Ataluren Treatment Improves Hematopoietic and Pancreatic Disorders in Patients with Shwachman-Diamond Syndrome

Ataluren Treatment Improves Hematopoietic and Pancreatic Disorders in Patients with Shwachman-Diamond Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication Ataluren Treatment Improves Hematopoietic and Pancreatic Disorders in Patients with Shwachman-Diamond Syndrome Marco Cipolli, Valentino Bezzerri, Anna Pegoraro, Anca Hristodor, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5231941/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Shwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and a high risk of myeloid malignancy. Most patients with SDS harbor nonsense mutations in Shwachman-Bodian-Diamond syndrome gene ( SBDS) , which encodes a ribosome assembly factor. We investigated the translational read-through effect of ataluren in three patients with SDS. The primary and secondary endpoints were restoring SBDS protein levels in hematopoietic cells and improving myelopoiesis, respectively. SBDS synthesis increased in hematopoietic cells, whereas the bone marrow showed improved cellularity with the maturation of myeloid progenitors. The exocrine pancreatic function also improved. Thus, this clinical study strongly encourages the further clinical development of ataluren to treat SDS. Health sciences/Medical research/Drug development Health sciences/Medical research/Genetics research Figures Figure 1 Figure 2 Full Text Shwachman-Diamond syndrome (SDS) is a rare genetic disease that results from biallelic mutations in the Shwachman-Bodian-Diamond syndrome ( SBDS ) gene. 1 SDS involves exocrine pancreatic insufficiency, skeletal abnormalities, cognitive impairment, and neutropenia. 2 Affected individuals may develop bone marrow aplasia or, more frequently, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). 3 SBDS interacts with elongation factor-like 1 (EFL1), a GTPase that facilitates the release of eukaryotic initiation factor (EIF6) and promotes the assembly of the large and small ribosomal subunits to form functional ribosomes. 4 Failure to produce full-length SBDS induces ribosomal stress, resulting in SDS. The two most common mutations in SBDS are c.258+2T>C and c.183-184TA>CT. The first is a splicing mutation, and the second introduces an in-frame stop codon (K62X). International registries of patients with SDS report that more than 50% of these patients are compound heterozygotes consisting of c.258+2T>C and c.183–184TA>CT mutations. 5 Ataluren 6 is an orally available translational read-through-inducing drug approved in Europe for the treatment of Duchenne muscular dystrophy (DMD). 7 Ataluren inhibits the activity of the release factor complex termination activity in ribosomes, forcing the suppression of nonsense mutations. 8 Cellular and animal models have demonstrated in vitro translational read-through efficacy of ataluren. 9-11 However, the clinical evaluation of ataluren remains understudied. In DMD, real-world treatment with ataluren delayed disease progression compared to patients undergoing standard care therapy. 7,12 We reported the beneficial effects of ataluren in ex vivo SDS cell models, showing a significant increase in neomorphic SBDS protein followed by an improved myelopoiesis and reduced expression of biomarkers, including TP53 and MTOR. 13 Based on these pre-clinical data, we initiated an ataluren compassionate program in three patients with SDS carrying the c.183-184TA>CT nonsense mutation in SBDS . During treatment, none of the three patients reported adverse events. The therapy was well-tolerated. SBDS protein levels were normalized in bone marrow mononuclear cells (BM-MNC) isolated from UPN26 and UPN58 patients ( Fig. 1a and Extended data Fig. 1a) . Because of decreased bone marrow cellularity, western blot analysis could not be performed on UPN74 BM-MNC. Therefore, we assessed the effects of ataluren therapy on PBMC isolated from this patient. Western blot analysis showed a remarkable increase in SBDS synthesis after either 3 or 6 months of therapy ( Extended data Fig. 1b ). Bone marrow biopsies obtained from UPN26, UPN58, and UPN74 before the initiation of ataluren therapy showed hypocellularity and decreased maturation of the hematopoietic lineages ( Fig. 1b ). Two study-blinded evaluators reviewed the embedded bone marrow tissues and agreed on the observation of a remarkable increase in cellularity, accompanied by improved maturation of myeloid elements after six months of treatment in all three patients ( Fig. 1b ). The colony assays consistently indicated a steady increase in myeloid colony formation ( Fig. 1c and Extended data Fig. 1c ). We determined the effect of the drug on peripheral blood cells. The number of neutrophils was significantly increased in UPN26 and UPN74 patients after 12 months of treatment compared to the mean of three analyses performed in the last two years before therapy initiation. In contrast, no effect was observed with UPN58 ( Extended data Fig. 2a-b ). The platelet count improved in all three patients treated with ataluren ( Extended data Fig. 2c-d ). No appreciable effect was observed on hemoglobin and reticulocytes ( Supplementary Table 1 ). These results correlated with those of erythroid colony assays ( Extended data Fig. 1c ) and with previously published data on ex vivo efficacy of ataluren in BM-MNC. 13 No statistically significant changes were observed in total leukocyte and monocyte counts after one year of treatment ( Supplementary Table 1 ). Patients with SDS have fewer B cells, mainly because of a severe deficiency in the B memory compartment. 14 In this study, we showed that ataluren restored the normal number of memory B cells (CD19 + and CD27 + ) in all patients who underwent therapy for 12 months ( Extended data Fig. 2e-g ). In particular, the number of non-switched (CD27 + , IgD + ) B memory cells was increased by ataluren. In parallel, exhausted B memory cells (CD27 - , IgD - ) decreased after treatment. The total number of B cells and T lymphocytes did not show substantial changes after 12 months of therapy ( Supplementary Table 1 ). SDS is associated with a hyper-phosphorylation of MTOR Ser2448 15 with possible activation of both MTORC1 and MTORC2 complexes, modulating MTOR pathway activation, which in turn may regulate cell growth and proliferation, survival, and metabolism. At the baseline, we observed a 2.4-fold increase in phospho-mTOR levels in SDS leukocytes compared to healthy donors. After six months of treatment, we observed a 34% decrease in mean mTOR levels in white blood cell count (WBC) ( Fig. 2a ). Phospho-mTOR levels were significantly reduced by 22% in lymphocytes ( Fig. 2b ), and by 38% in monocytes ( Fig. 2c ). We failed to observe a notable increase in phospho-mTOR levels in SDS neutrophils compared to those in healthy donors ( Fig. 2d ). To assess whether the inhibition of MTOR activation could rescue myelopoiesis, we evaluated the effects of the MTOR inhibitors everolimus (RAD001) and dactolisib (NVP-BEZ-235) on myeloid differentiation in bone marrow progenitor cells isolated from patients with SDS using colony assays. Inhibition of MTOR did not improve granulopoiesis or erythropoiesis ( Extended data Fig. 3 ), suggesting that ataluren-induced MTOR activation was a secondary effect due to the restoration of SBDS protein synthesis, followed by improved overall cellular fitness. All three patients reported exocrine pancreatic insufficiency at enrollment ( Table 1 ). After one year of treatment, the release of pancreatic enzymes fecal elastase-1 and amylase increased. Fecal elastase-1 levels displayed 1.3-, 4.6-, and 2.5-fold increases in subjects UPN26, UPN58, and UPN74, respectively ( Fig. 2e ). Serum total amylase levels improved similarly in all the patients ( Fig. 2f ). Currently, hematopoietic stem cell transplantation is the only effective therapy; however, it is not required for most patients. Herein, we report the first pharmacological treatment to improve hematopoiesis in SDS through nonsense suppression. Over the past decade, several studies on ataluren in rare diseases caused by nonsense mutations have yielded ambivalent results. However, patients with SDS carry only one specific nonsense mutation, which reduces the variability of the mutation-dependent efficacy of treatment. Despite the ubiquitous cellular need for ribosomes, one curious feature is that ribosomopathies have tissue-limited phenotypes. Ataluren efficacy can also vary in a tissue-dependent manner. 16 Here, we found an improvement in both hematopoietic and exocrine pancreatic tissues. In addition, the primary endpoint of this study measured the direct effect of the drug (i.e., restoration of SBDS protein in the BM and improved hematopoiesis), whereas previous clinical trials for DMD and CF determined only surrogate endpoints, such as cardiopulmonary function measured by the six-minute walk test and respiratory function measured as forced expiratory volume in the first second (FEV1). The major limitation of this study was its small sample size. Considering that SDS is ultra-rare, with an incidence of approximately 1:160,000 live births, 17 and that ataluren treatment can be administered only to patients carrying nonsense mutations, three patients should be considered a reliable starting point for designing larger multicenter clinical trials. Although previous clinical trials on the effect of ataluren on cystic fibrosis and DMD dampened enthusiasm for this drug, the results of our study should encourage both the scientific community and the pharmaceutical company owner of the patent to continue the drug repurposing of ataluren for SDS. Furthermore, decreased ribosomal stress due to neomorphic SBDS may reduce the lifetime risk of myeloid malignancies in patients with SDS. Declarations Acknowledgments. This work was supported in part by the Italian Association for Shwachman-Diamond Syndrome (AISS, to VB and MC), NIH R01DK132812 (to SJC), Lisa Dean Moseley Foundation, VeloSano, and 2nd Chance 4 Kids. We thank PTC Therapeutics Inc. (NJ, USA) for providing the drug covering manufacturing costs and Fondazione Telethon (Milan, Italy), which contributed to the clinical development of ataluren. We are grateful to Federica Quiri (Cystic Fibrosis Center, Verona, Italy) and Angela Mercuri (University of Verona) for their excellent technical assistance, Antonella Minelli, Cesare Danesino (University of Pavia, Italy), and Barouk Maurice Assael (Adult Cystic Fibrosis Center, IRCCS Ca’ Granda, Milan, Italy) for helpful discussions, Francesca Buniotto and Sandra Perobelli (Cystic Fibrosis Center, Verona, Italy) for her contribution to the psychological assessment of patients during therapy. Data availability De-identified patient data and source data are available in supplementary information. All original clinical data can be obtained by contacting the chief investigator (M.C.). Individual patient data will be shared in a de-identified and anonymized format, following our data-sharing process. References Boocock, G.R., et al. Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet 33, 97–101 (2003). Nelson, A.S. & Myers, K.C. Diagnosis, Treatment, and Molecular Pathology of Shwachman-Diamond Syndrome. Hematol Oncol Clin North Am 32, 687–700 (2018). Donadieu, J., et al. Classification of and risk factors for hematologic complications in a French national cohort of 102 patients with Shwachman-Diamond syndrome. Haematologica 97, 1312–1319 (2012). Warren, A.J. Molecular basis of the human ribosomopathy Shwachman-Diamond syndrome. Adv Biol Regul 67, 109–127 (2018). Thompson, A.S., et al. Shwachman Diamond syndrome: narrow genotypic spectrum and variable clinical features. Pediatric Research 92, 1671–1680 (2022). Welch, E.M., et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 447, 87–91 (2007). McDonald, C.M., et al. Ataluren delays loss of ambulation and respiratory decline in nonsense mutation Duchenne muscular dystrophy patients. J Comp Eff Res (2021). Huang, S., et al. Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination. Nat Commun 13, 2413 (2022). Sermet-Gaudelus, I., et al. Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care Med 182, 1262–1272 (2010). Li, M., Andersson-Lendahl, M., Sejersen, T. & Arner, A. Muscle dysfunction and structural defects of dystrophin-null sapje mutant zebrafish larvae are rescued by ataluren treatment. FASEB J 28, 1593–1599 (2014). Wang, X., et al. Efficacy of Postnatal In Vivo Nonsense Suppression Therapy in a Pax6 Mouse Model of Aniridia. Mol Ther Nucleic Acids 7, 417–428 (2017). Mercuri, E., et al. Safety and effectiveness of ataluren: comparison of results from the STRIDE Registry and CINRG DMD Natural History Study. J Comp Eff Res 9, 341–360 (2020). Cipolli, M., et al. Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells. Br J Haematol (2023). Bezzerri, V., et al. Peripheral blood immunophenotyping in a large cohort of patients with Shwachman-Diamond syndrome. Pediatr Blood Cancer 66, e27597 (2019). Vella, A., et al. mTOR and STAT3 Pathway Hyper-Activation is Associated with evated Interleukin-6 Levels in Patients with Shwachman-Diamond Syndrome: Further Evidence of Lymphoid Lineage Impairment. Cancers (Basel) 12(2020). Thada, V., Miller, J.N., Kovács, A.D. & Pearce, D.A. Tissue-specific variation in nonsense mutant transcript level and drug-induced read-through efficiency in the Cln1(R151X) mouse model of INCL. J Cell Mol Med 20, 381–385 (2016). Minelli, A., et al. Incidence of Shwachman-Diamond syndrome. Pediatr Blood Cancer 59, 1334–1335 (2012). Tables Table 1 Clinical and laboratory data of patients enrolled in the ataluren study T Age Sex Height (cm) Weight (Kg) BMI FE-1 (µg/g) Conc. Med. WBC (10 9 /L) RBC (10 12 /L) Hb (g/dL) ANC (10 9 /L) PLT (10 9 /L) MCV (fL) Cytogenetics BM [mitosis] aCGH UPN26 T0 20 M 174 58.8 19.4 77.8 Vit, PERT 2.44 4.98 15.1 0.63 97 90 46,XY [1] del(20)(q11.21-q11.23) ~ 19% T9 21 174 58.8 19.4 97.2 Vit, PERT 2.27 4.81 14.9 0.87 112 92.1 46,XY [9] del(20)(q11.21-q11.23) ~ 19% UPN58 T0 16 M 160 42.3 16.5 15 Vit, PERT 2.14 4.17 13.2 0.38 97 95.7 46,XY [6] dup(1q)(q21.1-q44) ~ 16% T9 17 161 42.1 16.2 68.5 Vit, PERT 2.33 4.09 13.3 0.51 136 98.8 N/A dup(1q)(q21.1-q44) ~ 18% UPN74 T0 13 M 152 37.5 16.2 14 Vit, PERT 2.88 4.56 14.3 0.94 113 91.2 46,XY,del(20)(q11-q13)[2]/46,XY[16 del(20)(q11.22-q13.1) ~ 10% T9 14 159 43.1 17 34.6 Vit, PERT 3.07 4.73 14.9 0.97 143 92.4 46,XY,del(20)(q11- q13)[1]/46,XY[12] del(20)(q11.22-q13.1) ~ 12% T0 (initiation of therapy), T9 (12 months of therapy); M, male; BMI, body mass index; FE-1, fecal elastase-1); Conc. Med., concomitant medications; WBC, white blood cell; RBC, erythrocytes; Hb, hemoglobin; ANC, absolute neutrophil count; PLT, platelet count; MCV, mean corpuscular volume; BM, bone marrow; aCGH, array comparative genomic hybridization; Vit, vitamin (A,D,E,K) supplementation; PERT, pancreatic enzyme replacement therapy; N/A, not available (unsuitable sample). Online Methods Patient recruitment . Three adolescent male patients with SDS ( Table 1 ) of the same genotype (c.183-184TA>CT/c.258+2T>C) were enrolled for receiving ataluren for 12 months under a compassionate program. For the colony assays, six additional patients and three healthy donors were recruited ( Supplementary Table 2 ). Informed consent was obtained from the local Ethics Committee (approval no. 4090 CESC and nr. 4182 CESC) in agreement with the Declaration of Helsinki. Study protocol . Patients will be evaluated in the clinic before treatment initiation (T0), after 2 weeks (T1), every month (T2–T7) for 6 months, then every three months until 12 months (T7–T9) of treatment. In addition to routine monitoring (vital signs, height and weight, and physical examinations), hematology laboratory assessment, liver and renal function tests, serum electrolytes, urinalysis, and a review of concomitant medications will be performed at each clinic visit. Purpose. The objective of this compassionate use is to offer drug access to ataluren to provide a potential clinical benefit to patients diagnosed with Shwachman-Diamond Syndrome who have a high unmet medical need according to the Italian Ministerial Directive (DM) 07.09.2017. Eligibility. Patients (males, females) with SDS diagnosis carrying the c183-184ta>ct mutation in at least one allele, >= 6 years will be eligible to receive the drug (ataluren) for six months and depending on the medical judgment, treatment with ataluren may be extended for another six months. The patient will start treatment with ataluren only after informed consent has been provided (including the Information Sheet For The Processing Of Personal Information) and if he/she fulfills all inclusion/exclusion criteria. Inclusion criteria Patients with a diagnosis of SDS carrying in at least one allele c183-184ta>ct mutation Age >= 6 yrs Neutrophil count < 1000 PMN/mm3 at T0 and at least one evaluation performed within twelve months before patient enrollment. Bone marrow evaluation within 12 months of the patient's enrollment Serum total bilirubin within normal limits; serum ALT, AST, or GGT <=2.0 times the ULN, creatinine, BUN within normal limits Evidence of signed and dated informed consent and assent documents (s). Note: If the patient is considered a child under local regulations, the parents or legal guardians must provide written consent, and the patient may be required to provide written consent. In patients who are sexually active, willingness to abstain from sexual intercourse or employ a barrier or medical method of contraception during ataluren administration and the 60-d follow-up period. Able to understand and comply with treatment requirements, restrictions and instruction (as judged by the treating physician). Exclusion criteria Presence of myelodysplasia or leukemia Bone marrow transplantation Blood transfusions in the past three months High value (> 4%) of HbF according to medical evaluation Neutrophil count < 300 PMN/mm3 Serologic evidence of hepatitis B or C, or of HIV Severe abnormalities of pulmonary or renal function Ongoing intravenous (IV) aminoglycoside or IV vancomycin Pregnancy or breastfeeding. Ongoing warfarin, phenytoin, or tolbutamide therapy. Hypersensitivity to any of the ingredients or excipients of the study drug (polydextrose, polyethylene glycol 3350, poloxamer 407, mannitol 25C, crospovidone XL10, hydroxyethyl cellulose, vanilla, colloidal silica, or magnesium stearate). History of solid organ or hematological transplantation Prior or ongoing medical conditions (such as concomitant illness, alcoholism, drug abuse, and psychiatric condition), medical history, physical findings, ECG findings, or laboratory abnormalities that, in the investigator’s opinion, could adversely affect the safety of the subject, making it unlikely that the course of treatment or follow-up would be completed or could impair the assessment of study results. Inability to understand the informed consent Eligibility assessments. Eligibility assessments should occur before the administration of ataluren. The following eligibility assessments should be performed and documented by the treating physician: Review of eligibility criteria Collection of signed informed consent and assent (as applicable) Review of medical history (including SDS genotype) Liver and renal function tests and serum electrolytes (including ALT, AST, GGT, ALP, total bilirubin, cystatin C, BUN, urine protein, serum Na+, serum K+, serum Mg2+, serum Ca2+, serum phosphorous, and serum HCO3-) Urine and serum pregnancy test for females of childbearing potential as judged by the treating physician Complete blood count and peripheral blood immunophenotyping will be performed on the first day of treatment (before the treatment) Review of prior and concomitant medications Assessment and monitoring during the therapy with ataluren. Treating physicians should refer to the investigator’s brochure to summarize the ataluren data. Patients should be evaluated in the clinic on the first day of treatment and after 2 weeks, 1, 2, 3, 4, 5, and 6 months. If treatment with ataluren is prolonged for another six months, patients should be evaluated at the third and sixth months of this extension period. In addition to routine monitoring (such as vital signs, height and weight, and physical examinations), additional monitoring may occur, if necessary, by the treating physician. Ataluren administration and management. The ataluren regimen may be dispensed only under the supervision of the treating physician or an authorized designee and only for administration to the patient participating in this compassionate use. Ataluren will be provided as granules for oral suspension with a white to off-white powder appearance. Drug substances and products are manufactured under cGMP conditions. The formulation includes a matrix, suspending agents, surfactants, and various excipients that aid manufacturing. The granules for oral suspension are packaged in aluminium foil and child-resistant sachets (packets) and supplied with doses of 125, 250, or 1000 mg of the active drug substance. The powder in the sachet may be mixed with water, fruit juice, fruit punch, milk (skim milk, 1% fat, 2% fat, whole milk, chocolate milk, soy milk, or lactose-free milk), or semisolid food (yogurt, pudding, or apple sauce). PTC Therapeutics, Inc. will provided the drug. Drug kits. Drug kits will be provided, each containing 30 sachets of one dose (125, 250, or 1000-mg). Sachets and cartons will be color coded to indicate the dosage strength (125 mg, brown; 250 mg, green; 1000 mg, dark blue). Drug dispensation. Ataluren will be administered three times per day (TID). The dose level to be administered is as follows: 10 mg/kg in the morning, 10 mg/kg at mid-day, and 20 mg/kg in the evening Storage and stability. Ataluren will be shipped and stored at room temperature (approximately 15 to 30°C). The available stability data from representative samples supported using the drug product for 48 months when stored at room temperature. Schedule of administration. Three doses should be administered per day – the 1st dose, 10 mg/kg, in the morning, the 2nd dose, 10 mg/kg, during the middle of the day (mid-day); and the 3rd dose, 20 mg/kg, in the evening. Ideally, each dose should be administered within approximately 30 min before or after a meal (e.g., at approximately 7:00 AM after breakfast, approximately 1:00 PM after lunch, and approximately 7:00 PM after dinner). Intervals for dosing should be ~6 h (±1 h) between morning and mid-day doses, approximately 6 h (±1 h) between mid-day and evening doses, and approximately 12 h (±1 h) between evening doses and the morning dose on the next day. Instructions for delays in dosing. Dosing delays should be handled as follows: If dosing of ataluren is delayed by ≤1 h, the planned dose should be taken with no changes to the subsequent dose schedules. If ataluren dosing is delayed by >1 h but ≤3 h (or more than 6 h after the evening dose), the planned dose should be taken; however, all future doses for that day should be shifted later by an approximately corresponding amount. If ataluren dosing is delayed by >3 h (or > 6 h after the evening dose), the dose should not be taken. Ataluren administration may continue, but the missed dose should not be administered, and the planned timing of subsequent study drug dosing should not be altered. Drug preparation and storage. Ataluren sachets should be stored at room temperature, away from the reach of the children, until reconstitution. They should only be opened at the time of dose preparation. The powder in the sachet may be mixed with water, milk (skim milk, 1% fat, 2% fat, whole milk, chocolate milk, soy milk, or lactose-free milk), or semi-solid food (yogurt, pudding, or apple sauce). The full contents of the sachets should be mixed with at least 30 mL of liquid (water, milk [skim, 1% fat, 2% fat, whole milk, chocolate milk, or lactose-free milk]), or three tablespoons of semi-solid food (yogurt, pudding, or apple sauce). The prepared dose should be thoroughly mixed before administration. The amount of liquid or semi-solid food can be increased based on patient preferences. Each prepared dose is best administered immediately after preparation. Laboratory abnormalities and adverse events requiring evaluation and potential drug interruption/modification. During ataluren treatment, the subjects will be closely monitored for adverse events or laboratory abnormalities. Renal abnormalities will be studied closely. For adverse events or laboratory abnormalities, treating physicians will use their judgment to determine whether the event or abnormality is clinically significant, whether a diagnostic evaluation is warranted, and whether potential interruption of the study drug treatment is appropriate. In general, life-threatening (grade 4) or severe (grade 3) adverse events or laboratory abnormalities should be considered clinically significant; however, recurrent or persistent moderate events (grade 2) may also be considered clinically significant in certain circumstances. The Common Terminology Criteria for Adverse Events (CTCAE) is used to grade the severity of adverse events and laboratory abnormalities. Concomitant and supportive therapy. Information regarding all concomitant medications will be collected and documented. Drugs metabolized by cytochrome P450 enzymes. As the primary route of ataluren metabolism is glucuronidation by UGT1A9, clinically significant interactions between ataluren and co-administered drugs metabolized by cytochrome P450 enzymes (CYPs) are unlikely. In particular, ataluren is not an inhibitor of CYP1A2, CYP2B6, CYP2C19, CYP2D6, or CYP3A4/5, and does not induce major CYP enzymes. Drugs that are metabolized by CYP2C8 or CYP2C9, which have low therapeutic indices (in particular, paclitaxel for CYP2C8 and coumarin anticoagulants [such as warfarin], phenytoin, or tolbutamide for CYP2C9), may be of particular concern. Patients who require the use of these drugs will not be enrolled in the study. Coumarin anticoagulants are cleared by CYP2C9, and increases in their plasma concentrations of coumarin anticoagulants may have serious clinical consequences. For patients who require anticoagulation therapy, the use of an alternative form of anticoagulation (e.g., fractionated heparin) should be considered. Phenytoin is metabolized by CYP2C9, and its concomitant use with ataluren may be of potential concern. For patients who require anticonvulsant therapy during the study, the use of alternative anticonvulsant drugs should be considered. The metabolism of losartan to its active metabolites may be mediated, in part, by CYP2C9. However, the concomitant use of losartan and CYP2C9 inhibitors remains unexamined. Because this drug does not have a narrow therapeutic window, the potential for mild-to-moderate changes in activity does not require dose modification. In vitro studies have shown that ataluren is a substrate for UGT1A9 and breast cancer-resistant protein (BCRP). Caution should be exercised when ataluren is co-administered with drugs that induce UGT1A9 (e.g., phenobarbital and rifampin) or inhibit BCRP (e.g., cyclosporine, eltrombopag, and gefitinib). For patients requiring systemic antibiotic therapy, intravenous aminoglycosides may be administered when necessary. Dietary restrictions. There are no specific dietary restrictions. Hydration. Because of the potential risk of renal dysfunction during periods of dehydration in patients receiving ataluren, it is important to encourage them to maintain adequate hydration throughout treatment. Subjects should be adequately hydrated before receiving any potentially nephrotoxic agent, and their hydration status should be carefully monitored throughout the administration of any agent with nephrotoxic characteristics. Treating physicians should be particularly vigilant of patients who experience nausea, vomiting, diarrhea, fever, or laboratory evidence of dehydration. AE and SAE documentation and reporting. An AE is defined as any untoward medical occurrence in a patient during treatment; the event does not necessarily have a causal relationship with the treatment. This includes any newly occurring event or worsening of a preexisting condition (e.g., an increase in its severity or frequency) after the ICF, and assent, if applicable, is signed. An SAE is any AE that meets any of the following criteria: Fatal (death, regardless of cause, that occurs during participation in compassionate use or death that occurs after participation in compassionate use and is suspected to be a delayed toxicity due to administration of ataluren). Life-threatening, such that the patient was at immediate risk of death from the reaction. •Inpatient hospitalization or prolonged hospitalization. •Persistent or significant disability/incapacity (disability is defined as a substantial disruption in a person’s ability to perform normal life functions). Congenital anomalies or birth defects. An important medical event that, based on appropriate medical judgment, may jeopardize the patient or require medical or surgical intervention to prevent one of the outcomes listed above (e.g., allergic bronchospasm requiring intensive treatment in an emergency room or at home). If a patient had a hospitalization or procedure (e.g., surgery) for an event or condition that occurred before the patient signed the ICF and/or provided assent, and the hospitalization or procedure was planned before the patient signed the ICF and/or provided assent, the hospitalization or procedure should not be considered to indicate an SAE unless an AE caused the hospitalization or procedure to be rescheduled sooner or prolonged relative to what was planned. In addition, hospitalizations not clearly associated with an AE (e.g., social hospitalization for respite care) should not be considered indicative of an SAE. Completion of safety information collection forms. The treating physician will complete the appropriate safety information collection form and submit it to PTC Therapeutics. Action taken with ataluren. The treating physician will classify the action taken with ataluren with regard to AE. AE outcome. An AE will be followed-up until the treating physician determines the final outcome. Reporting procedures for SAEs. The institution (Azienda Ospedaliera Universitaria Integrata di Verona) will alert PTC PV via the mailbox [email protected] when physicians have given products to patients, so they can track this internally. Institution will ensure that Physician notifies PTC Pharmacovigilance Department (“PTC PV”) immediately via the mailbox [email protected] within one business day of any serious adverse event (SAE), and to the extent permitted by applicable laws and regulations and to the extent able to do so under the circumstances, will reasonably cooperate with PTC in connection with any reports or filings to the competent authorities related to such SAE. Physicians and institutions will be responsible for submitting safety information according to the local EC requirements. The PTC and the physician shall manage all reports of suspicious adverse reactions according to the standards set out by the DM Salute, April 30, 2015, and manage all relevant information to the Ethics Committee. PTC shall promptly report to the Institution both during the Treatment Plan and for a reasonable period of time after the Treatment Plan is completed, any findings of PTC or its designees that could affect the safety or medical care of the patient, affect the willingness of the patient to continue participation in the Treatment Plan, influence the conduct of the Treatment Plan, or alter the EC’s approval to continue the Treatment Plan. Termination. PTC agrees to provide and deliver sufficient products for the treatment of the patients for six months at no cost to the physician and institution, for the sole purpose of treatment of the patients following the terms and conditions of this agreement (the “Initial Supply”). The PTC shall have no further obligation to supply products and no other financial or payment obligations whatsoever in connection with the patients, the Treatment Plan or this Agreement, except to the extent expressly set forth herein. After the Initial Supply, PTC will supply a product free of charge for an additional six months to patients, showing a proven clinical benefit at the end of the observational period. Under no circumstances shall the PTC have any obligation to provide products beyond these additional six months. At the end of the six-month period, the institution shall account for all quantities of product used for the patients and, unless otherwise agreed upon in writing by the parties, shall return or otherwise dispose of any remaining product per the PTC instructions. This compassionate use expires upon the completion of the Treatment Plan, except that any provisions would reasonably be expected to survive such a termination. Compassionate use will terminate prematurely if: - The patient withdraws consent and/or discontinues treatment with the product. - The physician determines that treatment with the product should be withdrawn based on the patient’s condition, inability to tolerate the Product or Physician’s clinical judgment. A Patient becomes eligible to participate in a clinical trial program initiated by the PTC in which the Product is the study drug. - Access to the product is discontinued by competent authorities or the PTC The product is commercially available in Italy under an approved label for treating Shwachman-Diamond Syndrome. - Either party materially breaches any terms of the agreement and fails to cure such breach within 30 d of the written notice of the same. - Physicians are no longer available at the institution to complete the Treatment Plan, and a replacement physician is not agreed to by the parties within 30 d of notification to PTC that Physician is no longer available at the institution; - PTC determines at its sole discretion whether the product is safe or ineffective for the patient. - The PTC no longer has access to the quantity or quality of products necessary to provide under the agreement. Review of concomitant medications. The concomitant medication use of the patient should be assessed during each clinic visit. Review of AEs and serious adverse events (SAEs). Treating physicians should review AEs and SAEs continuously throughout the participation of the patient (from signing the informed consent form [ICF] to assent, if applicable). Hematological assessment White blood cell counts with differential, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total red cell counts with morphology, and platelet count were performed at the section of Clinical Biochemistry, AOUI Verona, using the validated diagnostic procedures. The total white blood cell count (WBC) was determined by Hematology Analyzer XN- 9000 (Sysmex Europe GmbH, Norderstedt, Germany). Liver and renal function tests, serum electrolytes and urinalysis assessments Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyltranspeptidase (GGT), alkaline phosphatase (ALP), total bilirubin, cystatin C, nitrogenous products (BUN), urine protein, serum Na+, serum K+, serum Mg2+, serum Ca2+, serum phosphorous, and serum HCO3-, glucose, pH, specific gravity, ketones, blood, protein, creatinine, urobilinogen, bilirubin, nitrite, and leukocyte esterase were quantified using clinical chemistry and immunochemistry instruments (Architect C 16000 plus and Architect i2000sr plus; Abbott Diagnostics, Lake Forest, IL, USA). Bone marrow biopsies Bone marrow aspirates and biopsies were withdrawn after six months of treatment (approximately 12 months after the previous bone marrow biopsy) according to the standard follow-up for patients with SDS validated at AOUI Verona. Western Blot Total proteins were extracted from the bone marrow (BM-MNC) and peripheral blood (PBMC) mononuclear cells, separated by 11% SDS-PAGE, and electroblotted onto Immobilon P filters (Millipore, Billerica, MA, USA) previously blocked with 5% non-fat milk in TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl) supplemented with 0.05% Tween-20 (TBS/T). The membranes were probed with: i) anti-human SBDS rabbit polyclonal IgG antibody (Abcam, Cambridge, MA, dilution 1:1500); monoclonal anti-β-Actin clone AC-15 (Sigma-Aldrich, diluted 1:2000) in 5% non-fat milk TBS/T. Membranes were incubated overnight at 4°C and then incubated with the secondary antibody horseradish peroxidase-coupled anti-rabbit IgG (Sigma-Aldrich, dilution 1:15000), for 1 h. Immunocomplexes were detected using the ECL Plus Western Blotting Detection System (Amersham Biosciences, Little Chalfont, UK). mTOR Phosphorylation assay The phosphorylation level of mTOR (S2448) in peripheral blood leukocytes was tested using phospho-flow analysis at T0, T4, and T7. Briefly, total leukocytes from peripheral blood withdrawals were fixed and permeabilized using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience, San Diego, CA, USA) following the manufacturer's protocol. After permeabilization, leukocytes were washed once in flow buffer and stained with Pacific Blue-conjugated anti-p-S2448-mTOR (BD, Franklin Lakes, NJ, USA) or isotype control antibodies for 30 min. The cells were washed, and 13 colors, 4 lasers DX-Flex flow cytometer (Beckman Coulter). All acquired data files were analyzed using Kaluza software (Beckman Coulter). Immunophenotyping of peripheral blood leukocytes Blood samples were prepared according to the Clinical and Laboratory Standard Institute H42-A2 "Enumeration of Immunologically Defined Cell Populations by Flow Cytometry, 2nd Edition" Guidelines. Immunophenotyping was performed at T0, T4, and T7 using a 13-color DX-Flex flow cytometer (Beckman Coulter). The UK Neqas External Quality Assessment “Leucocyte Immunophenotyping” internal quality control (IQC) was used. The following fluorochrome-conjugated monoclonal antibodies were used: CD3-APC750, CD4-PC7, CD5-PC5, CD8-ECD, CD16-Pacific Blue, CD19-Chrome-Orange, CD23-ECD, CD27-PC5, CD38-APC750, CD45-APC700, CD56-PC5, and HLA-DR-PE (Beckman CA). Peripheral blood lymphocytes were gated into side-scatter (SSlow) and CD45 positive area. Within the lymphocytes, B cells were gated as CD19 positive events and subsequently divided into CD19+ CD5- (B2 or conventional B cells) and CD19+ CD5+ (B1a cells) subpopulations. T cells were defined as CD3+ events and further gated into CD4+ and CD8+ T cells; NK cells were identified as CD3/CD19 double-negative events expressing CD56 and/or CD16. B-cell subsets were separated into CD27- (naive B cells) and CD27+ (memory B cells), or CD23+ (activated B cells). CD4+ and CD8+ T lymphocytes (CD3+) were further distinguished using CD38 and HLA-DR (activation markers). A total of 25,000 events were detected during each run. Pancreatic activity test Levels of fecal elastase 1 (FE-1) were analyzed in stool samples (500 μg/g of wet stools) at T0, T7, and T9 by ELISA (ScheBo, Meridian Bioscience Europe, Milan, Italy). Stool samples were diluted in 50 μl assay buffer [0.1 M Tris·HCl (pH 8.0), 1 mM CaCl2, and 0.05% Tween 20] and added to the ELISA multi-wells containing the immobilized anti-elastase antibody. As blank, 50 µl of assay buffer were used. The plates were incubated at 22°C for 30 min. After washes, 50 µl of a biotinylated anti-elastase antibody were complexed with peroxidase-conjugated streptavidin and added to the wells. The samples were incubated at 22°C for 30 min in the dark. After washes, 100 µl of 2,2′-azino-bis(3-thylbenzothiazoline-6-sulfonic acid)-peroxidase substrate solution were added, and samples were incubated at 22°C for 20 min in darkness. Stop solution (100 µl) was added into each well and incubated for 15 min. The absorbance was measured at 405 nm using a Victor Nivo plate reader (Perkin Elmer, Waltham, MA, USA). Values were expressed as μg/g of wet stool. Colony assays BM-MNC freshly isolated from bone marrow biopsies were seeded at a density of 105 cell/ml in methylcellulose medium (StemMacs HSC-CFU human, Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 20ng/ml human recombinant G-CSF (Filgrastim). The medium was then supplemented with everolimus (350 nM, Selleckchem, Houston, TX, USA), dactolisib (NVP-BEZ-235, 300 nM, Selleckchem), or DMSO as the vehicle control (1:10000, Merck, Rahway, NJ). Spontaneous growth of CFU-GM and BFU-E was observed using an Axio Observer 7 (Zeiss, Oberkochen, Germany) inverted microscopy (magnification 5×) and colonies were counted after 7, 14, and 21 d of incubation at 37°C. Statistical analysis . The Shapiro–Wilk test was used to evaluate the normal distribution of samples, which enabled parametric or non-parametric tests to be selected. Independent group determination was performed using a two-tailed Student's t-test for paired or unpaired data. A p value < 0.05 was considered statistically significant. Statistical analyses were performed using Sigma Plot V14.0 (Systat Software Inc., San Jose, CA, USA). Additional Declarations Yes there is potential Competing Interest. Marco Cipolli and Valentino Bezzerri are inventors of the international patent WO2018/050706A1 "Method of treatment of Shwachman-Diamond syndrome". Other authors have no competing interests. Supplementary Files ExtendeddataBezzerrietalNatMed.docx SupplementaryInformationBezzerrietalNatMed.docx Cite Share Download PDF Status: Published Journal Publication published 02 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5231941","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Brief Communication","associatedPublications":[],"authors":[{"id":368702045,"identity":"1364615c-46bc-468a-80bd-9dc534bd05f1","order_by":0,"name":"Marco 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11:05:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5231941/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5231941/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-63137-3","type":"published","date":"2025-09-02T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68213738,"identity":"3fe884fc-c846-4df5-bec5-8bd31fa4edf6","added_by":"auto","created_at":"2024-11-04 18:27:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":397397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtaluren-dependent restoration of SBDS protein synthesis was followed by improved bone marrow cellularity and maturation in BM-MNC. \u003c/strong\u003eBone marrow biopsies and aspirates were performed after six months of therapy (T7). Total proteins were isolated from BM-MNC and paraffin-embedded bone marrow biopsies were processed for immunohistochemical analysis. Colony assays were performed, seeding 1×10\u003csup\u003e5\u003c/sup\u003e BM-MNC of each patient and healthy donor. \u003cstrong\u003ea\u003c/strong\u003e, Representative western blot analyses (UPN26) for SBDS before and after ataluren treatment. \u003cstrong\u003eb\u003c/strong\u003e, immunohistochemical analysis of bone marrow biopsies collected from each patient (UPN26, UPN58, and UPN74), before (T0) and after (T7) therapy. \u003cstrong\u003ec\u003c/strong\u003e, Representative colony assays demonstrating differences between patients with SDS and healthy donors (upper panel) and the effect of ataluren on SDS BM-MNC maturation at T7 (lower panel).\u003c/p\u003e","description":"","filename":"Figure1MSNatMedBriefFINAL.png","url":"https://assets-eu.researchsquare.com/files/rs-5231941/v1/b3311f9494dd97cab3fa9458.png"},{"id":68213737,"identity":"bfffbc67-e30a-457a-95fa-aab16e20053f","added_by":"auto","created_at":"2024-11-04 18:27:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37500,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAtaluren reduced the levels of phospho-MTOR S2248 and improved digestive enzyme production in patients\u003c/strong\u003e \u003cstrong\u003ewith\u003c/strong\u003e \u003cstrong\u003eSDS. \u003c/strong\u003ePeripheral blood and stool samples were collected before (T0) and after six (T7) and twelve (T9) months of therapy. Phospho-MTOR S2448 levels were analyzed by Phospho-Flow analysis in whole blood. Immunophenotypic markers were used to isolate different leukocyte populations. Data represent phospho-MTOR level in total leukocytes (\u003cstrong\u003ea\u003c/strong\u003e), lymphocytes (\u003cstrong\u003eb\u003c/strong\u003e), monocytes (\u003cstrong\u003ec\u003c/strong\u003e), and neutrophils (\u003cstrong\u003ed\u003c/strong\u003e) of UPN26 (red lines), UPN58 (blue lines), and UPN74 (green lines). Student’s t-test has been indicated (* p\u0026lt;0.05, *** p\u0026lt;0.001). \u003cstrong\u003ee\u003c/strong\u003e, Fecal elastase-1 levels were measured by ELISA in stool samples at T0, T7, and T9. Data are represented as a gain of Fecal elastase-1 release relative to T0. \u003cstrong\u003ef\u003c/strong\u003e, amylase levels released into serum in each patient throughout the 12 months of therapy.\u003c/p\u003e","description":"","filename":"Figure2MSNatMedBriefFINAL.png","url":"https://assets-eu.researchsquare.com/files/rs-5231941/v1/7de597fa5f4745f3cf44baa4.png"},{"id":90476576,"identity":"29d527e7-21c6-48e3-ac57-9b3824daf723","added_by":"auto","created_at":"2025-09-03 07:09:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1370091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5231941/v1/012b0949-9849-4797-950e-4db829dae838.pdf"},{"id":68213741,"identity":"f018b73c-6462-4d7e-aebb-a83f714cbf74","added_by":"auto","created_at":"2024-11-04 18:27:34","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":89329808,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendeddataBezzerrietalNatMed.docx","url":"https://assets-eu.researchsquare.com/files/rs-5231941/v1/703f30a3a145c5abc359e4c8.docx"},{"id":68213739,"identity":"44f3ddf1-1961-416f-8ffc-85e311fe9bc7","added_by":"auto","created_at":"2024-11-04 18:27:32","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":56013,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationBezzerrietalNatMed.docx","url":"https://assets-eu.researchsquare.com/files/rs-5231941/v1/2a0751dfe9ad3d3954dcd4aa.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nMarco Cipolli and Valentino Bezzerri are inventors of the international patent WO2018/050706A1 \"Method of treatment of Shwachman-Diamond syndrome\". Other authors have no competing interests.","formattedTitle":"Ataluren Treatment Improves Hematopoietic and Pancreatic Disorders in Patients with Shwachman-Diamond Syndrome","fulltext":[{"header":"Full Text","content":"\u003cp\u003eShwachman-Diamond syndrome (SDS) is a rare genetic disease that results from biallelic mutations in the Shwachman-Bodian-Diamond syndrome (\u003cem\u003eSBDS\u003c/em\u003e) gene.\u003csup\u003e1\u003c/sup\u003e SDS involves exocrine pancreatic insufficiency, skeletal abnormalities, cognitive impairment, and neutropenia.\u003csup\u003e2\u003c/sup\u003e Affected individuals may develop bone marrow aplasia or, more frequently, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eSBDS interacts with elongation factor-like 1 (EFL1), a GTPase that facilitates the release of eukaryotic initiation factor (EIF6) and promotes the assembly of the large and small ribosomal subunits to form functional ribosomes.\u003csup\u003e4\u003c/sup\u003e Failure to produce full-length SBDS induces ribosomal stress, resulting in SDS. \u0026nbsp;The two most common mutations in \u003cem\u003eSBDS\u003c/em\u003e are c.258+2T\u0026gt;C and c.183-184TA\u0026gt;CT. The first is a splicing mutation, and the second introduces an in-frame stop codon (K62X). International registries of patients with SDS report that more than 50% of these patients are compound heterozygotes consisting of c.258+2T\u0026gt;C and c.183\u0026ndash;184TA\u0026gt;CT mutations.\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAtaluren \u003csup\u003e6\u003c/sup\u003e is an orally available translational read-through-inducing drug approved in Europe for the treatment of Duchenne muscular dystrophy (DMD).\u003csup\u003e7\u003c/sup\u003e Ataluren inhibits the activity of the release factor complex termination activity in ribosomes, forcing the suppression of nonsense mutations.\u003csup\u003e8\u003c/sup\u003e Cellular and animal models have demonstrated \u003cem\u003ein vitro\u003c/em\u003e translational read-through efficacy of ataluren.\u003csup\u003e9-11\u003c/sup\u003e However, the clinical evaluation of ataluren remains understudied. In DMD, real-world treatment with ataluren delayed disease progression compared to patients undergoing standard care therapy.\u003csup\u003e7,12\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eWe reported the beneficial effects of ataluren in \u003cem\u003eex vivo\u003c/em\u003e SDS cell models, showing a significant increase in neomorphic SBDS protein followed by an improved myelopoiesis and reduced expression of biomarkers, including TP53 and MTOR.\u003csup\u003e13\u003c/sup\u003e Based on these pre-clinical data, we initiated an ataluren compassionate program in three patients with SDS carrying the c.183-184TA\u0026gt;CT nonsense mutation in \u003cem\u003eSBDS\u003c/em\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring treatment, none of the three patients reported adverse events. The therapy was well-tolerated. SBDS protein levels were normalized in bone marrow mononuclear cells (BM-MNC) isolated from UPN26 and UPN58 patients (\u003cstrong\u003eFig. 1a\u003c/strong\u003e and \u003cstrong\u003eExtended data Fig. 1a)\u003c/strong\u003e. Because of decreased bone marrow cellularity, western blot analysis could not be performed on UPN74 BM-MNC. Therefore, we assessed the effects of ataluren therapy on PBMC isolated from this patient. Western blot analysis showed a remarkable increase in SBDS synthesis after either 3 or 6 months of therapy (\u003cstrong\u003eExtended data Fig. 1b\u003c/strong\u003e). Bone marrow biopsies obtained from UPN26, UPN58, and UPN74 before the initiation of ataluren therapy showed hypocellularity and decreased maturation of the hematopoietic lineages (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). Two study-blinded evaluators reviewed the embedded bone marrow tissues and agreed on the observation of a remarkable increase in cellularity, accompanied by improved maturation of myeloid elements after six months of treatment\u0026nbsp;in all three patients (\u003cstrong\u003eFig. 1b\u003c/strong\u003e). The colony assays consistently indicated a steady increase in myeloid colony formation (\u003cstrong\u003eFig. 1c\u003c/strong\u003e and \u003cstrong\u003eExtended data Fig. 1c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe determined the effect of the drug on peripheral blood cells. The number of neutrophils was significantly increased in UPN26 and UPN74 patients after 12 months of treatment compared to the mean of three analyses performed in the last two years before therapy initiation. In contrast, no effect was observed with UPN58 (\u003cstrong\u003eExtended data Fig. 2a-b\u003c/strong\u003e). The platelet count improved in all three patients treated with ataluren (\u003cstrong\u003eExtended data Fig. 2c-d\u003c/strong\u003e). No appreciable effect was observed on hemoglobin and reticulocytes (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e). These results correlated with those of erythroid colony assays (\u003cstrong\u003eExtended data Fig. 1c\u003c/strong\u003e) and with previously published data on \u003cem\u003eex vivo\u003c/em\u003e efficacy of ataluren in BM-MNC.\u003csup\u003e13\u003c/sup\u003e No statistically significant changes were observed in total leukocyte and monocyte counts after one year of treatment (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003ePatients with SDS have fewer B cells, mainly because of a severe deficiency in the B memory compartment. \u003csup\u003e14\u003c/sup\u003e In this study, we showed that ataluren restored the normal number of memory B cells (CD19\u003csup\u003e+\u003c/sup\u003e and CD27\u003csup\u003e+\u003c/sup\u003e) in all patients who underwent therapy for 12 months (\u003cstrong\u003eExtended data Fig. 2e-g\u003c/strong\u003e). In particular, the number of non-switched (CD27\u003csup\u003e+\u003c/sup\u003e, IgD\u003csup\u003e+\u003c/sup\u003e) B memory cells was increased by ataluren. In parallel, exhausted B memory cells (CD27\u003csup\u003e-\u003c/sup\u003e, IgD\u003csup\u003e-\u003c/sup\u003e) decreased after treatment. The total number of B cells and T lymphocytes did not show substantial changes after 12 months of therapy (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eSDS is associated with a hyper-phosphorylation of MTOR Ser2448\u003csup\u003e15\u003c/sup\u003e with possible activation of both MTORC1 and MTORC2 complexes, modulating MTOR pathway activation, which in turn may regulate cell growth and proliferation, survival, and metabolism. At the baseline, we observed a 2.4-fold increase in phospho-mTOR levels in SDS leukocytes compared to healthy donors. After six months of treatment, we observed a 34% decrease in mean mTOR levels in white blood cell count (WBC) (\u003cstrong\u003eFig. 2a\u003c/strong\u003e). Phospho-mTOR levels were significantly reduced by 22% in lymphocytes (\u003cstrong\u003eFig. 2b\u003c/strong\u003e), and by 38% in monocytes (\u003cstrong\u003eFig. 2c\u003c/strong\u003e). We failed to observe a notable increase in phospho-mTOR levels in SDS neutrophils compared to those in healthy donors (\u003cstrong\u003eFig. 2d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo assess whether the inhibition of MTOR activation could rescue myelopoiesis, we evaluated the effects of the MTOR inhibitors everolimus (RAD001) and dactolisib (NVP-BEZ-235) on myeloid differentiation in bone marrow progenitor cells isolated from patients with SDS using colony assays. Inhibition of MTOR did not improve granulopoiesis or erythropoiesis (\u003cstrong\u003eExtended data Fig. 3\u003c/strong\u003e), suggesting that ataluren-induced MTOR activation was a secondary effect due to the restoration of SBDS protein synthesis, followed by improved overall cellular fitness.\u003c/p\u003e\n\u003cp\u003eAll three patients reported exocrine pancreatic insufficiency at enrollment (\u003cstrong\u003eTable 1\u003c/strong\u003e). After one year of treatment, the release of pancreatic enzymes fecal elastase-1 and amylase increased. Fecal elastase-1 levels displayed 1.3-, 4.6-, and 2.5-fold increases in subjects UPN26, UPN58, and UPN74, respectively (\u003cstrong\u003eFig. 2e\u003c/strong\u003e). Serum total amylase levels improved similarly in all the patients (\u003cstrong\u003eFig. 2f\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCurrently, hematopoietic stem cell transplantation is the only effective therapy; however, it is not required for most patients. Herein, we report the first pharmacological treatment to improve hematopoiesis in SDS through nonsense suppression.\u003c/p\u003e\n\u003cp\u003eOver the past decade, several studies on ataluren in rare diseases caused by nonsense mutations have yielded ambivalent results. However, patients with SDS carry only one specific nonsense mutation, which reduces the variability of the mutation-dependent efficacy of treatment. Despite the ubiquitous cellular need for ribosomes, one curious feature is that ribosomopathies have tissue-limited phenotypes. Ataluren efficacy can also vary in a tissue-dependent manner.\u003csup\u003e16\u003c/sup\u003e Here, we found an improvement in both hematopoietic and exocrine pancreatic tissues.\u003c/p\u003e\n\u003cp\u003eIn addition, the primary endpoint of this study measured the direct effect of the drug (i.e., restoration of SBDS protein in the BM and improved hematopoiesis), whereas previous clinical trials for DMD and CF determined only surrogate endpoints, such as cardiopulmonary function measured by the six-minute walk test and respiratory function measured as forced expiratory volume in the first second (FEV1).\u003c/p\u003e\n\u003cp\u003eThe major limitation of this study was its small sample size. Considering that SDS is ultra-rare, with an incidence of approximately 1:160,000 live births,\u003csup\u003e17\u003c/sup\u003e and that ataluren treatment can be administered only to patients carrying nonsense mutations, three patients should be considered a reliable starting point for designing larger multicenter clinical trials. Although previous clinical trials on the effect of ataluren on cystic fibrosis and DMD dampened enthusiasm for this drug, the results of our study should encourage both the scientific community and the pharmaceutical company owner of the patent to continue the drug repurposing of ataluren for SDS. Furthermore, decreased ribosomal stress due to neomorphic SBDS may reduce the lifetime risk of myeloid malignancies in patients with SDS.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments.\u003c/h2\u003e \u003cp\u003eThis work was supported in part by the Italian Association for Shwachman-Diamond Syndrome (AISS, to VB and MC), NIH R01DK132812 (to SJC), Lisa Dean Moseley Foundation, VeloSano, and 2nd Chance 4 Kids. We thank PTC Therapeutics Inc. (NJ, USA) for providing the drug covering manufacturing costs and Fondazione Telethon (Milan, Italy), which contributed to the clinical development of ataluren. We are grateful to Federica Quiri (Cystic Fibrosis Center, Verona, Italy) and Angela Mercuri (University of Verona) for their excellent technical assistance, Antonella Minelli, Cesare Danesino (University of Pavia, Italy), and Barouk Maurice Assael (Adult Cystic Fibrosis Center, IRCCS Ca\u0026rsquo; Granda, Milan, Italy) for helpful discussions, Francesca Buniotto and Sandra Perobelli (Cystic Fibrosis Center, Verona, Italy) for her contribution to the psychological assessment of patients during therapy.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eDe-identified patient data and source data are available in supplementary information. All original clinical data can be obtained by contacting the chief investigator (M.C.). Individual patient data will be shared in a de-identified and anonymized format, following our data-sharing process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBoocock, G.R., \u003cem\u003eet al.\u003c/em\u003e Mutations in SBDS are associated with Shwachman-Diamond syndrome. Nat Genet 33, 97\u0026ndash;101 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson, A.S. \u0026amp; Myers, K.C. Diagnosis, Treatment, and Molecular Pathology of Shwachman-Diamond Syndrome. Hematol Oncol Clin North Am 32, 687\u0026ndash;700 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonadieu, J., \u003cem\u003eet al.\u003c/em\u003e Classification of and risk factors for hematologic complications in a French national cohort of 102 patients with Shwachman-Diamond syndrome. Haematologica 97, 1312\u0026ndash;1319 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarren, A.J. Molecular basis of the human ribosomopathy Shwachman-Diamond syndrome. Adv Biol Regul 67, 109\u0026ndash;127 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson, A.S., \u003cem\u003eet al.\u003c/em\u003e Shwachman Diamond syndrome: narrow genotypic spectrum and variable clinical features. Pediatric Research 92, 1671\u0026ndash;1680 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWelch, E.M., \u003cem\u003eet al.\u003c/em\u003e PTC124 targets genetic disorders caused by nonsense mutations. Nature 447, 87\u0026ndash;91 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcDonald, C.M., \u003cem\u003eet al.\u003c/em\u003e Ataluren delays loss of ambulation and respiratory decline in nonsense mutation Duchenne muscular dystrophy patients. J Comp Eff Res (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, S., \u003cem\u003eet al.\u003c/em\u003e Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination. Nat Commun 13, 2413 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSermet-Gaudelus, I., \u003cem\u003eet al.\u003c/em\u003e Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis. Am J Respir Crit Care Med 182, 1262\u0026ndash;1272 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, M., Andersson-Lendahl, M., Sejersen, T. \u0026amp; Arner, A. Muscle dysfunction and structural defects of dystrophin-null sapje mutant zebrafish larvae are rescued by ataluren treatment. FASEB J 28, 1593\u0026ndash;1599 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X., \u003cem\u003eet al.\u003c/em\u003e Efficacy of Postnatal In Vivo Nonsense Suppression Therapy in a Pax6 Mouse Model of Aniridia. Mol Ther Nucleic Acids 7, 417\u0026ndash;428 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMercuri, E., \u003cem\u003eet al.\u003c/em\u003e Safety and effectiveness of ataluren: comparison of results from the STRIDE Registry and CINRG DMD Natural History Study. J Comp Eff Res 9, 341\u0026ndash;360 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCipolli, M., \u003cem\u003eet al.\u003c/em\u003e Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells. Br J Haematol (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBezzerri, V., \u003cem\u003eet al.\u003c/em\u003e Peripheral blood immunophenotyping in a large cohort of patients with Shwachman-Diamond syndrome. Pediatr Blood Cancer 66, e27597 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVella, A., \u003cem\u003eet al.\u003c/em\u003e mTOR and STAT3 Pathway Hyper-Activation is Associated with evated Interleukin-6 Levels in Patients with Shwachman-Diamond Syndrome: Further Evidence of Lymphoid Lineage Impairment. Cancers (Basel) 12(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThada, V., Miller, J.N., Kov\u0026aacute;cs, A.D. \u0026amp; Pearce, D.A. Tissue-specific variation in nonsense mutant transcript level and drug-induced read-through efficiency in the Cln1(R151X) mouse model of INCL. J Cell Mol Med 20, 381\u0026ndash;385 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinelli, A., \u003cem\u003eet al.\u003c/em\u003e Incidence of Shwachman-Diamond syndrome. Pediatr Blood Cancer 59, 1334\u0026ndash;1335 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":" Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eClinical and laboratory data of patients enrolled in the ataluren study\u003c/div\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eT\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eAge\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eSex\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHeight\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e(cm)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eWeight\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e(Kg)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eBMI\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eFE-1\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e(\u0026micro;g/g)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eConc. Med.\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eWBC\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e(10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eRBC (10\u003csup\u003e12\u003c/sup\u003e/L)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHb (g/dL)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eANC (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ePLT (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eMCV\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003e(fL)\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eCytogenetics\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003eBM [mitosis]\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eaCGH\u003c/div\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth colspan=\"13\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eUPN26\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eT0\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e20\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eM\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e174\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e58.8\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e19.4\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e77.8\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eVit, PERT\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.44\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4.98\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e15.1\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.63\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e97\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e90\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e46,XY [1]\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003edel(20)(q11.21-q11.23)\u0026thinsp;~\u0026thinsp;19%\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eT9\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e21\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e174\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e58.8\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e19.4\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e97.2\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eVit, PERT\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.27\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4.81\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14.9\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.87\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e112\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e92.1\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e46,XY [9]\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003edel(20)(q11.21-q11.23)\u0026thinsp;~\u0026thinsp;19%\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"13\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eUPN58\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eT0\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e16\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eM\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e160\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e42.3\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e16.5\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e15\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eVit, PERT\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.14\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4.17\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e13.2\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.38\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e97\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e95.7\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e46,XY [6]\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003edup(1q)(q21.1-q44)\u0026thinsp;~\u0026thinsp;16%\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eT9\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e17\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e161\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e42.1\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e16.2\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e68.5\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eVit, PERT\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.33\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4.09\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e13.3\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.51\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e136\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e98.8\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eN/A\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003edup(1q)(q21.1-q44)\u0026thinsp;~\u0026thinsp;18%\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"13\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Bold\"\u003eUPN74\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eT0\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e13\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd rowspan=\"2\" align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eM\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e152\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e37.5\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e16.2\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eVit, PERT\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e2.88\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4.56\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14.3\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.94\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e113\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e91.2\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e46,XY,del(20)(q11-q13)[2]/46,XY[16\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003edel(20)(q11.22-q13.1)\u0026thinsp;~\u0026thinsp;10%\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e\u003cspan class=\"Italic\"\u003eT9\u003c/span\u003e\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e159\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e43.1\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e17\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e34.6\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eVit, PERT\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e3.07\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e4.73\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e14.9\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e0.97\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e143\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e92.4\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003e46,XY,del(20)(q11-\u003c/div\u003e\n\u003cdiv class=\"SimplePara\"\u003eq13)[1]/46,XY[12]\u003c/div\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003edel(20)(q11.22-q13.1)\u0026thinsp;~\u0026thinsp;12%\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eT0 (initiation of therapy), T9 (12 months of therapy); M, male; BMI, body mass index; FE-1, fecal elastase-1); Conc. Med., concomitant medications; WBC, white blood cell; RBC, erythrocytes; Hb, hemoglobin; ANC, absolute neutrophil count; PLT, platelet count; MCV, mean corpuscular volume; BM, bone marrow; aCGH, array comparative genomic hybridization; Vit, vitamin (A,D,E,K) supplementation; PERT, pancreatic enzyme replacement therapy; N/A, not available (unsuitable sample).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Online Methods","content":"\u003cp\u003e\u003cstrong\u003ePatient recruitment\u003c/strong\u003e. Three adolescent male patients with SDS (\u003cstrong\u003eTable 1\u003c/strong\u003e) of the same genotype (c.183-184TA\u0026gt;CT/c.258+2T\u0026gt;C) were enrolled for receiving ataluren for 12 months under a compassionate program. For the colony assays, six additional patients and three healthy donors were recruited (\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e). Informed consent was obtained from the local Ethics Committee (approval no. 4090 CESC and nr. 4182 CESC) in agreement with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy protocol\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003ePatients will be evaluated in the clinic before treatment initiation (T0), after 2 weeks (T1), every month (T2\u0026ndash;T7) for 6 months, then every three months until 12 months (T7\u0026ndash;T9) of treatment. In addition to routine monitoring (vital signs, height and weight, and physical examinations), hematology laboratory assessment, liver and renal function tests, serum electrolytes, urinalysis, and a review of concomitant medications will be performed at each clinic visit.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003ePurpose. \u003c/u\u003eThe objective of this compassionate use is to offer drug access to ataluren to provide a potential clinical benefit to patients diagnosed with Shwachman-Diamond Syndrome who have a high unmet medical need according to the Italian Ministerial Directive (DM) 07.09.2017.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEligibility.\u003c/u\u003e Patients (males, females) with SDS diagnosis carrying the c183-184ta\u0026gt;ct mutation in at least one allele, \u0026gt;= 6 years will be eligible to receive the drug (ataluren) for six months and depending on the medical judgment, treatment with ataluren may be extended for another six months. The patient will start treatment with ataluren only after informed consent has been provided (including the Information Sheet For The Processing Of Personal Information) and if he/she fulfills all inclusion/exclusion criteria.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eInclusion criteria\u003c/u\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003ePatients with a diagnosis of SDS carrying in at least one allele c183-184ta\u0026gt;ct mutation\u003c/li\u003e\n\u003cli\u003eAge \u0026gt;= 6 yrs\u003c/li\u003e\n\u003cli\u003eNeutrophil count \u0026lt; 1000 PMN/mm3 at T0 and at least one evaluation performed within twelve months before patient enrollment.\u003c/li\u003e\n\u003cli\u003eBone marrow evaluation within 12 months of the patient's enrollment\u003c/li\u003e\n\u003cli\u003eSerum total bilirubin within normal limits; serum ALT, AST, or GGT \u0026lt;=2.0 times the ULN, creatinine, BUN within normal limits\u003c/li\u003e\n\u003cli\u003eEvidence of signed and dated informed consent and assent documents (s). Note: If the patient is considered a child under local regulations, the parents or legal guardians must provide written consent, and the patient may be required to provide written consent.\u003c/li\u003e\n\u003cli\u003eIn patients who are sexually active, willingness to abstain from sexual intercourse or employ a barrier or medical method of contraception during ataluren administration and the 60-d follow-up period.\u003c/li\u003e\n\u003cli\u003eAble to understand and comply with treatment requirements, restrictions and instruction (as judged by the treating physician).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cu\u003eExclusion criteria\u003c/u\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003ePresence of myelodysplasia or leukemia\u003c/li\u003e\n\u003cli\u003eBone marrow transplantation\u003c/li\u003e\n\u003cli\u003eBlood transfusions in the past three months\u003c/li\u003e\n\u003cli\u003eHigh value (\u0026gt; 4%) of HbF according to medical evaluation\u003c/li\u003e\n\u003cli\u003eNeutrophil count \u0026lt; 300 PMN/mm3\u003c/li\u003e\n\u003cli\u003eSerologic evidence of hepatitis B or C, or of HIV\u003c/li\u003e\n\u003cli\u003eSevere abnormalities of pulmonary or renal function\u003c/li\u003e\n\u003cli\u003eOngoing intravenous (IV) aminoglycoside or IV vancomycin\u003c/li\u003e\n\u003cli\u003ePregnancy or breastfeeding.\u003c/li\u003e\n\u003cli\u003eOngoing warfarin, phenytoin, or tolbutamide therapy.\u003c/li\u003e\n\u003cli\u003eHypersensitivity to any of the ingredients or excipients of the study drug (polydextrose, polyethylene glycol 3350, poloxamer 407, mannitol 25C, crospovidone XL10, hydroxyethyl cellulose, vanilla, colloidal silica, or magnesium stearate).\u003c/li\u003e\n\u003cli\u003eHistory of solid organ or hematological transplantation\u003c/li\u003e\n\u003cli\u003ePrior or ongoing medical conditions (such as concomitant illness, alcoholism, drug abuse, and psychiatric condition), medical history, physical findings, ECG findings, or laboratory abnormalities that, in the investigator\u0026rsquo;s opinion, could adversely affect the safety of the subject, making it unlikely that the course of treatment or follow-up would be completed or could impair the assessment of study results.\u003c/li\u003e\n\u003cli\u003eInability to understand the informed consent\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cu\u003eEligibility assessments. \u003c/u\u003eEligibility assessments should occur before the administration of ataluren.\u003c/p\u003e\n\u003cp\u003eThe following eligibility assessments should be performed and documented by the treating physician:\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eReview of eligibility criteria\u003c/li\u003e\n\u003cli\u003eCollection of signed informed consent and assent (as applicable)\u003c/li\u003e\n\u003cli\u003eReview of medical history (including SDS genotype)\u003c/li\u003e\n\u003cli\u003eLiver and renal function tests and serum electrolytes (including ALT, AST, GGT, ALP, total bilirubin, cystatin C, BUN, urine protein, serum Na+, serum K+, serum Mg2+, serum Ca2+, serum phosphorous, and serum HCO3-)\u003c/li\u003e\n\u003cli\u003eUrine and serum pregnancy test for females of childbearing potential as judged by the treating physician\u003c/li\u003e\n\u003cli\u003eComplete blood count and peripheral blood immunophenotyping will be performed on the first day of treatment (before the treatment)\u003c/li\u003e\n\u003cli\u003eReview of prior and concomitant medications\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cu\u003eAssessment and monitoring during the therapy with ataluren. \u003c/u\u003eTreating physicians should refer to the investigator\u0026rsquo;s brochure to summarize the ataluren data. Patients should be evaluated in the clinic on the first day of treatment and after 2 weeks, 1, 2, 3, 4, 5, and 6 months. If treatment with ataluren is prolonged for another six months, patients should be evaluated at the third and sixth months of this extension period.\u0026nbsp; In addition to routine monitoring (such as vital signs, height and weight, and physical examinations), additional monitoring may occur, if necessary, by the treating physician.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAtaluren administration and management.\u003c/u\u003e The ataluren regimen may be dispensed only under the supervision of the treating physician or an authorized designee and only for administration to the patient participating in this compassionate use. Ataluren will be provided as granules for oral suspension with a white to off-white powder appearance. Drug substances and products are manufactured under cGMP conditions. The formulation includes a matrix, suspending agents, surfactants, and various excipients that aid manufacturing. The granules for oral suspension are packaged in aluminium foil and child-resistant sachets (packets) and supplied with doses of 125, 250, or 1000 mg of the active drug substance. The powder in the sachet may be mixed with water, fruit juice, fruit punch, milk (skim milk, 1% fat, 2% fat, whole milk, chocolate milk, soy milk, or lactose-free milk), or semisolid food (yogurt, pudding, or apple sauce). PTC Therapeutics, Inc. will provided the drug.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDrug kits.\u003c/u\u003e Drug kits will be provided, each containing 30 sachets of one dose (125, 250, or 1000-mg). Sachets and cartons will be color coded to indicate the dosage strength (125 mg, brown; 250 mg, green; 1000 mg, dark blue).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDrug dispensation. \u003c/u\u003eAtaluren will be administered three times per day (TID). The dose level to be administered is as follows: 10 mg/kg in the morning, 10 mg/kg at mid-day, and 20 mg/kg in the evening\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eStorage and stability.\u003c/u\u003e Ataluren will be shipped and stored at room temperature (approximately 15 to 30\u0026deg;C). The available stability data from representative samples supported using the drug product for 48 months when stored at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eSchedule of administration. \u003c/u\u003eThree doses should be administered per day \u0026ndash; the 1st dose, 10 mg/kg, in the morning, the 2nd dose, 10 mg/kg, during the middle of the day (mid-day); and the 3rd dose, 20 mg/kg, in the evening. Ideally, each dose should be administered within approximately 30 min before or after a meal (e.g., at approximately 7:00 AM after breakfast, approximately 1:00 PM after lunch, and approximately 7:00 PM after dinner). Intervals for dosing should be ~6 h (\u0026plusmn;1 h) between morning and mid-day doses, approximately 6 h (\u0026plusmn;1 h) between mid-day and evening doses, and approximately 12 h (\u0026plusmn;1 h) between evening doses and the morning dose on the next day.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eInstructions for delays in dosing. \u003c/u\u003eDosing delays should be handled as follows:\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eIf dosing of ataluren is delayed by \u0026le;1 h, the planned dose should be taken with no changes to the subsequent dose schedules.\u003c/li\u003e\n\u003cli\u003eIf ataluren dosing is delayed by \u0026gt;1 h but \u0026le;3 h (or more than 6 h after the evening dose), the planned dose should be taken; however, all future doses for that day should be shifted later by an approximately corresponding amount.\u003c/li\u003e\n\u003cli\u003eIf ataluren dosing is delayed by \u0026gt;3 h (or \u0026gt; 6 h after the evening dose), the dose should not be taken. Ataluren administration may continue, but the missed dose should not be administered, and the planned timing of subsequent study drug dosing should not be altered.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cu\u003eDrug preparation and storage. \u003c/u\u003eAtaluren sachets should be stored at room temperature, away from the reach of the children, until reconstitution. They should only be opened at the time of dose preparation. The powder in the sachet may be mixed with water, milk (skim milk, 1% fat, 2% fat, whole milk, chocolate milk, soy milk, or lactose-free milk), or semi-solid food (yogurt, pudding, or apple sauce). The full contents of the sachets should be mixed with at least 30 mL of liquid (water, milk [skim, 1% fat, 2% fat, whole milk, chocolate milk, or lactose-free milk]), or three tablespoons of semi-solid food (yogurt, pudding, or apple sauce). The prepared dose should be thoroughly mixed before administration. The amount of liquid or semi-solid food can be increased based on patient preferences. Each prepared dose is best administered immediately after preparation.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eLaboratory abnormalities and adverse events requiring evaluation and potential drug interruption/modification. \u003c/u\u003eDuring ataluren treatment, the subjects will be closely monitored for adverse events or laboratory abnormalities. Renal abnormalities will be studied closely. For adverse events or laboratory abnormalities, treating physicians will use their judgment to determine whether the event or abnormality is clinically significant, whether a diagnostic evaluation is warranted, and whether potential interruption of the study drug treatment is appropriate. In general, life-threatening (grade 4) or severe (grade 3) adverse events or laboratory abnormalities should be considered clinically significant; however, recurrent or persistent moderate events (grade 2) may also be considered clinically significant in certain circumstances. The Common Terminology Criteria for Adverse Events (CTCAE) is used to grade the severity of adverse events and laboratory abnormalities.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConcomitant and supportive therapy. \u003c/u\u003eInformation regarding all concomitant medications will be collected and documented.\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eDrugs metabolized by cytochrome P450 enzymes. As the primary route of ataluren metabolism is glucuronidation by UGT1A9, clinically significant interactions between ataluren and co-administered drugs metabolized by cytochrome P450 enzymes (CYPs) are unlikely. In particular, ataluren is not an inhibitor of CYP1A2, CYP2B6, CYP2C19, CYP2D6, or CYP3A4/5, and does not induce major CYP enzymes. Drugs that are metabolized by CYP2C8 or CYP2C9, which have low therapeutic indices (in particular, paclitaxel for CYP2C8 and coumarin anticoagulants [such as warfarin], phenytoin, or tolbutamide for CYP2C9), may be of particular concern. Patients who require the use of these drugs will not be enrolled in the study. Coumarin anticoagulants are cleared by CYP2C9, and increases in their plasma concentrations of coumarin anticoagulants may have serious clinical consequences. For patients who require anticoagulation therapy, the use of an alternative form of anticoagulation (e.g., fractionated heparin) should be considered. Phenytoin is metabolized by CYP2C9, and its concomitant use with ataluren may be of potential concern. For patients who require anticonvulsant therapy during the study, the use of alternative anticonvulsant drugs should be considered. The metabolism of losartan to its active metabolites may be mediated, in part, by CYP2C9. However, the concomitant use of losartan and CYP2C9 inhibitors remains unexamined. Because this drug does not have a narrow therapeutic window, the potential for mild-to-moderate changes in activity does not require dose modification.\u003c/li\u003e\n\u003cli\u003eIn vitro studies have shown that ataluren is a substrate for UGT1A9 and breast cancer-resistant protein (BCRP). Caution should be exercised when ataluren is co-administered with drugs that induce UGT1A9 (e.g., phenobarbital and rifampin) or inhibit BCRP (e.g., cyclosporine, eltrombopag, and gefitinib).\u003c/li\u003e\n\u003cli\u003eFor patients requiring systemic antibiotic therapy, intravenous aminoglycosides may be administered when necessary.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cu\u003eDietary restrictions. \u003c/u\u003eThere are no specific dietary restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eHydration.\u003c/u\u003e Because of the potential risk of renal dysfunction during periods of dehydration in patients receiving ataluren, it is important to encourage them to maintain adequate hydration throughout treatment. Subjects should be adequately hydrated before receiving any potentially nephrotoxic agent, and their hydration status should be carefully monitored throughout the administration of any agent with nephrotoxic characteristics. Treating physicians should be particularly vigilant of patients who experience nausea, vomiting, diarrhea, fever, or laboratory evidence of dehydration.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAE and SAE documentation and reporting. \u003c/u\u003eAn AE is defined as any untoward medical occurrence in a patient during treatment; the event does not necessarily have a causal relationship with the treatment. This includes any newly occurring event or worsening of a preexisting condition (e.g., an increase in its severity or frequency) after the ICF, and assent, if applicable, is signed. An SAE is any AE that meets any of the following criteria:\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eFatal (death, regardless of cause, that occurs during participation in compassionate use or death that occurs after participation in compassionate use and is suspected to be a delayed toxicity due to administration of ataluren).\u003c/li\u003e\n\u003cli\u003eLife-threatening, such that the patient was at immediate risk of death from the reaction. \u0026bull;Inpatient hospitalization or prolonged hospitalization. \u0026bull;Persistent or significant disability/incapacity (disability is defined as a substantial disruption in a person\u0026rsquo;s ability to perform normal life functions). Congenital anomalies or birth defects.\u003c/li\u003e\n\u003cli\u003eAn important medical event that, based on appropriate medical judgment, may jeopardize the patient or require medical or surgical intervention to prevent one of the outcomes listed above (e.g., allergic bronchospasm requiring intensive treatment in an emergency room or at home).\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eIf a patient had a hospitalization or procedure (e.g., surgery) for an event or condition that occurred before the patient signed the ICF and/or provided assent, and the hospitalization or procedure was planned before the patient signed the ICF and/or provided assent, the hospitalization or procedure should not be considered to indicate an SAE unless an AE caused the hospitalization or procedure to be rescheduled sooner or prolonged relative to what was planned. In addition, hospitalizations not clearly associated with an AE (e.g., social hospitalization for respite care) should not be considered indicative of an SAE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCompletion of safety information collection forms. \u003c/u\u003eThe treating physician will complete the appropriate safety information collection form and submit it to PTC Therapeutics.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAction taken with ataluren.\u003c/u\u003e The treating physician will classify the action taken with ataluren with regard to AE.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAE outcome.\u003c/u\u003e An AE will be followed-up until the treating physician determines the final outcome.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eReporting procedures for SAEs. \u003c/u\u003eThe institution (Azienda Ospedaliera Universitaria Integrata di Verona) will alert PTC PV via the mailbox [email protected] when physicians have given products to patients, so they can track this internally. Institution will ensure that Physician notifies PTC Pharmacovigilance Department (\u0026ldquo;PTC PV\u0026rdquo;) immediately via the mailbox [email protected] within one business day of any serious adverse event (SAE), and to the extent permitted by applicable laws and regulations and to the extent able to do so under the circumstances, will reasonably cooperate with PTC in connection with any reports or filings to the competent authorities related to such SAE. Physicians and institutions will be responsible for submitting safety information according to the local EC requirements. The PTC and the physician shall manage all reports of suspicious adverse reactions according to the standards set out by the DM Salute, April 30, 2015, and manage all relevant information to the Ethics Committee. PTC shall promptly report to the Institution both during the Treatment Plan and for a reasonable period of time after the Treatment Plan is completed, any findings of PTC or its designees that could affect the safety or medical care of the patient, affect the willingness of the patient to continue participation in the Treatment Plan, influence the conduct of the Treatment Plan, or alter the EC\u0026rsquo;s approval to continue the Treatment Plan.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eTermination.\u0026nbsp; \u003c/u\u003ePTC agrees to provide and deliver sufficient products for the treatment of the patients for six months at no cost to the physician and institution, for the sole purpose of treatment of the patients following the terms and conditions of this agreement (the \u0026ldquo;Initial Supply\u0026rdquo;). The PTC shall have no further obligation to supply products and no other financial or payment obligations whatsoever in connection with the patients, the Treatment Plan or this Agreement, except to the extent expressly set forth herein. After the Initial Supply, PTC will supply a product free of charge for an additional six months to patients, showing a proven clinical benefit at the end of the observational period. Under no circumstances shall the PTC have any obligation to provide products beyond these additional six months. At the end of the six-month period, the institution shall account for all quantities of product used for the patients and, unless otherwise agreed upon in writing by the parties, shall return or otherwise dispose of any remaining product per the PTC instructions. This compassionate use expires upon the completion of the Treatment Plan, except that any provisions would reasonably be expected to survive such a termination.\u003c/p\u003e\n\u003cp\u003eCompassionate use will terminate prematurely if:\u003c/p\u003e\n\u003cp\u003e- The patient withdraws consent and/or discontinues treatment with the product.\u003c/p\u003e\n\u003cp\u003e- The physician determines that treatment with the product should be withdrawn based on the patient\u0026rsquo;s condition, inability to tolerate the Product or Physician\u0026rsquo;s clinical judgment.\u003c/p\u003e\n\u003cp\u003eA Patient becomes eligible to participate in a clinical trial program initiated by the PTC in which the Product is the study drug.\u003c/p\u003e\n\u003cp\u003e- Access to the product is discontinued by competent authorities or the PTC\u003c/p\u003e\n\u003cp\u003eThe product is commercially available in Italy under an approved label for treating Shwachman-Diamond Syndrome.\u003c/p\u003e\n\u003cp\u003e- Either party materially breaches any terms of the agreement and fails to cure such breach within 30 d of the written notice of the same.\u003c/p\u003e\n\u003cp\u003e- Physicians are no longer available at the institution to complete the Treatment Plan, and a replacement physician is not agreed to by the parties within 30 d of notification to PTC that Physician is no longer available at the institution;\u003c/p\u003e\n\u003cp\u003e- PTC determines at its sole discretion whether the product is safe or ineffective for the patient.\u003c/p\u003e\n\u003cp\u003e- The PTC no longer has access to the quantity or quality of products necessary to provide under the agreement.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eReview of concomitant medications.\u003c/u\u003e The concomitant medication use of the patient should be assessed during each clinic visit.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eReview of AEs and serious adverse events (SAEs).\u003c/u\u003e Treating physicians should review AEs and SAEs continuously throughout the participation of the patient (from signing the informed consent form [ICF] to assent, if applicable).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematological assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhite blood cell counts with differential, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total red cell counts with morphology, and platelet count were performed at the section of Clinical Biochemistry, AOUI Verona, using the validated diagnostic procedures. The total white blood cell count (WBC) was determined by Hematology Analyzer XN- 9000 (Sysmex Europe GmbH, Norderstedt, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiver and renal function tests, serum electrolytes and urinalysis assessments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLevels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), \u0026gamma;-glutamyltranspeptidase (GGT), alkaline phosphatase (ALP), total bilirubin, cystatin C, nitrogenous products (BUN), urine protein, serum Na+, serum K+, serum Mg2+, serum Ca2+, serum phosphorous, and serum HCO3-, glucose, pH, specific gravity, ketones, blood, protein, creatinine, urobilinogen, bilirubin, nitrite, and leukocyte esterase were quantified using clinical chemistry and immunochemistry instruments (Architect C 16000 plus and Architect i2000sr plus; Abbott Diagnostics, Lake Forest, IL, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBone marrow biopsies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow aspirates and biopsies were withdrawn after six months of treatment (approximately 12 months after the previous bone marrow biopsy) according to the standard follow-up for patients with SDS validated at AOUI Verona.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted from the bone marrow (BM-MNC) and peripheral blood (PBMC) mononuclear cells, separated by 11% SDS-PAGE, and electroblotted onto Immobilon P filters (Millipore, Billerica, MA, USA) previously blocked with 5% non-fat milk in TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl) supplemented with 0.05% Tween-20 (TBS/T). The membranes were probed with: i) anti-human SBDS rabbit polyclonal IgG antibody (Abcam, Cambridge, MA, dilution 1:1500); monoclonal anti-\u0026beta;-Actin clone AC-15 (Sigma-Aldrich, diluted 1:2000) in 5% non-fat milk TBS/T. Membranes were incubated overnight at 4\u0026deg;C and then incubated with the secondary antibody horseradish peroxidase-coupled anti-rabbit IgG (Sigma-Aldrich, dilution 1:15000), for 1 h. Immunocomplexes were detected using the ECL Plus Western Blotting Detection System (Amersham Biosciences, Little Chalfont, UK).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emTOR Phosphorylation assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phosphorylation level of mTOR (S2448) in peripheral blood leukocytes was tested using phospho-flow analysis at T0, T4, and T7. Briefly, total leukocytes from peripheral blood withdrawals were fixed and permeabilized using an Intracellular Fixation and Permeabilization Buffer Set (eBioscience, San Diego, CA, USA) following the manufacturer's protocol. After permeabilization, leukocytes were washed once in flow buffer and stained with Pacific Blue-conjugated anti-p-S2448-mTOR (BD, Franklin Lakes, NJ, USA) or isotype control antibodies for 30 min. The cells were washed, and 13 colors, 4 lasers DX-Flex flow cytometer (Beckman Coulter). All acquired data files were analyzed using Kaluza software (Beckman Coulter).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunophenotyping of peripheral blood leukocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were prepared according to the Clinical and Laboratory Standard Institute H42-A2 \"Enumeration of Immunologically Defined Cell Populations by Flow Cytometry, 2nd Edition\" Guidelines. Immunophenotyping was performed at T0, T4, and T7 using a 13-color DX-Flex flow cytometer (Beckman Coulter). The UK Neqas External Quality Assessment \u0026ldquo;Leucocyte Immunophenotyping\u0026rdquo; internal quality control (IQC) was used. The following fluorochrome-conjugated monoclonal antibodies were used: CD3-APC750, CD4-PC7, CD5-PC5, CD8-ECD, CD16-Pacific Blue, CD19-Chrome-Orange, CD23-ECD, CD27-PC5, CD38-APC750, CD45-APC700, CD56-PC5, and HLA-DR-PE (Beckman CA). Peripheral blood lymphocytes were gated into side-scatter (SSlow) and CD45 positive area. Within the lymphocytes, B cells were gated as CD19 positive events and subsequently divided into CD19+ CD5- (B2 or conventional B cells) and CD19+ CD5+ (B1a cells) subpopulations. T cells were defined as CD3+ events and further gated into CD4+ and CD8+ T cells; NK cells were identified as CD3/CD19 double-negative events expressing CD56 and/or CD16. B-cell subsets were separated into CD27- (naive B cells) and CD27+ (memory B cells), or CD23+ (activated B cells). CD4+ and CD8+ T lymphocytes (CD3+) were further distinguished using CD38 and HLA-DR (activation markers). A total of 25,000 events were detected during each run.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePancreatic activity test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLevels of fecal elastase 1 (FE-1) were analyzed in stool samples (500 \u0026mu;g/g of wet stools) at T0, T7, and T9 by ELISA (ScheBo, Meridian Bioscience Europe, Milan, Italy). Stool samples were diluted in 50 \u0026mu;l assay buffer [0.1 M Tris\u0026middot;HCl (pH 8.0), 1 mM CaCl2, and 0.05% Tween 20] and added to the ELISA multi-wells containing the immobilized anti-elastase antibody. As blank, 50 \u0026micro;l of assay buffer were used. The plates were incubated at 22\u0026deg;C for 30 min. After washes, 50 \u0026micro;l of a biotinylated anti-elastase antibody were complexed with peroxidase-conjugated streptavidin and added to the wells. The samples were incubated at 22\u0026deg;C for 30 min in the dark. After washes, 100 \u0026micro;l of 2,2\u0026prime;-azino-bis(3-thylbenzothiazoline-6-sulfonic acid)-peroxidase substrate solution were added, and samples were incubated at 22\u0026deg;C for 20 min in darkness. Stop solution (100 \u0026micro;l) was added into each well and incubated for 15 min. The absorbance was measured at 405 nm using a Victor Nivo plate reader (Perkin Elmer, Waltham, MA, USA). Values were expressed as \u0026mu;g/g of wet stool.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBM-MNC freshly isolated from bone marrow biopsies were seeded at a density of 105 cell/ml in methylcellulose medium (StemMacs HSC-CFU human, Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 20ng/ml human recombinant G-CSF (Filgrastim). The medium was then supplemented with everolimus (350 nM, Selleckchem, Houston, TX, USA), dactolisib (NVP-BEZ-235, 300 nM, Selleckchem), or DMSO as the vehicle control (1:10000, Merck, Rahway, NJ). Spontaneous growth of CFU-GM and BFU-E was observed using an Axio Observer 7 (Zeiss, Oberkochen, Germany) inverted microscopy (magnification 5\u0026times;) and colonies were counted after 7, 14, and 21 d of incubation at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e. The Shapiro\u0026ndash;Wilk test was used to evaluate the normal distribution of samples, which enabled parametric or non-parametric tests to be selected. Independent group determination was performed using a two-tailed Student's t-test for paired or unpaired data. A \u003cem\u003ep \u003c/em\u003evalue \u0026lt; 0.05 was considered statistically significant. Statistical analyses were performed using Sigma Plot V14.0 (Systat Software Inc., San Jose, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5231941/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5231941/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eShwachman-Diamond syndrome (SDS) is characterized by exocrine pancreatic insufficiency, neutropenia, and a high risk of myeloid malignancy. Most patients with SDS harbor nonsense mutations in Shwachman-Bodian-Diamond syndrome gene (\u003cem\u003eSBDS)\u003c/em\u003e, which encodes a ribosome assembly factor. We investigated the translational read-through effect of ataluren in three patients with SDS. The primary and secondary endpoints were restoring SBDS protein levels in hematopoietic cells and improving myelopoiesis, respectively. SBDS synthesis increased in hematopoietic cells, whereas the bone marrow showed improved cellularity with the maturation of myeloid progenitors. The exocrine pancreatic function also improved. Thus, this clinical study strongly encourages the further clinical development of ataluren to treat SDS.\u003c/p\u003e","manuscriptTitle":"Ataluren Treatment Improves Hematopoietic and Pancreatic Disorders in Patients with Shwachman-Diamond Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-04 18:27:28","doi":"10.21203/rs.3.rs-5231941/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3c9192c7-8af1-4537-adf9-897db3734da6","owner":[],"postedDate":"November 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39233176,"name":"Health sciences/Medical research/Drug development"},{"id":39233177,"name":"Health sciences/Medical research/Genetics research"}],"tags":[],"updatedAt":"2025-09-03T07:09:00+00:00","versionOfRecord":{"articleIdentity":"rs-5231941","link":"https://doi.org/10.1038/s41467-025-63137-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-09-02 04:00:00","publishedOnDateReadable":"September 2nd, 2025"},"versionCreatedAt":"2024-11-04 18:27:28","video":"","vorDoi":"10.1038/s41467-025-63137-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63137-3","workflowStages":[]},"version":"v1","identity":"rs-5231941","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5231941","identity":"rs-5231941","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}