Distinguishing acquired (idiopathic/immune) severe aplastic anemia (SAA) from inherited bone marrow failure syndromes (IBMFS) is critical since only acquired cases respond to immunosuppressive therapy (IST), the standard treatment in cases without a matched sibling donor. Differentiating SAA from IBMFS is particularly challenging in children younger than 3 years, in whom inherited disorders are more prevalent. No age threshold exists to guide IST use in this population. Thus, we evaluated IST outcomes in children aged

Diamond-Blackfan anemia (DBA) is a pure red blood cell aplasia with a predisposition towards cancer. In DBA erythroid progenitors, NLK is activated and mTORC1 activity is suppressed. Systemic leucine administration high enough to hyperstimulate mTORC1 improves erythroid expansion in patients and animal models of DBA. However, leucine at such high doses significantly increases malignant transformation of non-erythroid cells upon exposure to carcinogens, heightening cancer risk in these patients. Here we show that exosomal delivery of miR-26a restores basal mTORC1 activity in erythroid progenitors by targeting the NLK transcript for degradation. MiR-26a delivery does not hyperstimulate mTORC1, but removal of NLK-mediated mTORC1 suppression primes erythroid cells for mTORC1 hyperstimulation at a ten-fold lower systemic dose of leucine. Delivery of exosomal miR-26a supports optimal mTORC1 hyperstimulation and erythroid expansion with the administration of a leucine dose that does not increase the risk of transformation in nonerythroid cells, thus offering a safe, novel and effective treatment strategy for increasing erythropoiesis in DBA.

Background: Pyruvate kinase (PK) deficiency is a rare chronic hemolytic anemia caused by homozygous or compound heterozygous mutations in the PKLR gene. The PKLR gene encodes the red blood cell (RBC)-specific form of PK (PKR), an enzyme that is crucial in the final step of glycolysis. The clinical presentation of PK deficiency is variable, ranging from mild to severe anemia; it is associated with a spectrum of symptoms and complications which can be acute and long-term and start early in life. Registry data indicated an early age of onset of liver disease and iron overload (regardless of transfusion status), both of which were common in pediatric patients (pts). Supportive therapies in children include transfusions and splenectomy and can be associated with short- and long-term risks. No pharmacotherapies are approved for use in children with PK deficiency. Mitapivat is a first-in-class, oral, allosteric activator of PK, including the PKR and M2 (PKM2) isoforms, which act in glycolysis to generate adenosine triphosphate. Aims: Evaluate efficacy and safety of mitapivat versus placebo in pediatric pts with PK deficiency who were not regularly transfused. Methods: ACTIVATE-Kids (NCT05175105) is a phase 3, randomized, global, multicenter, double-blind, placebo-controlled study. Pts aged 1–<18 years (yrs) with PK deficiency who were not regularly transfused (defined as ≤5 transfusions in the 52-week [wk] period before providing informed consent/assent and no RBC transfusions ≤12 wks before administration of the first dose of study drug) were randomized 2:1 to receive twice daily oral mitapivat (1 mg to 5 mg based on age and weight, with potential escalation up to 10 mg to 50 mg) or placebo during the double-blind period (20 wks). Randomization was stratified by age (1 to <6 yrs, 6 to <12 yrs, 12 to <18 yrs). The primary endpoint was hemoglobin (Hb) response, defined as a ≥1.5 g/dL increase in Hb concentration from baseline, sustained at ≥2 scheduled assessments at Wks 12, 16, and 20 during the double-blind period. The primary endpoint was analyzed using Bayesian methodology that incorporated Hb response information from the adult ACTIVATE (NCT03548220) study. Secondary endpoints included: average change from baseline in Hb concentration, indirect bilirubin, and lactate dehydrogenase (LDH) at Wks 12, 16, and 20, and safety. Results: Thirty pts were randomized (mitapivat: N=19 and placebo: N=11). Mean age was 9.6 yrs. Baseline characteristics for the mitapivat and placebo arms: prior splenectomy (52.6% [10/19] and 36.4% [4/11]), prior iron chelation (21.1% [4/19] and 18.2% [2/11]), prior cholecystectomy (42.1% [8/19] and 36.4% [4/11]), mean (SD) baseline Hb (8.48 [1.085] and 8.46 [0.685] g/dL). The primary endpoint was met; observed Hb response rate was higher for pts in the mitapivat arm than in the placebo arm (31.6% [6/19] vs 0% [0/11]). Improvements in changes from baseline for Hb and markers of hemolysis were observed in the mitapivat arm compared to the placebo arm: average change from baseline at Wks 12, 16, and 20 (difference in least squares mean [95% CI]) in Hb concentration (0.90 g/dL [-0.07, 1.87]), indirect bilirubin (-27.25 umol/L [-59.65, 5.15]), and LDH (-154.08 U/L [-290.76, -17.40]). The proportion of pts with any treatment-emergent adverse events (TEAEs) was similar across treatment arms (mitapivat: 78.9% [15/19]; placebo: 90.9% [10/11]). TEAEs reported in ≥15% of pts on mitapivat include upper respiratory infection, headache, and initial insomnia. Serious TEAEs were reported in 5.3% (1/19) of pts on mitapivat and 9.1% (1/11) of pts on placebo, none were considered treatment related. No AEs led to discontinuation or death. Conclusions: ACTIVATE-Kids is the first study to demonstrate improvements in Hb and markers of hemolysis in children with PK deficiency who are not regularly transfused. Mitapivat, in tablets and pediatric granule formulation, was generally well tolerated and consistent with the safety profile observed for adults and regularly transfused children with PK deficiency. The efficacy and safety results from ACTIVATE-Kids together with the previous ACTIVATE-KidsT (NCT05144256) study support the potential for mitapivat to provide clinically substantial benefits in children with PK deficiency and may provide insight into the development of future clinical trials for pediatric pts with other hemolytic anemias.

Data from a large cohort of individuals referred for NGS testing evaluate the utility of next-generation sequencing in clinical practice for diagnosing hereditary haemolytic anaemias.

Introduction:Molecular analysis of red cell disorders has revolutionized diagnosis, however, there remain challenges. Main Symptoms:This patient presented with hemolytic anemia in the newborn period. He required chronic transfusions to maintain his hemoglobin level until 6 years of age. A splenectomy was performed at 3 years of age. Main Diagnoses:Using whole genome sequencing, we were able to identify a duplication upstream of the red cell promoter of HK1. Long-read RNA sequencing established aberrant expression off of this promoter. Conclusions:These non-coding variants remain challenging to identify. His promoter duplication may have a founder effect in South Asia.

Introduction: Pyruvate kinase (PK) deficiency is a rare, genetic disease that leads to chronic hemolytic anemia and potentially life-limiting complications. To facilitate best management and improve patient outcomes, international expert guidelines for the diagnosis and management of PK deficiency were published in 2024 (Al-Samkari). Aims: To characterize the disease monitoring and management practices among pediatric patients with PK deficiency and compare the findings to the 2024 guideline recommendations. Methods: This study was a retrospective analysis of real-world data for pediatric patients (aged 1–17 years at the last documented visit [index date]) from the observational PK Deficiency Natural History Study (NHS; NCT02053480; 2014–2019) and the Pyruvate Kinase Deficiency Global Longitudinal (Peak) Registry (NCT03481738; 2018–ongoing). Data from the two registries were merged where possible and summarized descriptively for patients overall and by transfusion status in the year prior to the index: not regularly transfused (NRT; <6 transfusions) and regularly transfused (RT; ≥6 transfusions). Results are specific to documentation of clinical monitoring and disease management activities documented during registry participation; patients were required to have at least 1 year of retrospective data to ensure adequate time for monitoring to occur. Data were compared with the 2024 guidelines for the monitoring and management of pediatric patients, including recommendations concerning iron overload (IO), chelation, vitamin D and bone health, endocrine function, and splenectomy. Results: As of May 2023 (data cut-off), 140 pediatric patients were included (NRT, n=107; RT, n=33). At the index date, the median age of patients was 8.5 years (range 1–17), 47.9% were female, 82.4% were White, and PKLR genotype distribution was 53.3% missense/missense, 31.4% missense/non-missense, and 15.3% non-missense/non-missense. Patients who were NRT averaged less than one transfusion in the year prior to the index date (mean 0.9; standard deviation [SD] 1.5); 61.4% had a history of IO. In contrast, patients who were RT received an average of 10.2 transfusions (SD: 3.5); 90.6% had a history of IO. At the index date, patients who were NRT were older than patients who were RT (median age 10 and 5 years, respectively), and this cohort had a higher rate of splenectomy (NRT: 43.3%; RT: 27.3%). The data obtained during the registry did not allow for assessment of all practice monitoring or management recommendations. However, analysis showed that all patients who were RT (33/33) received ferritin monitoring in alignment with the recommendation to screen for IO in patients ≥3 years of age or after 12 transfusions, whichever occurs first. In contrast, not all patients who were NRT aged ≥3 years received ferritin monitoring (mean percentage for ages 3 to <6, 83%; for 6 to <12, 85%; for 12 to <18 years, 77%). Annual liver T2* MRI is recommended in all patients receiving chelation therapy, but only 31.6% (12/38) of those patients with ongoing chelation received liver T2* MRI monitoring. One-third (31.6%; 6/19) of patients who were RT and 52.6% (10/19) of patients who were NRT had a cardiac T2* MRI whilst on chelation therapy. Annual vitamin D monitoring is recommended from age 1 for those not on regular supplementation; monitoring occurred in 24.7% of patients (20/81; Peak registry data only), and 27.2% of patients received vitamin D supplementation (22/81; Peak registry data only). Guidelines advise endocrine monitoring for all patients who are RT and non-transfused patients with IO: 36.8% (7/19) of patients who were RT and 51.9% (14/27) of patients who were NRT with IO received such monitoring (NHS registry data only; thyroid hormone and sex hormones measured). Conclusions: Our review of the real-world practices reported in the NHS and Peak Registry prior to the publication of the 2024 guidelines in PK deficiency identified notable areas for improvement in patient management including monitoring of bone health, endocrine dysfunction, and IO in patients who were NRT. The findings emphasize the need for consistent care and appropriate monitoring for all pediatric patients with PK deficiency, regardless of transfusion status or perceived severity of disease.

Diamond-Blackfan anemia (DBA) is a rare hematological disorder characterized by red blood cell aplasia. Advances in genomic studies have identified mutations in ribosomal protein genes (such as RPS19, RPL11, RPS26) and non-ribosomal genes (GATA1, EPO, ADA2, TSR2) as the underlying causes of DBA. In addition to anemia, DBA patients often present with bone development-associated defects, including short stature, thumb or craniofacial anomalies, and an increased risk of developing osteosarcoma. These findings suggest a dysregulated non-hematopoietic bone marrow microenvironment (BME) in DBA patients, which is not well understood. Mesenchymal stem cells (MSCs) are a crucial component of the BME and can differentiate into osteoblasts, adipocytes, and chondrocytes. In this study, we determined the molecular defects of DBA MSCs using both mouse and human RPL11 haploinsufficient MSCs. Mouse DBA MSCs were derived from RPL11+/fl carrying Cre-ERt2 and Mx1-Cre, in which the Rpl11 gene is deleted upon injecting tamoxifen or polyinosinic:polycytidylic acid (poly(I:C)), respectively. Additionally, we used cell-permeable Cre in vitro to delete the gene and observe the immediate effect of gene deletion. For human MSCs, we either knocked out the RPL11 gene using the CRISPR/Cas9 system or used DBA patient-derived induced pluripotent stem cells (iPSCs)-derived mesenchymal stem cells. Using these models, we identified a decrease in the proliferation of MSCs from RPL11 mutant mice and a DBA patient compared to healthy MSCs (7.04x104±1.01 and 3.62x104±1.00 in WT and DBA, respectively). The reduced proliferation observed in DBA MSCs is due to G2/M phase cell cycle arrest. Interestingly, DBA cells did not show elevated p53 compared to WT MSCs, which is often observed in DBA patients. DBA cells showed a significant reduction of G2/M phase-associated mRNA and proteins such as cyclin A, cyclin B, and CDK1. DBA MSCs showed an increased accumulation of binucleated cells (27.01%±7.501) compared to healthy MSCs (4.995%±1.596), suggesting cytokinesis failure as the underlying cause for G2/M phase arrest. Cytokinesis failure is known to activate the Hippo pathway in cells. When the Hippo pathway is activated, a core kinase called LATS1/2 (large tumor suppressor kinases 1/2) phosphorylates a downstream target called YAP (yes-associated protein), a transcription co-activator that mediates cell proliferation, survival, cytoskeleton arrangement, and osteogenic differentiation in MSCs. Phosphorylation of YAP inhibits the nuclear translocation of YAP through cytoplasmic retention or protein degradation. Western blot analysis and fluorescence microscopy showed a 30% increase in cytoplasmic YAP and a reduction in nuclear YAP in DBA MSCs compared to normal MSCs. As a result of YAP inactivation, actin remodeling and osteogenic differentiation potential were significantly reduced in DBA MSCs. Both mouse and human DBA MSCs showed increased cortical actin accumulation and loss of lamellipodia and stellate morphology. Furthermore, DBA MSCs expressed low RUNX2 (Runt-related transcription factor 2), which is an essential transcription factor for osteoblast differentiation. As expected, both mouse and human DBA MSCs show reduced osteogenic differentiation potential compared to healthy MSCs. Based on these results, we hypothesize that reactivating YAP nuclear translocation increases proliferation in DBA MSCs. To test this hypothesis, we treated mouse DBA and healthy MSCs with a LATS inhibitor at 10nM, 100nM, 1µM, 5 µM, and 10 µM. After 7 days of treatment, DBA MSCs partially restored proliferation compared to control MSCs, with statistical significance starting from 1 µM (p-value < 0.0001 compared to the DMSO control). Among other drugs tested, such as corticosteroids and L-leucine, the LATS inhibitor was the only effective drug that restored proliferation. In addition, the LATS inhibitor restored the lamellipodia formation and stellate morphology of DBA MSCs in a dose-dependent manner.In conclusion, we characterized and identified the impaired YAP signaling pathway of bone marrow mesenchymal stem cells in DBA. Results from this study will lay the groundwork for understanding the bone marrow microenvironment of DBA and identifying potential targets for more effective treatment in DBA.

Severe aplastic anemia (SAA) is characterized by pancytopenia and is either inherited or acquired (idiopathic). Accurate diagnosis is critical for proper treatment; however, the frequent overlap in clinical presentation between idiopathic SAA and inherited bone marrow failure syndromes (IBMFS) presents diagnostic and management challenges. For patients with idiopathic SAA, immunosuppressive therapy (IST) is considered the standard of care, when there is no available matched related donor. While 70-75% of pediatric patients with SAA respond to IST, patients with IBMFS do not. To our knowledge, there are no published data on a specific age cut-off that predicts a higher likelihood of an inherited disease, to guide treatment with IST or hematopoietic stem cell transplantation (HSCT). The aim of this study was to evaluate response to IST in children with SAA who are less than 3 years of age and to gain insight into the age below which response to IST is unlikely, suggesting an IBMFS. In a retrospective cohort study, we extracted data of patients diagnosed with SAA between 2002 and 2021 from 3 ethics board-approved multicenter databases: (1) North American Pediatric Aplastic Anemia Consortium (NAPAAC; 2002-2014), (2) Canadian Aplastic Anemia and Myelodysplasia Study (CAMS) and (3) Canadian Inherited Marrow Failure Registry (CIMFR). Patient characteristics, treatment and outcomes were collected. Patients were included if diagnosed with acquired SAA before the age of 3 years and received IST as a first line treatment for at least 3 months. Patients were excluded if they had physical malformations suggestive of an IBMFS, a first-degree relative with a history of bone marrow failure, or insufficient available data. Response rate was compared with published literature on pediatric patients with SAA over the age of 3 years. Descriptive statistics were utilized (SPSS V25). Among 31 patients aged 1.3-2.9 years (median 2.25) treated with IST as a first line therapy for SAA, 13 (42%) were male, 10 (32%) had hepatitis associated SAA, 3 (10%) had mild physical abnormalities (vesicoureteral reflux, patent ductus arteriosus, mild hearing impairment), none had short stature, and 7 (23%) had a family history of cancer (n=4), polycythemia vera (n=1), hyper-eosinophilic syndrome (n=1) or consanguinity (n=1). Twenty-nine patients (93%) were treated with horse anti-thymocyte globulin (ATG) and cyclosporine A (CSA), while 2 (7%) were treated with cyclophosphamide (CTX). Of the 31 patients, 24 (77%) had a complete response (CR) to IST (including the 2 patients post CTX). Twelve (50%), 22 (92%) and 24 (100%) were transfusion independent by 3, 6 and 12 months from the start of IST, respectively. CSA was discontinued after a median time of 20.4 months (range 5.6-55). No relapse was reported during long-term follow up (median 79 months, range 29-233). Seven patients (23%, age 1.95-2.83 years, median 2.44), had no response to a first IST course, 5 of whom received a second IST course with rabbit ATG. Of these 5 patients, 3 (age 1.95, 2.44 and 2.45 years) did not respond to the second IST and underwent HSCT, 1 (age 2.8 years) had a partial response and became transfusion-independent, 1 (age 2.83 years, who had a second IST with tacrolimus) achieved partial response and then switched to danazol with a CR. Two of the 7 patients who did not respond to the first IST did not receive a second IST before transplant; 1 of them (age 2.25 years) had an unsuccessful response to danazol and 1 (age 2.04 years) had no interim treatment. Complications during the first 3 months post IST included infections (bacteremia/sepsis, n=5; cellulitis, n=2; viral gastroenteritis, n=1), gross hematuria (n=1), hypertension (n=4) and gum hyperplasia (n=1). In the 3-6 months post-IST interval, 2 patients had bleeding events (gross hematuria, oral bleed) and 1 had a bacteremia episode. In the 6-12 months post-IST interval, 1 patient had bacteremia, 2 had hypertension and 1 developed a chronic kidney injury secondary to CSA. In conclusion, our study indicates for the first time a high complete response rate of 77% in SAA patients under 3 years of age who were treated with IST, a response rate comparable to what is reported in the literature for IST response in older pediatric patients. Although our study is limited by its retrospective nature and a relatively small cohort, due to the lack of previously published data, this research provides critical insight to help in managing this age group.

Diamond Blackfan Anemia (DBA) is a rare macrocytic red blood cell aplasia that usually presents within the first year of life. The vast majority of patients carry a mutation in one of approximately 20 genes that results in ribosomal insufficiency with the most significant clinical manifestations being anemia and a predisposition to cancers. Nemo-like Kinase (NLK) is hyperactivated in the erythroid progenitors of DBA patients and inhibition of this kinase improves erythropoiesis, but how NLK contributes to the pathogenesis of the disease is unknown. Here we report that activated NLK suppresses the critical upregulation of mitochondrial biogenesis required in early erythropoiesis. During normal erythropoiesis, mTORC1 facilitates the translational upregulation of Transcription factor A, mitochondrial (TFAM) and Prohibin 2 (PHB2) to increase mitochondrial biogenesis. In our models of DBA, active NLK phosphorylates the regulatory component of mTORC1, thereby suppressing mTORC1 activity and preventing mTORC1-mediated TFAM and PHB2 upregulation and subsequent mitochondrial biogenesis. Improvement of erythropoiesis that accompanies NLK inhibition is negated when TFAM and PHB2 upregulation is prevented. These data demonstrate that a significant contribution of NLK on the pathogenesis of DBA is through loss of mitochondrial biogenesis.

Pyruvate kinase (PK) deficiency is the most common cause of chronic congenital non-spherocytic haemolytic anaemia worldwide, with an estimated prevalence of one in 100 000 to one in 300 000 people. PK deficiency results in chronic haemolytic anaemia, with wide ranging and serious consequences affecting health, quality of life, and mortality. The goal of the International Guidelines for the Diagnosis and Management of Pyruvate Kinase Deficiency was to develop evidence-based guidelines for the clinical care of patients with PK deficiency. These clinical guidelines were developed by use of GRADE methodology and the AGREE II framework. Experts were invited after consideration of area of expertise, scholarly contributions in PK deficiency, and country of practice for global representation. The expert panel included 29 expert physicians (including adult and paediatric haematologists and other subspecialists), geneticists, laboratory specialists, nurses, a guidelines methodologist, patients with PK deficiency, and caregivers from ten countries. Five key topic areas were identified, the panel prioritised key questions, and a systematic literature search was done to generate evidence summaries that were used in the development of draft recommendations. The expert panel then met in person to finalise and vote on recommendations according to a structured consensus procedure. Agreement of greater than or equal to 67% among the expert panel was required for inclusion of a recommendation in the final guideline. The expert panel agreed on 31 total recommendations across five key topics: diagnosis and genetics, monitoring and management of chronic complications, standard management of anaemia, targeted and advanced therapies, and special populations. These new guidelines should facilitate best practices and evidence-based PK deficiency care into clinical practice.

Introduction: Pyruvate kinase (PK) deficiency is a rare, congenital, hemolytic anemia caused by mutations in the PKLR gene. Patients, including those never managed with blood transfusions, experience iron overload and other disease-related complications. In 2024, the first international expert guidelines for PK deficiency were published and included monitoring recommendations for patients never transfused (NT). Aim: Todescribe the reasons provided by clinicians for not transfusing patients with PK deficiency who were NT and the disease monitoring activities in these patients, using data from two real-world studies: Peak Registry (NCT03481738) and PK deficiency Natural History Study (NHS; NCT02053480). Methods: Both NHS and Peak were designed as global, longitudinal, observational studies enrolling patients with PK deficiency (NHS 2014-2017, Peak 2018-ongoing [data cut-off date: 15May2023]). This descriptive analysis used merged data from both studies and included patients aged ≥18 years, with a confirmed diagnosis of PK deficiency who were NT (defined as no lifetime history of blood transfusions before/during study follow-up). A subgroup analysis of patients with ≥12 months of retrospective data was performed to ensure adequate time for monitoring to occur. Results were evaluated relative to pertinent recommendations from the PK deficiency international expert guidelines. Results: A total of 55 adult NT patients were included in the analysis. Median age (min-max) at the last visit was 39 years (18-81), 43.6% were female, 86.0% were White, 10.2% were Hispanic/Latino. Most patients resided in Southern Europe (43.6%), followed by North America (18.2%), Northern Europe (18.2%), Asia (12.7%), and Central Europe (7.3%). Overall, 18.9% (10/53; 2 unknown) had previously undergone splenectomy. PKLR genotype distribution was 62.3% missense/missense, 34.0% missense/non-missense, and 3.8% non-missense/non-missense. Median (min-max) lab results at patients' last visit included hemoglobin 11.4 g/dL (6.8-18.3), reticulocytes 6.6% (2.6-63.0), and ferritin 312.0 ng/mL (16.6-6208.0). The most common reason for not being transfused was “anemia not very severe” (71.1%), followed by “anemia not symptomatic” (20.0%), “iron-overload risks” (11.1%), “patient objection” (4.4%), “immune-associated risks” (2.2%), and “injection-related risks” (2.2%); rationale was only captured in Peak (n=45) and multiple response options were allowed. Among the 42 NT patients with ≥12 months of retrospective data, clinical monitoring received during registry participation (and recent pre-baseline history) included lab assessments for hemoglobin (97.6%), reticulocytes (85.7%), and ferritin (95.2%). Bone health was monitored via 25-hydroxyvitamin D (23.8%, 10/42) and Dexa scan (12.9%, 4/31). Cardiovascular monitoring occurred in 37.5% and 19.4% of NHS and Peak participants, respectively. MRI for iron assessment (liver and/or cardiac) was performed for 20.8% of NHS patients and 25.0% of Peak patients. Among 5 patients with ongoing chelation therapy, all had registry documentation of ferritin monitoring, and none had a liver iron concentration evaluation via MRI. Complications among the 42 NT patients with ≥12 months of retrospective data included iron overload (40.0%), osteoporosis (12.1%), extramedullary hematopoiesis (8.6%), left ventricular hypertrophy (7.4%), diabetes (5.7%), thromboembolic events (4.3%), hepatic cirrhosis (2.8%), and arrhythmia (2.7%). These complications, except for hepatic cirrhosis, were also observed in the subgroup reporting anemia not very severe and/or not symptomatic. Conclusion: Among NT patients with PK deficiency, observed medical monitoring practices fall short of evidence-based recommendations in recently published guidelines. NT patients are at risk for complications, such as iron overload and osteoporosis, that require routine monitoring. Even patients classified as “not being very severe” and/or “not symptomatic” experienced disease complications. These findings emphasize the need for evidence-based disease monitoring to be consistently implemented for all patients with PK deficiency, enabling early detection and management of complications. Guidelines recommend annual screening for iron overload irrespective of transfusion status, highlighting that chelation therapy can potentially avoid further complications.

Pyruvate kinase (PK) deficiency, a rare, congenital haemolytic anaemia caused by mutations in the PKLR gene, is associated with many clinical manifestations, but the full disease burden has yet to be characterised. The Peak Registry (NCT03481738) is an observational, longitudinal registry of adult and paediatric patients with PK deficiency. Here, we described comorbidities and complications in these patients by age at most recent visit and PKLR genotype. As of 13 May 2022, 241 patients were included in the analysis. In total, 48.3% had undergone splenectomy and 50.5% had received chelation therapy. History of iron overload (before enrolment/during follow-up) was common (52.5%), even in never-transfused patients (20.7%). Neonatal complications and symptoms included jaundice, splenomegaly and hepatomegaly, with treatment interventions required in 41.5%. Among adults, osteopenia/osteoporosis occurred in 19.0% and pulmonary hypertension in 6.7%, with median onset ages of 37, 33 and 22 years, respectively. Biliary events and bone health problems were common across PKLR genotypes. Among 11 patients who had thromboembolic events, eight had undergone prior splenectomy. Patients with PK deficiency may have many complications, which can occur early in and throughout life. Awareness of their high disease burden may help clinicians better provide appropriate monitoring and management of these patients.

BACKGROUND:Identification of hemoglobin (Hb) variants is of significant value in the clinical diagnosis of hemoglobinopathy. However, conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of structural characterization. We describe the use of neutral-coating capillary electrophoresis coupled with high-resolution mass spectrometry (CE-HR-MS) to achieve high-performance top-down identification of Hb variants.METHODS:An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 capillary electrophoresis (CE) unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Deconvolution of raw mass spectrometry data was carried out to merge multiple charge states and isotopic peaks of an analyte to obtain its monoisotopic mass.RESULTS:The neutral-coating CE could baseline separate individual Hb subunits dissociated from intact Hb forms, and the HR-MS could achieve both intact-protein analysis and top-down analysis of analytes. A number of patient samples that contain Hb subunit variants were analyzed, and the variants were successfully identified using the CE-HR-MS method.CONCLUSIONS:The CE-HR-MS method has been demonstrated as a useful tool for top-down identification of Hb variants. With the ability to characterize the primary structures of Hb subunits, the CE-HR-MS method has significant advantages to complement or partially replace the conventional methods for the identification of Hb variants.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that presents during early childhood and is characterized by macrocytic anemia, congenital malformations, and predisposition to cancer. More than 90% of DBA patients are diagnosed during their first year of life (median age 12 weeks). The pathogenesis of DBA is linked to loss-of-function mutations in genes encoding ribosomal proteins (RP), although mutations in 3 non-RP genes ( GATA1, TSR2, and HEATR3) have been detected. DBA patients present with a high degree of clinical heterogeneity with varying severity and responses to steroid therapy. DBA provides a unique disease model to study how RP deficiency impacts the phenotype and pathogenesis of ribosomopathies. However, one challenge is the lack of animal models that faithfully recapitulate the clinical features of DBA in terms of disease severity and onset age. Heterozygous loss-of-function mutations in RPL11 are found in 5-20% of DBA patients, but previous mouse models carrying Rpl11 haploinsufficiency have only a mild anemia. Here, we report a novel mouse model of DBA with inducible haploinsufficient expression of Rpl11 which results in a severe macrocytic anemia in juvenile mice. Mx1-Cre Rpl11 +/flox experimental mice were generated by cross-breeding homozygous Rpl11 flox/flox and Mx1-cre breeders. Haploinsufficiency of Rpl11 ( Rpl11 +/Δ) was induced by intraperitoneal injection of polyinosinic-polycytidylic acidto mice with Mx1-Cre Rpl11 +/flox on postnatal day 8 and 10. Diseased mice with Rpl11 +/Δ showed macrocytic anemia at 2 weeks (wks) post-induction with hemoglobin (Hb) < 10 g/dL, eventually dying of severe anemia (Hb< 2 g/mL) by 30 wks (median survival 27 wks, p<0.001, n=7 )( Figure 1). We observed a 50% decreased expression of Rpl11 in blood neucleated cells from Rpl11 +/Δ mice and several characteristic clinical features of DBA. Erythrocyte adenosine deaminase was significantly elevated in the blood of all Rpl11 +/Δ mice when compared to wild type (WT) littermates (median 5 vs 1 EU/g Hg, p<0.001, n=6). Similarly, EPO concentrations were significantly elevated in Rpl11 +/Δ mice compared to WT mice (median 38752 vs 23 pg/mL, p<0.01, n=6). On postmortem examination, the DBA mice displayed splenomegaly. FACS analysis data demonstrated that the differentiation of erythropoiesis is significantly blocked in peripheral blood, bone marrow (BM), and spleen of Rpl11 +/Δ mice compared to WT mice (p<0.05, n = 5). In addition, methylcellulose colony assays revealed that CFU-E colonies were significantly decreased in BM from Rpl11 +/Δ mice in comparison to WT mice (median 23.5 vs 40.5 per 0.2 million BM cells in the presence of EPO at a concentration of 1 unit/mL, p<0.05, n=4). To determine whether BM hematopoietic stem cells/progenitor cells from Rpl11 +/Δ mice can transfer the DBA phenotype, we transplanted bone marrow cells of Rpl11+/ Δ mice (CD45.2+) into sublethally irradiated WT mice (CD45.1+). Our data demonstrate that the BM cells from Rpl11 +/Δ donor mice can induce lethal macrocytic anemia in WT recipient mice. Studies on the molecular mechanisms underlying the dysregulated erythropoiesis in Rpl11 +/Δ mice are ongoing. In conclusion, we have successfully generated the first DBA mouse model that recapitulates the hematologic features of DBA patients. This model provides an ideal tool to study the pathogenesis of DBA and other ribosomopathies.