Pyruvate Kinase Deficiency Research Articles

P1735: IMPROVEMENTS IN PATIENT-REPORTED OUTCOMES IN MITAPIVAT-TREATED PATIENTS WITH PYRUVATE KINASE DEFICIENCY: A DESCRIPTIVE ANALYSIS FROM THE PHASE 3 ACTIVATE TRIAL

Background: Pyruvate kinase (PK) deficiency is a rare, inherited, non-spherocytic hemolytic anemia associated with acute and long-term complications, as well as a spectrum of signs and symptoms including jaundice, fatigue, and dyspnea. PK deficiency has a profound wide-ranging impact on health-related quality of life (HRQoL). Mitapivat (AG-348), a first-in-class, oral, allosteric activator of PK, was shown to improve hemoglobin (Hb), hemolysis, and hematopoiesis in a global, phase 3, randomized, placebo (PBO)-controlled trial evaluating mitapivat efficacy and safety in adults with PK deficiency who were not regularly transfused (ACTIVATE; NCT03548220). Furthermore, significant improvements in patient-reported outcomes (PROs) (measured by disease-specific PRO instruments: the PK deficiency diary [PKDD] and the PK deficiency impact assessment [PKDIA]) were demonstrated in patients (pts) receiving mitapivat compared with PBO. The PKDD is a self-administered, 7-item daily diary to assess the signs and symptoms of PK deficiency (including jaundice, tiredness, and shortness of breath), while the PKDIA is a 12-item weekly measure assessing the impacts of PK deficiency (pts rated the frequency of occurrence or difficulty of various activities). Aims: To describe PKDD and PKDIA outcomes for the subset of pts in the ACTIVATE trial who achieved the primary endpoint of Hb response, defined as a ≥1.5 g/dL increase in Hb from baseline (BL), sustained at ≥2 scheduled assessments at weeks (wks) 16, 20, and 24. Methods: Eighty pts were randomized 1:1 to receive mitapivat (5/20/50 mg twice daily) or PBO for 24 wks in the ACTIVATE trial. Change from BL to wk 24 in PKDD and PKDIA scores were prespecified secondary endpoints. For this post hoc analysis, changes from BL in PKDD weekly mean scores and PKDIA scores measured at scheduled visits (wks 4, 8, 12, 16, 20, and 24) were summarized for Hb responders, the overall mitapivat treatment arm, and PBO, using mean and 95% confidence intervals (CIs). For both the PKDD and the PKDIA, a lower score represents a lower disease burden. Results: In the ACTIVATE trial, 16/40 (40%) pts receiving mitapivat met the primary endpoint of Hb response compared with none for PBO (0/40; 0%). At wk 24, mean (95% CI) change from BL in PKDD weekly mean score was ‒7.12 (‒10.98, ‒3.27) for pts who achieved Hb response, ‒5.43 (‒7.47, ‒3.40) for the overall mitapivat treatment arm, and ‒1.86 (–4.04, 0.32) for the PBO arm (Figure 1a). At wk 24, mean (95% CI) change from BL in PKDIA score was ‒8.07 (‒11.05, ‒5.08) for pts who achieved Hb response, ‒4.82 (‒7.18, ‒2.46) for the overall mitapivat treatment arm, and ‒1.06 (‒3.70, 1.59) for the PBO arm (Figure 1b). Across both PRO instruments, improvements among mitapivat pts who achieved Hb response were sustained over time. Image:Summary/Conclusion: In the ACTIVATE trial, mitapivat-treated pts demonstrated significant improvements in signs, symptoms, and impacts based on PK deficiency-specific PRO instruments, compared with PBO. This analysis further reveals that improvements were greater in the subset of mitapivat-treated pts who achieved the primary endpoint. Together, these data indicate that mitapivat has the potential to improve HRQoL in pts with PK deficiency who are not regularly transfused.

P1547: ACTIVATE-KIDS: MITAPIVAT IN CHILDREN WITH PYRUVATE KINASE DEFICIENCY WHO ARE NOT REGULARLY TRANSFUSED

Background: Pyruvate kinase (PK) deficiency is a rare, inherited disorder caused by PKLR gene mutations that is associated with chronic hemolytic anemia and lifelong complications. Clinical management includes red blood cell (RBC) transfusions and splenectomy, which only partially improves anemia. No pharmacotherapies are approved for use in children, and therapies targeting the underlying cause of hemolysis are needed. Mitapivat (AG-348), a first-in-class, oral, allosteric activator of the RBC PK enzyme, demonstrated significant and durable improvements in hemoglobin (Hb) and markers of hemolysis, hematopoiesis, and iron metabolism in adults with PK deficiency who were not regularly transfused (ACTIVATE NCT03548220, long-term extension study NCT03853798). Safety and efficacy data support the evaluation of mitapivat in children with PK deficiency who are not regularly transfused. Aims: Report the design of ACTIVATE-Kids (NCT05175105), a phase 3 study, which will evaluate the efficacy and safety of mitapivat in children with PK deficiency who are not regularly transfused. Methods: ACTIVATE-Kids, a global, multicenter, double-blind, placebo-controlled study, will stratify 30 children by age (1 to <6, 6 to <12, and 12 to <18 years); a minimum of 6 patients in each age group will be randomized (2:1) to mitapivat or placebo. The double-blind period comprises an 8-week dose-titration period and a 12-week fixed-dose period. A 5-year open-label period will follow the double-blind period. The study drug will be dosed (1–50 mg twice daily) according to patient age and weight, based on pharmacokinetic modeling data of adults with PK deficiency. Key inclusion criteria: ≥2 PKLR mutant alleles with ≥1 missense mutation, ≤5 RBC transfusions in the past year, Hb concentrations of ≤10 g/dL (12 to <18 years) or ≤9 g/dL (1 to <12 years). Key exclusion criteria: R479H homozygosity, 2 non-missense mutations without a missense mutation in PKLR, or prior stem cell transplantation. The primary endpoint is Hb response, defined as a ≥1.5 g/dL increase in Hb concentration from baseline that is sustained at ≥2 scheduled assessments at Weeks 12, 16, and 20 in the double-blind period. Secondary endpoints will evaluate the effect of mitapivat on Hb, hemolysis, erythropoiesis, iron metabolism and overload, safety, quality of life, and pharmacokinetics. Results: Global site recruitment is in-progress; an update on recruitment and patient enrollment will be provided. Summary/Conclusion: The phase 3 ACTIVATE-Kids study of children with PK deficiency who are not regularly transfused will evaluate treatment with mitapivat, a potential disease-modifying pharmacotherapy.

P1548: LONG-TERM TREATMENT WITH ORAL MITAPIVAT IS ASSOCIATED WITH NORMALIZATION OF HEMOGLOBIN LEVELS IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY

Background: Pyruvate kinase (PK) deficiency is a rare hereditary anemia caused by mutations in the PKLR gene encoding the red blood cell PK enzyme (PKR). Defects in PKR lead to chronic hemolytic anemia, which is associated with serious complications regardless of transfusion status. Mitapivat (AG-348), a first-in-class, oral, allosteric activator of PKR, has been shown in a placebo (PBO)-controlled, phase 3 clinical trial (ACTIVATE, NCT03548220) to significantly improve hemoglobin (Hb) concentration and markers of hemolysis and hematopoiesis in patients (pts) with PK deficiency who were not regularly transfused. These improvements were sustained in a long-term extension (LTE, NCT03853798) study that included pts who completed the fixed-dose period of the ACTIVATE study. Aims: This analysis focuses on normalization of Hb levels in pts treated in the ACTIVATE and ongoing LTE studies. Methods: The randomized, double-blind, PBO-controlled ACTIVATE study consisted of a 12-week dose-optimization period (5/20/50 mg twice daily) and a 12-week fixed-dose period. Eighty pts (≥18 years) with a diagnosis of PK deficiency who were not regularly transfused (≤4 transfusion episodes in prior year; none in the prior 3 months) were randomized 1:1 to receive mitapivat or PBO. The primary endpoint was Hb response, defined as ≥1.5 g/dL increase in Hb from baseline sustained at ≥2 scheduled assessments at weeks 16, 20, and 24 in the fixed-dose period. Pts who completed the fixed-dose period of ACTIVATE were eligible to continue in the LTE study, where all pts received mitapivat. In this analysis, the proportions of pts who achieved normal Hb at any time while receiving mitapivat and within specified time intervals were assessed, with data cutoff of 21Sep2021. Normal Hb levels achieved within 61 days after a transfusion were excluded. Results: In ACTIVATE, 16 of the 40 pts (40%) treated with mitapivat and none of the 40 patients randomized to PBO achieved an Hb response as defined in the study protocol. Across the ACTIVATE and LTE studies, 28/78 (35.9%) pts achieved a normal Hb level at least once during treatment with mitapivat. Among pts treated with mitapivat in both the ACTIVATE and LTE studies (M/M arm, mean Hb at baseline 8.66 g/dL), 15/40 (37.5%) achieved a normal Hb level at least once during treatment, the majority within the first few months. Similarly, among patients randomized to PBO in the ACTIVATE study and treated with mitapivat in the LTE study (P/M arm, mean Hb at baseline 8.47 g/dL), 13/38 (34.2%) achieved a normal Hb level at least once during the LTE, all within the first 4 months of treatment with mitapivat. Of the 16 M/M pts who achieved Hb response, 14 (87.5%) achieved a normal Hb level at least once during treatment with mitapivat either in the ACTIVATE study or the LTE; of the 15 P/M pts who achieved a protocol-defined Hb response during mitapivat treatment in the LTE, 12 (80.0%) achieved a normal Hb at least once. Seven pts had normal hemoglobin measurements at each time point assessed while on mitapivat treatment (Figure). Image:Summary/Conclusion: In this study, treatment with mitapivat was associated with early and robust Hb responses with approximately one-third of pts achieving normal Hb levels at least once. These results add to the evolving data demonstrating potential real-world benefits of mitapivat for patients with PK deficiency and supports mitapivat as an effective and disease-modifying therapy for this condition.

P1545: DURABILITY OF HEMOGLOBIN RESPONSE AND REDUCTION IN TRANSFUSION BURDEN IS MAINTAINED OVER TIME IN PATIENTS WITH PYRUVATE KINASE DEFICIENCY TREATED WITH MITAPIVAT IN A LONG-TERM EXTENSION STUDY

Background: Mitapivat (AG-348), a first-in-class, oral, allosteric activator of red blood cell (RBC) PK enzyme, demonstrated improvements in hemoglobin (Hb) and markers of hemolysis and hematopoiesis (ACTIVATE, NCT03548220), and reduction in transfusion burden (ACTIVATE-T, NCT03559699) in patients (pts) with PK deficiency. Aims: Report data from ACTIVATE, ACTIVATE-T, and their long-term extension (LTE) study (NCT03853798). Methods: In the double-blind, placebo (PBO)-controlled ACTIVATE study (12-week [wk] dose-optimization period [5/20/50 mg twice daily [BID]]; 12-wk fixed-dose period [FDP]), 80 pts (≥18 years [yrs]) with PK deficiency who were not regularly transfused (≤4 transfusion episodes in prior yr; none in prior 3 months [mos]) were randomized 1:1 to mitapivat or PBO. Primary endpoint was Hb response (≥1.5 g/dL increase in Hb from baseline [BL] sustained at ≥2 assessments at Wks 16, 20, and 24 in the FDP). In the single-arm, open-label ACTIVATE-T study (16-wk dose-optimization period [5/20/50 mg BID]; 24-wk FDP), 27 pts (≥18 yrs) with PK deficiency who were regularly transfused (≥6 transfusion episodes in prior yr) were treated with mitapivat. Primary endpoint was transfusion response (≥33% reduction in number of RBC units transfused during the FDP, compared with pt’s individual historical transfusion burden standardized to 24 wks). Secondary endpoint was achieving transfusion-free status (no transfusions in FDP). Pts who completed the FDP of ACTIVATE/ ACTIVATE-T were eligible to continue in the LTE, where all received mitapivat. The ACTIVATE/LTE analysis assessed duration of Hb response in 2 cohorts: 1) pts assigned to mitapivat who achieved a Hb response and continued to the LTE (mitapivat-to-mitapivat arm [M/M]); 2) pts assigned to PBO who switched to mitapivat in the LTE (PBO-to-mitapivat arm [P/M]) and then met Hb response criteria. The ACTIVATE-T/LTE analysis assessed transfusion response in the LTE, and transfusion-free duration among pts from ACTIVATE-T who achieved transfusion-free status. Results: In ACTIVATE, 40% of pts treated with mitapivat (N=40) achieved a Hb response; in the LTE, pts randomized to PBO in ACTIVATE showed similar improvements in Hb levels after switching to mitapivat. These improvements were sustained with continued treatment (Figure 1a). All 16 pts assigned to mitapivat in ACTIVATE who achieved Hb responses continued to the LTE; 15 M/M pts were evaluable for Hb assessment in the LTE. 13/15 M/M pts (86.7%) maintained ≥1.5 g/dL Hb increase from BL up to 19.5 mos at all time points; 2 pts maintained ≥1 g/dL Hb increase from BL at all time points. None of the pts assigned to PBO in ACTIVATE (N=40) had a Hb response; 17 P/M pts had sufficient time (24 wks of treatment) in the LTE for Hb response assessment. 6/17 pts (35%) achieved Hb responses in the LTE, and all maintained Hb responses during follow-up. In ACTIVATE-T (N=27), 37% of pts achieved a transfusion response and 22% of pts achieved transfusion-free status. In the LTE, 9 pts (33.3%) met criteria for a transfusion response. All 6 pts who achieved transfusion-free status in ACTIVATE-T maintained the status in the LTE up to 21.9 mos (Figure 1b). One additional pt, who met the primary endpoint, but was not transfusion free in ACTIVATE-T, did not receive any transfusions in the LTE. Image:Summary/Conclusion: Mitapivat improved Hb and reduced transfusion burden in pts with PK deficiency over time, demonstrating the consistency and long-term durability of treatment with mitapivat.

PB2247: PHARMACOKINETIC MODELING AND SIMULATION TO SUPPORT MITAPIVAT DOSE SELECTION USED IN PEDIATRIC PHASE III STUDIES

Background: Mitapivat (AG-348) is a first-in-class, oral, small molecule, allosteric activator of the red blood cell pyruvate kinase (PK) enzyme (PKR). The positive benefit-risk profile of mitapivat has been demonstrated in two recently completed phase 3 studies in adults with PK deficiency who are not regularly transfused (ACTIVATE, NCT03548220) or regularly transfused (ACTIVATE-T, NCT03559699). Currently, two phase 3 studies are being initiated to evaluate the efficacy and safety of mitapivat in pediatric subjects aged 1 to <18 years (yrs) with PK deficiency who are not regularly transfused (ACTIVATE-Kids, NCT05144256) or regularly transfused (ACTIVATE-KidsT, NCT05175105). Aims: Conduct a pharmacometrics analysis to support the mitapivat dose selection in the phase 3 studies in pediatric subjects. Methods: A three compartment population pharmacokinetic model with allometrically scaled parameters (clearances and volume of distribution) was applied. Dose simulations were conducted for various age subsets and body weight ranges using data from the National Health and Nutrition Examination Survey (NHANES) database. For subjects aged 1 to <2 yrs, in addition to allometric scaling, maturation factors (Salem F et al. Clin Pharmacokinet. 2014;53:625–36. Salem F et al. Clin Pharmacokinet. 2015;54:671) accounting for changes in cytochrome P450 (CYP) 3A activity were implemented. This approach was employed because mitapivat is metabolized primarily by CYP3A, and maturational changes in CYP3A ontogeny are known to occur during the first few yrs of life. The selected pediatric dose for each age subset and weight range was based on a dose that was predicted to achieve mitapivat exposure (ie, the area under the plasma concentration-time curve [AUC] during a dosing interval at steady state) similar to that achieved in adults receiving the recommended clinical dose for PK deficiency (dose titrations occurring every 4 weeks, from 5 mg twice daily [BID] to 20 mg BID, and then to the maximum recommended dose of 50 mg BID). Results: The proposed dosing scheme is shown in the Table. For subjects aged 12 to <18 yrs who weigh ≥40 kg, the recommended dose used in adults would provide a similar mitapivat AUC to that achieved in adults; the predicted difference is <20%, and therefore is not considered to be clinically meaningful. For subjects aged 12 to <18 yrs who weigh <40 kg, or subjects aged 2 to <12 yrs, dosing according to body weight (≥40 kg, ≥20 to <40 kg, and <20 kg) would yield similar mitapivat AUC ranges between pediatric and adult subjects. For subjects aged 1 to <2 yrs, the proposed doses shown in the Table are predicted to provide <5% difference in mitapivat AUC as compared with adults. Image:Summary/Conclusion: Population pharmacokinetic modeling and simulation was utilized to identify candidate doses for evaluation in mitapivat pediatric phase 3 studies. The selected doses for each pediatric age subset and body weight range are projected to provide mitapivat AUC similar to that achieved in adults receiving the recommended clinical dose.

Iron overload disorders.

Iron overload disorders represent a variety of conditions that lead to increased total body iron stores and resultant end‐organ damage. An elevated ferritin and transferrin‐iron saturation can be commonly encountered in the evaluation of elevated liver enzymes. Confirmatory homeostatic iron regulator (HFE) genetic testing for C282Y and H63D, mutations most encountered in hereditary hemochromatosis, should be pursued in evaluation of hyperferritinemia. Magnetic resonance imaging with quantitative assessment of iron content or liver biopsy (especially if liver disease is a cause of iron overload) should be used as appropriate. A secondary cause for iron overload should be considered if HFE genetic testing is negative for the C282Y homozygous or C282Y/H63D compound heterozygous mutations. Differential diagnosis of secondary iron overload includes hematologic disorders, iatrogenic causes, or chronic liver diseases. More common hematologic disorders include thalassemia syndromes, myelodysplastic syndrome, myelofibrosis, sideroblastic anemias, sickle cell disease, or pyruvate kinase deficiency. If iron overload has been excluded, evaluation for causes of hyperferritinemia should be pursued. Causes of hyperferritinemia include chronic liver disease, malignancy, infections, kidney failure, and rheumatic conditions, such as adult‐onset Still's disease or hemophagocytic lymphohistiocytosis. In this review, we describe the diagnostic testing of patients with suspected hereditary hemochromatosis, the evaluation of patients with elevated serum ferritin levels, and signs of secondary overload and treatment options for those with secondary iron overload.

Multicenter Study of Pyruvate Kinase Deficiency in Argentina

The red blood cell (RBC) pyruvate kinase deficiency (PKD) is the most common recessive congenital defect of glycolytic enzymes associated with non-spherocytic hemolytic anemia. It is a rare hereditary disorder caused by &gt;300 variants in the PKLR gene. This is a retrospective study of 19 patients from different centers from Argentina with confirmed molecular diagnosis of PKD. Clinical follow-up was carried out from birth in most cases. Five consanguineous patients from “gypsy” community, were homozygous for the “PK-Gypsy deletion” (PK-Gd). During the neonatal period they developed anemia with icterus. Transfusion exchange was required in 60%, light therapy in 80%, and RBC transfusion in 80%. During the follow-up iron overload was detected in the 100%, cholecystectomy was indicated in 40%, and splenectomy in 60%. Thirteen cases had 2 missense variants (MS), being the Mediterranean variant (p.Arg486Trp) the more frequent detected (26%).Only 1 patient had a missense-splicing mutation combination. During the neonatal period, 86% had anemia and icterus. Light therapy was required in 78%, transfusion exchange in 21% and RBC transfusion in 64%. During the follow-up iron overload was detected in 57% and splenectomy was indicated in 43%. Transfusions (pre-splenectomy and post-splenectomy) were more required in PK-Gd cases as compared with patients with point mutations (100%/60% vs 71%/29% respectively). Our data indicates a high clinical-therapeutic-molecular heterogeneity in PKD patients with the PK-Gd group presenting the most severe cases.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

Efficient and Fast Generation of Relevant Disease Mouse Models by In Vitro and In Vivo Gene Editing of Zygotes.

Knockout mice for human disease-causing genes provide valuable models in which new therapeutic approaches can be tested. Electroporation of genome editing tools into zygotes, in vitro or within oviducts, allows for the generation of targeted mutations in a shorter time. We have generated mouse models deficient in genes involved in metabolic rare diseases (Primary Hyperoxaluria Type 1 Pyruvate Kinase Deficiency) or in a tumor suppressor gene (Rasa1). Pairs of guide RNAs were designed to generate controlled deletions that led to the absence of protein. In vitro or in vivo ribonucleoprotein (RNP) electroporation rendered more than 90% and 30% edited newborn animals, respectively. Mice lines with edited alleles were established and disease hallmarks have been verified in the three models that showed a high consistency of results and validating RNP electroporation into zygotes as an efficient technique for disease modeling without the need to outsource to external facilities.

Genetic resistance to human malaria

Human beings are sometime expose to the same to predisposing factors of a given infectious disease, but the outcome in terms of disease manifestation differs greatly. This variation is mainly attributed to the genetic makeup of such individuals; this is because human genetic has long been associated with the variation in susceptibility to various infectious diseases, which is termed as genetic resistance. Therefore the aim of this paper was to review the state of knowledge on genetic resistance associated with malaria infection. Genetic resistance to malaria can be describe as an inherited alteration or changes in the genetic material of humans specifically DNA molecule and other vital biomolecules which increases the chances of resistance to malaria and thus, result in an increased survival of individuals with those genetic alterations. In addition such changes also affect the general wellbeing and survival of the parasite to the extent that the parasite cannot even multiply or replicate itself while in such infected erythrocyte. This is because such alteration in the DNA molecule interferes with some of the vital chemical and biochemical processes of the parasite (Plasmodim spp). Therefore, several genetic disorders and or trait which include: Sickle cell disease, Glocose-6-Phosphatedehyrogenase deficiency, Pyruvate Kinase deficiency, Duffy antigen, Ovalocytocytosis, Thalassemia and ABO blood group are known to offer special protection against malaria disease in individuals who possessed at least one of such disorders or trait.

Diagnosis, monitoring, and management of pyruvate kinase deficiency in children.

Pyruvate kinase (PK) deficiency is a rare, congenital red blood cell disorder caused by a single gene defect. The spectrum of genotypes, variants, and phenotypes are broad, commonly requiring a multimodal approach including enzyme and genetic testing for accurate and reliable diagnosis. Similarly, management of primary and secondary sequelae of PK deficiency varies, mainly including supportive care with transfusions and surgical interventions to improve symptoms and quality of life. Given the risk of acute and long-term complications of PK deficiency and its treatment, regular monitoring and management of iron burden and organ dysfunction is critical. Therefore, all children and adolescents with PK deficiency should receive regular hematology care with visits at least every 6months regardless of transfusion status. We continue to learn more about the spectrum of symptoms and complications of PK deficiency and best practice for monitoring and management through registry efforts (NCT03481738). The treatment of PK deficiency has made strides over the last few years with newer disease-modifying therapies being developed and studied, with the potential to change the course of disease in childhood and beyond.

Mitapivat versus Placebo for Pyruvate Kinase Deficiency

BackgroundPyruvate kinase deficiency is a rare, hereditary, chronic condition that is associated with hemolytic anemia. In a phase 2 study, mitapivat, an oral, first-in-class activator of erythrocyte pyruvate kinase, increased the hemoglobin level in patients with pyruvate kinase deficiency.MethodsIn this global, phase 3, randomized, placebo-controlled trial, we evaluated the efficacy and safety of mitapivat in adults with pyruvate kinase deficiency who were not receiving regular red-cell transfusions. The patients were assigned to receive either mitapivat (5 mg twice daily, with potential escalation to 20 or 50 mg twice daily) or placebo for 24 weeks. The primary end point was a hemoglobin response (an increase from baseline of ≥1.5 g per deciliter in the hemoglobin level) that was sustained at two or more scheduled assessments at weeks 16, 20, and 24. Secondary efficacy end points were the average change from baseline in the hemoglobin level, markers of hemolysis and hematopoiesis, and the change from baseline at week 24 in two pyruvate kinase deficiency–specific patient-reported outcome measures.ResultsSixteen of the 40 patients (40%) in the mitapivat group had a hemoglobin response, as compared with none of the 40 patients in the placebo group (adjusted difference, 39.3 percentage points; 95% confidence interval, 24.1 to 54.6; two-sided P<0.001). Patients who received mitapivat had a greater response than those who received placebo with respect to each secondary end point, including the average change from baseline in the hemoglobin level. The most common adverse events were nausea (in 7 patients [18%] in the mitapivat group and 9 patients [23%] in the placebo group) and headache (in 6 patients [15%] and 13 patients [33%], respectively). Adverse events of grade 3 or higher occurred in 10 patients (25%) who received mitapivat and 5 patients (13%) who received placebo.ConclusionsIn patients with pyruvate kinase deficiency, mitapivat significantly increased the hemoglobin level, decreased hemolysis, and improved patient-reported outcomes. No new safety signals were identified in the patients who received mitapivat. (Funded by Agios Pharmaceuticals; ACTIVATE ClinicalTrials.gov number, NCT03548220.)

Gene Editing for Inherited Red Blood Cell Diseases.

Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as β-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from β-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.

Next generation sequencing for diagnosis of hereditary anemia: Experience in a Spanish reference center

Background and aimsHereditary anemia (HA) encloses a wide group of rare inherited disorders with clinical and hematologic overlaps that complicate diagnosis. Materials and methodsA 48-gene panel was developed to diagnose HA by Next Generation Sequencing (NGS) in a large cohort of 165 patients from 160 unrelated families. ResultsPatients were divided in: A) patients who had a suspicion of a specific type of HA (n = 109), and B) patients who had a suspicion of HA but with no clear type (n = 56). Diagnostic performance was 83.5% in group A and a change of the initial diagnosis occurred in 11% of these patients. In group B, 35.7% of patients achieved a genetic diagnosis. NGS identified 6 cases of xerocytosis, 6 of pyruvate kinase (PK) deficiency, 4 of G6PD, and 1 case of phytosterolemia with no initial suspicion of these pathologies, which is clinically relevant since they have specific treatment. Five patients were found to carry variants associated to two different pathologies (4 of them combining a metabolic deficiency and a membrane defect), and 44 new variants were identified in 41 patients. ConclusionThe use of NGS is a sensitive technique to diagnose HA and it shows better performance when patients are better characterized.

Synthetic Red Blood Cell-Specific Glycolytic Intermediate 2,3-Diphosphoglycerate (2,3-DPG) Inhibits Plasmodium falciparum Development In Vitro.

Mechanisms of malaria parasite interaction with its host red blood cell may provide potential targets for new antimalarial approaches. Pyruvate kinase deficiency has been associated with resistance to malaria in both experimental models and population studies. Two of the major pyruvate kinase deficient-cell disorders are the decrease in ATP and the increase in 2,3-biphosphoglycerate (2,3-BPG) concentration. High levels of this metabolite, only present in mammalian red blood cell, has an inhibitory effect on glycolysis and we hypothesized that its accumulation may also be harmful to the parasite and be involved in the mechanism of protection provided by that enzymopathy. We examined the effect of a synthetic form, 2,3-DPG, on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. Results showed an impairment of parasite growth with a direct effect on parasite maturation as significant lower progeny emerged from parasites that were submitted to 2,3-DPG. Further, adding the compound to the culture medium did not result in any effect on the host cell, but instead the metabolic profile of an infected cell became closer to that of a non-infected cell.

A207 COMMON BILE DUCT OBSTRUCTION FROM RADIOGRAPHICALLY OCCULT LITHIASIS: A CASE REPORT AND CAUTIONARY TALE

BackgroundBiliary obstruction from choledocholithiasis (CDL) can lead to many serious complications, such as cholangitis and pancreatitis. Among noninvasive diagnostic tests for CDL, MRCP is superior to US and CT with a sensitivity of 89–95%. Despite this, MRCP may not always identify the cause of obstruction, leading to diagnostic dilemmas.AimsWe aim to review the various ways that CDL can hide on imaging, specifically MRCP.MethodsA 22-year-old male with pyruvate kinase deficiency was admitted to General Surgery for acute cholecystitis, for which he had a laparoscopic cholecystectomy. Preoperative MRCP showed a 3 mm common bile duct (CBD) with no stones; however, gross inspection of the gallbladder showed multiple green stones. Around one month later, he was readmitted to General Surgery for symptomatic biliary obstruction, with total bilirubin 923, ALP 457 and ALT 559. He was not febrile and did not require antibiotics. An MRCP showed a CBD of 10 mm with intra- and extrahepatic biliary dilation, but no obstructing focus was seen. Gastroenterology was consulted for undifferentiated hyperbilirubinemia. After further discussion, an ERCP with sphincterotomy was done the next day which also did not show obstruction. An abdominal US done the day after ERCP showed improvement in biliary dilation. After ERCP, the patient’s symptoms improved. Total bilirubin, ALP, and ALT one month later decreased to 121, 134, and 139 respectively. The hypothesis was that he had an obstructing gallstone that camouflaged within bile on imaging.Consent was obtained from the patient to present this case report.ResultsAlthough MRCP is one of the best noninvasive diagnostic tests for CDL, some stones may not appear on imaging. On MRCP, stones usually appear as hypointense spots surrounded by hyperintense bile on T2-weighted imaging. However, sludge can be isointense compared to bile. Additionally, stones impacted at the ampulla of Vater typically are not surrounded by bile and thus may evade detection on MRCP. Small stones <4 mm can be missed as well. CBD dilation greater than 10 mm is also associated with reduced sensitivity of MRCP for CDL. Even with ERCP, small stones may be missed unless seen on direct optical visualization after a blind balloon sweep.Even if no obstructing lesion is seen on imaging, other clinical features can still suggest CDL requiring ERCP. The ASGE 2019 Guidelines on CDL recommend prompt ERCP for patients with total bilirubin >68.4 µmol/L and CBD dilation on imaging. Other radiographic signs suggesting CDL include CBD dilation, papillitis, and pericholecystic fat infiltration on CT, and MRCP findings of CDL-associated inflammation such as biliary wall thickening and periductal edema.ConclusionsEven if MRCP does not show obvious obstruction, ERCP should still be considered if the likelihood of CDL remains high.Funding AgenciesNone

A novel homozygous missense variant p.D339N in the PKLR gene correlates with pyruvate kinase deficiency in a Pakistani family: a case report

BackgroundPyruvate kinase deficiency is an exceptionally rare autosomal recessive Mendelian disorder caused by bi-allelic pathogenic variants in the PKLR gene. It is mainly characterized by chronic nonspherocytic hemolytic anemia though other symptoms such as splenomegaly, hepatomegaly, pallor, fatigue, iron overload, shortness of breath, hyperbilirubinemia, and gallstones might also prevail.Case presentationWe present here a novel genetic defect in the PKLR gene that correlates with pyruvate kinase deficiency phenotype in a consanguineous family from North-Western Pakistan. The family included three affected individuals who were all born to consanguineous parents. The proband, a 13-year-old female of Pashtun ethnicity, showed chronic nonautoimmune hemolytic anemia since birth, extremely low hemoglobin (7.6 g/dL) and pyruvate kinase (12.4 U/g Hb) levels, splenomegaly, and hepatomegaly. Bone marrow aspirate showed a markedly decreased myeloid to erythroid ratio and hypercellular marrow particles due to hyperplasia of the erythroid elements. Molecular characterization of the proband’s genomic DNA uncovered a likely pathogenic homozygous missense variant p.[D339N] in exon 7 of the PKLR gene. In-depth in silico analysis and familial cosegregation implies p.[D339N] as the likely cause of pyruvate kinase deficiency in this family. Further in vitro or in vivo studies are required to validate the impact of p.[D339N] on protein structure and/or stability, and to determine its role in the disease pathophysiology.ConclusionsIn summary, these findings suggest a novel genetic defect in the PKLR gene as a likely cause of pyruvate kinase deficiency, thus further expanding the mutational landscape of this rare Mendelian disorder.

Novel PKLR missense mutation (A300P) causing pyruvate kinase deficiency in an Omani Kindred—PK deficiency masquerading as congenital dyserythropoietic anemia

We report herein a child with transfusion‐dependent chronic anemia, the cause of which was difficult to establish because of his transfusion dependency. The clinical and laboratory features suggested a chronic nonspherocytic hemolytic anemia (CNSHA) with bone marrow features suggestive of congenital dyserythropoietic anemia (CDA). DNA studies, however, revealed the underlying condition to be due to a novel mutation in the PKLR gene responsible for pyruvate kinase deficiency (PKD). Molecular investigations by a targeted next‐generation sequencing (t‐NGS) using a custom panel of 71 genes involved in the red blood cell (RBC) disorders revealed that the patient was homozygous for a novel missense mutation c.898G>C, p.Ala300Pro, whereas both his parents were heterozygous for the same mutation.

Haemoglobin Bristol-Alesha in a child with non-spherocytic severe haemolytic anaemia and marked anisochromic poikilocytosis with basophilic stippling and amorphous intracellular content

Here we describe a retrospective study of a 10-year-old girl, adopted from India, and referred to the Rare Anemias Unit for the diagnosis of a severe haemolytic anaemia of unknown etiology. Blood film examination revealed markedly abnormal red cell morphology characterised by a mixture of very pale (hypochromic) cells with basophilic stippling and macrocytic cells containing coarse basophilic dots and an amorphous material of unknown origin. With a presumptive diagnosis of pyruvate kinase deficiency (PK), the patient had been splenectomised at 7 years of age with a partial recovery of the anaemia and a decrease of the blood transfusion rate. Three years after splenectomy, the patient was revisited and a haemoglobin stability test was performed with a positive result. Accordingly, the correct diagnosis was an unstable haemoglobinopathy. Targeted next generation sequencing (t-NGS) revealed haemoglobin Bristol-Alesha, a hyper unstable haemoglobinopathy associated with severe haemolytic anaemia. Since unstable haemoglobins do not necessarily have specific red cell morphological abnormalities, our findings reinforce the need to include, the haemoglobin stability test, in the first diagnostic approach of hemolytic anaemias of unknown etiology,

Clinical Outcome and Genotype Analysis of 4 Chinese Children with Pyruvate Kinase Deficiency

Clinical Outcome and Genotype Analysis of 4 Chinese Children with Pyruvate Kinase Deficiency