] at each condition. Probabilities for differences were reported in figures when above 65% (weak evidence), 75% (moderate evidence), and 90% (strong evidence). In addition, the group-level parameters for the varying-intercepts for year and varying-slopes for year by N treatments, hybrids, and transgenic events were examined using their 90% credible intervals to determine whether each variable varied significantly across years (Supplementary Fig. Bayesian ‘mixed-effects’ models were fitted to the data to quantify the likelihood for differences between treatments in the experiment. Detailed information on the model adjustments, choice of priors, and Markov chain Monte Carlo settings are provided in the Supplementary Information (Supplementary Note Further information on research design is available in the Supplementary informationReporting Summary


However, these trials have failed to induce robust drug-free tolerance and/or significantly reduce the level of immunosuppression needed to prevent solid organ transplant (SOTx) rejection. Improving Treg therapy effectiveness may require increasing Treg persistence or orchestrating Treg migration to secondary lymphatic tissues or places of inflammation. In this review, we describe current clinical Treg manufacturing methods used for clinical trials. We also highlight current strategies being implemented to improve delivered Treg ACT persistence and migration in preclinical studies.Based on successes in preclinical animal transplant models, adoptive cell therapy (ACT) with regulatory T cells (Tregs) is a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection. Extensive work has been done in optimizing the best approach to manufacture Treg cell products for testing in transplant recipients. Collectively, clinical evaluations have demonstrated that large numbers of Tregs can be expanded  These mechanisms include the production of anti-inflammatory cytokines such as TGF-β (transforming growth factor beta) and IL-10 and the expression of membrane-bound molecules such as CTLA-4 (cytotoxic T cell-associated antigen), LAG-3 (lymphocyte activation gene 3), TIGIT (T cell immune receptor with Ig and ITIM domains), and CD39 (ectonucleoside triphosphate diphosphohydrolase 1).+CD25+ Tregs in inhibiting the development of autoimmune disease.8 It was then demonstrated that naturally occurring CD4+CD25+ Treg populations could be expanded ex vivo to treat autoimmune diseases in mice by suppressing and modulating effector T cells.9 Thereafter, using adoptively transferred CD4+CD25+ Tregs in a series of preclinical sentinel model studies in solid organ transplantation (SOTx) and graft-versus-host disease (GvHD) after allogeneic hematopoietic cell transplantation (AlloHCT) further supported Treg therapeutic potential to induce immunological tolerance to non-self-antigens to prevent allograft rejection and GvHD. GvHD is a life-threatening complication of AlloHCT, which is used to treat hematological and non-hematological diseases.10 GvHD is an alloreactive donor T cell-mediated response driven immunologically by differences between donor and recipient. In seminal studies completed by Taylor et al., they demonstrated that depletion of murine CD4+CD25+ cells from the BM donor increased GvHD, which was then reduced upon infusion of fresh donor CD4+CD25+ cells.11 Since then, utilizing Treg to prevent/delay GvHD has been routinely demonstrated in preclinical murine models.12–14Interest in harnessing the suppressive capacity of Tregs for immunotherapy originates from early studies which demonstrated the importance of CD4ex vivo expanded CD4+CD25+ Tregs in wild-type mice.15 Additionally, studies using humanized murine models demonstrated prolonged allograft survival in pancreatic islet and skin Tx following adoptive transfer of huTreg.16–18 What would further translate to huTregs was the finding that murine Treg function was more potent than CD4+CD25+ Treg when sorted based on low expression of CD127.19 Due to their immunological similarity to humans, the use of non-human primate models (NHP) has also been important in evaluating adoptive cell therapy (ACT) with Treg in SOTx. Ex vivo expanded Treg in NHP models are well characterized and adoptive transfer has been reported to suppress renal allograft rejection and prolong survival.20–22The therapeutic potential of Tregs to support allograft survival after SOTx has also been demonstrated using preclinical models of skin and heart Tx. For example, heart allograft survival was prolonged following transfer of 23–25 These clinical studies have also shown that large numbers of CD4+CD25+ Treg can be expanded in the presence of high doses of IL-2 ex vivo and infused safely.26–34 The number of clinical trials utilizing Treg cell therapy in transplantation and other fields are expected to increase. In this review, we describe current clinical GMP methods used to isolate and expand Tregs ex vivo for clinical trials in solid organ Tx and treatment/prevention of GvHD. We then discuss in vivo and ex vivo strategies being used to improve adoptive Treg cell therapy persistence and migration to increase the efficacy of Treg therapy in preclinical studies. While numerous preclinical publications exist describing manufacturing methods of Treg cell products, few of these publications provide a correlation with completed clinical studies in SOTx. Therefore, to improve and enhance Treg cell therapy in the clinic, it is important to highlight manufacturing protocols specific to completed and recruiting clinical trials when possible. In this review, we aim to briefly introduce general concepts around Treg and then highlight clinical manufacturing methods used to generate Treg cell products used in SOTx referencing published clinical trial protocols, manuscripts, and preclinical protocol development. It is also noteworthy that even in preclinical animal transplant models Treg ACT has only provided prolonged SOTx survival. Thus, even at the preclinical level, Treg ACT is far from optimized. All clinical trials to date unfortunately have not reduced the levels of immunosuppressive drugs needed to prevent SOTx rejection.33 Thus, we also provide examples of ways investigators are working to improve Treg ACT and move the needle toward the field’s goal of maintenance of normal graft function without the use of immunosuppression, also known as drug-free tolerance or operational tolerance (OT).Recently, clinical studies have been completed or underway assessing Treg to improve outcomes after SOTx or with AlloHCT to prevent GVHD.24 For example, calcineurin inhibitors, antiproliferative agents, and mTOR  inhibitors can control the immune response early post-transplant, but have detrimental side effects such as cardiovascular diseases, kidney failure, and susceptibility to opportunistic infections.24 Efforts to improve long-term outcomes and 10-year survival remain unsuccessful due to the failure of multi-drug immunosuppression to address chronic rejection despite their toxic side effects.35 Other therapeutic strategies such as co-stimulation-blockade (CoSB) or monoclonal antibody (Ab) therapy to target cytokines and other co-stimulatory molecules have only had limited success at reducing the use of immunosuppressive drugs.21 Therefore, utilizing Tregs or other regulatory immune cells as a potential immune cell therapy may be a promising alternative to reduce the use of immunosuppressive drugs and even induce allograft tolerance based on data obtained from various preclinical murine and NHP studies.Treg ACT has developed due to the need for alternative therapeutic agents to limit the need for immunosuppression (IS) after SOTx, and ideally support the routine induction and maintenance of immune transplant tolerance in recipients of allogeneic materials. SOTx remains the only effective lasting treatment for end-stage organ diseases. Strategies to increase short-term outcomes after SOTx have relied on better patient donor selection and improved immunosuppressive regimens.10 Thus, alternative therapies such as Tregs are being investigated to prevent the development of GVHD after AlloHCT. If prophylaxes therapies can successfully prevent GVHD, this could also expand the use of AlloHCT beyond cancer treatment and for routine use as a means to induce transplant tolerance after SOTx.36–38Despite new prophylaxis strategies, GVHD is a common side effect of AlloHCT where alloreactive T cells destroy host tissues. Current treatments for GVHD involve nonspecific multi-drug immunosuppression, particularly corticosteroids, that often leads to morbidity and mortality due to cancer relapse or secondary infection.39 In contrast to tTreg cells that originate from the thymus, pTreg develop from conventional CD4+ T cells in the periphery after antigen encounter along with TGF-β, especially in the gut.23 Similarly, investigators have “induced” Foxp3+ Treg (iTreg) in vitro from CD4+ Foxp3- populations by expanding CD4+CD25− cells in the presence of IL-2, rapamycin, and TGF-β.40 To date, the majority of the Tregs used in clinical trials for SOTx, however, were isolated from autologous peripheral blood mononuclear cells (PBMC) or Umbilical cord blood (UCB). Unfortunately, there are no consistently reliable specific markers to distinguish between tTreg and pTreg in mice and humans, thus making specific origination of Treg isolated from these sources imprecise. There is evidence to support the use of allogenic Tregs in humans; however, that is beyond the scope of this review.30 Therefore, this review will focus discussions on the detailed manufacturing of autologous Tregs from human subjects.The donor source of Tregs is important when designing and describing Treg cell therapy products. Tregs are typically classified as thymic-derived Tregs (tTreg) and peripherally-derived Tregs (pTreg).+ T cells in healthy adults. Thus, Treg isolation and ex vivo expansion is required to reach the numbers of Treg predicted in animal studies to be therapeutic after adoptive transfer.42 As introduced above, the Tregs for clinical trials have been most commonly isolated from PBMC or UCB.43–45 A recent novel strategy used pediatric thymuses which are routinely discarded during pediatric heart transplantation as a Treg source.47 Tregs develop in the thymus and thus the thymus represents an attractive alternative source to isolate Tregs for cellular therapies. In a study involving 11 different donors, roughly 12% of the CD4+ cells were CD3+ CD4+ CD8− CD25+ CD127− with 85% of these cells expressing the FoxP3.46 This approach is an exciting alternative to isolate large numbers of Tregs; however, it is so far limited to pediatric heart transplant recipients or when third-party Tregs are utilized. The clinical feasibility to isolate and expand functional tTregs is currently being explored in an ongoing clinical trial  where tTregs isolated from the thymus are being tested for their ability to prevent rejection after pediatric heart transplantation.46Human Tregs are relatively rare in the PBMCs and comprise only 2–10% of peripheral CD4ex vivo expansion.48 Although not yet tested in clinical trials, the use of Granulocyte Colony-Stimulating Factor (G-CSF)-mobilized peripheral blood stem cells (G-PBSC) may be another potential source that could be used to isolate Tregs for patients receiving HCT.50 In this case, Treg isolation and expansion would be performed after CD34 selection utilizing the typically discarded CD34 negative fraction. Application of G-CSF significantly increased Treg yield while preserving suppressive function and phenotype.50 To date, only PBMC and UBC have been clinically tested in SOTx and AlloHCT.Treg isolation from leukapheresis has also been evaluated as a potential alternative source and offers increased starting cell density to improve Treg yields for subsequent 52 Of these, common isolation techniques include fluorescence-activated cell sorting (FACS), direct or untouched magnetic cell separation, or a combination of both. In the case of magnetic cell separation, these systems utilize magnetic microbeads to bind cells of interest  or to selectively deplete unwanted cells (“negative” selection). Additionally, regardless of the methodology or source, high surface expression of CD25 remains the primary marker used to distinguish human Tregs from non-target cells along with low expression of CD127.53 Unfortunately, there is no singular surface marker for Tregs that distinguish them from other immune cells, and Foxp3 is an intracellular transcription factor that cannot be utilized for the isolation of live cells. Therefore, multiple surface parameters are needed to isolate Treg from blood products. Many groups have utilized cell sorting based on parameters of CD3+CD4+CD25+CD127low.54–56 While this process yields highly pure Tregs, it is less efficient as run times can take many hours depending on the purity and cell concentration of the starting product. Additionally, there can be considerable cell loss with traditional sorting via FACS or positive and negative bead selection. Such loss impacts the overall product yield and increases the need for robust ex vivo expansion. These shortcomings have driven advancements that have been made in streamlining GMP-compliant protocols to generate clinical grade Tregs.There has been rapid progress in the development of Good Manufacturing Practice (GMP) grade protocols to isolate and expand large numbers, and recent reviews have detailed the strategies used to isolate Tregs from various blood products for use in clinical trials.57 These systems utilize magnetic microbeads to enrich for Tregs or to selectively deplete non-Treg . Like flow cytometric cell sorting, this platform allows for multiparameter selection, and numerous protocols specifically for Tregs have been developed and established. Currently, the approaches most cited are CD25 enrichment with or without prior selective depletion of CD8+ and/or CD19+ cells.58–63One of the most notable advancements in this field has been with Miltenyi’s closed system magnetic cell separators CliniMACS Prodigy and CliniMACS Plus.+CD25+ CD127low selection using the closed cartridge, low cost MACSQuant® Tyto® . This strategy works by using a clinical GMP grade CD25 reagent (like the CD25 Enrichment microbeads) that is also biotinylated which allows the user to fluorescently label CD25 positive cells with phycoerythrin (PE) and perform purification using the MACSQuant Tyto sorter following the CD25 enrichment step. In addition, other fluorescent markers in addition to CD25, such as CD4, CD127, or CD45RA, can be used for subsequent sorting if desired. This approach significantly reduces the processing time while also preserving the yield and purity of the product. If optimized, this type of processing approach could limit product variability and increase reliability. Investigators have demonstrated the feasibility of using the CliniMACS for CD4+CD25+ Treg enrichment; however, in this case, the purity was suboptimal.65 In theory, subsequent sorting for CD4+CD25+CD127low could significantly improve the purity and allow for robust ex vivo expansions of pure Treg populations.Another notable strategy that is becoming more common for Treg isolation allowing for a high purity Treg product is CD25 enrichment utilizing Miltenyi’s CliniMACS systems as described above in combination with a purification based on CD4ex vivo Treg expansion. There have been numerous different reagents and approaches used for generating clinical Treg products for the use in kidney transplantation . There is variation among protocols, however. IL-2 is typically added at day zero at 1000 U/mL and replenished as needed.52 There is no standard IL-2 concentration; however, a recent review has provided a detailed summary of reported concentrations (300–1000 U/mL) used for expansion in clinical trials.44 The use of mTOR inhibitor rapamycin, an immunosuppressive drug used to prevent graft rejection, can also be used in clinical grade Treg expansion protocols.66 Rapamycin selectively supports the ex vivo expansion of CD4+CD25+FoxP3+ Tregs while limiting the proliferation of contaminating non-Treg. Likewise, rapamycin promotes the stability and functional capacity of expanded Treg to suppress the proliferation of both autologous and allogenic CD4+ and CD8+ T cells in vitro.67 This was also confirmed in preclinical rodent studies demonstrating that Tregs could be expanded ex vivo in the presence of rapamycin, and rapamycin delivery with Treg ACT can support tolerance to SOTx in rodents.68–71 Therefore, in cases with less than ideal Treg purity following isolation, it may be beneficial to expand Tregs in the presence of rapamycin to block proliferation of unwanted CD4+CD25− cells.Human Tregs can be expanded in several ways all of which are dependent on IL-2 for proliferation and maintenance of the Treg phenotype  mice.78 Additional observations in rodents and humans also demonstrate the importance of CD28 signaling for Treg proliferation.80–83 As such, most clinical trials have expanded nonspecific polyclonal Tregs (polyTregs) using CD3/CD28 expander beads which provide both TCR and CD28 co-stimulation. There are several published GMP compatible methods used to ex vivo expand Tregs for cell therapy which have been previously described.52 The optimal way to stimulate Tregs remains unclear; however, there are two main approaches to ex vivo expand polyTregs for clinical trials. The most common approach to expand human polyTregs is to use polyclonal stimulation with anti-CD3 and anti-CD28 coated microbeads.85 GMP grade anti-CD3 and anti-CD28-coated nanoparticles are available for clinical use from Miltenyi Biotec (ExpAct™ Treg kit) and Invitrogen (CTS Dynabeads™ CD3/CD28). These beads are typically added at the start of culture and are expanded over a period of 14–36 days. It is unclear if multiple restimulations are required. Of those reported, fold expansions have been variable and can range from 100 to 2000 with IL-2 alone.63 Tregs can also be activated using artificial antigen-presenting cells (aAPCs). These cells have been developed to replace natural antigen-presenting cells (APCs) which mediate T cell effector function. Using lentiviral vector technology, aAPCs can be generated to more closely replicate the features of DCs that deliver CD28 co-stimulation.86 For example, aAPCs expressing CD64 and CD86 were used to expand human UCB-derived Tregs.27 These cells are advantageous because they do not require removal post expansion as cells are lethally irritated prior to use. K562 cells expressing CD86 and CD64 that are loaded with soluble anti-CD3 have been used in clinical trials.40 It has been reported that these aAPCs are highly effective at expanding Treg.60 For example, Brunstein et al. were able to achieve a 10,000-fold expansion of Tregs from UBC in 2 weeks using the aAPCs that expressed CD64 and CD86. In the presence of rapamycin, 3000-fold expansion was achieved.87T cell receptor (TCR) engagement is required for Treg cell differentiation and the induction of Foxp3.88 Generally, darTreg are generated by isolating recipient Tregs and culturing them with donor alloantigen-expressing APCs from the transplant donor, such as monocyte-derived DC and B cells.90 The global Treg pool contains a small fraction (10–20%) of darTregs, and generating them in large quantities has been a barrier for successful implementation.91–94 Despite manufacturing challenges due to low frequency of darTregs, experimental models of transplantation have demonstrated that donor antigen-specific Tregs (darTregs) are more potent than polyTregs in promoting allograft survival.95 Therefore, due to increased specificity for alloreactive effector T cells and increased potency, it is likely that fewer darTreg are required to induce tolerance compared to polyTregs.In addition to manufacturing polyclonal Tregs through the process discussed above, generating donor-antigen-specific/reactive Tregs (darTreg) has been gaining traction in recent attempts to induce tolerance after SOTx. In comparison to polyTregs, darTregs are expanded in the presence of donor cells and exhibit donor specificity after alloantigen exposure by selectively targeting alloreactive effector T cells that are detrimental to the allograft.85 Patients who underwent living donor liver Tx (LDLT) were lymphodepleted 8–10 days post liver Tx and received a single infusion of the darTreg-enriched product. Of the 10 patients infused, 7 met the trial-defined endpoint of tolerance and were successfully weaned off immunosuppression within the study period.85 Overall, infusions of 100–300 million cells were well tolerated without significant adverse event and the mean number of Tregs (CD4+CD25+FoxP3+) infused were 24.8% of the CD4+cells, which is a low Treg concentration compared to other clinical studies (85 The investigators measured suppressive function of the expanded cells in vitro using the mixed lymphocyte reaction (MLR) and inhibited recipient T cell proliferation by donor antigen stimulation. Yet, it is unclear what cell populations in the darTreg-enriched product were indeed responsible for tolerance induction in vivo and suppressive function in vitro. Nonetheless, these were impressive and exciting studies. As the Tregs made up a very small fraction of the total product, this would suggest that lower numbers of darTreg are needed to induce tolerance or at the least weaning of immunosuppressive regimens. Additionally, it suggests that possibly other regulatory cells in addition to darTregs may be beneficial, contributing to tolerance induction or reduction of immunosuppression. Todo et al.’s study has facilitated the development of protocols to expand antigen-specific or darTregs from humans.97 Several clinical trials are testing darTregs in SOTx . Tregs were assessed longitudinally in 16 patients at pre-Tx, 6 months post-Tx, and 2 years post-Tx to assess darTreg and conventional T cell reactivity to donor antigen. Overall, authors showed reduced proliferation and selective reduction of donor reactivity in all T cells after liver Tx starting 6 months post liver Tx persisting until 2 years after transplant.94 The differences in epigenetic regulation, transcription, and donor reactivity of darTregs in liver Tx highlight important insights to the difficulties of generating successful Treg products and underpin challenges faced when manufacturing enough darTregs. Future longitudinal studies should investigate both darTreg and polyTreg transcriptional changes and donor reactivity pre- and post-Tx when possible, which may offer solutions to improve clinical manufacturing of Treg products that are functionally potent and persist after infusion.Recently, results of the Artemis Phase I/II clinical trial (NCT02474199) have been published. The objective of this study was to determine safety and efficacy of a single dose of darTreg to facilitate reduction of immunosuppression in patients 2–7 years post liver Tx. Nine participants initiated immunosuppression reduction and were eligible for a single darTreg infusion; however, only 5 of the 9 products manufactured met release criteria.44 However, clinical trials attempting to limit recipient anti-AlloAg responses have yet failed to translate to reduced use of immunosuppressants after SOTx.33 One factor potentially leading to the limited efficacy of Treg ACT may be due to their reduced persistence in vivo. Results from clinical trials demonstrate that Tregs expanded in culture with high doses of IL-2 decrease rapidly in vivo and methods of tracking infused Tregs are limited.99 Thus, alternative methods such as administering low-dose IL-2 (ld-IL-2) to increase their persistence in vivo are being investigated. As discussed in earlier sections, Tregs require the growth factor IL-2 for proliferation, survival, and functional activity.43 Additionally, Treg ACT requires extensive ex vivo expansion with high doses of IL-2 which may affect their stability, phenotype, and survival once transferred in vivo. It is thought that using IL-2 at low doses in vivo in combination with adoptive Treg cell therapy could promote Treg survival and improve therapeutic efficacy. Use of IL-2 in combination with adoptive Treg cell therapy has not been approved and clinical trials are underway investigating safety, dosing, and efficacy of low-dose IL-2 and Treg infusion for GvHD and T1D.99–104 More recently, it has also been clinically tested in stable liver transplant recipients patients 2–6 years post-transplant.105 Unfortunately, it is clear that using ld-IL-2 to increase Treg persistence is not without its risks, as there is clear evidence for activation of effector cells such as cytotoxic CD8+ T cells and NK cells in the recipient.105 In the context of SOTx, recently published results from a clinical trial (NCT02949492) that used ld-IL-2 to expand endogenous Tregs in LTx recipients reported rejection episodes in 4 of 5 participants who initiated immunosuppression withdrawal.105 These episodes were mild to moderate and tended to resolve after initiating immunosuppression. One participant, however, developed T cell mediated rejection that was unresponsive to steroids and the individual required re-transplantation.105 Due to these negative outcomes, it is very unlikely that ld-IL-2 can be easily developed as an adjuvant to Treg ACT supporting tolerance induction. As discussed below, however, several groups are developing IL-2 orthologs to target transferred Tregs more precisely without impact on other immune cells responsive to the natural endogenous cytokine.106Multiple clinical trials have demonstrated the safety and tolerability of adoptive Treg cell therapy after AlloHCT or SOTx.in vivo may be a promising new approach to improve clinical outcomes. In a recent proof-of-principle study, it was demonstrated that poly lactic-co-glycolic acid (PLGA) microparticles (MP) could generate a chemokine gradient of C-C-Motif Chemokine 22 (CCL22) to recruit CCR4-expressing Tregs in vivo.107 These CCL22 MP prolonged hindlimb allograft survival and promoted donor-specific tolerance.108 Additionally, synthetic human CCL22 MP induced human Treg migration in vitro demonstrating that this technology has the potential to improve Treg cell therapy efficacy by delivering chemokines, like CCL22, that directs them to the graft, or by providing other stimuli  that support their survival or functions in the graft.The rapid loss of infused Treg may also suggest that they undergo generalized and unfocused migration into the tissues or are lost in the absence of signals supporting their survival and required functions. As such, harnessing mechanisms that can orchestrate Treg ACT recruitment and function 109 Currently, the use of low dose IL-2 in combination with Treg cell therapy is being evaluated to promote Treg persistence and endogenous Treg expansion.99 A limitation to this is that this approach is nonspecific and can have systemic off target effects. Additionally, a recently published study found that administration of low-dose IL-2 in liver Tx patients did increase the number of Tregs but failed to induce transplantation tolerance.105 An alternative to this would be to provide local extended release of these cytokines and drugs at the graft site to promote tolerance. In one study, Treg inducing microparticles (TRI-MP) were engineered to release TGF-β1, IL-2, and rapamycin to induce differentiation from naïve T cells.110 Using a rat hindlimb vascular composite allotransplantation (VCA) model, TRI-MP prolonged allograft survival without the use of immunosuppression. This system also enriched Treg and reduced inflammatory Th1 populations. The studies above suggest that microparticles can be engineered to support Treg localization, survival, and persistence after adoptive transfer to prevent allograft rejection and promote tolerance. Microparticles delivering supportive or stimulatory signals are an attractive potential method to add value to Treg ACT by boosting the efficacy of a product that, to date, has shown minimum impact.The use of IL-2, TGF-β, and rapamycin has been shown to favor Treg suppressive function and development.+ and CD8+ cytotoxic effectors as well as NK cells making it difficult for Tregs to persist. Therefore, cytokine-cytokine receptor engineering such as the IL-2 cytokine receptor complex on Tregs may be an innovative approach to induce allograft tolerance and Treg survival without supporting effector T cell and NK cells that may contribute to GvHD response.As described earlier, low dose IL-2 in combination with adoptive Treg transfer is an approach currently being evaluated in clinical trials to promote Treg survival and persistence. However, low dose IL-2 may support an inflammatory environment consisting of activated CD4+GFP+-BALB/c Tregs (green fluorescent protein under the control of the mouse Foxp3 promotor) were transduced to express an orthogonal IL-2 (oIL-2) receptor β chain (oIL-2Rβ). Transduced Tregs were adoptively transferred along with C57BL/6 bone marrow cells (BMC) into wild type BALB/c recipients. Recipients that received oIL-2Rβ Tregs and treated with oIL-2 had significantly improved engraftment and increased percentage of FoxP3+GFP+ in CD4+ T cells without increasing CD8+ T cells. In addition, those same recipients had improved acceptance of heart allografts from C57BL/6 donor mice demonstrating donor-specific tolerance.111In a murine mixed chimerism model, FoxP3in vivo and retained their ability to migrate to the gastrointestinal tract and lymph nodes.112 These preclinical data support the potential of using cytokine-cytokine receptor engineering to improve adoptive Treg therapy efficacy for the treatment of Tx and GvHD. These data demonstrate the specificity of cytokine-cytokine receptor interactions avoiding interactions with the natural cytokine response, thus reducing potentially harmful off target expansion of effectors, improving allograft tolerance.Another study used a murine major mismatch acute GvHD model to investigate the suppressive function of this Treg orthogonal IL-2/IL-2 receptor complex. Briefly, irradiated BALB/c recipients received T cell-depleted BMC from C57Bl/6 mice with or without fresh C57Bl/6 FoxP3 Treg cells or with oIL-2Rβ Treg. On Day 2, C57Bl/6 T responder cells were injected to induce GvHD. Increased survival was observed in recipients that received oIL-2Rβ+ Tregs with oIL-2. Additionally, oIL-2 selectively expanded oIL-2Rβ Treg ex vivo expansion for clinical use have been outlined here and in other reviews.113 However, it is clear from this discussion that there is still work required to get to the generation of a functionally therapeutic product for clinical use that matches the effectiveness observed in preclinical studies. Clinical trials using Treg-based therapeutics for the prevention of GvHD after HCT or rejection after SOTx have been described and consistently shown that infusion of ex vivo expanded Tregs is well tolerated and safe.52 Technical advancements in clinical manufacturing of Treg cell products for ACT have improved the isolation of these cells, with many coming online that allow isolation in closed systems as part of GMP processes. This includes positive selection for CD25 followed by subsequent sorting for CD4+CD127loCD25hi using Miltenyi’s closed system magnetic cell separator (CliniMACS Plus) and sorter (MACSQuant Tyto)  . Variatisues see .Figure


Large structural chromosomal deletions and duplications, referred to as copy number variants (CNVs), play a role in the pathogenesis of neurodevelopmental disorders (NDDs) through effects on gene dosage. This review focuses on our current understanding of genomic disorders that arise from large structural chromosome rearrangements in patients with NDDs, as well as difficulties in overlap of clinical presentation and molecular diagnosis. We discuss the implications of epigenetics, specifically DNA methylation (DNAm), in NDDs and genomic disorders, and consider the implications and clinical impact of copy number and genomic DNAm testing in patients with suspected genetic NDDs. We summarize evidence of global methylation episignatures in CNV-associated disorders that can be used in the diagnostic pathway and may provide insights into the molecular pathogenesis of genomic disorders. Finally, we discuss the potential for combining CNV and DNAm assessment into a single diagnostic assay. Neurodevelopmental disorders (NDDs) are a class of neurological and neuropsychiatric conditions that manifest in childhood during the developmental phase and persist throughout life . They afNDDs present with a broad range of genetic and phenotypic heterogeneity, and clinical presentations are often non-specific. Genetics plays an important role in the etiology of hereditary NDDs. Genetic mutations associated with NDDs vary in size from single nucleotide variants (SNVs) to whole chromosome aneuploidies . Due to Microarray platforms can be customized to increase coverage and resolution in clinically relevant regions and regions associated with well-defined genomic syndromes. In addition to probe-coverage enriched regions, clinical use microarray platforms have probes equally spaced across the rest of the genome, termed “backbone” coverage. The combination of high probe densities and optimized targeted design is aimed at reducing the rate of ambiguous findings termed variants of uncertain significance (VUSs), since, at this time, there is a limited understanding of the impact of CNVs outside of protein coding regions.Structural variants, defined as “alterations that involve segments of DNA larger than 1 kb” , includeGenomic disorders are a group of genetic conditions caused by CNVs affecting dosage sensitive genes or genes critical for normal development or maintenance and/or their regulatory elements . RecurreSegmental duplications  are blocks of DNA ranging from 1–400 kb that occur throughout the genome and typically share a high level (>90%) of sequence identity ,24. ManyA recent study assessed the prevalence and inheritance of CNVs associated with NDDs and estimated recurrent CNVs present in approximately 1 in 200 live births . These rNSD1) gene contained within this region is shown, on its own, to be causative for Sotos [RAI1) gene contained within this region [Considerable research to date has focused on genes within the CNV regions of genomic disorders and how dosage sensitivity may be responsible for the observed phenotypes. While this research has identified some causative or candidate genes for specific Mendelian disorders and phenotypes, the genetic contribution for the majority of the observed clinical phenotypes in CNV disorders are not well defined . One exaor Sotos . This iss region ,26,27,28Overall, CNVs may contribute to the clinical features observed in genomic disorders through dosage sensitivity, via haploinsufficiency (as described for Sotos and Smith–Magenis), triplosensitivity  or imprinting effects (Prader-Willi and Angelman syndromes), or through disruption of gene expression via positional effects, including disruption of transcriptional regulatory elements and changes in the chromatin structure.CMA screening for CNVs in patients with NDDs has an estimated diagnostic yield of approximately 15–20% ,27,28,29FMR1) gene, resulting in promotor DNA hypermethylation and gene silencing [FMR1 gene [This ‘diagnostic odyssey’, the time from initial consultation to diagnosis, often involves multiple clinical evaluations and laboratory tests spanning years ,36, resuilencing . FRX canMR1 gene . ReflexiMR1 gene ,38,39, aMR1 gene ,41. TherMR1 gene . Many recent studies have demonstrated disruption of genomic DNAm as a functional consequence of genetic defects in patients with NDDs ,44,45,46Epigenetics refers to mitotically heritable gene regulatory mechanisms without changes in the DNA sequence ,48. EpigAn increasing number of chromatin and epigenetic regulatory genes are becoming implicated in a variety of NDDs. Mutations in these genes result in DNAm episignatures, whole genome methylation changes, which are routinely detectable in the peripheral blood of patients affected by these disorders . An episKMT2D) [KMT2B), set domain-containing protein 2 (SETD2), creb-binding protein (CREBBP), lysine acetyltransferase 6A (KAT6A) and lysine demethylase 4B (KDM4B) [KDM5C) in Claes–Jensen syndrome [Histone modifications refer to the chemical modification of histone tails by processes including methylation, acetylation, phosphorylation and ubiquitination. Histone tails are loosely structured protein segments that can mediate interaction between nucleosomes, and their modifications can result in either condensed or more relaxed chromatin, ultimately exhibiting an effect on gene transcription, as well as accessibility of DNA to other chromatin remodeling factors, including those involved in DNA methylation. Our group and others have identified unique episignatures in multiple NDDs that are the consequence of mutations in genes associated with histone modification , includiKMT2D) . We have (KDM4B) . In addisyndrome . HistoneDNMT1, DNMT3A and DNMT3B [DNMT1, hereditary sensory neuropathy with dementia and hearing loss (HSNDHL), and autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCADN) ([DNMT3A result in an episignature in Tatton–Brown–Rahman syndrome (TBRS) [DNMT3B, which cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome, also result in genomic defects in DNAm [TET3), an “eraser” gene that opposes the writer function of DNMT1 [The DNAm reaction is catalyzed by enzymes known as DNA methyltransferases (DNMT), which are responsible for mediating the transfer of the methyl group from S-adenosylmethionine (SAM) to cytosine residues. Robust episignatures have been reported in NDDs caused by mutations in the DNA methyltransferase genes d DNMT3B ,55. ThesADCADN) ,57), whi, whiDNMTe (TBRS) ,58. Muta in DNAm . We receof DNMT1 . Mutatioof DNMT1 . Throughof DNMT1 .SRCAP) result in an episignature specific for Floating-Harbor syndrome [ATRX in alpha-thalassemia X-linked intellectual disability syndrome [SMARCA2)—a gene reported in multiple individuals with NCBRS. This new syndrome was identified based on unique methylation patterns observed in individuals with intragenic variants located in the helicase domain of the SMARCA2 gene compared to individuals with pathogenic variants located outside the helicase domain [SMARCA2 helicase domain mutations exhibited a common phenotype distinct from NCBRS. Similarly, functional studies in yeast supported a different molecular mechanism underlying these two disorders [To date, chromatin remodeling genes comprise the largest group of epigenetic modifier genes with mapped episignatures, e.g., truncating mutations in the SNF2-related CBP activator protein gene , which has chromatin regulatory functions [Interestingly, two distinct domain-specific episignatures have also been described in Helsmoortel-van der Aa syndrome associated with dominant negative truncating mutations in the activity-dependent neuroprotector homeobox gene (unctions . These sunctions .UBE2A) and spermine synthase (SMS) in X-linked syndromic forms of mental retardation—Nascimento and Snyder–Robinson types, respectively [UBE2A gene at Xq24 encodes the RAD6 ubiquitin-conjugating enzyme and has been recently shown to be involved in histone modifications that control gene expression [SMS gene at Xp22.11 encodes for an enzyme involved in polyamine synthesis and recycling and is directly related to decarboxylated SAM. Previous studies have suggested that alterations in this polyamine synthesis could result in an excess of SAM and may lead to aberrant DNAm status [Genes whose primary function is not associated with epigenetic and chromatin regulatory mechanisms, such as ubiquitin-conjugating enzyme E2 A  and extent  of the observed DNAm changes . Some diWhen analyzing methylation effects, it is important to consider confounding biological factors such as age, sex and blood cell composition, which are known to be associated with changes in methylation patterns in healthy individuals ,74. MethTo identify regions containing methylation changes, referred to as differentially methylated regions (DMRs), a ‘bump hunting’ approach  can be uThe complex bioinformatic pipeline required to identify episignatures, and to overcome previously mentioned confounding variables, relies heavily on a large-scale tissue-specific reference DNAm database, as well as bioinformatic and clinical genetic expertise . BroadenEpiSign is a clinical genome-wide DNAm test that has been available since 2019 that can detect over 60 disorders in more than 80 genes associated with Mendelian disorders through assessment of peripheral blood DNA . A list In parallel with screening for episignatures, EpiSign also permits concurrent detection of FRX in males  and commThe cost of completing a methylation array is comparable to the cost of most CMAs, and the results are highly reproducible. The assay uses peripheral blood, similar to current CMA platforms, allowing streamlined adaption in the clinical setting given the overlap in equipment and laboratory techniques.The ability to detect episignatures in patients with NDDs has been shown to increase the diagnostic yield, helping to resolve the diagnostic odyssey. A recent study assessing the clinical impact of EpiSign for patients with rare Mendelian disorders demonstrated a 27.6% diagnostic yield among patients with previous ambiguous/inconclusive genetic findings including genetic VUSs . As episChanges in DNAm profiles, or episignatures, in patients with large CNV defects associated with genomic disorders have not been systematically studied, and it is plausible that large CNVs, much like gene specific variants, may exhibit unique diagnostic methylation signatures in patients with NDDs. SHANK3). Intragenic variants in SHANK3 alone are responsible for a broad range of the phenotypic features observed in PHMDS [SHANK3 gene level variants (BRD1), a gene involved in epigenetic mechanisms and a likely candidate gene for the methylation signature observed in these patients  or duplication of the same region (7q11.23 duplication syndrome) , highligSD1 gene . A DNAm signature was reported in a cohort with the genomic disorder 16p11.2 deletion syndrome (16p11.2DS) —a disordSETD1B), and demonstrated that patients who harbored point mutations within SETD1B shared the same methylation episignature [Our group recently described an aberrant DNAm pattern in patients with deletions of 12q24.31 encompassing the known histone modifier gene SET domain-containing protein 1B  gene in 1q21.1 deletions and duplications, a gene that has a role in chromatin remodeling following DNA damage [Only a few of the most prevalent genomic disorders have a candidate gene considered responsible for the entire phenotypic spectrum. Interestingly, where these candidate genes have been identified, they are predominantly involved in epigenetic regulation including chromatin remodeling or histone modification, e.g., syndrome  and NSD1syndrome  . The ability to integrate the detection of genetic and epigenetic findings can provide a more complete view of underlying pathogenic mechanisms. Recent studies have shown it is possible to detect CNVs by applying computational methods to data obtained from DNAm arrays, such as the Illumina 450K and EPIC Bead Chip arrays ,90,91. MWe applied a similar computational approach using the DNAcopy package (Bioconductor.org) to our PHMDS cohort, and confirmed we could detect breakpoint coordinates similar to those obtained from conventional clinical CMA at the time of original diagnosis ; these fCombining these detection methods is not without challenges, most notably in coverage of the genome, as CpG sites are not uniformly distributed throughout the genome and therefore methylation arrays lack the “backbone coverage” observed in high-density SNP arrays. However, it is plausible that, with modifications, a combined array could be developed containing a combination of copy number and CpG targeted probes to produce a clinically targeted array enabling accurate episignature and CNV analysis on a single platform. This has the potential to impact healthcare resource utilization by reducing concurrent testing in NDD patients, and decreasing the need for reflexive testing for disorders such as those associated with imprinting. There would continue to be limitations in the ability to detect low level mosaicism, as seen with existing CNV platforms; however, studies have shown the ability to detect mosaicism from methylation arrays in Kabuki syndrome 1 , imprintAdditional benefits of a combined testing platform include those to the patient; a combined array would permit screening for more disorders in a single assay, thereby potentially increasing diagnostic yield over that of the current first-tier clinical test (chromosome microarray), and shortening the time spent in the diagnostic odyssey. This approach could concurrently reduce the burden on clinical services and genetic counselling by providing results for CMA, FRX, imprinting and methylation in a single report, leading to a reduction in requisitions and clinic visits. A combined platform would also benefit oncology studies, where limitations in tumor sample availability can often impact research and diagnosis; this would permit the detection of CNVs and methylation status from the same volume of tissue as traditional testing.The identification of episignatures in genomic disorders associated with CNVs could facilitate the expansion of screening capabilities for patients with NDDs, improving the diagnostic yield of clinical testing. This work may also provide novel insights into the pathogenesis of genomic disorders and provide targets for future therapies. The ability to combine CNV and episignature detection into a single assay would reduce the overall cost of testing, increase the number of disorders being screened for, and contribute to the reduction in alternative reflexive and concurrent genetic tests ordered. The benefit to patients and families in reducing wait times and increasing screened disorders, as well as reducing clinician and laboratory burden via fewer clinic visits and fewer genetic tests ordered, would have significant impacts on healthcare resource utilization and the costs associated with the diagnosis of NDDs.


Is subclinical postpartum depressed maternal mood associated with how the young infant’s brain processes speech information?In this population-based cohort study, more depressed maternal mood at 2 months after birth was associated with a statistically significant smaller change in infants’ electrophysiological brain responses to syllable pitch perception between ages 2 and 6.5 months.This study’s findings suggest an association between aberrant maturation patterns of infant speech perception and postpartum depressed maternal mood at subclinical levels. Language development builds on speech perception, with early disruptions increasing the risk for later language difficulties. Although a major postpartum depressive episode is associated with language development, this association has not been investigated among infants of mothers experiencing a depressed mood at subclinical levels after birth, even though such a mood is frequently present in the first weeks after birth. Understanding whether subclinical depressed maternal mood after birth is associated with early language development is important given opportunities of coping strategies for subclinical depressed mood.To examine whether depressed maternal mood at subclinical levels 2 months after birth is associated with infant speech perception trajectories from ages 2 to 6.5 months.In this longitudinal cohort study conducted between January 1, 2018, and October 31, 2019, 46 healthy, monolingual German mother-infant dyads were tested. The sample was recruited from the infants database of the Max Planck Institute for Human Cognitive and Brain Sciences. Initial statistical analysis was performed between January 1 and March 31, 2021; the moderation analysis (results reported herein) was conducted between July 1 and July 31, 2022.Mothers reported postpartum mood via the German version of the Edinburgh Postnatal Depression Scale  when their infants were 2 months old.Electrophysiological correlates of infant speech perception (mismatch response to speech stimuli) were tested when the infants were aged 2 months  and 6.5 months (follow-up).P = .04).A total of 46 mothers  and their 2-month-old children  participated at the initial assessment, and 36 mothers  and their then 6.5-month-old children  participated at follow-up. Moderation analyses revealed that more depressed maternal subclinical postpartum mood  was associated with weaker longitudinal changes of infants’ electrophysiological brain responses to syllable pitch speech information from ages 2 to 6.5 months (coefficient: 0.68; 95% CI, 0.03-1.33; The results of this cohort study suggest that infant speech perception trajectories are correlated with subclinical depressed mood in postpartum mothers. This finding lays the groundwork for future research on early support for caregivers experiencing depressed mood to have a positive association with children’s language development. This cohort study examines whether depressed maternal mood at subclinical levels 2 months after birth is associated with infant speech perception trajectories from ages 2 to 6.5 months. Clinical studies on postpartum depression4 indicate that a child’s language development can be negatively affected by caregivers’ depression.9 Given that (1) women experience depressive symptoms along a wide spectrum after giving birth, (2) most mothers currently experience subclinical depressed mood in the postpartum period,10 and (3) subclinical levels of depressive symptoms in the early postpartum period increase the likelihood of later deterioration of mood,11 investigating whether subclinical levels of depressive symptoms are associated with early infant language development is important for a child’s linguistic development.Early human infancy is a critical period for studying language development, as infants already show perceptual capacities for linguistic input.12 making depressed maternal mood a potential risk factor associated with early language acquisition milestones. Because altered infant speech perception is associated with the risk of later language impairments,14 it is important to identify factors negatively influencing infants’ speech perception as early as possible. However, it remains unknown whether subclinical depressed mood in otherwise healthy mothers who have recently given birth is associated with early infant speech perception, and whether such an association can be detected in very early infancy.Foundations of language are already established during the first weeks after birth,15 or mismatch response indicates the brain’s neural response to speech that is the response to deviant stimuli compared with the response to standard stimuli, without requiring an infant’s attention. Although mismatch negativity among adults is negative in polarity, a shift from an “immature” positive to a more adultlike mismatch response is typically observed during the first year of life.18 Of note, a delayed polarity shift of an infant’s mismatch response has been associated with the risk of developing language impairments later in life.14 Hence, the mismatch response is well suited to study trajectories of early linguistic development.Infant speech perception can be investigated by electrophysiological markers because electroencephalography can be performed relatively easily in young infants. The event-related potential mismatch negativity17 We hypothesized that a more depressed maternal mood during the early postpartum period is associated with a lower likelihood of developing a more mature mismatch response across infancy compared with infants of mothers with more positive mood.The present study investigated whether maternal mood at 2 months after birth is associated with infants’ longitudinal speech perception development. We analyzed mismatch response patterns to different acoustic speech features among infants aged 2 months and 6.5 months, to cover the age range during which a mismatch response polarity shift is expected.19 mismatch response variance with amount of language exposure among 27 infants,20 and mismatch response variance with infant-directed speech quality among 17 infants.21 As we were specifically interested in maternal postpartum mood at subclinical levels, we used the German version22 of the Center for Epidemiologic Studies Depression Scale23 to screen for and exclude mothers with (past) clinical episodes of major depressive disorder. Exclusion criteria for infants were gestational age less than 37 weeks, birth weight less than 2700 g, and diagnosed hearing deficits or neurologic conditions. Parents were informed of the aim and procedure and provided written parental consent. Mothers were compensated with 7.50€ and could pick a small toy for their infants. The study followed American Psychological Association standards according to the Declaration of Helsinki24 and was approved by the medical faculty’s ethics committee of Leipzig University.This cohort study was conducted between January 1, 2018, and October 31, 2019. The sample, recruited from the infants database of the Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany, consisted of 46 dyads of mothers and their 2-month-old infants at initial assessment. Thirty-six mother-infant dyads completed the follow-up assessment when the infants were aged 6.5 months. Infant studies of comparable sample sizes have explained mismatch response variance by factors of the learning environment, such as mismatch response variance with quantity of language input among 37 infants,26 was used (German version27). Scores range from 1 to 30; higher scores indicate higher levels of depressed mood, with a cutoff of 13 points indicating a high probability of clinical depression. This cutoff ensures high specificity but lower sensitivity,28 as we were specifically interested in identifying mothers with subclinical levels of depressed mood. We also assessed maternal subjective perceived stress using the Perceived Stress Scale (PSS-10)29 (German version30), with higher scores indicating higher levels of perceived stress. Furthermore, mothers were screened for any psychiatric or neurologic diseases and any current medications.Mothers completed mood and stress questionnaires at 2 months after birth. For maternal postpartum mood, the Edinburgh Postnatal Depression Scale (EPDS)Identical protocols were used for 2-month-old and 6.5-month-old infants to assess speech perception in an electrophysiology experiment, conducted in a quiet, infant-friendly room (each appointment lasted 60 minutes). We recorded the electroencephalogram from 21 active electrodes  at standard positions (10-20 system), referenced online to electrode Cz and a ground electrode at position Fp1. Electrooculograms were recorded from electrodes at the outer canthi of both eyes and orbital ridges of the right eye. Electrode impedances were largely less than 10 kΩ and always less than 20 kΩ. The electroencephalogram signal was amplified , digitized online at 500 Hz, and recorded . During experiments, infants lay (aged 2 months) or sat (aged 6.5 months) on their parent’s lap and, if necessary, were entertained using silent toys or fed by their mothers.31 with semisynthesized syllables , /bu/ (vowel deviant34), raised the pitch by 16 Hz /ba+/ (syllable pitch [F0] deviant35), and lengthened the vowel by 100 ms /ba:/ . Stimuli were recorded by a female native German speaker  and adjusted using Praat, version 6.0.2836 . Standard and deviant syllables were presented in alternation . Offline, data were algebraically re-referenced to the mean of mastoids and bandpass filtered  and semiautomatically scanned for artifacts  for subsequent independent-component analysis. Calculated independent components38 were visually scanned and artifact-related components were determined, saved, and applied to continuous data bandpass filtered at 0.5 to 30 Hz , typically used for mismatch response analysis.39 Data epochs of 700 milliseconds postsyllable onset  were obtained from the corrected data and again semiautomatically scanned for artifacts. Finally, individual means for deviants  and standard (/ba/) were calculated, and grand means were computed.Electrophysiological data were processed using EEGLAB, version 13.5.4b40 central , and posterior  regions of interest. Analyses were performed for six 100-millisecond time windows (starting 100 milliseconds after stimulus onset), while controlling for infants’ physical maturation , time-window , and region .Statistical analysis was performed with SPSS software, version 24 (IBM Corp) between January 1 and March 31, 2021  and between July 1 and July 31, 2022 . We tested for significance of the mismatch response as an indicator of infant speech perception at ages 2 and 6.5 months for all contrasts by comparing the mean event-related potential amplitude of the standard stimulus with those of the deviant stimuli at frontal ,41 and applying the Johnson-Neyman procedure43 for identifying boundaries of significance when the moderation effect of maternal mood on the change of the mismatch response amplitude from ages 2 to 6.5 months becomes significant.To investigate the association of postpartum maternal mood with longitudinal changes in infant speech perception from ages 2 to 6.5 months, moderation analyses were performed for statistically significant mismatch response effects found in the repeated-measures analyses of covariance. Within-participant factor was the mismatch response amplitude change (from ages 2 to 6.5 months) and the moderator was maternal mood (EPDS scores). Significant association of maternal mood with speech perception development from ages 2 to 6.5 months was probed by using the SPSS toolbox MEMOREP values are 2-sided, and effects are considered to be statistically significant at P < .05. P values were Bonferroni corrected for multiple comparisons.Assumptions for metric statistical tests were checked; if not otherwise specified, data were normally distributed (Kolmogorov-Smirnov test). Reported All infants met the criteria of at least 50% valid trials in the electrophysiological experiment. All mothers were under the clinical cutoff for a major depressive episode. From the first to second assessment, 10 infant-mother dyads withdrew.All 46 infants  and 46 mothers  were German monolingual, healthy, and from families with middle to high socioeconomic backgrounds; mothers did not smoke and had no history of neurotoxin use. All mothers were on maternity leave at both assessment points and reported a joint household with both parents .28 Five mothers participating in the first assessment and 2 mothers in the final sample were taking l-thyroxin and in a stable euthyroid state with this treatment. After controlling for additional factors associated with maternal mood, we did not find significant correlations between maternal EPDS scores and socioeconomic background  or number of children in the family   was revealed but was not significantly associated with infant age as a control variable. Post hoc pairwise comparisons (contrasting each deviant with the standard condition at each region and time window) showed this interaction to be associated with a positive frontal mismatch response to all deviant types, as well as a positive central mismatch response to the vowel length deviant  was again revealed, with no significant association of infant age with outcomes. Post hoc pairwise comparisons showed this interaction to be associated with a positive frontal mismatch response to all deviant types, as well as positive central mismatch response to the syllable pitch, consonant, and vowel deviant types , with a nonsignificant intercept , suggesting a mismatch response change from positive to more negative amplitudes between ages 2 and 6.5 months. We also found that maternal EPDS scores were significantly associated with syllable pitch mismatch response change . Probing the moderation association of maternal mood with mismatch response development revealed that the Johnson-Neyman transition point for a significant association of maternal mood with infant speech perception change is at an EPDS score of 8.57  in 6.5-month-old infants compared with 2-month-old infants, infants of mothers with more depressed mood 2 months after birth showed overall more positive, less mature mismatch response polarity at both ages. This finding suggests that the main caregiver’s mood is a pace-setting factor for infant mismatch response maturation. This finding is corroborated by the observation that the developmental shift toward more negative mismatch responses at age 6.5 months when compared with age 2 months was more likely for infants of mothers with a less depressed mood. Given that infants with a prolonged period of positive “immature” mismatch responses during the first months of life are at risk of later language difficulties12 and immature mismatch responses to pitch variations were found among individuals experiencing language developmental difficulties,49 mothers’ more depressed postpartum mood can be considered a risk factor for infants experiencing language difficulties later in life.Within the first year of life, the brain’s signature of processing an auditory mismatch is expected to develop from positive, immature mismatch responses to a more adultlike mismatch negativity.50 They align with previous work suggesting that a reduction of infant-directed speech use among mothers with depressed mood is associated with children’s developmental trajectories.51 Infant-directed speech uses greater pitch variability and slower speech rate compared with adult-directed speech53 and is critical for encouraging early language perception.57 However, depressed mothers often show a reduction in vocal pitch modulation when directing speech toward their infants.60 Our findings lend support to the role of pitch modulations in the mother-infant relationship, even in subclinical mood deterioration: it was the response to the syllable pitch and not the other speech deviants that showed associations with infant mismatch response negativity and maternal mood. However, associations might have been missed because of the relatively small sample.Our findings extend previous reports on negative associations between maternal postpartum clinically depressed mood and children’s language development by exploring the subclinical range of maternal mood.59 and to promote infant speech perception,8 an alternative approach of supporting women experiencing postpartum subclinical depressed mood might be the involvement of other close caregivers. As fathers also use increased pitch variability in infant-directed speech,61 paternal pitch variability may be one option for a low-threshold coping strategy to support infants’ language development during the postpartum period.62Although further studies with infant-caregiver dyads are needed to fully characterize the association of the caregiver’s mood with infant language development, the present findings suggest that the postpartum period is a critical time for infant speech perception. This finding provides the foundation for future studies testing the hypothesis that early support of mothers with postpartum depressed mood may also be beneficial for their infants’ speech perception and language development. Although pharmacologic interventions for postpartum depression have been shown to effectively encourage infant-directed speech use59 we did not test whether our main findings were associated specifically with infant-directed speech use by mothers. Future research should include assessments of mothers’ infant-directed speech characteristics to disentangle the specific aspects underlying the association between maternal postpartum mood, maternal pitch modulation, and infant mismatch response to syllable pitch deviants. Moreover, we assessed maternal mood only when infants were 2 months old. To reliably conclude that maternal mood 2 months after birth moderates less maturational change of infant speech perception from ages 2 months to 6.5 months, only a second assessment would allow for excluding that mothers continue to experience a more depressed mood, which might also be associated with infants’ speech perception at 6.5 months.This study has some limitations. The longitudinal design offers the advantage of establishing a sequence of events; however, correlation over time does not imply causation and allows only for speculation on the underpinnings of the association between postpartum maternal mood and infant syllable pitch perception. Although a reduced range in the vocal pitch of mothers with postpartum depression has been reliably shown,Our study offers, to our knowledge, the first evidence of an association of postpartum maternal mood at a subclinical level and infant speech perception. Future research will need to replicate these findings in a larger, more heterogeneous sample with families of broader socioeconomic backgrounds to allow for generalizability of the present results. In the present study, family-related variables such as socioeconomic background and number of siblings did not explain maternal postpartum mood. However, even in our limited sample with mostly middle to high socioeconomic status, the observed variance in mood showed an association with infant speech perception.The results of this cohort study suggest an association of postpartum depressed maternal mood at subclinical levels with less maturational change of speech perception among young infants between ages 2 and 6.5 months. Our observation that maternal mood at 2 months post partum was associated with infant speech perception longitudinally emphasizes the importance of considering the role of postpartum maternal mood in shaping infants’ brain responses to speech stimuli. This finding advocates for systematic early screenings for mothers’ postpartum mood and for future studies exploring whether early support for caregivers experiencing depressed mood can have a positive association with infant language development.


Amyotrophic lateral sclerosis (ALS) is genetically diverse, with numerous variants associated with either causing or increasing risk of developing the condition . The incATXN2 gene have been associated with increased susceptibility for ALS [ATXN2 is currently in a Phase 1 clinical trial for people living with ALS, both with and without polyQ expansions (NCT04494256). To date, studies report varying incidence of ATXN2 intermediate repeats in ALS patients [ATXN2 repeat expansion testing now available on several commercial ALS testing panels, implementation of ATXN2 genetic testing provides an opportunity to identify suitable participants for clinical trials.Ataxin-2, a polyglutamine (polyQ) protein, is a modifier of TDP-43 toxicity in ALS models, and intermediate polyQ repeats in the NCT0449426. To datATXN2 genetic testing as part of routine clinical practice.Here, we outline our clinic’s early experience with implementing SOD1 and C9orf72 testing have been consistently offered to all patients for several years. Initiation of a clinical trial targeting ATXN2 intermediate repeat expansions prompted an evolution of the clinic’s genetic testing practices to include offering ATXN2 testing to all patients.The ALS clinic at the Montreal Neurological Institute-Hospital (The Neuro) has been offering genetic testing to all newly diagnosed ALS patients since 2015. A tiered testing approach is used—for patients with a family history of ALS, FTD, related disorders, or an atypical presentation (such as a young age of onset), a comprehensive panel of ALS genes is ordered; and, for isolated ALS, only those genes for which there are targeted, ongoing clinical trials are ordered. With this testing algorithm, both ATXN2 testing to their offering in October 2020. Subsequently, all patients newly diagnosed with ALS would be offered ATXN2 repeat expansion testing as part of the clinic’s standard practice. To date, 61 newly diagnosed ALS patients have had ATXN2 genetic testing.Clinical genetic testing at The Neuro’s ALS clinic is performed by PreventionGenetics, who added n = 116). This request was approved by their Tissue and Data Committee under the ethical framework approved by the McGill University Health Centre’s Research Ethics Board—Neuroscience and Psychiatry Panel (MUHC-15-944 (CRU)/2017-330, 15-944-MUHC).For previously diagnosed patients, for whom genetic testing was completed prior to October 2020, repeat testing would consume considerable clinical and financial resources. However, patients at The Neuro’s ALS clinic are given the opportunity to consent to the Clinical Biospecimen Imaging and Genetic (C-BIG) Repository, where their DNA is banked for future analysis and scientific research. A request was made to C-BIG to access samples from ALS patients actively being followed in the clinic (ATXN2 CAG repeat region was amplified following published methodology [DNA was extracted from blood samples using standard procedures. The hodology , with mihodology . Repeat ATXN2 intermediate repeat expansion testing to the clinic’s standard genetic testing practices, five patients have been identified (Table Since adding ed Table : four weATXN2 are associated with increased risk of developing ALS, as opposed to a monogenetic, inherited cause. Should a test result indicate that intermediate repeats are present, this introduces challenges in conveying the risk to family members in post-test counselling, as the risk and implications are unclear. Genetic counselling, provided by an appropriate specialist, should always accompany the offer of genetic testing for people living with ALS.Intermediate repeats in ATNX2 testing in our clinical setting identified people living with ALS who were potentially eligible for a targeted genetic therapy trial. Use of banked DNA samples to conduct a retrospective screen of those patients who had previously had genetic testing reduced the burden on both financial and human resources, in our public healthcare setting. This model could be applied in other clinical settings, and allows for adaptability in clinical practice in an evolving therapeutic landscape.Genetics has long contributed to our understanding of ALS, and is translating into the development of targeted therapies at an increasing rate. As such, genetic testing practices in ALS need to evolve with emerging clinical trials. Implementation of routine