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While it is widely thought that de novo mutations (DNMs) occur randomly, we previously showed that some DNMs are enriched because they are positively selected in the testes of aging men. These "selfish" mutations cause disorders with a shared presentation of features, including exclusive paternal origin, significant increase of the father's age, and high apparent germline mutation rate. To date, all known selfish mutations cluster within the components of the RTK-RAS-MAPK signaling pathway, a critical modulator of testicular homeostasis. Here, we demonstrate the selfish nature of the SMAD4 DNMs causing Myhre syndrome (MYHRS). By analyzing 16 informative trios, we show that MYHRS-causing DNMs originated on the paternally derived allele in all cases. We document a statistically significant epidemiological paternal age effect of 6.3 years excess for fathers of MYHRS probands. We developed an ultra-sensitive assay to quantify spontaneous MYHRS-causing SMAD4 variants in sperm and show that pathogenic variants at codon 500 are found at elevated level in sperm of most men and exhibit a strong positive correlation with donor's age, indicative of a high apparent germline mutation rate. Finally, we performed in vitro assays to validate the peculiar functional behavior of the clonally selected DNMs and explored the basis of the pathophysiology of the different SMAD4 sperm-enriched variants. Taken together, these data provide compelling evidence that SMAD4, a gene operating outside the canonical RAS-MAPK signaling pathway, is associated with selfish spermatogonial selection and raises the possibility that other genes/pathways are under positive selection in the aging human testis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Pathogenic LZTR1 variants cause schwannomatosis and dominant/recessive Noonan syndrome (NS). We aim to establish an association between heterozygous loss-of-function LZTR1 alleles and isolated multiple café-au-lait macules (CaLMs)., Methods: A total of 849 unrelated participants with multiple CaLMs, lacking pathogenic/likely pathogenic NF1 and SPRED1 variants, underwent RASopathy gene panel sequencing. Data on 125 individuals with heterozygous LZTR1 variants were collected for characterizing their clinical features and the associated molecular spectrum. In vitro functional assessment was performed on a representative panel of missense variants and small in-frame deletions., Results: Analysis revealed heterozygous LZTR1 variants in 6.0% (51/849) of participants, exceeding the general population prevalence. LZTR1-related CaLMs varied in number, displayed sharp or irregular borders, and were generally isolated but occasionally associated with features recurring in RASopathies. In 2 families, CaLMs and schwannomas co-occurred. The molecular spectrum mainly consisted of truncating variants, indicating loss-of-function. These variants substantially overlapped with those occurring in schwannomatosis and recessive NS. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate mitogen-activated protein kinase signaling., Conclusion: Our findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive NS, occur in individuals with isolated multiple CaLMs., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Purpose: KBG syndrome (KBGS) is a rare neurodevelopmental syndrome caused by haploinsufficiency of ANKRD11. The childhood phenotype is extensively reported but limited for adults. Thus, we aimed to delineate the clinical features of KBGS., Methods: We collected physician-reported data of adults with molecularly confirmed KBGS through an international collaboration. Moreover, we undertook a systematic literature review to determine the scope of previously reported data., Results: The international collaboration identified 36 adults from 31 unrelated families with KBGS. Symptoms included mild/borderline intellectual disability (n = 22); gross and/or fine motor difficulties (n = 15); psychiatric and behavioral comorbidities including aggression, anxiety, reduced attention span, and autistic features (n = 26); nonverbal (n = 3), seizures with various seizure types and treatment responses (n = 10); ophthalmological comorbidities (n = 20). Cognitive regression during adulthood was reported once. Infrequent features included dilatation of the ascending aorta (n = 2) and autoimmune conditions (n = 4). Education, work, and residence varied, and the diversity of professional and personal roles highlighted the range of abilities seen. The literature review identified 154 adults reported across the literature, and we have summarized the features across both data sets., Conclusion: Our study sheds light on the long-term neurodevelopmental outcomes, seizures, behavioral and psychiatric features, and education, work, and living arrangements for adults with KBGS., Competing Interests: Conflict of Interest The authors declare no conflicts of interests., (Copyright © 2024 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Abstract: In physiological conditions, few circulating hematopoietic stem/progenitor cells (cHSPCs) are present in the peripheral blood, but their contribution to human hematopoiesis remain unsolved. By integrating advanced immunophenotyping, single-cell transcriptional and functional profiling, and integration site (IS) clonal tracking, we unveiled the biological properties and the transcriptional features of human cHSPC subpopulations in relationship to their bone marrow (BM) counterpart. We found that cHSPCs reduced in cell count over aging and are enriched for primitive, lymphoid, and erythroid subpopulations, showing preactivated transcriptional and functional state. Moreover, cHSPCs have low expression of multiple BM-retention molecules but maintain their homing potential after xenotransplantation. By generating a comprehensive human organ-resident HSPC data set based on single-cell RNA sequencing data, we detected organ-specific seeding properties of the distinct trafficking HSPC subpopulations. Notably, circulating multi-lymphoid progenitors are primed for seeding the thymus and actively contribute to T-cell production. Human clonal tracking data from patients receiving gene therapy (GT) also showed that cHSPCs connect distant BM niches and participate in steady-state hematopoietic production, with primitive cHSPCs having the highest recirculation capability to travel in and out of the BM. Finally, in case of hematopoietic impairment, cHSPCs composition reflects the BM-HSPC content and might represent a biomarker of the BM state for clinical and research purposes. Overall, our comprehensive work unveiled fundamental insights into the in vivo dynamics of human HSPC trafficking and its role in sustaining hematopoietic homeostasis. GT patients' clinical trials were registered at ClinicalTrials.gov (NCT01515462 and NCT03837483) and EudraCT (2009-017346-32 and 2018-003842-18)., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors., (© 2024. The Author(s).)

ANKRD11 (ankyrin repeat domain 11) is a chromatin regulator and the only gene associated with KBG syndrome, a rare neurodevelopmental disorder. We have previously shown that Ankrd11 regulates murine embryonic cortical neurogenesis. Here, we show a novel olfactory bulb phenotype in a KBG syndrome mouse model and two diagnosed patients. Conditional knockout of Ankrd11 in murine embryonic neural stem cells leads to aberrant postnatal olfactory bulb development and reduced size due to reduction of the olfactory bulb granule cell layer. We further show that the rostral migratory stream has incomplete migration of neuroblasts, reduced cell proliferation as well as aberrant differentiation of neurons. This leads to reduced neuroblasts and neurons in the olfactory bulb granule cell layer. In vitro, Ankrd11-deficient neural stem cells from the postnatal subventricular zone display reduced migration, proliferation, and neurogenesis. Finally, we describe two clinically and molecularly confirmed KBG syndrome patients with anosmia and olfactory bulb and groove hypo-dysgenesis/agenesis. Our report provides evidence that Ankrd11 is a novel regulator of olfactory bulb development and neuroblast migration. Moreover, our study highlights a novel clinical sign of KBG syndrome linked to ANKRD11 perturbations in mice and humans., (© 2024. The Author(s).)

Quiescent human hematopoietic stem cells (HSC) are ideal targets for gene therapy applications due to their preserved stemness and repopulation capacities; however, they have not been exploited extensively because of their resistance to genetic manipulation. We report here the development of a lentiviral transduction protocol that overcomes this resistance in long-term repopulating quiescent HSC, allowing their efficient genetic manipulation. Mechanistically, lentiviral vector transduction of quiescent HSC was found to be restricted at the level of vector entry and by limited pyrimidine pools. These restrictions were overcome by the combined addition of cyclosporin H (CsH) and deoxynucleosides (dNs) during lentiviral vector transduction. Clinically relevant transduction levels were paired with higher polyclonal engraftment of long-term repopulating HSC as compared with standard ex vivo cultured controls. These findings identify the cell-intrinsic barriers that restrict the transduction of quiescent HSC and provide a means to overcome them, paving the way for the genetic engineering of unstimulated HSC., Competing Interests: Declaration of interests E.V., G.U., F.P., and A.K.-R. are inventors on pending and issued patents on lentiviral gene transfer filed by the Telethon Foundation and the San Raffaele Scientific Institute., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

High-throughput clonal tracking in patients under hematopoietic stem cell gene therapy with integrating vector is instrumental in assessing bio-safety and efficacy. Monitoring the fate of millions of transplanted clones and their progeny across differentiation and proliferation over time leverages the identification of the vector integration sites, used as surrogates of clonal identity. Although γ-tracking retroviral insertion sites (γ-TRIS) is the state-of-the-art algorithm for clonal identification, the computational drawbacks in the tracking algorithm, based on a combinatorial all-versus-all strategy, limit its use in clinical studies with several thousands of samples per patient. We developed the first clonal tracking graph database, InCliniGene (https://github.com/calabrialab/InCliniGene), that imports the output files of γ-TRIS and generates the graph of clones (nodes) connected by arches if two nodes share common genomic features as defined by the γ-TRIS rules. Embedding both clonal data and their connections in the graph, InCliniGene can track all clones longitudinally over samples through data queries that fully explore the graph. This approach resulted in being highly accurate and scalable. We validated InCliniGene using an in vitro dataset, specifically designed to mimic clinical cases, and tested the accuracy and precision. InCliniGene allows extensive use of γ-TRIS in large gene therapy clinical applications and naturally realizes the full data integration of molecular and genomics data, clinical and treatment measurements and genomic annotations. Further extensions of InCliniGene with data federation and with application programming interface will support data mining toward precision, personalized and predictive medicine in gene therapy. Database URL:  https://github.com/calabrialab/InCliniGene., (© The Author(s) 2023. Published by Oxford University Press.)

Competing Interests: None declared.

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome., (© 2023. The Author(s).)

Adeno-associated virus (AAV) vectors have been successfully exploited in gene therapy applications for the treatment of several genetic disorders. AAV is considered an episomal vector, but it has been shown to integrate within the host cell genome after the generation of double-strand DNA breaks or nicks. Although AAV integration raises some safety concerns, it can also provide therapeutic benefit; the direct intrathymic injection of an AAV harboring a therapeutic transgene results in integration in T-cell progenitors and long-term T-cell immunity. To assess the mechanisms of AAV integration, we retrieved and analyzed hundreds of AAV integration sites from lymph node-derived mature T cells and compared these with liver and brain tissue from treated mice. Notably, we found that although AAV integrations in the liver and brain were distributed across the entire mouse genome, >90% of the integrations in T cells were clustered within the T-cell receptor α, β, and γ genes. More precisely, the insertion mapped to DNA breaks created by the enzymatic activity of recombination activating genes (RAGs) during variable, diversity, and joining recombination. Our data indicate that RAG activity during T-cell receptor maturation induces a site-specific integration of AAV genomes and opens new therapeutic avenues for achieving long-term AAV-mediated gene transfer in dividing cells., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Longitudinal clonal tracking studies based on high-throughput sequencing technologies supported safety and long-term efficacy and unraveled hematopoietic reconstitution in many gene therapy applications with unprecedented resolution. However, monitoring patients over a decade-long follow-up entails a constant increase of large data volume with the emergence of critical computational challenges, unfortunately not addressed by currently available tools. Here we present ISAnalytics, a new R package for comprehensive and high-throughput clonal tracking studies using vector integration sites as markers of cellular identity. Once identified the clones externally from ISAnalytics and imported in the package, a wide range of implemented functionalities are available to users for assessing the safety and long-term efficacy of the treatment, here described in a clinical trial use case for Hurler disease, and for supporting hematopoietic stem cell biology in vivo with longitudinal analysis of clones over time, proliferation and differentiation. ISAnalytics is conceived to be metadata-driven, enabling users to focus on biological questions and hypotheses rather than on computational aspects. ISAnalytics can be fully integrated within laboratory workflows and standard procedures. Moreover, ISAnalytics is designed with efficient and scalable data structures, benchmarked with previous methods, and grants reproducibility and full analytical control through interactive web-reports and a module with Shiny interface. The implemented functionalities are flexible for all viral vector-based clonal tracking applications as well as genetic barcoding or cancer immunotherapies., (© The Author(s) 2022. Published by Oxford University Press.)

KBG syndrome (KBGS) is characterized by distinctive facial gestalt, short stature and variable clinical findings. With ageing, some features become more recognizable, allowing a differential diagnosis. We aimed to better characterize natural history of KBGS. In the context of a European collaborative study, we collected the largest cohort of KBGS patients (49). A combined array- based Comparative Genomic Hybridization and next generation sequencing (NGS) approach investigated both genomic Copy Number Variants and SNVs. Intellectual disability (ID) (82%) ranged from mild to moderate with severe ID identified in two patients. Epilepsy was present in 26.5%. Short stature was consistent over time, while occipitofrontal circumference (median value: -0.88 SD at birth) normalized over years. Cerebral anomalies, were identified in 56% of patients and thus represented the second most relevant clinical feature reinforcing clinical suspicion in the paediatric age when short stature and vertebral/dental anomalies are vague. Macrodontia, oligodontia and dental agenesis (53%) were almost as frequent as skeletal anomalies, such as brachydactyly, short fifth finger, fifth finger clinodactyly, pectus excavatum/carinatum, delayed bone age. In 28.5% of individuals, prenatal ultrasound anomalies were reported. Except for three splicing variants, leading to a premature termination, variants were almost all frameshift. Our results, broadening the spectrum of KBGS phenotype progression, provide useful tools to facilitate differential diagnosis and improve clinical management. We suggest to consider a wider range of dental anomalies before excluding diagnosis and to perform a careful odontoiatric/ear-nose-throat (ENT) evaluation in order to look for even submucosal palate cleft given the high percentage of palate abnormalities. NGS approaches, following evidence of antenatal ultrasound anomalies, should include ANKRD11., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features., (© 2022 Wiley Periodicals LLC.)

Mutations in the SZT2 gene have been associated with developmental and epileptic encephalopathy-18, a rare severe autosomal recessive neurologic disorder, characterized by psychomotor impairment/intellectual disability, dysmorphic facial features and early onset of refractory seizures. Here we report the generation of the first induced pluripotent stem cell (iPSC) lines from a patient with treatment-resistant epilepsy, carrying compound heterozygous mutations in SZT2 (Mut1: c.498G>T and Mut2: c.6553C>T), and his healthy heterozygous parents. Peripheral blood mononuclear cells were reprogrammed by a non-integrating Sendai virus-based reprogramming system. The generated human iPSC lines exhibited expression of the main pluripotency markers, the potential to differentiate into all three germ layers and presented a normal karyotype. These lines represent a valuable resource to study neurodevelopmental alterations, and to obtain mature, pathology-relevant neuronal populations as an in vitro model to perform functional assays and test the patient’s responsiveness to novel antiepileptic treatments.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA., Competing Interests: K.P., D.M.M., and P.M.T. are/have been employees of Sernova Corp., which holds the patent US20190240375A1., (© 2021 The Authors.)