Mutations in ATP-dependent chromatin remodeler CHD8 cause one of the most frequent monogenetic forms of autism and are associated with brain overgrowth. Nevertheless, the activities of CHD8 in autism-relevant cell types are still poorly understood. Here, we purify the CHD8 protein from human neural stem cells and determine its interaction partners using mass spectrometry. We identify the TRRAP complex, a coactivator of MYC and E2F transcription factors, as a prominent CHD8 interaction partner. CHD8 colocalizes genome-wide with TRRAP and binds together at MYC and E2F target gene promoters in human neural stem cells. Depletion of CHD8 or TRRAP in human neural stem cells shows downregulation of MYC and E2F target genes as the most prominent gene-regulatory events. Depletion of CHD8 diminishes cell-cycle entry into S-phase. MYC and E2F transcription factors are established oncogenes and regulate cell growth. Our results link CHD8 to TRRAP in facilitating the regulation of MYC and E2F target genes in human neural stem cells.

Transcription pause-release constitutes a key step in the regulation of gene expression. Pathogenic variants in various pausing regulators have been identified in neurodevelopmental disorders, but how transcription pause-release regulates neural development remains largely unknown. Here we show that a patient-specific in-frame deletion in Integrator subunit 8 (INTS8) that prevents association of this subunit with the Integrator complex results in a global increase in nascent transcription and precocious expression of neuronal genes in progenitor cells. Consequently, differentiating neural progenitors fail to sustain a proliferating progenitor cell population during cortical neuronal network formation, resulting in network collapse. We show that attenuation of RNApol2 pause-release by targeted degradation of BRD4 can revert neuronal gene activation and prevent premature progenitor cell loss. Our data demonstrate that a fine-tuned balance between RNApol2 pausing and elongation is crucial for normal neuronal development and suggest transcription pause-release as a druggable target for neurological defects in developmental disorders. ### Competing Interest Statement The authors have declared no competing interest.

Genomic profiles and prognostic biomarkers in patients with acute myeloid leukemia (AML) from ancestry-diverse populations are underexplored. We analyzed the exomes and transcriptomes of 100 patients with AML with genomically confirmed African ancestry (Black; Alliance) and compared their somatic mutation frequencies with those of 323 self-reported white patients with AML, 55% of whom had genomically confirmed European ancestry (white; BeatAML). Here we find that 73% of 162 gene mutations recurrent in Black patients, including a hitherto unreported PHIP alteration detected in 7% of patients, were found in one white patient or not detected. Black patients with myelodysplasia-related AML were younger than white patients suggesting intrinsic and/or extrinsic dysplasia-causing stressors. On multivariable analyses of Black patients, NPM1 and NRAS mutations were associated with inferior disease-free and IDH1 and IDH2 mutations with reduced overall survival. Inflammatory profiles, cell type distributions and transcriptional profiles differed between Black and white patients with NPM1 mutations. Incorporation of ancestry-specific risk markers into the 2022 European LeukemiaNet genetic risk stratification changed risk group assignment for one-third of Black patients and improved their outcome prediction. Analysis of exomes and transcriptomes from 100 African American patients with acute myeloid leukemia identifies ancestry-related variation in mutation profiles and survival. Refined risk classification suggests clinical relevance of these ancestry-associated differences.

Introduction The revised 5th edition of the World Health Organization Classification of Haematolymphoid Tumours and the 2022 European LeukemiaNet (ELN) genetic-risk classification have led to profound changes in the classification, risk assignment, and associated treatment options for AML pts. One of the major changes affects AML-MR. Pts with MR-mutations are now considered to have adverse-risk, except when MR-mutations co-occur with favorable-risk AML subtypes. However, AML-MR is a clinically and molecularly heterogeneous group with diverse clinical outcomes when treated with intensive chemotherapy.Although gene mutations and recurrent cytogenetic abnormalities are equally included as AML-MR-defining genetic events, the associations of specific genetic lesions with pretreatment features and clinical outcome may differ. Thus, data-driven support for the definition of AML-MR is needed to improve risk assignment and subsequent treatment guidance for pts. We hypothesized that better portrayal of tumor biology characterized by gene expression-based genetic phenotype may help refine AML-MR classification. Methods We performed transcriptome-based gene-expression profiling and t-distributed stochastic neighbor embedding (t-SNE) data visualization on a clinically and molecularly well-characterized cohort of 971 newly diagnosed AML pts who were similarly treated with intensive chemotherapy on CALGB/Alliance frontline chemotherapy-based protocols. Using transcriptome-based gene-expression profiles of pts diagnosed with myelodysplastic neoplasms, we defined an MDS similarity score, which was almost exclusively found in a relatively large, central t-SNE cluster enriched for pts with AML-MR-defining genetic lesions. We recognized 3 pt groups (n=377 pts): pts with both AML-MR-defining genetic lesions and MR-expression profile (n=149), pts who had the MR-expression profile but not AML-MR-defining genetic lesions (n=99) and pts who harbored AML-MR-defining genetic lesions but did not have the MR-expression profile (n=129). Clinical and molecular characteristics and treatment outcomes of pts in these 3 groups were then compared. Results Treatment outcomes of pts with MR-expression profile with and of those without AML-MR-defining genetic lesions were equally poor, whereas pts with AML-MR-defining genetic lesions but without the MR-expression profile had superior complete remission (CR) rates (43% vs 45% vs 69%, P<.001), and overall survival (OS; 3-y rates: 18% vs 14% vs 39%, P<.001). Because the outcome of pts with MR-expression profile who harbored AML-defining genetic lesions was very similar to outcome of pts with MR-expression profile who did not, we next combined these 2 pt groups into one MR-expression group (termed “MR-e”, n=248) and compared this group with the “mutation only” (“MR-mut”, n=129) group. Pts in the MR-e group were older than pts in the MR-mut group (median, 57 vs 48 y, P<.001). We next performed cell type quantifications using Dampened Weighted Least Squares (DWLS) based on our recently established single cell labels (Lasry et al, 2022) for deconvolution. Here, MR-e patients had a more HSC-like phenotype and a larger fraction of CD4+ T cells. In comparative survival analyses, MR-e patients had lower CR rates (44% vs 69%, P<.001), and worse OS (3-y rates: 15% vs 39% P<.001) than MR-mut pts. Within the 2022 ELN Favorable genetic-risk group, although pts with MR-expression profile had similar CR rates (60% vs 81%, P=.21), their OS was worse (3-y rates: 11% vs 60%, P<.001) than OS of MR-mut pts. In multivariable analyses, the presence of MR-e independently associated with lower CR rates (P=.004), adjusting for SF3B1 mutations and shorter OS (P<.001), adjusting for IDH1/2 mutations). Co-existing IDH1/2 mutations were the only identified genetic feature outweighing the negative prognostic effect of MR-e. Conclusions We have identified an expression-based MDS-similarity score (MR-e) as a possible phenotypic correlate for AML-MR pts that also includes patients without AML-MR defining genetic lesions. Its presence portends very poor treatment outcome, especially in pts classified in the 2022 ELN Favorable genetic-risk group. In contrast, the absence of MR-e associates with better CR and OS in pts carrying AML-MR-defining lesions. Validation of these findings in an independent data set of intensive chemotherapy treated AML-MR patients is ongoing.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive single cell RNA-Sequencing census of the bone marrow immune microenvironment in adult and pediatric AML patients. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B cell subtype enriched in high-inflammation AML patients, as well as an increase in CD8+ GZMK+ and regulatory T cells, accompanied by a reduction in T cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in AML patients. Addition of the iScore refines current risk stratifications for AML patients and may enable identification of patients in need of more aggressive treatment. This work provides a first framework for classifying AML patients based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings. Citation Format: Audrey Lasry, Bettina Nadorp, Maarten Fornerod, Deedra Nicolet, Huiyun Wu, Christopher J Walker, Zhengxi Sun, Matthew T Witkowski, Anastasia N Tikhonova, Maria Guillamot, Geraldine Cayanan, Anna Yeaton, Tanja A Gruber, Ann-Kathrin Eisfeld, Iannis Aifantis. Inflammation remodels the immune microenvironment in acute myeloid leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A20.

Genome wide expression profiles of pediatric myeloid malignancies can be a powerful resource to inform on disease subtype, leukemia stemness and associated risk. However, individual mRNA expression data are difficult to interpret in the absence of a reference cohort due to batch and scaling effects. We recently developed an expression based, 381 gene classifier for pediatric myeloid leukemias using a 435 patient cohort spanning a wide variety of subsets. These include myeloid, megakaryoblastic, early T-cell progenitor and mixed phenotype acute leukemias. We now have developed an web application which allows analysis of individual pediatric myeloid leukemia samples in a scale and batch independent manner, predicting transcriptional identity and leukemia stemness. We extensively test the application, which is robust to genome wide ex-pression analysis platform and sample purity. We also show that leukemic cells from a CBF2AT3-GLIS2 genetically engineered human xenograft model transcriptionally match CBF2AT3-GLIS2 acute megakaryoblastic patient derived cells. Thus, using this application (http://pamland.org), determination of transcriptional identity and leukemia stemness can sig-nificantly supplement existing patient characteristics, such as mutation status and karyotype, and can be used to evaluate experimental myeloid leukemia systems.

Background Our knowledge of the genomics of acute myeloid leukemia (AML), which serves as the basis of clinically used prognostic biomarkers and therapeutic advances (including targeted therapies), is almost exclusively based on data from patients (pts) of European ancestry. Methods We analyzed the exomes and transcriptomes of 100 Black AML pts at diagnosis who received intensive induction chemotherapy on Alliance for Clinical Trials in Oncology (Alliance) protocols. African Ancestry was confirmed by SNP analysis. We established their genetic landscape including somatic gene mutations, structural variants, gene fusions and associated gene expression. Somatic mutation frequency was compared to whole-exome sequencing of 741 White pts at diagnosis from the Beat AML cohort analyzed by identical workflow. Survival of Black pts was compared to 1,519 White pts treated on the same Alliance protocols. Multivariable analysis was used to identify clinical and molecular features associated with outcomes. Results We identified 162 recurrently mutated genes in Black AML pts. We detected different mutation frequencies for AML-associated genes based on ancestry, and mutations in genes not previously implicated in AML, including PHIP alterations in 7% of this cohort. 38 genes were mutated in 3-5% of Black pts but in <1% of White pts . Of the 162 recurrently mutated genes in Black pts, only 41 (25%) were recurrently mutated in White pts, while 121 genes (75%) were mutated in ≤1 White patient. Only 1/741 White AML pts did not have a mutation in at least 1/ 56 recurrently mutated AML genes assessed by clinical NGS panels. By comparison, 10% of Black pts had no mutations in these genes. Analysis of gene fusions showed recurrent, cryptic CBFA2T3 fusions in 4 pts and a recurrent GGNBP2:: MYO19 fusion involving chromosome 17 not previously described in AML in 2 pts. The CBFA2T3 fusions did not cluster with CBF-AMLs by gene expression, suggesting biological differences. t-SNE visualization of gene expression showed that Black and White AML pts clustered together by known driver mutations. We identified a t-SNE cluster enriched for pts with myelodysplasia-related (MR) mutations. Notably, Black pts in this cluster were significantly younger than White pts [50 vs 58-years (y), p=0.04], suggestive of intrinsic and/or extrinsic dysplasia-causing stressors. When matched for age, performance status and study date, Black pts had higher relapse rates (63% vs 48%, p=0.05), worse disease-free (DFS, 3-y rates, 30% vs 47%, p=0.01) and overall survival (OS, 3-y rates, 31% vs 47%, p=0.007) compared to White pts. In a multivariable analysis, MR mutations (HR=2.06, p=0.03) and mutations in NPM1 (HR=2.29, p=0.009) and NRAS (HR=1.95, p=0.04) were associated with shorter DFS. Using the recently published iScore as a proxy for inflammation, we detected an enrichment of high inflammation in Black pts harboring NPM1 mutations compared to Whites (iScore high vs low; Black, 69% vs 31%, White, 45% vs 55%; p=0.08). Furthermore, when we classified pts into LSC17 prognostic groups, only 13% of Black pts in the prognostically favorable low LSC17 group were NPM1-mutated, compared with to 38% of White pts, suggesting ancestry-associated differences in the biological impact of NPM1 mutations. Mutations in IDH1/2 associated with shorter OS (HR=1.73, p=0.05) in Black pts identifying ancestry-specific risk markers. Finally, we show that the current ELN 2022 risk stratification system can be significantly improved by including NPM1, NRAS and IDH1/2 mutations as ancestry-specific adverse risk markers for Black pts ( Figure 2). Mutations in NPM1, NRAS, and IDH1/2 were enriched in a cohort of 43 Black relapsed/refractory (R/R)-AML pts via MSK-IMPACT supporting their adverse prognostic risk in Black pts. Conclusions Our work demonstrates ancestry-related differences in the genomic profiles of AML pts and calls for additional studies of ancestry-diverse populations to understand the landscape of somatic genetic alterations in AML. Further, our results emphasize the need to include ancestry-associated variants in clinical testing panels and to refine genetic risk assessment of AML by incorporating ancestry-specific prognostic biomarkers. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224. https://acknowledgments.alliancefound.org.

In addition to its structural role in enclosing and protecting the genome, the nuclear envelope (NE) forms a highly adaptive communication interface between the cytoplasm and the nuclear interior in eukaryotic cells. The double membrane of the NE is perforated by nuclear pores lined with large multi-protein structures, called nuclear-pore complexes (NPCs), which selectively allow the bi-directional transport of ions and macromolecular cargo. In order to nucleate a pore, the inner and outer nuclear membrane have to fuse at the site of NPC insertion, a process requiring both lipid bilayers to be deformed into highly curved structures. How this curvature is achieved and which factors are involved in inducing and stabilizing membrane curvature at the nuclear pore remain largely unclear. In this review, we will summarize the molecular mechanisms thought to be involved in membrane curvature generation, with a particular emphasis on the role of lipids and lipid metabolism in shaping the nuclear pore membrane.

Adult and pediatric acute myeloid leukemia (AML) are clinical entities separated by an arbitrary age cutoff, yet they share substantial cytogenetic and morphological features. A 21-year-old newly diagnosed AML patient with a KMT2A-MLLT3 translocation who presents at an adult hospital will be offered a stem cell transplant in first remission if a suitable donor is available based on European LeukemiaNet (ELN) risk stratification whereas a pediatric facility will treat with chemotherapy alone provided they achieve MRD negative status after two cycles. To understand whether age defines molecular groups or alternatively informs risk, we compiled a cohort of 1307 clinically and molecularly well characterized AML patients with long-term follow-up who were similarly treated with anthracycline-based induction chemotherapy (pediatric patients, n=435, adult patients, n=872, median age 38 [range: 0-84 years]). Biological diversity of AML is best captured by gene expression signatures of highly variant genes. We therefore used a previously characterized gene expression classifier to integrate the pediatric and adult cohorts, yielding subsets with mixed adult and pediatric composition. Notably, a previously unrecognized pediatric subset merged with one of the dominant adult subsets, characterized by mutations present in myelodysplastic syndrome (MDS) and secondary AML (sAML) such as RUNX1, ASXL1/2, TP53, DNMT3A, TET2 and SRSF2. Males were represented at a higher frequency in this subset (p=.006), consistent with the higher proportion seen in the clonal hematopoiesis, MDS, and sAML clinical entities. Additionally, clinically confirmed adult sAML patients from a previously published cohort clustered with this subset, indicating that AML in these patients may have developed from a subclinical myelodysplastic state. Other major iso-transcriptional subsets with a pediatric to adult composition include RUNX1-RUNX1T1, KMT2A rearrangements, CBFB-MYH11, CEBPA, Acute Myeloid and T-Cell precursor Leukemia (AMTL) and a subset with high HOX cluster gene expression characterized by NPM1 mutations, nucleoporin rearrangements and/or FLT3 internal tandem duplications (ITD). To understand the association of age and risk, we analyzed an expanded cohort of 2217 AML patients that belonged to one of 8 molecular subsets: CEBPA, CBFB-MYH11, RUNX1-RUNX1T1, KMT2A-MLLT3, NPM1 single mutant, FLT3-ITD single mutant, NPM1/FLT3-ITD double mutant, and MDS-like (pediatric patients, n=790, adult patients, n=1427, median age 36 [range: 0-59 years]). Treating age as a continuum, we found sharp or gradual decreases in overall survival, depending on AML subset. An optimal cut algorithm was used to identify age cut-offs that distinguish good and poor risk prognoses within molecular subgroups which varied significantly. Patients with KMT2A-MLLT3 that were greater than 12 years of age were identified as having an inferior prognosis whereas age greater than 45 years was associated with poor outcomes for FLT3-ITD positive patients (Figure A, B). Mutational analysis of the entire cohort revealed a higher burden of cooperating mutations in certain molecular subsets in the older risk groups. For example, older patients carrying NPM1 mutations in the absence of FLT3-ITD had a higher occurrence of DNMT3A and single CEBPA mutations. In CEBPA double mutated, KMT2A-MLLT3 and CBFB-MYH11 subsets, significant age risk associating variables included an increased transcriptional resemblance to fetal liver hematopoietic progenitor cells, suggesting an additional age dependent role of quiescence potential in disease outcome. Age risk thus may be a function of both mutational timing and oncogenic driver lesion. In conclusion, we propose that pediatric and adult AML can be integrated, with common biological subsets in which age plays different roles. Both pediatric and adult clinical practice of treating AML may benefit from this approach of age integrated analysis. M.F. and D.N. contributed equally. Co-corresponding authors: A-K.E. T.A.G. Support: U10CA180821, U10CA180882, U24CA196171, https://acknowledgments.alliancefound.org. Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Elucidating genetic aberrations in pediatric acute myeloid leukemia (AML) provides insight in biology and may impact on risk-group stratification and clinical outcome. This study aimed to detect such aberrations in a selected series of samples without known (cyto)genetic aberration using molecular profiling. A cohort of 161 patients was selected from various study groups: DCOG, BFM, SJCRH, NOPHO and AEIOP. Samples were analyzed using RNA sequencing (n=152), whole exome (n=135) and/or whole genome sequencing (n=100). In 70 of 156 patients (45%), of whom RNA sequencing or whole genome sequencing was available, rearrangements were detected, 22 of which were novel; five involving ERG rearrangements and four NPM1 rearrangements. ERG rearrangements showed self-renewal capacity in vitro, and a distinct gene expression pattern. Gene set enrichment analysis of this cluster showed upregulation of gene sets derived from Ewing sarcoma, which was confirmed comparing gene expression profiles of AML and Ewing sarcoma. Furthermore, NPM1-rearranged cases showed cytoplasmic NPM1 localization and revealed HOXA/B gene overexpression, as described for NPM1 mutated cases. Single-gene mutations as identified in adult AML were rare. Patients had a median of 24 coding mutations (range, 7-159). Novel recurrent mutations were detected in UBTF (n=10), a regulator of RNA transcription. In 75% of patients an aberration with a prognostic impact could be detected. Therefore, we suggest these techniques need to become standard of care in diagnostics.

SMPD4 is a neutral sphingomyelinase implicated in a specific type of congenital microcephaly. Although not intensively studied, SMPD4 deficiency has also been found to cause cell division defects. This suggests a role for SMPD4 in cell-cycle and differentiation. In order to explore this role, we used proximity ligation to identify the partners of SMPD4 in vivo in HEK293T cells. We found that these partners localize near the endoplasmic reticulum (ER) and the nuclear membrane. Using mass spectrometry, we could identify these partners and discovered that SMPD4 is closely associated with several nucleoporins, including NUP35, a nucleoporin directly involved in pore membrane curvature and pore insertion. This suggests that SMPD4 may play a role in this process.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with poor prognosis and limited treatment options. Here we provide a comprehensive census of the bone marrow immune microenvironment in adult and pediatric patients with AML. We characterize unique inflammation signatures in a subset of AML patients, associated with inferior outcomes. We identify atypical B cells, a dysfunctional B-cell subtype enriched in patients with high-inflammation AML, as well as an increase in CD8 + GZMK + and regulatory T cells, accompanied by a reduction in T-cell clonal expansion. We derive an inflammation-associated gene score (iScore) that associates with poor survival outcomes in patients with AML. Addition of the iScore refines current risk stratifications for patients with AML and may enable identification of patients in need of more aggressive treatment. This work provides a framework for classifying patients with AML based on their immune microenvironment and a rationale for consideration of the inflammatory state in clinical settings.

Abstract Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell–like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. Significance: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549

The increasing pace of gene discovery in the last decade has brought a major change in the way the genetic causes of brain malformations are being diagnosed. Unbiased genomic screening has gained the first place in the diagnostic protocol of a child with congenital (brain) anomalies and the detected variants are matched with the phenotypic presentation afterwards. This process is defined as “reverse phenotyping”. Screening of DNA, through copy number variant analysis of microarrays and analysis of exome data on different platforms, obtained from the index patient and both parents has become a routine approach in many centers worldwide. Clinicians are used to multidisciplinary team interaction in patient care and disease management and this explains why the majority of research that has led to the discovery of new genetic disorders nowadays proceeds from clinical observations to genomic analysis and to data exchange facilitated by open access sharing databases. However, the relevance of multidisciplinary team interaction has not been object of systematic research in the field of brain malformations. This review will illustrate some examples of how diagnostically driven questions through multidisciplinary interaction, among clinical and preclinical disciplines, can be successful in the discovery of new genes related to brain malformations. The first example illustrates the setting of interaction among neurologists, geneticists and neuro-radiologists. The second illustrates the importance of interaction among clinical dysmorphologists for pattern recognition of syndromes with multiple congenital anomalies. The third example shows how fruitful it can be to step out of the “clinical comfort zone”, and interact with basic scientists in applying emerging technologies to solve the diagnostic puzzles.

ObjectiveExamine the associations of training volume and longest endurance run with (half‐)marathon performance and running‐related injuries (RRIs) in recreational runners.Materials and MethodsDuring the preparation for and directly after the running event, 556 participants of a half marathon and 441 participants of a marathon completed three questionnaires on RRIs, average weekly training volume and the longest endurance run. With finish time, decline in pace during the running event and RRIs as dependent variables, linear and logistic regression analyses were performed to test the associations with weekly training volume and the longest endurance run.ResultsIn half‐marathon runners, a high training volume (>32 km/wk) (β −4.19, 95% CI: −6.52;−1.85) and a long endurance run (>21 km) (β −3.87, 95% CI: −6.31;‐−1.44) were associated with a faster finish time, while a high training volume (β −1.81, 95% CI: −3.49;−0.12) and a long endurance run (β −1.89, 95% CI: −3.65;−0.12) were also related to less decline in pace. In marathon runners, a low training volume (<40 km/wk) was related to a slower finish time (β 6.33, 95% CI: 0.18;12.48) and a high training volume (>65 km/wk) to a faster finish time (β −14.09, 95% CI: −22.47;−5.72), while a longest endurance run of <25 km was associated with a slower finish time (β 13.44, 95% CI: 5.34;21.55). No associations between training characteristics and RRIs were identified.ConclusionsPreparation for a (half‐)marathon with a relatively high training volume and long endurance runs associates with a faster finish time, but does not seem related to an increased injury risk.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation, dendritic and axonal growth. The relevance of ER-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by biallelic loss of function variants in Thioredoxin (TRX)Related Transmembrane-2 ( TMX2 ), detected by exome sequencing in fourteen affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria and other migration disorders. TMX2 encodes one of the five TMX proteins of the Protein Disulfide Isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER Mitochondria-Associated-Membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2 -deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration and organization in the developing brain. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Sophia Children's Hospital Department of Clinical Genetics Postal address P.O. box 2060 3000 CB Rotterdam, NL Visiting address Dr. Molewaterplein 40 3015 GD Rotterdam, NL Contact & route www.erasmusmc.nl Head of Department Prof. dr. R.M.W. Hofstra, PhD Heads of section Mr R.J.H. Galjaard, MD, PhD www.erasmusmc.nl To The Editor of the American Journal of Human Genetics Dr Sara Cullinan Direct dial +31-10-7036915 Fax number +31-10-7043072 Internal postal address Ee2018 E-mail ervo@erasmusmc.nl Our reference AJHG-D-19-00366-R4 Date October 3, 2019 Concerning AJHG-D-19-00366R3 , manuscript title: TMX2 is a key regulator of cellular redox state and its dysfunction causes severe brain developmental abnormalities.